# Patent

## Title
Compositions and method for inhibiting hepcidin antimicrobial peptide (HAMP) or HAMP-related gene expression

## Abstract

The invention relates to lipid formulated double-stranded ribonucleic acid (dsRNA) targeting a hepcidin antimicrobial peptide (HAMP) and/or HAMP-related gene, and methods of using the dsRNA to inhibit expression of HAMP and/or HAMP-related genes.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application Ser. No. 61/499,516, filed Jun. 21, 2011, and claims the benefit of U.S. Provisional Application Ser. No. 61/569,054, filed Dec. 9, 2011; each of which are incorporated herein by reference, in their entirety, for all purposes.


REFERENCE TO SEQUENCE LISTING
This application includes a Sequence Listing submitted electronically as a text file named 20072PCT_CRF_.txt, created on Jul. 17, 2012, with a size of 519,357 bytes. The sequence listing is incorporated by reference.
FIELD
The disclosure relates to double-stranded ribonucleic acid (dsRNA) targeting HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1, and methods of using dsRNA to inhibit expression of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1.
BACKGROUND
The discovery of the hepcidin peptide and characterization of its gene, HAMP, has led to the revision of previous models for the regulation of iron homeostasis and the realization that the liver plays a key role in determining iron absorption from the gut and iron release from recycling and storage sites. In summary, the hepcidin model proposes that the rate of iron efflux into the plasma depends primarily on the plasma level of hepcidin; when iron levels are high the synthesis of hepcidin increases and the release of iron from enterocytes and macrophages is diminished. Conversely when iron stores drop, the synthesis of hepcidin is down-regulated and these cells release more iron. Hepcidin directly binds to ferroportin and decreases its functional activity by causing it to be internalized from the cell surface and degraded.
Hepcidin provides a unifying hypothesis to explain the behavior of iron in two diverse but common clinical conditions, the anemia of chronic disease and both HFE and non-HFE haemochromatosis. The pathophysiology of hepcidin has been sufficiently elucidated to offer promise of therapeutic intervention in both of these situations. Administering either hepcidin or an agonist could treat haemochromatosis, where the secretion of hepcidin is abnormally low.
The anemia of inflammation, commonly observed in patients with chronic infections, malignancy, trauma, and inflammatory disorders, is a well-known clinical entity. Until recently, little was understood about its pathogenesis. It now appears that the inflammatory cytokine IL-6 induces production of hepcidin, an iron-regulatory hormone that may be responsible for most or all of the features of this disorder. (Andrews N C. J Clin Invest. 2004 May 1; 113(9): 1251-1253). As such, down regulation of hepcidin in anemic patients will lead to a reduction in inflammation associated with such anemia.
Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
The following publications disclose dsRNA (siRNA) targeting the HAMP gene and are herein incorporated by reference for all purposes: WO 2008/036933 (International application no. PCT/US2007/079212, filed Sep. 21, 2007); US 2009-0209478 (U.S. patent application Ser. No. 11/859,288, filed Sep. 21, 2007); US 2010-0204307 (U.S. patent application Ser. No. 12/757,497, filed Apr. 9, 2010); US 2011-0269823 (U.S. patent application Ser. No. 13/184,087, filed Jul. 15, 2011).



DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the HAMP1 mRNA levels in mouse liver following various dosages of siRNA and the serum iron concentration (μg/dL) following various dosages of siRNA in mice.
FIG. 2 shows the HAMP mRNA levels in liver following siRNA administration as well as the serum iron concentration (μg/dL) and the HAMP serum protein concentration (mg/mL) following siRNA administration to non-human primates.
FIG. 3 shows the HAMP1 and TFR2 mRNA levels in mouse liver following various dosages of siRNA and the percent (%) transferrin saturation following various dosages of siRNA.
FIG. 4 shows the HAMP1 and TFR2 mRNA levels in mouse liver following administration of siRNA and the percent (%) transferrin saturation over a 30 day time course.
FIG. 5 shows the HAMP1 and TFR2 mRNA levels in rat liver following administration of siRNA. FIG. 5 also shows the serum iron and Hb concentrations in rats at various time points.
FIG. 6 shows the level of HAMP mRNA reduction in the liver of each animal following siRNA administration, compared to PBS controls.
FIG. 7 shows the level of TFR2 mRNA reduction in the liver of each animal following siRNA administration, compared to PBS controls.
FIG. 8 shows that serum iron concentration was increased in each animal after 1 mg/kg AD-52590 siRNA administration.
FIG. 9 shows that the HAMP serum protein concentration was decreased in each animal following 1 mg/kg AD-52590 siRNA administration.
FIG. 10 shows combinatorial use of dsRNAs targeting different HAMP-related mRNAs (HFE and TFR2) in vivo. FIG. 10A shows relative mRNA levels for HFE (left bar), TFR2 (middle bar), and HAMP (right bar) for each group. FIG. 10B shows UIBC (μg/dL) for each group. FIG. 10C shows the percent transferring saturation for each group. FIG. 10D shows serum iron concentration (μg/dL) for each group.



SUMMARY
Disclosed herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of hepcidin antimicrobial peptide (HAMP), wherein said dsRNA is selected from the dsRNAs listed in Table 2, 3, 4, or 5 with a start position of 379, 380, 382, or 385. In some aspects, the dsRNA consists of a dsRNA listed in Table 2, 3, 4, or 5 with a start position of 382.
Also described herein is a dsRNA for inhibiting expression of HAMP, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a HAMP mRNA transcript, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 2, 3, 4, or 5.
In some aspects, the region of complementarity is at least 17 nucleotides in length. In some aspects, the region of complementarity is between 19 and 21 nucleotides in length. In some aspects, the region of complementarity is 19 nucleotides in length. In some aspects, the region of complementarity consists of one of the antisense strand sequences of Table 2, 3, 4, or 5.
In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 2, 3, 4, or 5. In some aspects, the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the sense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the sense strand comprises one of the sense strand sequences of Table 2, 3, 4, or 5. In some aspects, the antisense strand comprises one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the sense strand comprises one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand comprises one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the sense strand consists of one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand consists of one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the dsRNA mediates degradation of HAMP mRNA.
In some aspects, said dsRNA further comprises at least one modified nucleotide. In some aspects, at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. In some aspects, said modified nucleotide is chosen from the group consisting of: a 2′-fluoro modified nucleotide, a 2′-fluoro modified nucleoside, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
In some aspects, each strand is no more than 30 nucleotides in length. In some aspects, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In some aspects, at least one strand comprises a 3′ overhang of at least 2 nucleotides. In some aspects, each strand comprises a 3′ overhang of 2 nucleotides.
In some aspects, a dsRNA described above further comprises a ligand. In some aspects, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA. In some aspects, the dsRNA further comprises an N-Acetyl-Galactosamine (GalNac) conjugate.
In some aspects, a dsRNA described above is formulated in a nucleic acid lipid particle formulation. In some aspects, the nucleic acid lipid particle formulation is selected from Table A. In some aspects, the nucleic acid lipid particle formulation comprises MC3.
Also described herein is a cell comprising a dsRNA described above.
Also described herein is a vector encoding at least one strand of a dsRNA described above.
Also described herein is a cell comprising a vector described above.
Also described herein is a pharmaceutical composition for inhibiting expression of a HAMP gene comprising a dsRNA described above. In some aspects, the composition further comprises a lipid formulation. In some aspects, the lipid formulation is a nucleic acid lipid particle formulation.
Also described herein is a dsRNA for inhibiting expression of hemojuvelin (HFE2), wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a HFE2 mRNA transcript, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10A.
In some aspects, the region of complementarity is at least 17 nucleotides in length. In some aspects, the region of complementarity is between 19 and 21 nucleotides in length. In some aspects, the region of complementarity is 19 nucleotides in length. In some aspects, the region of complementarity consists of one of the antisense strand sequences of Table 10A.
In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10A. In some aspects, the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10A. In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10A and the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10A. In some aspects, the sense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the sense strand sequences of Table 10A and the antisense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the antisense strand sequences of Table 10A. In some aspects, the sense strand comprises one of the sense strand sequences of Table 10A. In some aspects, the antisense strand comprises one of the antisense strand sequences of Table 10A. In some aspects, the sense strand comprises one of the sense strand sequences of Table 10A and the antisense strand comprises one of the antisense strand sequences of Table 10A. In some aspects, the sense strand consists of one of the sense strand sequences of Table 10A and the antisense strand consists of one of the antisense strand sequences of Table 10A. In some aspects, the dsRNA mediates degradation of HFE2 mRNA.
In some aspects, said dsRNA further comprises at least one modified nucleotide. In some aspects, at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. In some aspects, said modified nucleotide is chosen from the group consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
In some aspects, each strand is no more than 30 nucleotides in length. In some aspects, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In some aspects, at least one strand comprises a 3′ overhang of at least 2 nucleotides. In some aspects, each strand comprises a 3′ overhang of 2 nucleotides.
In some aspects, a dsRNA described above further comprises a ligand. In some aspects, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA. In some aspects, a dsRNA described above further comprises a GalNac conjugate.
In some aspects, the dsRNA is formulated in a nucleic acid lipid particle formulation. In some aspects, the nucleic acid lipid particle formulation is selected from Table A. In some aspects, the nucleic acid lipid particle formulation comprises MC3.
Also described herein is a cell comprising a dsRNA described above.
Also described herein is a vector encoding at least one strand of a dsRNA described above.
Also described herein is a cell comprising a vector described above.
Also described herein is a pharmaceutical composition for inhibiting expression of a HFE2 gene comprising a dsRNA described above. In some aspects, the composition further comprises a lipid formulation. In some aspects, the lipid formulation is a nucleic acid lipid particle formulation.
Also described herein is a dsRNA for inhibiting expression of transferrin receptor 2 (TFR2), wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a TFR2 mRNA transcript, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10B or 13.
In some aspects, the region of complementarity is at least 17 nucleotides in length. In some aspects, the region of complementarity is between 19 and 21 nucleotides in length. In some aspects, the region of complementarity is 19 nucleotides in length. In some aspects, the region of complementarity consists of one of the antisense strand sequences of Table 10B or 13.
In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10B or 13. In some aspects, the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10B or 13. In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10B or 13 and the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10B or 13. In some aspects, the sense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the sense strand sequences of Table 10B or 13 and the antisense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the antisense strand sequences of Table 10B or 13. In some aspects, sense strand comprises one of the sense strand sequences of Table 10B or 13. In some aspects, the antisense strand comprises one of the antisense strand sequences of Table 10B or 13. In some aspects, the sense strand comprises one of the sense strand sequences Of Table 10B or 13 and the antisense strand comprises one of the antisense strand sequences of Table 10B or 13. In some aspects, the sense strand consists of one of the sense strand sequences of Table 10B or 13 and the antisense strand consists of one of the antisense strand sequences of Table 10B or 13. In some aspects, the dsRNA mediates degradation of TFR2 mRNA.
In some aspects, said dsRNA further comprises at least one modified nucleotide. In some aspects, at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. In some aspects, said modified nucleotide is chosen from the group consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
In some aspects, each strand is no more than 30 nucleotides in length. In some aspects, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In some aspects, at least one strand comprises a 3′ overhang of at least 2 nucleotides. In some aspects, each strand comprises a 3 overhang of 2 nucleotides.
In some aspects, a dsRNA described above further comprises a ligand. In some aspects, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA. In some aspects, a dsRNA described above further comprises a GalNac conjugate.
In some aspects, the dsRNA is formulated in a nucleic acid lipid particle formulation. In some aspects, the nucleic acid lipid particle formulation is selected from Table A. In some aspects, the nucleic acid lipid particle formulation comprises MC3.
Also described herein is a cell comprising a dsRNA described above.
Also described herein is a vector encoding at least one strand of a dsRNA described above.
Also described herein is a cell comprising a vector described above.
Also described herein is a pharmaceutical composition for inhibiting expression of a TFR2 gene comprising a dsRNA described above. In some aspects, the composition further comprises a lipid formulation. In some aspects, the lipid formulation is a nucleic acid lipid particle formulation.
Also described herein is a composition comprising a first dsRNA for inhibiting expression of a HAMP gene and a second dsRNA for inhibiting expression of an HFE2 gene, wherein the first dsRNA comprises a first sense strand and an first antisense strand, the first antisense strand comprising a region of complementarity to a HAMP mRNA transcript, wherein the first antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 2, 3, 4, or 5; and wherein the second dsRNA comprises a second sense strand and a second antisense strand, the second antisense strand comprising a region of complementarity to a HFE2 mRNA transcript, wherein the second antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10A.
Also described herein is a composition comprising a first dsRNA for inhibiting expression of a HAMP gene and a second dsRNA for inhibiting expression of an TFR2 gene, wherein said first dsRNA comprises a first sense strand and a first antisense strand, the first antisense strand comprising a region of complementarity to a HAMP mRNA transcript, wherein the first antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 2, 3, 4, or 5; and wherein said second dsRNA comprises a second sense strand and a second antisense strand, the second antisense strand comprising a region of complementarity to a TFR2 mRNA transcript, wherein the second antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10B or 13.
Also described herein is a composition comprising a first dsRNA for inhibiting expression of a TFR2 gene and a second dsRNA for inhibiting expression of a HFE2 gene, wherein said first dsRNA comprises a first sense strand and a first antisense strand, the first antisense strand comprising a region of complementarity to a TFR2 mRNA transcript, wherein the first antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10B or 13; and wherein said second dsRNA comprises a second sense strand and a second antisense strand, the second antisense strand comprising a region of complementarity to a HFE2 mRNA transcript, wherein the second antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10A.
Also described herein is a composition comprising a plurality of dsRNAs selected From the dsRNAs described above.
Also described herein is a method of inhibiting HAMP expression in a cell, the Method comprising: (a) introducing into the cell a dsRNA described above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a HAMP gene, thereby inhibiting expression of the HAMP gene in the cell. In some aspects, the HAMP expression is inhibited by at least 30%. In some aspects, the HAMP expression is inhibited by at least 80%.
Also described herein is a method of treating a disorder associated with HAMP expression comprising administering to a subject in need of such treatment a therapeutically effective amount of a dsRNA described above.
In some aspects, the subject has anemia. In some aspects, the subject has refractory anemia. In some aspects, the subject has anemia of chronic disease (ACD). In some aspects, the subject has iron-restricted erythropoiesis. In some aspects, the subject is a human.
In some aspects, the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.
In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.
Also described herein is a method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of a HAMP dsRNA described above.
In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is administered intravenously. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.
In some aspects, the subject is a primate or a rodent. In some aspects, the subject is a human.
In some aspects, the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.
In some aspects, the subject has fatigue, shortness of breath, headache, dizziness, or pale skin. In some aspects, the subject has reduced iron levels compared to a subject without anemia. In some aspects, the subject has haemoglobin (Hb) levels <9 g/dL. In some aspects, the subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA). In some aspects, the subject has reduced renal erythropoietin (EPO) synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD. In some aspects, the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD. In some aspects, the subject has a reticulocyte Hb of <28 pg. In some aspects, the subject has >10% hypochromic red blood cells (RBCs). In some aspects, the method further comprises determining the complete blood count (CBC), serum iron concentration, Transferrin (Tf) saturation, or ferritin levels of the subject.
In some aspects, administering results in an increase in iron levels in the subject. In some aspects, administering results in a 2-fold increase in iron levels in the subject. In some aspects, administering results in an increase in Tf saturation in the subject.
In some aspects, the method further comprises determining the iron level in the subject. In some aspects, the method further comprises administering intravenous iron or ESAs to the subject.
Also described herein is a method of inhibiting HFE2 expression in a cell, the method comprising: (a) introducing into the cell a dsRNA described above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a HFE2 gene, thereby inhibiting expression of the HFE2 gene in the cell. In some aspects, the HFE2 expression is inhibited by at least 30%. In some aspects, the HFE2 expression is inhibited by at least 80%.
Also described herein is a method of treating a disorder associated with HFE2 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of a dsRNA described above.
In some aspects, the subject has anemia. In some aspects, the subject has refractory anemia. In some aspects, the subject has anemia of chronic disease (ACD). In some aspects, the subject has iron-restricted erythropoiesis. In some aspects, the subject is a human.
In some aspects, the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.
In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.
Also described herein is a method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of a HFE2 dsRNA described above.
In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is administered intravenously. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.
In some aspects, the subject is a primate or a rodent. In some aspects, the subject is a human.
In some aspects, the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.
In some aspects, the subject has fatigue, shortness of breath, headache, dizziness, or pale skin. In some aspects, the subject has reduced iron levels compared to a subject without anemia. In some aspects, the subject has haemoglobin (Hb) levels <9 g/dL. In some aspects, the Subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA). In some aspects, the subject has reduced renal erythropoietin (EPO) synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD. In some aspects, the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD. In some aspects, the subject has a reticulocyte Hb of <28 pg. In some aspects, the subject has >10% hypochromic red blood cells (RBCs). In some aspects, the method further comprises determining the complete blood count (CBC), serum iron concentration. Transferrin (Tf) saturation, or ferritin levels of the subject.
In some aspects, administering results in an increase in iron levels in the subject. In some aspects, administering results in a 2-fold increase in iron levels in the subject. In some aspects, administering results in an increase in Tf saturation in the subject.
In some aspects, the method further comprises determining the iron level in the subject. In some aspects, the method further comprises administering intravenous iron or ESAs to the subject.
Also described herein is a method of inhibiting TFR2 expression in a cell, the method comprising: (a) introducing into the cell a dsRNA described above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a TFR2 gene, thereby inhibiting expression of the TFR2 gene in the cell. In some aspects, the TFR2 expression is inhibited by at least 30%. In some aspects, the TFR2 expression is inhibited by at least 80%.
Also described herein is a method of treating a disorder associated with TFR2 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of a dsRNA described above.
In some aspects, the subject has anemia. In some aspects, the subject has refractory anemia. In some aspects, the subject has anemia of chronic disease (ACD). In some aspects, the subject has iron-restricted erythropoiesis. In some aspects, the subject is a human.
In some aspects, the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.
In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.
Also described herein is a method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of a TFR2 dsRNA described above.
In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is administered intravenously. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.
In some aspects, the subject is a primate or a rodent. In some aspects, the subject is a human.
In some aspects, the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.
In some aspects, the subject has fatigue, shortness of breath, headache, dizziness, or Pale skin. In some aspects, the subject has reduced iron levels compared to a subject without anemia. In some aspects, the subject has haemoglobin (Hb) levels <9 g/dL. In some aspects, the subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA). In some aspects, the subject has reduced renal erythropoietin (EPO) synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD. In some aspects, the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD. In some aspects, the subject has a reticulocyte Hb of <28 pg. In some aspects, the subject has >10% hypochromic red blood cells (RBCs). In some aspects, the method further comprises determining the complete blood count (CBC), serum iron concentration, Transferrin (Tf) saturation, or ferritin levels of the subject.
In some aspects, administering results in an increase in iron levels in the subject. In some aspects, administering results in a 2-fold increase in iron levels in the subject. In some aspects, administering results in an increase in Tf saturation in the subject.
In some aspects, the method further comprises determining the iron level in the subject. In some aspects, the method further comprises administering intravenous iron or ESAs to the subject.
Also described herein is a method of inhibiting HAMP, HFE2, and/or TFR2 expression in a cell, the method comprising: (a) introducing into the cell a plurality of dsRNAs selected from the dsRNAs described above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a HAMP, HFE2, and/or TFR2 gene, thereby inhibiting expression of the HAMP, HFE2, and/or TFR2 gene in the cell.
In some aspects, the plurality of dsRNAs are introduced simultaneously. In some aspects, the plurality of dsRNAs are introduced concurrently. In some aspects, the plurality of dsRNAs are introduced individually. In some aspects, the plurality of dsRNAs are introduced together. In some aspects, the expression is inhibited by at least 30%. In some aspects, the expression is inhibited by at least 80%.
Also described herein is a method of treating a disorder associated with HAMP, HFE2, and/or TFR2 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of a plurality of dsRNAs selected from the dsRNAs described above.
In some aspects, the plurality of dsRNAs are administered to the subject simultaneously. In some aspects, the plurality of dsRNAs are administered to the subject concurrently. In some aspects, the plurality of dsRNAs are administered to the subject individually. In some aspects, the plurality of dsRNAs are administered to the subject together.
In some aspects, the subject has anemia. In some aspects, the subject has refractory anemia. In some aspects, the subject has anemia of chronic disease (ACD). In some aspects, the subject has iron-restricted erythropoiesis. In some aspects, the subject is a human. In some aspects, the plurality is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.
Also described herein is a method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of a plurality of dsRNAs selected from the dsRNAs described above.
In some aspects, the plurality of dsRNAs are administered to the subject simultaneously. In some aspects, the plurality of dsRNAs are administered to the subject concurrently. In some aspects, the plurality of dsRNAs are administered to the subject individually. In some aspects, the plurality of dsRNAs are administered to the subject together.
In some aspects, the plurality is lipid formulated. In some aspects, the plurality is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the plurality is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the plurality is administered intravenously. In some aspects, the plurality is lipid formulated in a formulation selected from Table A. In some aspects, the plurality is conjugated to GalNac. In some aspects, the plurality is conjugated to GalNac and administered subcutaneously. In some aspects, the plurality is administered subcutaneously.
In some aspects, the subject is a primate or a rodent. In some aspects, the subject is a human.
In some aspects, the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.
In some aspects, the subject has fatigue, shortness of breath, headache, dizziness, or pale skin. In some aspects, the subject has reduced iron levels compared to a subject without anemia. In some aspects, the subject has haemoglobin (Hb) levels <9 g/dL. In some aspects, the subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA). In some aspects, the subject has reduced renal erythropoietin (EPO) synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD. In some aspects, the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD. In some aspects, the subject has a reticulocyte Hb of <28 pg. In some aspects, the subject has >10% hypochromic red blood cells (RBCs). In some aspects, the method further comprises determining the complete blood count (CBC), serum iron concentration, Transferrin (Tf) saturation, or ferritin levels of the subject.
In some aspects, administering results in an increase in iron levels in the subject. In some aspects, administering results in a 2-fold increase in iron levels in the subject. In some aspects, administering results in an increase in Tf saturation in the subject.
In some aspects, the method further comprises determining the iron level in the subject. In some aspects, the method further comprises administering intravenous iron or ESAs to the subject.
DETAILED DESCRIPTION
The details of one or more embodiments are set forth in the description below. Other features, objects, and advantages will be apparent from the description and the drawings, and from the claims.
Provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of HAMP in a cell or a mammal where the dsRNA targets HAMP. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of HAMP. A HAMP dsRNA directs the sequence-specific degradation of HAMP mRNA.
Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of HFE2 in a cell or a mammal where the dsRNA targets HFE2. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of HFE2. A HFE2 dsRNA directs the sequence-specific degradation of HFE2 mRNA.
Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of HFE in a cell or a mammal where the dsRNA targets HFE. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of HFE. A HFE dsRNA directs the sequence-specific degradation of HFE mRNA.
Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting The expression of TFR2 in a cell or a mammal where the dsRNA targets TFR2. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of TFR2. A TFR2 dsRNA directs the sequence-specific degradation of TFR2 mRNA.
Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of BMPR1a in a cell or a mammal where the dsRNA targets BMPR1a. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of BMPR1a. A BMPR1a dsRNA directs the sequence-specific degradation of BMPR1a mRNA.
Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of SMAD4 in a cell or a mammal where the dsRNA targets SMAD4. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of SMAD4. A SMAD4 dsRNA directs the sequence-specific degradation of SMAD4 mRNA.
Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting The expression of IL6R in a cell or a mammal where the dsRNA targets IL6R. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of IL6R. An IL6R dsRNA directs the sequence-specific degradation of IL6R mRNA.
Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of BMP6 in a cell or a mammal where the dsRNA targets BMP6. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of BMP6. A BMP6 dsRNA directs the sequence-specific degradation of BMP6 mRNA.
Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting The expression of NEO1 in a cell or a mammal where the dsRNA targets NEO1. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of NEO1. A NEO1 dsRNA directs the sequence-specific degradation of NEO1 mRNA.
Definitions
For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
As used herein, “HAMP” refers to the hepcidin antimicrobial peptide gene, transcript, or protein (also known as LEAP). A human mRNA sequence for HAMP is Genbank accession NM—021175.2, included below as SEQ ID NO:1. Other examples of mammalian HAMP sequences are shown in Table B.
As used herein. “HFE2” refers to hemojuvelin gene, transcript, or protein. Examples of mammalian HFE2 sequences are shown in Table B.
As used herein. “TFR2” refers to transferrin receptor 2 gene, transcript, or protein. Examples of mammalian TFR2 sequences are shown in Table B.
As used herein, “HFE” refers to hemochromatosis gene, transcript, or protein. Examples of mammalian HFE sequences are shown in Table B.
As used herein, “BMPR1a” refers to bone morphogenetic protein receptor, type 1A gene, transcript, or protein. Examples of mammalian BMPR1a sequences are shown in Table B.
As used herein, “SMAD4” refers to SMAD family member 4 gene, transcript, or protein. Examples of mammalian SMAD4 sequences are shown in Table B.
As used herein, “IL6R” refers to interleukin 6 receptor gene, transcript, or protein. Examples of mammalian IL6R sequences are shown in Table B.
As used herein, “BMP6” refers to bone morphogenetic protein 6 gene, transcript, or protein. Examples of mammalian BMP6 sequences are shown in Table B.
As used herein, “NEO1” refers to neogenin homolog 1 gene, transcript, or protein. Examples of mammalian NEO1 sequences are shown in Table B.
As used herein, “HAMP-related” refers to a HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, transcript, or protein.
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, including mRNA that is a product of RNA processing of a primary transcription product.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
This includes base-pairing of the oligonucleotide or polynucleotide comprising the First nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as complementary with respect to a second sequence herein, the two sequences can be fully complementary, or they may be “substantially complementary,” e.g., they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.
“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing.
The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1) including a 5′ UTR, an open reading frame (ORF), or a 3′ UTR. For example, a polynucleotide is complementary to at least a part of a HAMP mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding HAMP.
The term “double-stranded RNA” or “dsRNA,” as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.
The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. The term “siRNA” is also used herein to refer to a dsRNA as described above.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.
The term “antisense strand” refers to the strand of a dsRNA which includes a region that is complementary, e.g., fully complementary or substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is complementary, e.g., fully or substantially complementary to a region of the antisense strand.
The term “start position” refers to a nucleotide position on the target mRNA where the 5′ most nucleotide of a dsRNA sense strand aligns with the nucleotide position on the target mRNA. For example, a dsRNA with a start position of 382 on NM—021175.2 (SEQ ID NO:1) would include AD-11459 because position 382 on NM—021175.2 (SEQ ID NO: 1)is G and the sense sequence of AD-11459 is 5′-GAAcAuAGGucuuGGAAuAdTsdT-3′ (SEQ ID NO: 30), where G is the 5′ most nucleotide of the sense strand of AD-11459; thus G at position 382 on NM—021175.2 (SEQ ID NO:1) is the start position of AD-11459.
As used herein, the term “nucleic acid lipid particle” includes the term “SNALP” and refers to a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as a dsRNA or a plasmid from which a dsRNA is transcribed. Nucleic acid lipid particles, e.g., SNALP are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and U.S. Ser. No. 61/045,228 filed on Apr. 15, 2008. These applications are hereby incorporated by reference.
“Introducing into a cell,” when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein or known in the art.
The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of” and the like in as far as they refer to a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, herein refer to the at least partial suppression of the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, as manifested by a reduction of the amount of mRNA which may be isolated from a first cell or group of cells in which a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R. BMP6, and/or NEO1 gene is transcribed and which has or have been treated such that the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of




  
    
      
        
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Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression, e.g., the amount of protein encoded by a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g., apoptosis. In principle, HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a HAMP, HFE2, HFE, TFR2. BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide featured in the invention.
As used herein in the context of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.
As used herein, the phrases “effective amount” refers to an amount that provides a benefit in the treatment, prevention, or management of pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression or an overt symptom of pathological processes mediated by HAMP, HFE2, HFE, TFR2. BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression. The specific amount that is effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression, the patient's history and age, the stage of pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression, and the administration of other anti-pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression agents.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a pharmacologically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter. For example, a pharmacologically effective amount of a dsRNA targeting HAMP can reduce HAMP serum levels by at least 25%.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
Double-Stranded Ribonucleic Acid (dsRNA)
As described in more detail herein, the invention provides double-stranded Ribonucleic acid (dsRNA) molecules for inhibiting the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene in a cell or mammal, where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, and where the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and where said dsRNA, upon contact with a cell expressing said HAMP, HFE2, HFE, TFR2. BMPR1a, SMAD4, IL6R. BMP6, and/or NEO1 gene, inhibits the expression of said HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene by at least 30% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. Expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene can be reduced by at least 30% when measured by an assay as described in the Examples below. For example, expression of a HAMP gene in cell culture, such as in Hep3B cells, can be assayed by measuring HAMP mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by ELISA assay. The dsRNA of the invention can further include one or more single-stranded nucleotide overhangs.
The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. The dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, the other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 base pairs in length. In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different dsRNAs are used in combination, the duplex lengths can be identical or can differ. In one embodiment, the antisense strand of the dsRNA is sufficiently complementary to a target mRNA (e.g., a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 mRNA) so as to cause cleavage of the target mRNA.
Each strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each is strand is 25-30 nucleotides in length. Each strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ.
The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the sense strand. In further embodiments, the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the antisense strand. The dsRNA can include a 3′ overhang of 2 nucleotides on both the sense and antisense strands.
A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs can have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
In one embodiment, a HAMP gene is a human HAMP gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Tables 2, 3, 4,and 5, and the antisense strand is one of the antisense sequences of Tables 2, 3, 4, and 5. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 2, 3, 4, and 5 can readily be determined using the target sequence and the flanking HAMP sequence.
The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2, 3, 4, and 5, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Tables 2, 3, 4,and 5 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 2, 3, 4, and 5, and differing in their ability to inhibit the expression of a HAMP gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired HAMP target sequence can readily be made using the corresponding HAMP antisense sequence and a complementary sense sequence.
In addition, the dsRNAs provided in Tables 2, 3, 4, and 5 identify a site in a HAMP that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 2, 3, 4, and 5 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a HAMP gene.
In one embodiment, a HFE2 gene is a human HFE2 gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Table 10A, and the antisense strand is one of the antisense sequences of Table 10A. Alternative antisense agents that target elsewhere in the target sequence provided in Table 10A can readily be determined using the target sequence and the flanking HFE2 sequence.
In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 10A, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 10A minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 10A, and differing in their ability to inhibit the expression of a HFE2 gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired HFE2 target sequence can readily be made using the corresponding HFE2 antisense sequence and a complementary sense sequence.
In addition, the dsRNAs provided in Table 10A identify a site in a HFE2 that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 10A coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a HFE2 gene.
In one embodiment, a TFR2 gene is a human TFR2 gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Table 10B or 13, and the antisense strand is one of the antisense sequences of Table 10B or 13. Alternative antisense agents that target elsewhere in the target sequence provided in Table 10B or 13 can readily be determined using the target sequence and the flanking TFR2 sequence.
In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 10B or 13, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 10B or 13 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 10B or 13, and differing in their ability to inhibit the expression of a TFR2 gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired TFR2 target sequence can readily be made using the corresponding TFR2 antisense sequence and a complementary sense sequence.
In addition, the dsRNAs provided in Table 10B or 13 identify a site in a TFR2 that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 10B or 13 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a TFR2 gene.
In one embodiment, a SMAD4 gene is a human SMAD4 gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Table 15 or 16,and the antisense strand is one of the antisense sequences of Table 15 or 16. Alternative antisense agents that target elsewhere in the target sequence provided in Table 15 or 16 can readily be determined using the target sequence and the flanking SMAD4 sequence.
In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 15 or 16, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 15 or 16 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 15 or 16, and differing in their ability to inhibit the expression of a SMAD4 gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired SMAD4 target sequence can readily be made using the corresponding SMAD4 antisense sequence and a complementary sense sequence.
In addition, the dsRNAs provided in Table 15 or 16 identify a site in a SMAD4 that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 15 or 16 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a SMAD4 gene.
In one embodiment, a NEO1 gene is a human NEO1 gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Table 17 or 18, and the antisense strand is one of the antisense sequences of Table 17 or 18. Alternative antisense agents that target elsewhere in the target sequence provided in Table 17 or 18 can readily be determined using the target sequence and the flanking NEO1 sequence.
In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 17 or 18, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 17 or 18 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 17 or 18, and differing in their ability to inhibit the expression of a NEO1 gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired NEO1 target sequence can readily be made using the corresponding NEO1 antisense sequence and a complementary sense sequence.
In addition, the dsRNAs provided in Table 17 or 18 identify a site in a NEO1 that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 17 or 18 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a NEO1 gene.
In one embodiment, a BMP6 gene is a human BMP6 gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Table 21, and the antisense strand is one of the antisense sequences of Table 21. Alternative antisense agents that target elsewhere in the target sequence provided in Table 21 can readily be determined using the target sequence and the flanking BMP6 sequence.
In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 21, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 21 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 21, and differing in their ability to inhibit the expression of a BMP6 gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired BMP6 target sequence can readily be made using the corresponding BMP6 antisense sequence and a complementary sense sequence.
In addition, the dsRNAs provided in Table 21 identify a site in a BMP6 that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 21 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a BMP6 gene.
With regard to Tables 4, 10A, 10B, 13, 16, 18, and 21: It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention. “Unmodified version” refers to a sequence that does not include one or more chemical modifications, e.g., a 2′-O methyl group, a phosphorothioate, and/or a 2′-fluoro group. For example, included in the invention are unmodified versions of AD-47391, which targets HFE2. See Table 10A. Unmodified sense strand versions of AD-47391 include: AGAGUAGGGAAUCAUGGCUdTdT (SEQ ID NO: 31) and AGAGUAGGGAAUCAUGGCU (SEQ ID NO: 32). Unmodified antisense strand versions of AD-47391 include: AGCCAUGAUUCCCUACUCUdTdT (SEQ ID NO: 33)and AGCCAUGAUUCCCUACUCU (SEQ ID NO: 34). As another example, included in the invention are unmodified versions of AD-47826, which targets TFR2. See Table 10B. Unmodified sense strand versions of AD-47826 include: CAGGCAGCCAAACCUCAUUdTdT (SEQ ID NO: 35)and CAGGCAGCCAAACCUCAUU (SEQ ID NO: 36). Unmodified antisense strand versions of AD-47826 include: AAUGAGGUUUGGCUGCCUG (SEQ ID NO: 37)and AAUGAGGUUUGGCUGCCUGdTdT (SEQ ID NO: 38).
Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. The cleavage site on the target mRNA of a dsRNA can be determined using methods generally known to one of ordinary skill in the art, e.g., the 5′-RACE method described in Soutschek et al., Nature; 2004, Vol. 432, pp. 173-178 (which is herein incorporated by reference for all purposes). Included in the invention are dsRNA that cleave the RNA target at the same location as the dsRNA described in the tables herein.
The dsRNA featured in the invention can contain one or more mismatches to the target sequence. In one embodiment, the dsRNA featured in the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of a HAMP gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R. BMP6, and/or NEO1 gene is important, especially if the particular region of complementarity in a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is known to have polymorphic sequence variation within the population.
Modifications
In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Specific examples of dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference
Modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. In some instances, dsRNAs can be made with “Light Fluoro” chemical modifications as follows: all pyrimidines (cytosine and uridine) in the sense strand can be replaced with corresponding 2′-Fluoro bases (2′ Fluoro C and 2′-Fluoro U). In the antisense strand, pyrimidines adjacent to (towards 5′ position) ribo A nucleoside can be replaced with their corresponding 2-Fluoro nucleosides.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
In other suitable dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, a dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of a dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331: and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
Other embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F: O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3. SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2, also described in examples herein below.
Other preferred modifications include 2′-methoxy(2′-OCH3), 2′-aminopropoxy(2′-OCH2CH2CH2NH2) and 2′-fluoro(2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
dsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
Conjugates
Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330: Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
In some embodiments of the compositions and methods of the invention, an dsRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated dsRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g. C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).
In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as




In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:













Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,




(Formula XXIII), when one of X or Y is an oligonucleotide, the other is a hydrogen.
In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.
In some embodiments, the conjugate or ligand described herein can be attached to an dsRNA oligonucleotide with various linkers that can be cleavable or non cleavable.
The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.
A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.
A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.
Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular dsRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.
In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.
In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.
In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)— (SEQ ID NO: 13), where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.
In one embodiment, an dsRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of dsRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,







(Formula XXX), when one of X or Y is an oligonucleotide, the other is a hydrogen.
In certain embodiments of the compositions and methods of the invention, a ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.
In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):




wherein:
q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;
P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4A, T5B, T5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;
Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R′)═C(R′′), C≡C or C(O);
R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5C are each independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), —C(O)—CH(Ra)—NH—, CO, CH═N—O,




or heterocyclyl;

L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Ra is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):




wherein L5A, L5B and L5C represent a monosaccharide, such as GalNAc derivative.
Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II VII, XI, X, and XIII.
Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within a dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region.
In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306: Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.
Vector Encoded dsRNAs
In another aspect, HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 dsRNA molecules are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114,and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. NatI. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
For example, lentiviral vectors featured in the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors featured in the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
Viral vectors can be derived from AV and AAV. In one embodiment, the dsRNA featured in the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
A suitable AV vector for expressing the dsRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
The promoter driving dsRNA expression in either a DNA plasmid or viral vector featured in the invention may be a eukaryotic RNA polymerase I (e.g., ribosomal RNA promoter), RNA polymerase II (e.g., CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g., U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKOTM). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single HAMP, HFE2, HFE, TFR2. BMPR1a, SMAD4, IL6R. BMP6, and/or NEO1 gene or multiple HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 genes over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
Pharmaceutical Compositions Containing dsRNA
In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, such as pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.
The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 genes.
In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.0059 mg/kg, 0.01 mg/kg, 0.0295 mg/kg, 0.05 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.628 mg kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.
In one embodiment, the dosage is between 0.01 and 0.2 mg/kg. For example, the dsRNA can be administered at a dose of 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg 0.08 mg/kg 0.09 mg/kg, 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14 mg kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg kg, or 0.20 mg/kg.
In one embodiment, the dosage is between 0.005 mg/kg and 1.628 mg/kg. For example, the dsRNA can be administered at a dose of 0.0059 mg/kg, 0.0295 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, or 1.628 mg/kg.
In one embodiment, the dosage is between 0.2 mg/kg and 1.5 mg kg. For example, the dsRNA can be administered at a dose of 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, or 1.5 mg/kg.
The dsRNA can be administered at a dose of 0.03 mg/kg, or 0.03, 0.1, 0.2, or 0.4 mg/kg.
The pharmaceutical composition may be administered once daily or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The effect of a single dose on HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 levels is long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals, or at not more than 5, 6, 7, 8, 9, or 10 week intervals.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing human HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
The dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Administration
The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds featured in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration.
The dsRNA can be delivered in a manner to target a particular tissue.
Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the dsRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearoylphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-10 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.
Liposomal Formulations
There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising NovasomeTM I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NovasomeTM II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B 1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
Nucleic Acid Lipid Particles
In one embodiment, a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 dsRNA featured in the invention is fully encapsulated in the lipid formulation, e.g., a nucleic acid lipid particle, e.g., SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. Nucleic acid lipid particles typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). Nucleic acid lipid particles are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site).
The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.
In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. In some embodiments the lipid to dsRNA ratio can be about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 11:1.
In general, the lipid-nucleic acid particle is suspended in a buffer, e.g., PBS, for administration. In one embodiment, the pH of the lipid formulated siRNA is between 6.8 and 7.8, e.g., 7.3 or 7.4. The osmolality can be, e.g., between 250 and 350 mOsm/kg, e.g., around 300, e.g., 298, 299, 300, 301, 302, 303, 304, or 305.
The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200 or Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Cl6), or a PEG-distearyloxypropyl (C18). Other examples of PEG conjugates include PEG-cDMA (N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine), mPEG2000-DMG (mPEG-dimyrystylglycerol (with an average molecular weight of 2,000) and PEG-C-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxlpropyl-3-amine). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % of the total lipid present in the particle.
In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol % of the total lipid present in the particle.
LNP01
LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
Additional exemplary formulations are described in Table A.









TABLE A







cationic lipid/non-cationic




lipid/cholesterol/PEG-lipid conjugate



Cationic
Mol % ratios



Lipid
Lipid:siRNA ratio







SNALP
DLinDMA
DLinDMA/DPPC/Cholesterol/PEG-cDMA




(57.1/7.1/34.4/1.4)




lipid:siRNA ~7:1


S-XTC
XTC
XTC/DPPC/Cholesterol/PEG-cDMA




57.1/7.1/34.4/1.4




lipid:siRNA ~7:1


LNP05
XTC
XTC/DSPC/Cholesterol/PEG-DMG




57.5/7.5/31.5/3.5




lipid:siRNA ~6:1


LNP06
XTC
XTC/DSPC/Cholesterol/PEG-DMG




57.5/7.5/31.5/3.5




lipid:siRNA ~11:1


LNP07
XTC
XTC/DSPC/Cholesterol/PEG-DMG




60/7.5/31/1.5,




lipid:siRNA ~6:1


LNP08
XTC
XTC/DSPC/Cholesterol/PEG-DMG




60/7.5/31/1.5,




lipid:siRNA ~11:1


LNP09
XTC
XTC/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA 10:1


LNP10
ALN100
ALN100/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA 10:1


LNP11
MC3
MC-3/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA 10:1


LNP12
C12-200
C12-200/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA 10:1


LNP13
XTC
XTC/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 33:1


LNP14
MC3
MC3/DSPC/Chol/PEG-DMG




40/15/40/5




Lipid:siRNA: 11:1


LNP15
MC3
MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG




50/10/35/4.5/0.5




Lipid:siRNA: 11:1


LNP16
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP17
MC3
MC3/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP18
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 12:1


LNP19
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/35/5




Lipid:siRNA: 8:1


LNP20
MC3
MC3/DSPC/Chol/PEG-DPG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP22
XTC
XTC/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1









SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.
XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.
MC3 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), (e.g., DLin-M-C3-DMA) comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and International Application No. PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated by reference.
ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine) comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.
C12-200, i.e., Tech G1, comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.
Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. For a nucleic acid lipid formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Emulsions
The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199: Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335, Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids.
Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
Penetration Enhancers
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92: Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Carriers
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
Excipients
In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Other Components
The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more dsRNA compounds and (b) one or more anti-cytokine biologic agents which function by a non-RNAi mechanism. Examples of such biologics include, biologics that target IL1β (e.g., anakinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab, or certolizumab).
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration, as discussed above, the dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Methods for Inhibiting Expression of a HAMP, HFE2, HFE, TFR2. BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 Gene
In yet another aspect, the invention provides a method for inhibiting the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene in a mammal. The method includes administering a composition featured in the invention to the mammal such that expression of the target HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is silenced.
When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection.
Methods for Treating Diseases Caused by Expression of a HAMP, HFE2, HFE, TFR2,BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 Gene
The invention relates in particular to the use of a dsRNA targeting HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 and compositions containing at least one such dsRNA for the treatment of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1-mediated disorder or disease. For example, the compositions described herein can be used to treat anemia and other diseases associated with lowered iron levels.
Methods of Using dsRNAs Targeting HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1
In one aspect, the invention provides use of a siRNA for inhibiting the expression of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 in a mammal. The method includes administering a composition of the invention to the mammal such that expression of the target HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is decreased. In some embodiments, HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R. BMP6, and/or NEO1 expression is decreased for an extended duration, e.g., at least one week, two weeks, three weeks, or four weeks or longer. For example, in certain instances, expression of the HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a siRNA described herein. In some embodiments, the HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 60%, 70%, or 80% by administration of the siRNA. In some embodiments, the HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide.
The methods and compositions described herein can be used to treat diseases and conditions that can be modulated by down regulating HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression. For example, the compositions described herein can be used to treat anemia and other forms of iron imbalance such as refractory anemia, refractory anemia of chronic disease (ACD), iron-restricted erythropoiesis, and the pathological conditions associated with these disorders. In some aspects, ACD subjects are those who are refractory to ESAs and i.v. iron administration. In some embodiments, the method includes administering an effective amount of a siRNA disclosed herein to a patient having lower iron levels relative to a control patient.
Therefore, the invention also relates to the use of a siRNA for the treatment of a disorder or disease mediated by or related to HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression. For example, a siRNA is used for treatment of anemia.
The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression preferably results in an enhancement of iron mobilization in the mammal. In some embodiments, iron mobilization is enhanced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.
The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression preferably results in an Hb increase in the mammal. In some embodiments, Hb is increased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.
The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression preferably results in a serum iron increase in the mammal. In some embodiments, serum iron is increased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.
The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression preferably results in a transderrin (Tf) saturation increase in the mammal. In some embodiments, Tf saturation is increased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.
The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression preferably results in decreased levels of HAMP in the mammal. In some embodiments, HAMP is decreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.
The method includes administering a siRNA to the subject to be treated. The subject to be treated is generally a subject in need thereof. When the subject to be treated is a mammal, such as a human, the composition can be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, and airway (aerosol) administration. In some embodiments, the compositions are administered by intravenous infusion or injection.
The method includes administering a siRNA, e.g., a dose sufficient to depress levels of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 mRNA for at least5, more preferably 7, 10, 14, 21, 25, 30 or 40 days; and optionally, administering a second single dose of dsRNA, wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the target gene in a subject.
In one embodiment, doses of siRNA are administered not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administrations can be maintained for one, two, three, or six months, or one year or longer.
In another embodiment, administration can be provided when Hb levels reach or drop lower than a predetermined minimal level, such as less than 8 g/dL, 9 g/dL, or 10 g/dL. In some aspects, administration is continued until Hb levels are >11 g/dL, e.g, 12 g/dL.
In another embodiment, administration can be provided when a patient presents with various known symptoms of disorders such as anemia. These can include fatigue, shortness of breath, headache, dizziness, or pale skin.
In another embodiment, administration can be provided when a patient is diagnosed with anemia via CBC.
In general, the siRNA does not activate the immune system, e.g., it does not increase cytokine levels, such as TNF-alpha or IFN-alpha levels. For example, when measured by an assay, such as an in vitro PBMC assay, such as described herein, the increase in levels of TNF-alpha or IFN-alpha, is less than 30%, 20%, or 10% of control cells treated with a control dsRNA, such as a dsRNA that does not target HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1.
In an aspect, a subject can be administered a therapeutic amount of siRNA, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The siRNA can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. The administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the siRNA can reduce HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.
Before administration of a full dose of the siRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or IFN-alpha) levels.
A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given siRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.
Additional Agents and Co-Administration
In further embodiments, administration of a siRNA is administered in combination an additional therapeutic agent. The siRNA and an additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.
In one embodiment, the siRNA is administered to the patient, and then the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the siRNA and the additional therapeutic agent are administered at the same time.
In some aspects, the additional agent can include one or more Erythropoiesis-stimulating agents (ESAs). ESAs are generally known in the art. ESAs can include Erythropoietin (EPO), Epoetin alfa (Procrit/Epogen), Epoetin beta (NeoRecormon), Darbepoetin alfa (Aranesp), and Methoxy polyethylene glycol-epoetin beta (Micera). ESAs can be administered in various doses, e.g., 7,000 U/week to 30,000 U/week.
In some aspects, the additional agent can include intravenous iron. Iron can be administered in various doses known in the art.
In some aspects, two or more dsRNAs are co-administered to a subject. In one embodiment, a first dsRNA is administered to the patient, and then a second dsRNA is administered to the patient (or vice versa). In another embodiment, the first dsRNA and the second dsRNA are administered at the same time.
In some aspects, a HAMP dsRNA is co-administered with one or more dsRNAs selected from HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 dsRNAs.
In some aspects, a HFE2 dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1dsRNAs.
In some aspects, a HFE dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1dsRNAs.
In some aspects, a TFR2 dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1dsRNAs.
In some aspects, a BMPR1a dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, TFR2, SMAD4, IL6R, BMP6, and/or NEO1 dsRNAs.
In some aspects, a SMAD4 dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, TFR2, BMPR1a, IL6R. BMP6, and/or NEO1 dsRNAs.
In some aspects, an IL6R dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, BMP6, and/or NEO1dsRNAs.
In some aspects, a BMP6 dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/or NEO1dsRNAs.
In some aspects, a NEO dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/or BMP6 dsRNAs.
In another aspect, the invention features, a method of instructing an end user, e.g., a caregiver or a subject, on how to administer a siRNA described herein. The method includes, optionally, providing the end user with one or more doses of the siRNA, and instructing the end user to administer the siRNA on a regimen described herein, thereby instructing the end user.
Identification of Subjects in need of dsRNA Administration
In one aspect, the invention provides a method of treating a patient by selecting a patient on the basis that the patient is in need of iron mobilization. The method includes administering to the patient a siRNA in an amount sufficient to increase the patient's iron mobilization.
In one aspect, the invention provides a method of treating a patient by selecting a patient on the basis that the patient is in need of increased Hb levels. Such a subject can have Hb levels of <9 g/dL. The method includes administering to the patient a siRNA in an amount sufficient to increase the patient's Hb levels. Typically target Hb levels are >11 g/dL, e.g., 11 g/dL or 12 g/dL.
In some aspects, a subject is identified as having anemia. In some aspects, a subject is identified as having a refractory form of anemia. In some aspects, a subject is identified as having ACD. Such subjects can be in need of administration of a dsRNA described herein. ACD can include a form of anemia wherein the subject is refractory to ESAs and/or i.v. iron administration. Typical clinical presentation of ACD includes fatigue, shortness of breadth, headache, dizziness, and/or pale skin. ACD can also be diagnosed via a CBC test, which is generally known in the art. ACD can also be diagnosed via serum iron levels, Tf saturation, and/or ferritin levels. ACD is typically diagnosed in certain settings such as subjects with CKD, cancer, chronic inflammatory diseases such as RA, or IRIDA. In some aspects, a subject with ACD has Hb levels of less than 9 g/dL. Such subjects typically become symptomatic for ACD.
CKD can result in reduced renal EPO synthesis, dietary hematinic deficiencies, blood loss, and/or elevated hepcidin levels. The elevation in hepcidin levels can be due to decreased renal excretion and/or low grade inflammation characterized by, e.g., interleukin (IL)-6.
In some aspects, a subject is identified as having iron-restricted erythropoiesis (IRE). Such subjects can be in need of administration of a dsRNA described herein. IRE can be assessed via reticulocyte Hb (CHr). Typically a result of <28 pg suggests IRE, where normal is in the range of 28-35 pg. IRE can also be assessed via percent (%) hypochromic RBCs. Typically a result of >10% suggests IRE, where 1-5% is generally considered normal.
A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering a siRNA. In addition, a test may be performed to determine a geneotype or phenotype. For example, a DNA test may be performed on a sample from the patient, e.g., a blood sample, to identify the relevant genotype and/or phenotype before a dsRNA is administered to the patient. In another embodiment, a test is performed to identify a related genotype and/or phenotype.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the dsRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
dsRNA Synthesis
Source of Reagents
Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
siRNA Synthesis
Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim. Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use.
Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 1.







TABLE 1







Abbreviations










Abbreviation
Nucleotide(s)






A
adenosine-3′-phosphate



C
cytidine-3′-phosphate



G
guanosine-3′-phosphate



U
uridine-3′-phosphate



N
any nucleotide (G, A, C, or T)



a
2′-O-methyladenosine-3′-phosphate



c
2′-O-methylcytidine-3′-phosphate



g
2′-O-methylguanosine-3′-phosphate



u
2′-O-methyluridine-3′-phosphate



T, dT
2′-deoxythymidine-3′-phosphate



sT; sdT
2′-deoxy-thymidine-5′phosphate-phosphorothioate



Af
2′-fluoroadenosine-3′-phosphate



Cf
2′-fluorocytidine-3′-phosphate



Gf
2′-fluoroguanosine-3′-phosphate



Uf
2′-fluorouridine-3′-phosphate









Example 2
HAMP siRNA Design
Transcripts
siRNA design was carried out to identify siRNAs targeting human, cynomolgus monkey (Macaca fascicularis; herein “cyno”), mouse, and rat HAMP transcripts annotated in the NCBI Gene database ncbi.nlm.nih.gov/gene website. In mouse, the HAMP gene is duplicated, yielding distinct HAMP1 and HAMP2 loci; duplex designs targeted only HAMP1. Design used the following transcripts from the NCBI RefSeq and GenBank collections: Human—NM—021175.2 (SEQ ID NO:1); Cyno—EU076443.1; Mouse—NM—032541.1; Rat—NM—053469.1. Due to the short length of the HAMP transcripts and the high degree of primate/rodent HAMP sequence divergence, siRNA duplexes were designed in multiple separate batches. The separate batches are listed below and matched the various species as follows:

    
    
        human and cyno HAMP, exactly;
        only human HAMP, exactly;
        human and cyno HAMP, with mismatches to HAMP in both species allowed at sense-strand position 19 when a G or C HAMP targeting-nucleotide was replaced with a U or A, i.e. “UA-swap”;
        human and cyno HAMP, with exact match to human HAMP and mismatches to cyno HAMP allowed at sense-strand positions 1, 2, and 19, i.e. “mismatch-to-cyno”;
        mouse HAMP1, exactly;
        only rat HAMP, exactly.
    
    


All siRNA duplexes were designed that shared 100% identity with all listed human, cyno, mouse, or rat transcripts with the exception(s) of designated mismatched-to-target bases. Unless otherwise noted, duplexes themselves were 100% complementary and double-stranded.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.
siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand.
Table 2 provides the sequences of the sense and antisense strands of 42 duplexes targeting the 3′UTR of the human HAMP gene.
Table 3 provides the sequences of the sense and antisense strands of 47 duplexes targeting the CDS of the human HAMP gene.
Table 4 provides the sequences of the sense and antisense strands of the modified duplexes targeting the HAMP gene.
Table 5 provides the sequences of the sense and antisense strands of the unmodified version of the duplexes shown in Table 4.
The antisense-derived human/cyno, mouse, rat, UA-swap, and mismatch-to-cyno oligonucleotides shown in Tables 3-4 were synthesized and formed into duplexes.
In some instances the duplexes contained no chemical modifications (unmodified).
In some instances the duplexes contained modifications (modified). For example, some duplexes were made with “Light Fluoro” chemical modifications as follows: all pyrimidines (cytosine and uridine) in the sense strand were replaced with corresponding 2′-Fluoro bases (2′ Fluoro C and 2′-Fluoro U). In the antisense strand, pyrimidines adjacent to (towards 5′ position) ribo A nucleoside was replaced with their corresponding 2-Fluoro nucleosides.
Example 3
HAMP siRNA Screening
Cell Culture and Transfections:
Dual Luciferase System:
COS 7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in DMEM (Gibco) supplemented with 10% FBS before being released from the plate by trypsinization. Cells were transfected with a psiCHECK2 vector (Promega) containing the human HAMP open reading frame (ORF). The ORF was introduced following the stop codon in the renilla luciferase sequence. Plasmid transfection was carried out by adding 19.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and 2.5 ng plasmid into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media containing  ̃2×104 COS7 cells were then added. Cells were incubated for three hours, after which the media was removed from the wells and replaced with 80 μl of complete growth media. Transfection of siRNA was accomplished out by preparing adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a new 96-well plate and incubated at room temperature for 15 minutes. The 20 μl volumes containing the lipoplexes were then added over the culture plates and incubated for 48 hours. Single dose experiments were performed at final concentrations of 10 nM and 0.1 nM. An additional concentration of 0.01 nM was performed for selected duplexes. Final duplex concentrations for dose response experiments were 10, 1.67, 0.278, 0.046, 0.0077, 0.0012, 0.0002, and 0.000035 nM.
Endogenous System (Human):
For HAMP, HepG2 cells were used. HepG2 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in MEM (Gibco) supplemented with 10% FBS before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing  ̃2×104 HepG2 cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at final concentrations of 10 nM and 0.1 nM. An additional concentration of 0.0 nM was performed for selected duplexes. Final duplex concentrations for dose response experiments were 10, 1.67, 0.278, 0.046, 0.0077, 0.0012, 0.0002, and 0.000035 nM.
Endogenous System (Cynomolgus):
Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. Primary cynomolgus hepatocytes (M003055-P, Celsis) were thawed and prepared in InVitroGRO CP plating medium (Z99029, Celsis). 80 μl of complete growth media without antibiotic containing  ̃2×104 cynomolgus hepatocytes were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at final concentrations of 10 nM and 0.1 nM. Final duplex concentrations for dose response experiments were 10, 1.67, 0.278, 0.046, 0.0077, 0.0012, 0.0002, and 0.000035 nM.
Dual Luciferase Assay (Promega Part E2980):
For cells transfected with the psiCHECK2 vector containing the human HAMP ORF, the Dual Luciferase assay was performed to measure reduction in HAMP levels. Forty-eight hours after transfection, the media was removed over the cells, and cells received 150 uL of a 1:1 mixture of complete growth medium and Dual-Glo Luciferase Reagent. As a control, these reagents were also added to empty wells; data derived from these samples were thus used as a blank measurement. Cells were then incubated for 30 minutes at room temperature on a shaker, protected from light. At this time, luminescence was determined using a SpectraMax M5 (Molecular Devices) with an integration time of 500 ms, and resulting data defined as the firefly luciferase signal. Following measurement, 75 μL of Dual-Glo Stop & Glo Reagent was added and the plates incubated in the dark at room temperature, without shaking. After an additional 10 minutes luminescence was again measured as above, and resulting data defined as the renilla luciferase signal. Data were background-subtracted, and the renilla values normalized to the firefly Luciferase values. Data were then expressed as percent mock-transfected or percent AD-1955.
Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12):
Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):
A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
Real Time PCR:
For human HAMP, 2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl HAMP TaqMan probe (Applied Biosystems cat #Hs00221783_m1 https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=ABAssayDe tailDisplay&assayed=Hs01127366 m1) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). For cynomolgus HAMP, 2 μl of cDNA were added to a master mix containing 0.5 μl 18s TaqMan Probe (Applied Biosystems Cat #4319413 E), 0.1 μl 10× custom cynomolgus HAMP probe (Forward primer: CTCCGTTTTCCCACAACA (SEQ ID NO: 39); Reverse primer: CAGCACATCCCACACTTT (SEQ ID NO: 40); Probe: ACCCACTTCCCCATCTGCATT (SEQ ID NO: 41)), and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.
To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 100 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with 10 nM AD-1955, mock transfected, or to the average lowest dose.
Table 6 shows the HAMP single dose screening data of the modified duplexes using the dual luciferase assay. Data are expressed as a percent of mock or AD-1955.
Table 7 shows the HAMP single dose screening data of the unmodified duplexes using the human endogenous assay. Data are expressed as a percent of mock.
Table 8 shows the HAMP single dose screening data of the modified duplexes using the human endogenous assay. Data are expressed as a percent of mock.
Table 9 shows the HAMP dose response data of modified and unmodified duplexes using the dual luciferase assay. Cells used included HepG2 and Cyno primary hepatocytes.
Example 4
HFE2 siRNA Design
siRNA design was carried out to identify siRNAs targeting human, rhesus (Macaca mulatta), mouse, and rat HFE2 transcripts annotated in the NCBI Gene database website noted above. There are at least 4 annotated human HFE2 transcripts and at least 3 annotated rhesus transcripts. Accordingly, we focused on the shortest annotated transcript for human, and the rhesus transcript which shared the greatest number of orthologous human exons, and designed on sequences held in common by the alternate transcripts. Design used the following transcripts from the NCBI RefSeq collection: Human—NM—213652.3; Rhesus—XM—001092987.1; Mouse —NM—027126.4; Rat—NM—001012080.1. Due to high primate/rodent sequence divergenge, siRNA duplexes were designed in two separate batches. The first batch matched human and rhesus; the second matched mouse and rat. All siRNA duplexes were designed that shared 100% identity with all listed human/rhesus or mouse/rat transcripts.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.
siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 4 or more Us or As in the seed region.
siRNA Sequence Selection
A total of 47 sense and 47 antisense derived human/rhesus, and 40 sense and 40 antisense derived mouse/rat siRNA oligos were synthesized and formed into duplexes.
Table 10A provides the sequences of the sense and antisense strands of the duplexes targeting the HFE2 gene.
Example 5
TFR2 siRNA Design
Transcripts
siRNA design was carried out to identify siRNAs targeting human, rhesus (Macaca mulatta), mouse, and rat TFR2 transcripts annotated in the NCBI Gene database website noted above. Design used the following transcripts from the NCBI RefSeq collection: Human—NM—003227.3, NM—001206855.1; Rhesus—XM—001113151.2; Mouse—NM—015799.3; Rat—NM—001105916.1. Due to high primate/rodent sequence divergenge, siRNA duplexes were designed in three separate batches. The first batch matched human and rhesus; the second matched human, rhesus, and mouse; the last batch matched mouse and rat. All siRNA duplexes were designed that shared 100% identity with all listed human/rhesus, human/rhesus/mouse, or mouse/rat transcripts.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.
siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 3 or more Us or As in the seed region.
siRNA Sequence Selection
A total of 40 sense and 40 antisense derived human/rhesus, 5 sense and 5 antisense derived human/rhesus/mouse, and 45 sense and 45 antisense derived mouse/rat siRNA oligos were synthesized and formed into duplexes.
Table 10B provides the sequences of the sense and antisense strands of the duplexes targeting the TFR2 gene.
Example 6
HFE2 and TFR2 siRNA Screening
Cell Culture and Transfections:
Endogenous system (Human): For TFR2, HepG2 cells were used; Hep3b cells were used for HFE2. HepG2 and Hep3b cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in MEM (Gibco) supplemented with 10% FBS before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing  ̃2 ×104 HepG2 cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at final concentrations of 10 nM and 0.1 nM. An additional concentration of 0.01 nM was performed for selected duplexes. Final duplex concentrations for dose response experiments were 10, 1.67, 0.278, 0.046, 0.0077, 0.0012, 0.0002, and 0.000035 nM.
Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12):
Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif. Cat #4368813):
A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
Real Time PCR:
2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl HFE2 or TFR2 probes, and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). HFE2 and TFR2 probes were Applied Biosystems cat #Hs02378779_s1 and Hs00162690_m1, respectively. Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.
To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with 10 nM AD-1955, mock transfected, or to the average lowest dose.
Table 11 shows the HFE2 and TFR2 single dose screening data of the duplexes using the human endogenous assay.
Table 12 shows the HFE2 and TFR2 dose response data of the duplexes.
Example 7
HFE siRNA Design
Transcripts
siRNA design was carried out to identify siRNAs targeting human, rhesus (Macaca mulatta), mouse, and rat HFE transcripts annotated in the NCBI Gene database website noted above. There are at least 9 annotated human HFE transcripts, at least 5 annotated rhesus transcripts, and at least 4 annotated rat transcripts. Accordingly, we focused on the shortest annotated transcripts for human, rhesus, and rat HFE, and designed on sequences held in common by the alternate transcripts. Design used the following transcripts from the NCBI RefSeq collection: Human—NM—139006.2; Rhesus—XM—001085598.2; Mouse—NM—010424.4; Rat—NM—001173435.1. Due to high primate/rodent sequence divergenge, siRNA duplexes were designed in two separate batches. The first batch matched human and rhesus; the second matched mouse and rat. All siRNA duplexes were designed that shared 100% identity with all listed human/rhesus or mouse/rat transcripts.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.
siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 3 or more Us or As in the seed region.
siRNA Sequence Selection
A total of 46 sense and 46 antisense derived human/rhesus, and 24 sense and 24 antisense derived mouse/rat siRNA oligos are synthesized and formed into duplexes. The duplexes are screened using the methods described above. One or more duplexes are selected for further testing.
Example 8
BMPR1a siRNA Design
Transcripts
siRNA design was carried out to identify siRNAs targeting mouse and rat BMPR1A transcripts annotated in the NCBI Gene database website noted above. Design used the following transcripts from the NCBI RefSeq collection: Mouse—NM—009758.4; Rat—NM—030849.1. All siRNA duplexes were designed that shared 100% identity with all listed mouse/rat transcripts.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.
siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 4 or more Us or As in the seed region.
siRNA Sequence Selection
A total of 46 sense and 46 antisense derived mouse/rat siRNA oligos are synthesized and formed into duplexes. The duplexes are screened using the methods described above. One or more duplexes are selected for further testing.
Example 9
SMAD4 siRNA Design
Transcripts
siRNA design was carried out to identify siRNAs targeting human and mouse SMAD4 transcripts annotated in the NCBI Gene database website noted above. Design used the following transcripts from the NCBI RefSeq collection: Human—NM—005359.5; Mouse—NM—008540.2. All siRNA duplexes were designed that shared 100% identity with all listed human/mouse transcripts.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.
siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 4 or more Us or As in the seed region.
siRNA Sequence Selection
Tables 15-16 provide the sequences of the sense and antisense strands of the duplexes targeting SMAD4 mRNA at the indicated locations. Some duplexes were modified as indicated. These siRNA oligos were synthesized and formed into duplexes for further testing as described below.
Example 10
IL6R siRNA Design
Transcripts
siRNA design was carried out to identify siRNAs targeting mouse and rat IL6R transcripts annotated in the NCBI Gene database website noted above. Design used the following transcripts from the NCBI RefSeq collection: Mouse—NM—010559.2; Rat—NM—017020.3. All siRNA duplexes were designed that shared 100% identity with all listed mouse/rat transcripts.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.
siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position and had 2 or more Us or As in the seed region.
siRNA Sequence Selection
A total of 44 sense and 44 antisense derived mouse/rat siRNA oligos are synthesized and formed into duplexes. The duplexes are screened using the methods described above. One or more duplexes are selected for further testing.
Example 11
BMP6 siRNA Design
Transcripts
siRNA design was carried out to identify siRNAs targeting human, rhesus (Macaca mulatta), mouse, and rat BMP6 transcripts annotated in the NCBI Gene database website noted above. Design used the following transcripts from the NCBI RefSeq collection: Human—NM—001718.4; Rhesus—XM—001085364.2; Mouse—NM—007556.2; Rat—NM—013107.1. Due to high primate/rodent sequence divergenge, siRNA duplexes were designed in three separate batches. The first batch matched human and rhesus; the second matched human, rhesus, and mouse: the last batch matched mouse and rat. All siRNA duplexes were designed that shared 100% identity with all listed human/rhesus, human/rhesus/mouse, or mouse/rat transcripts.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.
siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 3 or more Us or As in the seed region.
siRNA Sequence Selection
Table 21 provides the sequences of the sense and antisense strands of the duplexes targeting BMP6 mRNA. Some duplexes were modified as indicated. These siRNA oligos were synthesized and formed into duplexes for further testing using the methods described herein.
Example 12
Neo1 siRNA Design
Transcripts
siRNA design was carried out to identify siRNAs targeting human and mouse NEO1 transcripts annotated in the NCBI Gene database website noted above. There are 2 annotated mouse NEO1 transcripts. Accordingly, we focused on the shortest annotated transcripts for mouse NEO1, and designed on sequences held in common by the alternate transcripts. Design used the following transcripts from the NCBI RefSeq collection: Human—NM—002499.2; Mouse—NM—001042752.1. All siRNA duplexes were designed that shared 100% identity with all listed human/mouse transcripts.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.
siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 3 or more Us or As in the seed region.
siRNA Sequence Selection
Tables 17-18 provide the sequences of the sense and antisense strands of the duplexes targeting NEO1 mRNA at the indicated locations. Some duplexes were modified as indicated. These siRNA oligos were synthesized and formed into duplexes for further testing as described below.
Example 13
Activity of Murine siRNA in Vivo
The efficacy of one or more siRNAs described above is determined in mice, e.g., normal 10 week old 129s6/svEvTac mice using AD-1955 targeting luciferase as a control. The siRNAs are formulated as described herein and administered, e.g., through i.v. bolus at a dose of e.g., 10 mg/kg. Forty eight hours after injection, the liver and serum samples are harvested. The liver mRNA levels of the target mRNA are determined by qRT-PCR using gene specific primers and serum iron levels are determined using Feroxcine (Randox Life Sciences) and Hitachi 717 instrument.
siRNA that result in lowering of mRNA are selected for further evaluation.
Examples 14
Activity of Murine Hepcidin siRNA in Vivo
The efficacy of an HAMP siRNA AD-10812 was determined in mice using AF-011 formulated control siRNA and PBS as controls. Each siRNA was formulated with AF-011. AF-011 is also known as LNP11 (See Table A above; MC-3/DSPC/Cholesterol/PEG-DMG (50/10/38.5/1.5); Lipid:siRNA 10.1)).















position







in mouse







access.







#

SEQ

SEQ



NM_03254
sense strand
ID
antisense strand
ID
duplex


1.1
sequence (5′-3′)
NO
sequence (5′-3′)
NO
name







245-263
uGcuGuAAcAAuucccAGuTsT
42
ACUGGGAAUUGUuAcAGcATsT
43
AD-10812









PBS and the siRNAs were administered at various dosages to the mice as shown in FIG. 1: 1 mg/kg, 0.3 mg/kg, 0.1 mg/kg, 0.03 mg/kg, 0.01 mg/kg, and 0.003 mg/kg. A single siRNA dose was administered to each mouse. After injection, liver and serum samples were harvested from the mice. the liver Hamp1 mRNA levels were determined by qRT-PCR using Hamp1 specific primers and serum iron levels were determined. FIG. 1 shows the HAMP1 mRNA levels in mouse liver following various dosages of siRNa and the serum iron concentration (μg/dL) following various dosages of siRNA.
Administration of AD-10812 HAMP siRNA to mice resulted in lowering of HAMP mRNA by >80% following a single dose. Adminidtration of AD-10812 HAMP siRNA to mice resulted in an approximatrely 2-fold increase in serum iron following a single dose.
Example 15
Activity of Hepcidin siRna in Nonhuman Primated (NHPs) in Vivo
The efficacy of an HAMP siRNA AD-11459 was determined in male cynomolgus monkeys using AF-011 siRNA as a control.























SEQ


SEQ



Duplex
Start
Sense
Sense
ID
Antisense
Antisense
ID


Target
ID
Position
Name
Sequence
NO
Name
Sequence
NO







HAMP
AD-
382
A-18280.2
GAAcAuAGGucuuG
30
A-18304.1
uAuUCcAAGACCuAuGuUC
44



11459


GAAuAdTsdT


dTsdT









Each siRNA was formulated with AF-011. The siRNA were administered intravenously at a dose of 1 mg/kg via a 15 minute infusion. A single siRNA dose was administered to each monkey. After injection, liver and serum samples were harvested. Liver samples were taken at 48 hours (h) post-injection. Serum samples were taken at Day-9, Day-6, Day-3, 24h post-injection, and 48h post-injection. The liver Hamp mRNA levels were determined by qRT-PCR using Hamp specific primers and serum iron levels were determined. Serum HAMP protein levels were also determined. FIG. 2 shows the HAMP mRNA levels in liver following siRNA administration as well as the serum iron concentration (μg/dL) and the HAMP serum protein concentration (mg/mL) following siRNA administration.
Single administration of LNP-siRNA AD-11459 resulted in rapid refuction of hepcidin mRNA and protein levels and elevation of serum iron levels in NHPs.
Examples 16
Silencing of Murine TFR2 Via siRNA in Vivo
The efficacy of TFR2 siRNA AD-47882 (see table below for sequences) was determined in C57BL6 mice using AF-011 siRNA and PBS as controls. Each siRNA was formulated with AF-011. PBS and the siRNAs were administered at various dosages to the mice as shown in FIG. 3: 1 mg/kg, 0.3 mg/kg, 0.1 mg/kg, and 0.03 mg/kg. After injection, liver and serum samples were harvested from the mice. The liver Hamp1 and TFR2 mRNA levels were determined by qRT-PCR using gene specific primers and transferrin saturation were determine at 48 hours post-injection. FIG. 3 shows the HAMP1 and TFR2 mRNA levels in mouse liver following various dosages of siRNA and the percent (%) transferrin saturation following various dosages of siRNA.


















SEQ

SEQ



Duplex
Sense
ID
Antisense
ID


Target
ID
Sequence
NO
Sequence
NO







TFR2
AD-
ccAcGuGAuucuccu
45
AGAAAGGAGAAUcACG
46



47882
uucudTsdT

UGGdTsdT









Administration of AD-47882 siRNA to mice resulted in lowering of HAMP and TFR2 mRNA levels. Administration of AD-47882 siRNA to mice resulted in an increase in trasferrin saturation.
Example 17
Silencing of Murine TFR2 Via siRNA in Vivo
The duration of TFR2 siRNA AD-47882 was determined in C57BL6 mice. The siRNA were administered at in a single 0.3 mg/kg dose intravenously. Each siRNA was formulated with AF-011. After injection, liver and serum samples were harvested from the mice at various time points shown in FIG. 4. The liver Hamp1 and TFR2 Mrna levels were determined by qRT-PCR using gene specific primers and transferrin saturation were determined. FIG. 4 shows the HAMP1 and TFR2 mRNA levels in mouse liver following administration of siRNA and the percent (%) transferrin saturation over a 30 day time course.
Administration of AD-47882 siRNA to mice resulted in lowering of HAMP and TFR2 mRNA levels. Administration of AD-47882 siRNA to mice resulted in an increase in transferrin saturation.
Example 18
Silencing of Rat TFR2 Via siRNA In Vivo
The duration and efficacy of TFR2 siRNA AD-47882 was determined in male Lewis rats using the anemia of chronic disease (ACD) model described in Coccia et al., Exp. Hematology, 2001. Briefly, anemia was initiated in the rats with a single intraperaoneal (i.p.) injection of PG-APS (polymers from Group A Streptococci). The rats were then treated 3× per week with AD-47882 siRNA, AF-011 control siRNA, or saline control starting at day 21 post PG-APS. Each siRNA was formulated with AF-011. Serum and hematology parameters were measured biweekly and at 48 hours post final treatment. Serum samples were harvested from the rats at various time points as shown in FIG. 5. Liver mRNA measurement was taken at 48 hours post final treatment. FIG. 5 shows the HAMP1 and TFR2 mRNA levels in rat liver following administration of siRNA. FIG. 5 also shows the serum iron and Hb concentrations at various time points.
Administration of AD-47882 siRNA resulted in lowering of HAMP and TFR2 mRNA levels. Administration of AD-47882 siRNA resulted in an approximate 2× increase in serum iron upon treatment. Administration of AD-47882 siRNA resulted in an increase in Hb levels to 11-12 g/dL with treatment.
Example 19
TFR2 siRNA Selection and Screening
siRNA Sequence Selection
Table 13 provides the sequences of the sense and antisense strands of the duplexes targeting the TFR2 gene at the indicated locations (64 or 239). These siRNA oligos were synthesized and formed into duplexes.
Cell Culture and Transfections:
Endogenous system (Human): For TFR2, HepG2 cells were used. HepG2 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in MEM (Gibco) supplemented with 10% FBS before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing  ̃2×104 HepG2 cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at final concentrations of 10 nM and 0.1 nM and 0.01 nM. An additional concentration of 0.01 nM was performed for selected duplexes.
Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12):
Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):
A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
Real Time PCR:
2 μl of cDNA were added to a master mix containing 0.511 GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl TFR2 probes, and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). TFR2 probes were Applied Biosystems cat #Hs02378779_s1 and Hs00162690_m1, respectively. Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.
To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with 10 nM AD-1955, mock transfected, or to the average lowest dose.
Table 14 shows the TFR2 dose response data of the duplexes.
Example 20
Activity of TFR2 and HAMP siRNA in Non-Human Primates (NHPs) In Vivo
The efficacy of AD-52590, AD-51707, and AD-48141 was determined in separate cynomolgus monkeys (3 each) using PBS as a control. The sequence of AD-52590, AD-51707, and AD-48141 are shown below and in Table 4, 10B, and 13.



















SEQ

SEQ




Duplex
Start
ID

ID



Target
ID
Position
NO
Sense Sequence
NO
Antisense Sequence







TFR2
AD-
239
35
cAGGcAGcCAAAcCuCAuUdTsdT
38
AAUGAGGUuUGGCUGcCugdTsdT



52590







 


TFR2
AD-
105
47
ccuucAAucAAAcccAGuudTsdT
48
AACuGGGUuUGAuUGAAGGdTsdT



51707







 


HAMP
AD-
382
30
GAAcAuAGGucuuGGAAuAdTdT
44
UAuUCcAAGACCuAuGuUCdTdT



48141









Each siRNA was formulated with AF-011. The siRNAs were administered intravenously as indicated (0.1 mg/kg, 0.03 mg/kg, or 1 mg/kg) via a 15 minute infusion. A single siRNA dose was administered to each monkey. After injection, liver and serum samples were harvested. Liver biopsy samples were taken at 48 hours (h) post-injection. Serum samples were taken at Day -9, Day -6, Day -3, 24 h post-injection, and 48 h post-injection. The liver Hamp mRNA levels were determined by qRT-PCR using Hamp specific primers. The liver TFR2 mRNA levels were determined by qRT-PCR using TFR2 specific primers. Serum iron levels were determined and are shown in μg/dL. Serum HAMP protein levels were also determined and are shown in ng/mL.
FIG. 6 shows HAMP mRNA levels in the liver of each animal following siRNA administration, relative to PBS controls. FIG. 7 shows TFR2 mRNA levels in the liver of each animal following siRNA administration, relative to PBS controls. FIG. 8 shows that serum iron concentration was increased in each animal after 1 mg/kg AD-52590 siRNA administration. FIG. 9 shows that the HAMP serum protein concentration was decreased in each animal following 1 mg/kg AD-52590 siRNA administration.
Single administration of AD-52590 resulted in rapid reduction of hepcidin mRNA and protein levels, TFR2 mRNA levels, and elevation of serum iron levels in NHPs.
Example 21
NEO1 and SMAD4 Duplex Screening
Human/mouse cross-reactive Neo1 and Smad4 siRNAs were screened in primary mouse hepatocytes. Duplexes are shown in Tables 15, 16, 17, and 18.
Cell Culture and Transfections:
Freshly isolated primary mouse hepatocytes (PMH) were transfected by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of primary hepatocyte media containing  ̃2×104 PMH cells were then added to the siRNA mixture. Cells were incubated for either 24 prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration.
Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12):
Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):
A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
Real Time PCR:
2 μl of cDNA were added to a master mix containing 0.5 μl of mouse GAPDH TaqMan Probe (Applied Biosystems Cat #4352932E), 0.5 μl Neo1 or SMAD4 TaqMan probe (Applied Biosystems cat #Neo 1-Mm00476326_m1 or SMAD4 Mm03023996_m1) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections and each transfection was assayed in duplicate, unless otherwise noted.
To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose.
Table 19 shows the percent remaining mRNA remaining for each SMAD4 duplex tested at 0.1 nM and 10 nM. Controls were 10 nM AD-1955, mock transfected. Table 20 shows the percent remaining mRNA remaining for each NEO1 duplex tested at 0.1 nM and 100 nM. Controls were 10 nM AD-1955, mock transfected.
Example 22
In Vivo Combinatorial Use of dsRNAs Targeting HAMP-Related mRNAs
The efficacy of TFR2 siRNA AD-47882 and HFE siRNA AD-47320 (see table below for sequences) alone and in combination was determined in C57BL6 female mice using AF-011-Luc siRNA and PBS as controls. Each siRNA was formulated with AF-011. PBS and the siRNAs were administered at various (mg/kg) dosages to the mice as shown on the X-axis of each subfigure (A-D) of FIG. 10. 48 hours after injection, liver and serum samples were harvested from the mice.




















SEQ

SEQ



Duplex
Accession
Sense
ID
Antisense
ID


Target
ID
Number
Sequence
NO
Sequence
NO







HFE
AD-
NM_010424.4
uuuucuccAGuuAAG
49
UGAACUuAACUGGAGA
51



47320

uucAdTsdT

AAAdTsdT



 


HFE
Unmo
NM_010424.4
UUUUCUCCAGUUA
50
UGAACUUAACUGGAGA
52



d AD-

AGUUCA

AAA




47320









The liver Hamp1, HFE, and TFR2 mRNA levels were determined by qRT-PCR using gene specific primers. Blood was processed into serum to measure serum iron, transferrin saturation, and UIBC. FIG. 10A shows the HAMP1, HFE, and TFR2 mRNA levels in mouse liver following various dosages of each siRNA group or PBS. FIGS. 10B-D shows serum iron concentration, transferrin saturation, and UIBC concentration in the serum of each group tested.
Example 23
Inhibition of HAMP in Humans
A human subject is treated with a siRNA targeted to a HAMP gene to inhibit expression of the HAMP gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene.
A subject in need of treatment is selected or identified.
The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.
At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
Example 24
Inhibition of HFE2 in Humans
A human subject is treated with a siRNA targeted to a gene to inhibit expression of the HFE2 gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene.
A subject in need of treatment is selected or identified.
The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.
At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
Example 25
Inhibition of HFE in Humans
A human subject is treated with a siRNA targeted to a gene to inhibit expression of the HFE gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene.
A subject in need of treatment is selected or identified.
The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.
At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
Example 26
Inhibition of TFR2 in Humans
A human subject is treated with a siRNA targeted to a gene to inhibit expression of the TFR2 gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene.
A subject in need of treatment is selected or identified.
The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.
At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
Example 27
Inhibition of BMPR1a in Humans
A human subject is treated with a siRNA targeted to a gene to inhibit expression of the BMPR1a gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, TFR2, SMAD4, IL6R, BMP6, and/or NEO1 gene.
A subject in need of treatment is selected or identified.
The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.
At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
Example 28
Inhibition of SMAD4 in Humans
A human subject is treated with a siRNA targeted to a gene to inhibit expression of the SMAD4 gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, TFR2, BMPR1a, IL6R, BMP6, and/or NEO1 gene.
A subject in need of treatment is selected or identified.
The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.
At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
Example 29
Inhibition of IL6R in Humans
A human subject is treated with a siRNA targeted to a gene to inhibit expression of the IL6R gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, BMP6, and/or NEO1 gene.
A subject in need of treatment is selected or identified.
The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.
At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
Example 30
Inhibition of BMP6 in Humans
A human subject is treated with a siRNA targeted to a gene to inhibit expression of the BMP6 gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/or NEO1 gene.
A subject in need of treatment is selected or identified.
The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.
At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
Example 31
Inhibition of NEO1 in Humans
A human subject is treated with a siRNA targeted to a gene to inhibit expression of the NEO1 gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/or BMP6 gene.
A subject in need of treatment is selected or identified.
The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.
At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
Tables









TABLE B





SEQ




ID




NO
DESCRIPTION
SEQUENCE







 1
Human HAMP -
gactgtcactcggtcccagacaccagagcaagctcaagacccagcagtgggacagcc



NM_021175.2
agacagacggcacgatggcactgagctcccagatctgggccgcttgcctcctgctcc




tcctcctcctcgccagcctgaccagtggctctgttttcccacaacagacgggacaac




ttgcagagctgcaaccccaggacagagctggagccagggccagctggatgcccatgt




tccagaggcgaaggaggcgagacacccacttccccatctgcattttctgctgcggct




gctgtcatcgatcaaagtgtgggatgtgctgcaagacgtagaacctacctgccctgc




ccccgtcccctcccttccttatttattcctgctgccccagaacataggtcttggaat




aaaatggctggttctttcgttttccaaaaaa


 


 2
Cyno HAMP -
tcaagacctagcagtgggacagccagacagacggcacgatggcactgagctcccaga



EU076443.1
tctgggccacttgcctcctcctccttctcctcctcgccagcctgaccagtggctccg




ttttcccacaacagacgggacaacttgcagagctgcaacctcaggacagagctggag




ccagggccagctggacgcccatgctccagaggcgaaggaggcgagacacccacttcc




ccatctgcattttctgctgcggctgctgtcatcgatcaaagtgtgggatgtgctgca




ggacgtagaaccttcctgccctgcccccatcccctcccttccttatttattcctgct




gccccagaacacaggtcttggaataaaacggctgattcttttgttttcc


 


 3
HAMP -
agtccttagactgcacagcagaacagaaggcatgatggcactcagcactcggaccca



NM_032541.1
ggctgcctgtctcctgcttctcctccttgccagcctgagcagcaccacctatctcca




tcaacagatgagacagactacagagctgcagcctttgcacggggaagaaagcagggc




agacattgcgataccaatgcagaagagaaggaagagagacaccaacttccccatctg




catcttctgctgtaaatgctgtaacaattcccagtgtggtatctgttgcaaaacata




gcctagagccacatcctgacctctctacacccctgcagcccctcaaccccattattt




attcctgccctccccaccaatgaccttgaaataaagacgattttattttcaaaaaaa




aaaaaaaaaaa


 


 4
Rat HAMP -
cacgagggcaggacagaaggcaagatggcactaagcactcggatccaggctgcctgt



NM_053469.1
ctcctgcttctcctcctggccagcctgagcagcggtgcctatctccggcaacagacg




agacagactacggctctgcagccttggcatggggcagaaagcaagactgatgacagt




gcgctgctgatgctgaagcgaaggaagcgagacaccaacttccccatatgcctcttc




tgctgtaaatgctgtaagaattcctcctgtggtctctgttgcataacatagagagcc




aagagccttgtcctgacctctcaacacactgcctcccctccgccccattatttattc




ctgtcctaccccagcaatgaccttg


 


 5
Human HEFE2 -
accgtcaactcagtagccacctccctccctgctcagctgtccagtactctggccagc



NM_213652.3
catatactcccccttccccccataccaaaccttctctggttccctgacctcagtgag




acagcagccggcctggggacctgggggagacacggaggaccccctggctggagctga




cccacagagtagggaatcatggctggagaattggatagcagagtaatgtttgacctc




tggaaacactcaccatcatatttaagaacatgcaggaatgcattgatcagaaggtgt




atcaggctgaggtggataatcttcctgtagcctttgaagatggttctatcaatggag




gtgaccgacctgggggatccagtttgtcgattcaaactgctaaccctgggaaccatg




tggagacccaagctgcctacattggcacaactataatcattcggcagacagctgggc




agctctccttctccatcaaggtagcagaggatgtggccatggccttctcagctgaac




aggacctgcagctctgtgttggggggtgccctccaagtcagcgactctctcgatcag




agcgcaatcgtcggggagctataaccattgatactgccagacggctgtgcaagaaag




ggcttccagtggaagatgcctacttccattcctgtgtctttgatgctttaatttctg




gtgatcccaactttaccgtggcagctcaggcagcactggaggatgcccgagccttcc




tgccagacttagagaagctgcatctcttcccctcagatgctggggttcctctttcct




cagcaaccctcttagctccactcctttctgggctctttgttctgtggctttgcattc




agtaaggggaccatcagccccattactagtttggaaatgatttggagatacagattg




gcatagaagaatgtaaagaatcattaaaggaagcagggcctaggagacacgtgaaac




aatgacattatccagagtcagatgaggctgcagtccagggttgaaattatcacagaa




taaggattctgggcaaggttactgcattccggatctctgtggggctcttcaccaatt




tttccagcctcatttatagtaaacaaattgttctaatccatttactgcagatttcac




ccttataagtttagaggtcatgaaggttttaatgatcagtaaagatttaagggttga




gatttttaagaggcaagagctgaaagcagaagacatgatcattagccataagaaact




caaaggaggaagacataattagggaaagaagtctatttgatgaatatgtgtgtgtaa




ggtatgttctgctttcttgattcaaaaatgaagcaggcattgtctagctcttaggtg




aagggagtctctgcttttgaagaatggcacaggtaggacagaagtatcatccctacc




ccctaactaatctgttattaaagctacaaattcttcacaccatcaaaaaaaaaaaaa




aaaaaa


 


 6
Rhesus HFE2 -
cttctctggctccctgacctcagtgagacagcagccggcctggggacctgggggaga



XM_001092987
catggagaaagagacggaggaccccctggctggagctgacccacagagtagggaatc



.1
atggctggagaattggatagcagagtaatgtttgacctctggaaacaccaaatttct




tttttcagtcacttacagggcttccggtcaaaattcactaggtaggagggtcatcag




ctgggaagaaccggcgcctggggaacctggctggataggtatgggggagcaaggcca




gtcccctagtcccaggtcctcccatggcagccccccaactctaagcactctcactct




cctgctgctcctctgtggacatgctcattcccaatgcaagatcctccgctgcaatgc




tgagtatgtatcgtccactctgagccttagaggtggcggttcatcaggagcacttcg




aggaggaggaggaggaggaggccggggtggaggggtgggctctggcggcctctgtcg




agccctccgctcctatgcgctctgcactcggcgcaccgcccgcacctgccgtgggga




cctcgccttccatccggcggtacatggcatcgaagacctgatgatccagcacaactg




ctcgcgccagggccctacagcccctcccccgccccggggccccgcccttccaggcgc




aggctccggcctccctgccccggacccttgtgactatgaaggccggtttccccggct




gcatggtcgtcccccggggttcttgcattgcgcttccttcggggacccccatgtgcg




cagcttccaccaccattttcacacatgccgtgtccaaggagcttggcctctactgga




taacgacttcctccttgtccaagccaccagctcccccatggcgttgggggccaacgc




taccgctacccggaagctcaccatcatatttaagaacatgcaggaatgcattgatca




gaaggtctatcaggctgaggtggataatcttcctgcagcctttgaagatggttctgt




caatggaggtgaccgacctgggggatccagtttgtcgattcaaactgctaaccctgg




gaaccacgtggagatccaagctgcctacattggcacaactataatcattcggcagac




agctgggcagctctccttctccatcaaggtagcagaggatgtggccatggccttctc




agctgaacaggacctgcagctctgtgttggggggtgccctccaagtcagcgactctc




tcgatcagagcgcagtcgtccgggagctataaccattgatactgccagacggctgtg




taaggaagggcttccagtggaagatgcttacttccattcctgtgtctttgatgtttt




aatttctggtgatcccaactttactgtggcagctcaggcagcactggaggatgcccg




agccttcctgccagacttagataagctgcatctcttcccttcagatgctggggtttc




tctttcctcagcaaccttcctagccccactcctttctgggctctttgttctgtggct




ttgcattcagtaaggaagccatcagtcctattactagtttggaaatgatttggggat




agagattggcatagaagaatgtaaacaatcattaaaggaagcagggcccagaagaca




catgaaacaatgacatcatccagagtcagatgaggctgcagtccagggttgaaatga




tcacagaataaggattctgggcaaggtttctgcattccagacctcttcgccaaattt




tccagccccatttacagtaaacaaattgttctttccatttactgcagatttcaccct




ataagcttagaggtcatgaaggttttaacaatcagtaaagacttaagggttgagatt




tttaagaggcaagagctgaaagcagaagacatgatcattagccataagaaactcaaa




ggaagaagaaataattagggaaagaagtctatttgatgaatatgtgtgtgtaaggta




tgttctgctttcttggttcaaaaatgaagcgggcgttgtctagctcttaggtgaagg




gagtctctgctttggaagaacggcacaggtaggacagaagtatcatccctaccccta




actgatctgttattaaagctacaaattcttcacaccgtc


 


 7
Mouse HFE2 -
ggctctctgacctgagtgagactgcagccattccggggcaatcatggagaaagagat



NM_027126.4
gggggaccccctggctggagcagaccaacagaataggcaactatggctcgagaaccc




agtatcagagtaatgcttgacctcgggaaacatcacagaagtacccagagaaattca




ctaggtaggaggctcatcatctgggaagaaccggtgcctggggggacctggctggat




aggtatgggccagtcccctagtccccggtccccccacggcagccctccaactccaag




caccctcactctcctgctgctcctctgtggacaggctcactcccagtgcaagatcct




ccgctgcaatgccgagtatgtctcgtccactctgagtcttcggggaggtggctcacc




ggacacgccgcgtggaggcggccgtggtgggctggcctcaggtggcttgtgtcgcgc




cctgcgctcctacgctctctgcacgcggcgcacggcccgcacctgccgcggggacct




tgctttccactctgcggtgcatggcatagaggacctgatgatccagcacaactgctc




acgccagggtcccacggccccgcccccggcccggggccccgccctgcccggggccgg




gccagcgcccctgaccccagatccctgtgactatgaggcccggttttccaggctgca




cggtcgagccccgggcttcttgcattgcgcatcctttggagatccccatgtgcgcag




tttccacaaccaatttcacacatgccgtgtccaaggagcttggcccttgctagataa




cgacttcctctttgtccaggccaccagctccccggtttcgtcgggagccaccgctac




caccatccggaagatcactatcatatttaaaaacatgcaggaatgcattgaccagaa




agtctaccaggctgaggtggacaatcttcctgcagcctttgaagatggttctatcaa




tgggggcgaccgacctgggggctcgagtttgtccattcaaactgctaaccttgggag




tcacgtggagattcgagctgcctacattggaacaactatcatcattcgacagacagc




tgggcagctctccttctccatcagggtagcagaggatgtggcgcgggccttctccgc




agagcaggacctacagctgtgtgttgggggatgccctccgagccagcgactctctcg




ctcagagcgcaaccgccgtggggctatagccatagatactgccagaaggctgtgtaa




ggaagggcttccggttgaagatgcctacttccaatcctgcgtctttgatgtttcagt




ctccggtgaccccaactttactgtggcagctcagacagctctggacgatgcccgaat




cttcttgacggatttagagaacttacatctctttccctcagatgcggggcctcccct




ctctcctgccatctgcctagtcccgcttctttcggccctctttgttctgtggctttg




cttcagtaagtaggccagcaacccatgactggtttggaaacgatttgaggatagagg




ttggtgtgagaaaccacaaagatgtgccaaaggaaacagcggggacaggagacaaca




cttacccaatcagatgaggttgcagtccagggctgaaatgaccctagaataaagatt




ctgggccagggttttgcactccagaccttggtgtgggctattcaccatggatttccc




agttagtgatttcccacttgtaatgaaattccactctccatacacctataccactcc




ctacaagcctagagattgtgagagtgctaatgaccagtgaaacattaaaggactgag




atatcgtaaaggcaaaaacatgattctctttgagaaagtcaaaagaggagaagctaa




ttaggaaaagcttttggttcagaaacgaagtgggcattgtctggcagaggaagtcag




cttttggagactggcaccaactcagaaacgggcatttccatcccttcctaatctgtt




attaaagcgattagttctccatcctg


 


 8
Rat HFE2 -
cggggacagacatggagaaggagatggaggaccccctggctggagcagaccaacaga



NM_001012080
ataggcaactatggctggagaaccgggtatcagagtaatgcttgacctcgggaaaca



.1
ccaaatttcttcttccgatcgcagaagtagtactcggcgaaattcactaggtaggag




gctcctcatctgggaagaaccggtgcctggggggacctggctggataggtatggggg




atcgaggccggtcccctagtctccggtccccccatggcagtcctccaactctaagca




ccctcactctcctgctgctcctctgtggacaggctcactcccagtgcaagatcctcc




gctgcaatgccgagtacgtctcgtccactctgagccttcggggagggggctcaccgg




acacgccacatggaggcggccgtggtgggccggcctcaggtggcttgtgtcgcgccc




tgcgctcctacgctctctgcacgcggcgcaccgcccgcacctgccgcggggacctcg




ctttccactccgcggtgcatggcatagaggacctgatgatccagcacaactgctcac




gccagggtcccacggcctcgcccccggcccggggtcctgccctgcccggggccggcc




cagcgcccctgaccccagatccctgtgactatgaagcccggttttccaggctgcacg




gtcgaaccccgggtttcttgcattgtgcttcctttggagacccccatgtgcgcagct




tccacaatcactttcacacatgccgcgtccaaggagcttggcccctactagataacg




acttcctctttgtccaagccaccagctccccggtagcatcgggagccaacgctacca




ccatccggaagatcactatcatatttaaaaacatgcaggaatgcattgaccagaaag




tctaccaggctgaggtagacaatcttcctgcagcctttgaagatggttctgtcaatg




ggggcgaccgacctgggggctcgagtttgtccattcaaactgctaaccttgggagcc




acgtggagattcgagctgcctacattggaacaactataatcgttcgtcagacagctg




gacagctctccttctccatcagggtagcggaggatgtggcacgggccttctctgctg




agcaggatctacagctgtgtgttgggggatgccctccgagccagcgactctctcgct




cagagcgcaatcgccgtggggcgatagccatagatactgccagaaggttgtgtaagg




aagggcttccggttgaagatgcctacttccaatcctgcgtctttgatgtttcagtct




ccggtgaccccaactttactgtggcagctcagtcagctctggacgatgcccgagtct




tcttgaccgatttggagaacttgcaccttttcccagtagatgcggggcctcccctct




ctccagccacctgcctagtccggcttctttcggtcctctttgttctgtggttttgca




ttcagtaagtaggccagcaacccgtgactagtttggaaacggtttgaggagagaggt




tgatgtgagaaaacacaaagatgtgccaaaggaaacagtggggacaggagacaacga




ccttactcaatcacacgaggttgcagtccagggctgaaatgaccctagaataaagat




tctgagacagggttttgcactccagaccttggtatgggctccccatgaatttcccca




ttagtgatttcccacttgtagtgaaattctactctctgtacacctgatatcactcct




gcaaggctagagattgtgagagcgctaagggccagcaaaacattaaagggctgagat




atcttaaaggcagaaactagaaaaggggaaaccatgattatctataagaaaatcaaa




agaggggtttgggaatttagctcagtggtagagcacttgcctagcaagcgcaaggcc




ctgggttcggtccccagctcctaaaaaagaaaaaaaaaatcaaaagagaaaaaacta




attaaggcaagctttttggttcagaaatgaagtgggcattgtctggcagaggaagtc




agcttttggagactggcaccaacatctccacccttcctactctgttattaaagtgac




gaattccccaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagg


 


 9
Human TFR2 -
cgctgggggacagcctgcaggcttcaggaggggacacaagcatggagcggctttggg



NM_003227.3
gtctattccagagagcgcaacaactgtccccaagatcctctcagaccgtctaccagc




gtgtggaaggcccccggaaagggcacctggaggaggaagaggaagacggggaggagg




gggcggagacattggcccacttctgccccatggagctgaggggccctgagcccctgg




gctctagacccaggcagccaaacctcattccctgggcggcagcaggacggagggctg




ccccctacctggtcctgacggccctgctgatcttcactggggccttcctactgggct




acgtcgccttccgagggtcctgccaggcgtgcggagactctgtgttggtggtcagtg




aggatgtcaactatgagcctgacctggatttccaccagggcagactctactggagcg




acctccaggccatgttcctgcagttcctgggggaggggcgcctggaggacaccatca




ggcaaaccagccttcgggaacgggtggcaggctcggccgggatggccgctctgactc




aggacattcgcgcggcgctctcccgccagaagctggaccacgtgtggaccgacacgc




actacgtggggctgcaattcccggatccggctcaccccaacaccctgcactgggtcg




atgaggccgggaaggtcggagagcagctgccgctggaggaccctgacgtctaccgcc




cctacagcgccatcggcaacgtcacgggagagctggtgtacgcccactacgggcggc




ccgaagacctgcaggacctgcgggccaggggcgtggatccagtgggccgcctgctgc




tggtgcgcgtgggggtgatcagcttcgcccagaaggtgaccaatgctcaggacttcg




gggctcaaggagtgctcatatacccagagccagcggacttctcccaggacccaccca




agccaagcctgtccagccagcaggcagtgtatggacatgtgcacctgggaactggag




acccctacacacctggcttcccttccttcaatcaaacccagttccctccagttgcat




catcaggccttcccagcatcccagcccagcccatcagtgcagacattgcctcccgcc




tgctgaggaagctcaaaggccctgtggccccccaagaatggcaggggagcctcctag




gctccccttatcacctgggccccgggccacgactgcggctagtggtcaacaatcaca




ggacctccacccccatcaacaacatcttcggctgcatcgaaggccgctcagagccag




atcactacgttgtcatcggggcccagagggatgcatggggcccaggagcagctaaat




ccgctgtggggacggctatactcctggagctggtgcggaccttttcctccatggtga




gcaacggcttccggccccgcagaagtctcctcttcatcagctgggacggtggtgact




ttggaagcgtgggctccacggagtggctagagggctacctcagcgtgctgcacctca




aagccgtagtgtacgtgagcctggacaacgcagtgctgggggatgacaagtttcatg




ccaagaccagcccccttctgacaagtctcattgagagtgtcctgaagcaggtggatt




ctcccaaccacagtgggcagactctctatgaacaggtggtgttcaccaatcccagct




gggatgctgaggtgatccggcccctacccatggacagcagtgcctattccttcacgg




cctttgtgggagtccctgccgccgagttctcctttatggaggacgaccaggcctacc




cattcccgcacacaaaggaggacacttatgagaacctgcataaggcgctgcaaggcc




gcctgcccgccgtggcccaggccgtggcccagctcgcagggcagctcctcatccggc




tcagccacgatcgcctgctgcccctcgacttcggccgctacggggacgtcgtcctca




ggcacatcgggaacctcaacgagttctctggggacctcaaggcccgcgggctgaccc




tgcagtgggtgtactcggcgcggggggactacatccgggcggcggaaaagctgcggc




aggagacctacagctcggaggagagagacgagcgactgacacgcatgtacaacgtgc




gcataatgcgggtggagttctacttcctttcccagtacgtgtcgccagccgactccc




cgctccgccacatcttcacgggccgtggagaccacacgctgggcgccctgctggacc




acctgcggctgctgcgctccaacagctccgggacccccggggccacctcctccactg




gcttccaggagagccgtttccggcgtcagccagccctgctcacctggacgctgcaag




gggcagccaatgcgcttagcggggatgtctggaacattgataacaacttctgaggcc




ctggggatcctcacatccccgtcccccagtcaagagctcctctgctcctcgcttgaa




tgattcagggtcagggaggtggctcagagtccacctctcattgctgatcaatttctc




attacccctacacatctctccacggagcccagaccccagcacagatatccacacacc




ccagccctgcagtgtagctgaccctaatgtgacggtcatactgtcggttaatcagag




agtagcatcccttcaatcacagccccttcccctttctggggtcctccatacctagag




accactctgggaggtttgctaggccctgggacctggccagctctgttagcgggagag




atcgctggcaccatagccttatggccaacaggtggtctgtggtgaaaggggcgtgga




gtttcaatatcaataaaccacctgatatcaataagccaaaa


 


10
Human TFR2 -
ccctgcccctggcgaccccacgtctctggcatccctccctcttccctccctctcctc



NM_001206855
cgggcgcccagaaaagtccccacctctccccgcttaggcaaaccagccttcgggaac



.1
gggtggcaggctcggccgggatggccgctctgactcaggacattcgcgcggcgctct




cccgccagaagctggaccacgtgtggaccgacacgcactacgtggggctgcaattcc




cggatccggctcaccccaacaccctgcactgggtcgatgaggccgggaaggtcggag




agcagctgccgctggaggaccctgacgtctactgcccctacagcgccatcggcaacg




tcacgggagagctggtgtacgcccactacgggcggcccgaagacctgcaggacctgc




gggccaggggcgtggatccagtgggccgcctgctgctggtgcgcgtgggggtgatca




gcttcgcccagaaggtgaccaatgctcaggacttcggggctcaaggagtgctcatat




acccagagccagcggacttctcccaggacccacccaagccaagcctgtccagccagc




aggcagtgtatggacatgtgcacctgggaactggagacccctacacacctggcttcc




cttccttcaatcaaacccagttccctccagttgcatcatcaggccttcccagcatcc




cagcccagcccatcagtgcagacattgcctcccgcctgctgaggaagctcaaaggcc




ctgtggccccccaagaatggcaggggagcctcctaggctccccttatcacctgggcc




ccgggccacgactgcggctagtggtcaacaatcacaggacctccacccccatcaaca




acatcttcggctgcatcgaaggccgctcagagccagatcactacgttgtcatcgggg




cccagagggatgcatggggcccaggagcagctaaatccgctgtggggacggctatac




tcctggagctggtgcggaccttttcctccatggtgagcaacggcttccggccccgca




gaagtctcctcttcatcagctgggacggtggtgactttggaagcgtgggctccacgg




agtggctagaaggctacctcagcgtgctgcacctcaaagccgtagtgtacgtgagcc




tggacaacgcagtgctgggggatgacaagtttcatgccaagaccagcccccttctga




caagtctcattgagagtgtcctgaagcaggtggattctcccaaccacagtgggcaga




ctctctatgaacaggtggtgttcaccaatcccagctgggatgctgaggtgatccggc




ccctacccatggacagcagtgcctattccttcacggcctttgtgggagtccctgccg




tcgagttctcctttatggaggacgaccaggcctacccattcctgcacacaaaggagg




acacttatgagaacctgcataaggtgctgcaaggccgcctgcccgccgtggcccagg




ccgtggcccagctcgcagggcagctcctcatccggctcagccacgatcgcctgctgc




ccctcgacttcggccgctacggggacgtcgtcctcaggcacatcgggaacctcaacg




agttctctggggacctcaaggcccgcgggctgaccctgcagtgggtgtactcggcgc




ggggggactacatccgggcggcggaaaagctgcggcaggagatctacagctcggagg




agagagacgagcgactgacacgcatgtacaacgtgcgcataatgcgggtggagttct




acttcctttcccagtacgtgtcgccagccgactccccgttccgccacatcttcatgg




gccgtggagaccacacgctgggcgccctgctggaccacctgcggctgctgcgctcca




acagctccgggacccccggggccacctcctccactggcttccaggagagccgtttcc




ggcgtcagctagccctgctcacctggacgctgcaaggggcagccaatgcgcttagcg




gggatgtctggaacattgataacaacctctgaggccctggggatcctcacatccccg




tcccccagtcaagagctcctctgctcctcgcttgaatgattcagggtcagggaggtg




gctcagagtccacctctcattgctgatcaatttctcattacccctacacatctctcc




acggagcccagaccccagcacagatatccacacaccccagccctgcagtgtagctga




ccctaatgtgacggtcatactgtcggttaatcagagagtagcatcccttcaatcaca




gccccttcccctttctggggtcctccatacctagagaccactctgggaggtttgcta




ggccctgggacctggccagctctgttagtgggagagatcgctggcaccatagcccta




tggccaacaggtggtctgtggtgaaaggggcgtggagtttcaatatcaataaaccac




ctgatatcaataagccaaaa


 


11
Rhesus TFR2 -
accccaggacctgcgctcagggagcaggcaggtgtggggctgtggagagattggcag



XM_001113151
gggagagcacagccgcttgtgctctggcctggactcaggggccacgtctggaaggtt



.2
ggaccgaggccaggactgtgcccccacccttgggggtggtaaggagcagccttggct




caggctttctgccagggctgataaggagccctcctggggctcccacaaacggtttat




cggtttatcactggggacagcctgcaggcttcaggagggggcacaagcatggagcag




ctttggggtctactccagagagcgcaacaactgtccccaagatcctctcagaccgtc




taccagcgtgtggaaggcccccagaaagggcacctggaggaggaagaggaagacggg




gaggagacactggcccacttctgccccatggagctgaagggccctgagcccctgggc




tctagacccaggcagccaaacctcattccctgggcagcagcaggacggagggctgcc




ccctacctggtcctgactgctctactgatcttcactggggccttccttctgggctac




gtcgccttccgagggtcctgccagacatgcggagactccgtgttggtggtcagtgag




gacgtcaactatgagcctgacctggatttccaccggggcacactgtactggagcgac




ctccaggccatgttcctgcagttcccgggggaggggcgcctggaggacaccatcagg




caaaccagccctcgggaacgggtggcaggctcggccgggatggccgctctgactcag




gatatccgcgcggcgctctctcgccagaaactggaccacgtgtggaccgacacgcac




tacgtggggctgcaattcccggacccggctcaccccaacaccctgcactgggtcgat




gaggccgggaaggtcggagagcagctgccgctagaggaccctgacgtctactgcccc




tacagcgccatcggcaacgtcacgggagagctggtgtacgcccactacgggcggccc




gaagacctgcaggacctgcgggccaggggcgtggacccagcgggccgcctgctgcta




gtgcgcgtgggggtgatcagcttcgcccagaaggtgaccaatgctcaggactttggg




gctcaaggagtgctcatatacccagagccagcggacttctcccaggacccacacaag




ccaagcctgtccagccagcaggctgtgtatggacatgtgcacctgggaactggagac




ccctacacgcctggcttcccttccttcaatcaaacccagttccctccagttgcatca




tcgggccttcccagcatcccagcccagcccatcactgcagacattgcctcccgcctg




ctgaggaagctcaaaggccctgcggccccccaggaatggcaggggagcctcctaggc




tccccttatcacctgggccccgggccacgactgcggctagcggtcaacaaccacagg




acctccacccccatcaacaacatctttggctgcatcgaaggccgctcagagccagat




cactatgttgtcatcggggcccagagggatgcgtggggcccaggagcagctaaatcc




gctgtggggacagctatactcctggagctggtgcggaccttttcctccatggtgagc




aacggcttccggccccgcagaagtctcctcttcatcagctgggatggcggtgacttt




gggagcgtgggctccacagagtggctagagggctacctcagtgtgctgcacctcaaa




gctgtagtgtacgtgagcctggacaacgcagtgccgggggatgacaagtttcatgcc




aagaccagcccccttctgacaagtctcattgagagtgtcctgaaacaggcaagagca




ccccaggaatggctgaccctgcagtgggtgtactccgcgcggggggactacatccgg




gcggcggagaagctgcggcaggagatctacagctcggaggagagagacgagcgactg




acacgcatgtacaacgtgcgcataatgcgggtggagttctacttcctttcccagtac




gtgtcgccggccgactccccgttccgccacatcttcatgggccgcggagaccacacg




ctgggcgccctgctggaccacctgcggctgctgcgctccaacagctccgggaccccc




ggggccacctcctccgccgtcttccaggagagtcgcttccggcgtcagctagccctg




ctcacctggacgctgcaaggggcagccaatgcgcttagcggggacgtctggaacatt




gataacaacttctgagaccctggggatcctcagatccccctgtccccttgtcgagag




ctcctctgctcctcgcttcaatgattcagggtcagggaggtggctcagagtccacct




ctcattgctgatcgacttctcattacccctacacgtctctccacggagcccagactg




cagcacagatatccacacaccccagccctgcagtgtagctgactctaatgtgatggt




catactgtcggttaatcagagagcagtatcccttcaatcacaaccccttcccctttc




tggggtcctccatacctagagactaggccttgggacctggccagctctcttagcggg




agagatcgctggcaccatagccttatggccaacaggtggtctgtggtgaaaggggca




tggagtttcaatgtc


 


12
Mouse TRF2 -
gagcatggtccaagaaacccagagacctgttgctgagctgaacttggctgctgtgtc



NM_015799.3
ttcccactcaggactcggctttgacagctgcaggtcctggtgtcttcgtcgcggctt




ggatttcaaactggaggagttcaggagggggcacaagcatggagcaacgttggggtc




tacttcggagagtgcaacagtggtccccaagaccctctcagaccatctacagacgcg




tggaaggccctcagctggagcacctggaggaggaagacagggaggaaggggcggagc




ttcctgcccagttctgccccatggaactcaaaggccctgagcacttaggctcctgtc




ccgggaggtcaattcccataccctgggctgcagcaggtcgaaaggctgccccctatc




tggtcctgatcaccctgctaatcttcactggggccttcctcctaggctacgtggcct




ttcgagggtcctgccaggcgtgtggggactccgtgttggtggtcgatgaagatgtca




accctgaggactccggccggaccacgttgtactggagcgacctccaggccatgtttc




tccggttccttggggaggggcgcatggaagacaccatcaggctgaccagcctccggg




aacgcgtggctggctcagccagaatggccaccctggtccaagatatcctcgataagc




tctcgcgccagaagctggaccacgtgtggactgacacgcactacgtgggacttcagt




tcccagatccggctcacgctaacaccctgcactgggtggatgcagacgggagcgtcc




aggagcagctaccgctgaaggatccggaagtctactgcccctacagcgccaccggca




acgccacgggcaagccggtgtacgcccactacgggcggtcggaggacctacaggacc




taaaagccaagggcgtggagctggccggcagcctcctgctagcgcgagttggaatta




ctagcttcgcccagaaggtagccgttgcccaggactttggggctcaaggagtgctga




tataccctgacccatcagacttctcccaggatccccacaagccaggcctgtctagcc




accaggctgtgtacggacatgcgcacctgggaactggagacccttacacacctggct




tcccgtccttcaatcaaacccagttccctccagtagaatcatcaggccttcccagca




tccccgcccagcccatcagtgctgacattgctgaccaattgctcaggaaactcacag




gccccgtggctccccaggagtggaaaggtcacctctcaggctctccttatcggctgg




gacctgggcccgacttacgccttgtggtcaacaaccacagagtctctacccccatca




gtaacatctttgcgtgcatcgagggctttgcagagccagatcactatgttgtcattg




gggcccagagggatgcatggggcccaggagcagccaagtctgcagtggggactgcca




tcctgctggagctggttcggaccttctcttccatggtcagcaatgggttcagacctc




gaagaagtcttttgttcatcagctgggacggaggtgactttggcagcgtgggagcca




cagagtggttggagggctacctcagcgtgctacacctcaaagctgttgtgtacgtga




gcctggacaactccgtgttgggagatggcaaattccatgctaagaccagcccccttc




tcgtcagcctcattgagaatatcttgaagcaggtggactcccctaaccatagtggac




agaccctctatgaacaagtggcactcacccaccccagctgggatgctgaagtgattc




agcccctgcccatggacagcagtgcatattccttcacagcctttgcgggggtcccag




ccgtggagtcctccttcatggaggacgatcgggtgtacccattcctgcacacgaagg




aggacacatacgagaatctgcacaagatgctgcgaggtcgcccgcccgccgtggtcc




aggcagtggctcagctcgcgggccagctcctcatccgactgagccacgatcacctac




tgccgctagacttcggccgctatggagacgcggttctcaggcacatcggcaacctca




atgagttctctggggacctcaaggagcgcgggctgaccctgcagtgggtgtactctg




caaggggggactacatccgtgcggcggaaaagctgcggaaggagatttacagctcgg




agcggaacgatgagcgtctgatgcgcatgtacaacgtgcgcatcatgagggtggagt




tctacttcctgtcccagtatgtgtcgccagccgactccccattccgccacattttcc




taggccaaggcgaccacactttgggtgccctggtagaccacctgcggatgctgcgcg




ccgatggctcaggagccgcctcttcccggttgacagcaggtctgggcttccaggaga




gtcgcttccggcgccagctggcgctgctcacctggacactgcagggggcagccaacg




ctctcagtggcgacgtttggaacattgacaataacttttgaagccaaaagccctcca




tgggccccacgtgattctcctttctccctctttgagtggtgcaggcaaaggaggcgc




ctgagattgtaacctattcttaacacccttggtcctgcaatgctggtgcgccatatt




ttctcagtgtggttgtcatgccgttgcttacccagaaagcggttttcttcccatcac




aggcccttctgtcttcaggagcaaagttccccatatctagagactatctagatgctg




ggatctgatcagctctcttagagagtgagatggacagcgtcattattttatgacaca




tgagctacggtatgtgagcagcccaaggggattagatgtcaataaaccaattgtaac




ccctgttgtccatacgcaa


 


13
Rat TFR2 -
aaatccagagacctgttgctgagttgaacttggctgctgtgtcttcccactcaggac



NM_001105916
tcggctttgacagacacgaggcagggactggggtgagcccctacctctcagatcttt



.1
ctggacctggctgcgggtcctgggatcttcagcgcggcttggatttcaaactggagg




ggttcaggagggggcacaagcatggaacaacgttggggtctacttcggaaagtgcaa




cagtggtccccaagaccctctcagaccatctacagacgtgtggaaggccctcaactg




gagaacctagaggaggaagatagggaggaaggggaggagcttcctgcccagttctgc




cccatggaactcaaaggccctgagcgcttaggctcctgtcctgggaggtccattccc




ataccctgggctgcagcaggtcgaaaggctgctccctatctggtcctgaccaccctg




ctaatcttcactggggccttcctcctgggctacgtggcctttcgagggtcctgccag




gcatgtggggactctgtgttggtggttggtgaagatgtcaactctgaggactccagc




cggggcacgttgtactggagtgacctccaggacatgtttctccggttccttggggag




ggacgcatggaggacaccatcaggctgaccagcctccgggaacgcgtggccggctca




gccagaatggccaccctggtccaagacatcctcgataagctctcgcgccagaagctg




gaccacgtgtggactgacacgcactatgtgggacttcagttcccggacccggctcac




cctaacaccctgcactgggtgggtgcagacgggagcgtccaagagcagctaccgctg




gaggatccggaagtctactgtccctacagcgccacgggcaacgccacgggcaagctt




gtgtacgcccactacgggcggcgggaggacctgcaggacctgaaagccaaggacgtg




gagctggccggcagcctcctgctagtgcgcgctgggattacaagcttcgcccagaag




gtagccattgcccaggactttggggcccacggagtgctgatataccctgacccagcg




gacttctcccaagacccccacaagccaggcctgcctagtgacagggctgcgtatgga




catgtgcacctgggaactggggacccttacacgcctggcttcccgtccttcaatcaa




acccagttccctccagtagaatcatcggggcttcccaacatccctgcccagcccatc




agtgccgacgttgctgatcgcttgctcaggaaactcacaggtcccgtggctcctcag




gaatggaagggtcgcctctcagactctccgtatcgcctgggacctgggccaggctta




cgccttgtggtcaacaaccacagaacctctactcccatcagtaacatctttgcgtgc




atcgagggcttcgcagagccagatcactatgtcgttatcggggcccagagggatgcc




tggggcccaggagcagccaagtctgcagtggggactgccatcctcctggagctggtt




cggaccttttcctccatggtcagcagtggctttagacctcgaagaagtcttttgttc




atcagctgggacggaggtgactttggcagcgtgggagccacggagtggttggagggc




tacctcagcgtgctacacctcaaagctgtcgtgtatgtgagcctggacaactccgtg




ttgggagacggcaaattccatgctaagaccagcccccttctcgtcagcctcattgag




aatatcctgaagcaggtggattcccctaaccacagtggacagacactctacgatcaa




gtggcattcacccacccaagctgggatgctgaagcgatccagcccctgcccatggac




agcagcgcatattccttcacagcttttgcgggcgtcccagctgtggagttctccttc




atggaggacgatagggtgtacccattcctgcacacgaaggaggacacgtatgagaat




ctgcacaagatgctgcgaggtcgcctgcccgccgtggtcctagcagtggctcagctc




gctggtcagctcctcatccgactgagccacgatcacctactgccgctggacttcggc




cgctacggagacgtggtcctcaggcacatcggcaaccttaatgagttctctggggac




ctcaaggcgcgcgggctgaccctgcagtgggtgtactctgcaaggggggactatatc




cgggcggcggagaagctgcggaaggagatttacagctcggagcagagcgatgagcgt




ctgatgcgcatgtacaacgtgcgcatcatgagggtggagttctacttcctgtcccag




tacgtgtcgccggccgactccccattccgccacattttcctaggccaaggcgaccac




actttgggtgccctggtggaacacctacggatgctgcgctccgatggctcaggagct




gcctcttctgggttgagcccaggtctgggcttccaggagagtcgcttccggcgacag




ctggcgctgctcacgtggacgctacagggggcagccaacgcactcagtggcgacgtt




tggaacatcgacaataacttttgaggccagaagtcctccatgggccccacgtgattc




tcctttctccctatttgagtggtgcaggcaacggaggtgcctgagagcaacctatcc




tcattaacaaccttggtcctgcaacgccagtgagacatattttctcagtgtgactgt




tataccactgtttatccagaaagcggttttcttcccatcactggcctctctgccttc




aggagcatagttccccatatctagaaaccatctagacactgggatccagctctctta




gcgggtgagatggatagcgtcatttccttatgacacacaagtggtatgtgggtggcc




caagggggattagatgtcaataaaccatttacctggtaacctctgttgtccataagc


 


14
Human HFE -
ctaaagttctgaaagacctgttgcttttcaccaggaagttttactgggcatctcctg



NM_139006.2
agcctaggcaatagctgtagggtgacttctggagccatccccgtttccccgcccccc




aaaagaagcggagatttaacggggacgtgcggccagagctggggaaatgggcccgcg




agccaggccggcgcttctcctcctgatgcttttgcagaccgcggtcctgcaggggcg




cttgccgcgttcacactctctgcactacctcttcatgggtgcctcagagcaggacct




tggtctttccttgtttgaagctttgggctacgtggatgaccagctgttcgtgttcta




tgatcatgagagtcgccgtgtggagccccgaactccatgggtttccagtagaatttc




aagccagatgtggctgcagctgagtcagagtctgaaagggtgggatcacacgttcac




tgttgacttctggactattatggaaaatcacaaccacagcaaggagtcccacaccct




gcaggtcatcctgggctgtgaaatgcaagaagacaacagtaccgagggctactggaa




gtacgggtatgatgggcaggaccaccttgaattctgccctgacacactggattggag




agcagcagaacccagggcctggcccaccaagctggagtgggaaaggcacaagattcg




ggccaggcagaacagggcctacctggagagggactgccctgcacagctgcagcagtt




gctggagctggggagaggtgttttggaccaacaagtgaccactctacggtgtcgggc




cttgaactactacccccagaacatcaccatgaagtggctgaaggataagcagccaat




ggatgccaaggagttcgaacctaaagacgtattgcccaatggggatgggacctacca




gggctggataaccttggccgtaccccctggggaagagcagagatatacgtgccaggt




ggagcacccaggcctggatcagcccctcattgtgatctgggagccctcaccgtctgg




caccctagtcattggagtcatcagtggaattgctgtttttgtcgtcatcttgttcat




tggaatcttgttcataatattaaggaagaggcagggttcaagaggagccatggggca




ctacgtcttagctgaacgtgagtgacacgcagcctgcagactcactgtgggaaggag




acaaaactagagactcaaagagggagtgcatttatgagctcttcatgtttcaggaga




gagttgaacctaaacatagaaattgcctgacgaactccttgattttagccttctctg




ttcatctcctcaaaaagatttccccatttaggtttctgagttcctgcatgccggtga




tccctagctgtgacctctcccctggaactgtctctcatgaacctcaagctgcaccta




gaggcttccttcatttcctccgtcacctcagagacatacacctatgtcatttcattt




cctatttttggaagaggactccttaaatttgggggacttacatgattcattttaaca




tctgagaaaagctttgaaccctgggacgtggctagtcataaccttaccagattttta




cacatgtatctatgcattttctggacccgttcaacttttcctttgaatcctctctct




gtgttacccagtaactcatctgtcaccaagccttggggattcttccatctgattgtg




atgtgagttgcacagctatgaaggctgtacactgcacgaatggaagaggcacctgtc




ccagaaaaagcatcatggctatctgtgggtagtatgatgggtgtttttagcaggtag




gaggcaaatatcttgaaaggggttgtgaagaggtgttttttctaattggcatgaagg




tgtcatacagatttgcaaagtttaatggtgccttcatttgggatgctactctagtat




tccagacctgaagaatcacaataattttctacctggtctctccttgttctgataatg




aaaattatgataaggatgataaaagcacttacttcgtgtccgactcttctgagcacc




tacttacatgcattactgcatgcacttcttacaacaattctacgagataggtactat




tatccccatttcttttttaaatgaagaaagtgaagtaggccgggcacggtggctcac




gcctgtaatcccag


 


15
Rhesus HFE -
ttttactgggcatctcctgagcctaggcaatagctgtagggtgacttctggagccat



XM_001085598
cgccgtttccccgccccaccaaagaagcggagacttaaaggggacgtgcagtcagag



.2
ctggggaaatgggcccgcgagccaggccggcgcttctcctcctgatgcttttgcaga




ccgcggtcctgcaggggcgcttgctgcgttcacactctctgcactacctcttcatgg




gttcctcagagcaggaccttggtctttccctgtttgaagctttgggctatgtggacg




accagctgttcgtgttccatgatcacgagagtcgccgtgtggagccccgaactccat




gggtttccggtagaacgtcaagccagatgtggctgcagctgagtcagagtctgaaag




ggtgggatcacatgttcactgttgacttctggactattatggaaaatcacaaccaca




gcaaggagtcccacaccctgcaggtcatcctgggctgcaaaatgcaagaggacaaca




gtaccgagggcttctggaagtacgggcacgatgggcaggaccaccttgaattctgcc




ctgacacactggattggagagcagcagaacccagggcctggcccaccaagctggagt




gggaaaggcacaaaattcgggccaggcagaacagggcctacctcgagagggactgcc




ctgtgcagctgcagcagttgctggagctggggagaggtgttttcgaccggccagtga




ccactctacggtgtcgggccctgaactactacccccagaacatcaccatgaagtggc




tgaaggataggcagtcaatggatgccaaggaggtcgaacctaaagacgtattgccca




atggggatgggacctaccagggctggataaccttgactgtacccccaggggaagaac




agagatatacttgccaggtggagcacccaggcctggatcagcccctccttgctttct




gggagccctcaccatctagaactctagtcattggagtcatcagtggaattgctgttt




ttgtcatcatcttgttcattggaattttgttcataatattaaggaagaggcagactt




caagaggagtcatggggcactacgtcttagctgaacgtgagtgacacg


 


16
Mouse HFE -
ctgagaggtctggaacctcagcaatggctacagggtgacttcttggatcctccacgt



NM_010424.4
ttccagatcctagtgaagaccggtggacccagctgaggacatgagcctatcagctgg




gctccctgtgcggccgctgctgctgctgctgctactgctgtggtccgtggccccgca




ggcactgccaccgcgttcacattctccaagatacctcttcatgggtgcctcagagcc




agacctcgggctgcctttgtttgaggctaggggctatgtggatgaccagctctttgt




gtcctacaatcatgagagtcgccgtgctgagcccagggccccgtggatcttggagca




aacctcaagccagctgtggctgcatctgagtcagagcctgaaagggtgggactacat




gttcatagtagacttctggaccatcatgggcaactataaccacagtaaggtcacgaa




gttgggagtggtgtccgagtcccacatcctgcaggtggtcctaggctgtgaggtgca




tgaagacaacagtaccagcggcctctggagatatggttatgacgggcaagatcacct




ggaattctgccccaagacactaaactggagcgcagccgagccaggggcctgggccac




caaggtggaatgggacgagcacaagatccgtgccaaacagaacagggactacctgga




gaaggactgccccgagcagctgaaacggctcctggagctggggagaggcgttctggg




acagcaagtgcctactttggtgaaagtgactcgccactgggcctctacggggacctc




tctaaggtgtcaggctctggacttcttcccccagaacatcactatgaggtggttgaa




ggacaaccaaccactggatgccaaagatgtcaaccccgagaaggtgctacctaacgg




ggatgagacctatcaaggctggctgacattggccgtggcccctggggacgagacaag




gttcacctgtcaagtggagcacccaggcctggaccagcctctcactgcctcttggga




gcccttgcaatctcaggccatgattatcggaatcatcagtggagtcaccgtctgtgc




catctccttggttggaattctgttcctaatcttaaggaaaaggaaggcttcaggagg




aaccatgggtggctatgtcttaacagactgtgagtgatctgcagcctgctgaaccac




ggaagagagaaaactcagccaaagacttggagggggcacacttgctccactgtagga




cacagttggacctaacacacagaaactgcctgagaactgtgctcttagctttctctg




ttcactttcttaaggtgttttctccagttaagttcagttcctgaatagtagtgattg




caccagttgcaacctctccctccagaactggtctcatgattcttaggctgcttcttg




gaagcatcctatgtttccttcatgcacctagactccatatgtctacgtaaagagccc




ctctaagtttagtggatacatgattcgtttccacatctgaagaagttgtgaaccttc




atccggggatgctcacacatacttgagccagaatttttcacctatatcctagaatcc




aggacccactcaactatcctccatctgttatagagtgactcctctgtcaccatgccc




tgacttctctgccattggagtgttatatatatggatcatcaataaagccatgaaggc




tacacaactgtg


 


17
Rat HFE -
tcagcaatggctacagggtgacttcttggatcctccacgtttccaggtcctagtgaa



NM_001173435
aaccggtggacccagctggaggcatggaccgatcagctgggctccctgtgcggctgc



.1
tattgctgctgctgttgttgctgctgtggtccgtggccccgcaggcgctgcggcccg




tgcctacttcggtgaaagtgactcgccactgggcctctacagggacctccctaaggt




gtcaggctctgaatttcttcccccagaacatcactatgaggtggttgaaggacagcc




agcccctagatgccaaggatgtcaaccctgagaacgtgctgccaaatggggatggga




cctatcagggctggctgaccttggctgtggcccctggagaagagacaaggttcagct




gtcaagtggagcacccaggcctggatcagcctctcactgccacttgggagccctcac




ggtctcaggacatgattattggaatcataagtgggatcaccatttgtgccatcttct




ttgttggaattctgatcctagtcttaaggaaaaggaaggtttcaggaggaaccatgg




gtgactatgtcttaacagagtgtgagtgacctgcagcatgcagaagcacagaagaga




gaagactcagccaaagacttggaggggacacacttgctccattctagaacacagctg




gacctaacacacagaaactgcctgaggactctgcccttagctttcctgtttgctttc




ttaaggtgttttctccagttaagttcagttcctgaataatagtgactgccccagctg




caacctctcccttcagaaccagtctcatgatctttaagctgctacttgcaggcatcc




ttcgttttctgcatccacctagacttcgtatgtctacttaaaaagccccactaaatt




tgggggacacatgattcatttccacatctgaagaagttatgaaccttcatcctggga




tgcacacattcttgtgccagaatttttcatacatatcctaggacccattcaattgtc




atttgagcccctctatccgttagtgactactctgacttctctgccattggagtgtta




tggcaataaagctatgaacgtta


 


18
Mouse BMPR1a -
ccgcgcgagacgacgactgtacggccgcgcgaggggcgaccgggcccgggccgctgc



NM_009758.4
acgccgagggcggaggccgagccgggccccgccgccccgcggctgtccgtgcccgcc




cgcgccgagcgccggaggatgagtttctcgggatcccgatttatgaaaatatgcatc




gctttgatactgtctggaattccatgagatggaagcataggtcaaagctgttcggag




aaattggaactacagttttatctagccacatctctgagaattctgaagaaagcagca




ggtgaaagtcattgccaagtgattttgttctgtaaggaagcctccctcattcactta




caccagtgagacagcaggaccagtcattcaaagggccgtgtacaggacgcgtgcgaa




tcagacaatgactcagctatacacttacatcagattactgggagcctgtctgttcat




catttctcatgttcaagggcagaatctagatagtatgctccatggcactggtatgaa




atcagacttggaccagaagaagccagaaaatggagtgactttagcaccagaggatac




cttgcctttcttaaagtgctattgctcaggacactgcccagatgatgctattaataa




cacatgcataactaatggccattgctctgccatcatagaagaagatgatcagggaga




aaccacattaacttctgggtgtatgaagtatgaaggctctgattttcaatgcaagga




ttcaccgaaagcccagctacgcaggacaacagaatgtcgtcggaccaatttgtgcaa




ccagtatttgcagcctacactgccccctgttgttataggtccgttctttgatggcag




catccgatggctggttgtgctcatttccatggctgtctgtatagttgctatgatcat




cttctccagctgcttttgctataagcattattgtaagagtatctcaagcaggggtcg




ttacaaccgtgatttggaacaggatgaagcatttattccagtaggagaatcattgaa




agacctgattgaccagtcccaaagctctgggagtggatctggattgcctttattggt




tcagcgaactattgccaaacagattcagatggttcggcaggttggtaaaggccgcta




tggagaagtatggatgggtaaatggcgtggtgaaaaagtggctgtcaaagtgttttt




taccactgaagaagctagctggtttagagaaacagaaatctaccagacggtgttaat




gcgtcatgaaaatatacttggttttatagctgcagacattaaaggcactggttcctg




gactcagctgtatttgattactgattaccatgaaaatggatctctctatgactccct




gaaatgtgccacactagacaccagagccctactcaagttagcttattctgctgcttg




tggtctgtgccacctccacacagaaatttatggtacccaagggaagcctgcaattgc




tcatcgagacctgaagagcaaaaacatccttattaagaaaaatggaagttgctgtat




tgctgacctgggcctagctgttaaattcaacagtgatacaaatgaagttgacatacc




cttgaataccagggtgggcaccaagcggtacatggctccagaagtgctggatgaaag




cctgaataaaaaccatttccagccctacatcatggctgacatctatagctttggttt




gatcatttgggaaatggctcgtcgtcgtattacaggaggaatcgtggaggaatatca




attaccatattacaacatggtgcccagtgacccatcctatgaggacatgcgtgaggt




tgtgtgtgtgaaacgcttgcggccaatcgtgtctaaccgctggaacagcgatgaatg




tcttcgagcagttttgaagctaatgtcagaatgttgggcccataatccagcctccag




actcacagctttgagaatcaagaagacacttgcaaaaatggttgaatcccaggatgt




aaagatttgacaattaaacaattttgagggagaatttagactgcaagaacttcttca




cccaaggaatgggtgggattagcatggaataggatgtcgacttggtttccagactcc




ttcctctacatcttcacaggctgctaacagtaaaccttaccgtactctacagaatac




aagattggaacttggaacttcaaacatgtcattctttatatatggacagctttgttt




taaatgtggggtttttttgttttgctttttttgttttgttttggttttgatgctttt




ttggtttttatgaactgcatcaagactccaatcctgataagaagtctctggtcaacc




tctgggtactcactatcctgtccataaagtggtgctttctgtgaaagccttaagaaa




attaatgagctcagcagagatggaaaaaggcatatttgccttctaccagagaaaaca




tctgtctgtgttctgtctttgtaaacagcctatagattatgatctctttgggatact




gcctggtttatgatggtgcaccatacctttgatatgcataccagaattctctgctgc




cctagggcttagaagacaagaatgtaaaggttgcacaggaaggtatttgtggccagt




ggtttaaatatgcaatatctagttgacaatcgccaatttcataaaagccatccacct




tgtaactgtagtaacttctccactgactttatttttagcataatagttgtgaaggcc




aaactccatgtaaagtgtccatagacttggactgttttcccccagtcaccattttgt




tctccttttggtaattatttttgttataaaaagccacctatccagaattggagctct




ctgtcttgaaccatactttgaaagaaacgcctcttccgtactgcatctgatcacaat




gtgcatacctatgatcaaattctggagtctttgttctcggtacctcctaaaaaggaa




agttgattcttgtgcaacatgcttttattttcagaacctgcacagctgtcattctag




ccatgttttacctacacactcagttctacacaagacagcccatacactctgtctcac




atctgatccttggtgggaagtgttttaaagtagaactatgtatgaatttcagaattc




atgcattttaaaacttcactaagatattgtctcatatctttatgagaatgtcagctg




acttttcaactaacagtaaatgtattttagatatctaaatcttttgaaatttggttt




tacaatttctggtccctaattgtgaagacaagaggcagaagtacccagtcactaccc




atatttacactgaacgttattaaataaaatgatgtgtattttattataaaataaata




taggccttgttatctcaaaaaacagatctggttcaaacttattataccaatatcata




ctatttaaatgttctaagtaaacaagccatgtgagcatcaagtggcattggctcttt




ggatgaaacataaacttaaggtgattgtatcaacacatagagtgactgaaattaaat




gggaggcaggtagagcatatgtccatctgtccacctacaggcatgactaaactacag




ctcatattccacaaatttgagatttgtcttgcctggtttgtttagtgagtctcatct




gatgtacctaaagcctgagagtactgaggtctgattttatatctttcccgaataaac




taaatcttttttgtcacttatcatcttaatgatatacctaaggaataattctttggc




atgtttcagttgtgcgtggcagccactgtaatgactcttctctaagaaaggctgtca




ggagttaattataaggcaggcagtgagcgctctagtcactgccttcccacgctgcca




tcactgcattcatgggaatcagtgacgttctcgaaatggcaaacgctgctgcttttc




cttatttggaatcctaaaatcaaaagttgcattaaacttactgtgttctcttatccc




tctcagccataaatgtaaaattcagtaagtaaaaatatttaaagagtgtatcagccc




tttggccagtgagatagctcagtagataaaggcatttgctgccaagtcctcaacctc




aattcagactctgggggacacatggcgaagggaggagccaactaccccatcattgtc




ctctgacttccacacactccatggcttgcgcccctccccacagacacacaccatgta




ctccacaaaagtagtttaaaggaaaaaaagaatagaacccactgtgtaatggaataa




gtattatgtagttacttaacaacttgtaaaaatctggaaactacgatttggttcccc




tttgaatctagagtttaaaaaacagatggctaaaatcagccatcatttaaataatta




aaaataaaagccccaaaccccaaactgcctaaataaataccaagtaatccaggaagc




cgtcatgtgtggtttgtatgaccagtagttctctggtacagagcatgttaagatttg




ccccagcctgattctctgaggtctctgcattactgagtactgtcctgagtataaaat




ctgaactgatttctctagaaatactgtaacaaaaaggtattttagtcagcatgttat




gttaacccttccactgtctagaaacttgaataagcacataaagacaccttttgctgt




catcatctgttgtcctggaatgtgccagttttaatttattcattctaatgatattca




atttgcttttcttttttagatgtttttcttgtttagagtaaaaggacgaatttttca




agaaccttgcatctctgatttggcctaaggtcaaattggatattgagtagtctattc




cagggcagatttcctaagcaatacttgtcttttcagctatgtattgttttgaaatgt




ttccatttcaacagaggtgtaagtcatgtgaaaagaaaggtggtgtagcccttgtgg




taatgacacaagttgacttgcgtcagatgttaagcagggacagttctcccacctcct




ggctgtaaggagtggaaactaggcaagcagtgtatcagtccacagaggacaggaagg




gtcatcccataaagaaagcctgtgagtatggctttggcaaaaaattagacataatac




tgtccttttaggttgtgctctgttctttcctttcagtggaattatttaagctcttta




gtggcctttgtttttcccacttaaaaactaaaatgtagcatatattgtataaaatgg




aaatattaatagcttagggaaactgtacataaggcattgacaggtttaaaaaaagca




tttttattatgcagttgtaaaacaccaaaaatatagattcatcttgatatgtaacac




taagtgtattttgtacagcatctgatttgaaaggtgccttatgaagtttaccattaa




ttgctttgttctatatacagattatgtccaatgtatcatttttcagtaaataacctt




attttagta


 


19
Rat BMPR1a -
gaattcatgagatggaaacataggtcaaagctgtttggagaaattggaactacagtt



NM_030849.1
ttatctagccacatctctgagaagtctgaagaaagcagcaggtgaaagtcattgtca




agtgattttgttcttctgtaaggaaacctcgttcagtaaggccgtttacttcagtga




aacagcaggaccagtaatcaaggtggcccggacaggacacgtgcgaattggacaatg




actcagctatacacttacatcagattactgggagcctgtctgttcaccatttctcat




gttcaagggcagaatctagatagtatgctccatggtactggtatgaaatcagacgtg




gaccagaagaagccggaaaatggagtgacgttagcaccagaggacaccttacctttc




ttaaaatgctattgctcaggacactgcccagatgacgctattaataacacatgcata




actaatggccattgctttgccattatagaagaagatgatcagggagaaaccacgtta




acttctgggtgtatgaagtatgaaggctctgattttcaatgcaaggattcaccaaaa




gcccagctacgcaggacaatagaatgttgtcggaccaatttgtgcaaccaatatttg




cagcctacactgccccctgtcgttataggcccattctttgatggcagcgtccgatgg




ctggctgtgctcatctctatggctgtctgtattgtcgccatgatcgtcttctccagc




tgcttctgttacaaacattactgtaagagtatctcaagcagaggtcgttacaaccgt




gacttggaacaggatgaagcatttattccagtaggagaatcactgaaagacctgatt




gaccagtcacaaagctctggtagtggatctggattacctttattggttcagcgaact




attgccaaacagattcagatggttcggcaggttggtaaaggccggtatggagaagta




tggatgggtaaatggcgtggtgaaaaagtggctgtcaaagtattttttaccactgaa




gaagctagctggtttagagaaacagaaatctaccagacggtgttaatgcgtcatgaa




aatatacttggttttatagctgcagacattaaaggcaccggttcctggactcagctg




tatttgattactgattaccatgagaatgggtctctctatgacttcctgaaatgtgcc




accctggacaccagagccctactcaagttagcttattctgctgcctgtggtctgtgc




cacctccacacagaaatttatggcacgcaaggcaagcctgcaattgctcatcgagac




ctgaagagcaaaaacatccttattaagaaaaatggtagttgctgtattgctgacctg




ggcctagctgttaaattcaacagtgacacaaatgaagttgacatacccttgaacacc




agggtgggcaccaggcggtacatggctccagaagtgctggacgagagcctgagtaaa




aaccatttccagccctacatcatggctgacatctacagctttggtttgatcatttgg




gagatggcccgtcgctgtattacaggaggaatcgtggaggaatatcaattaccatat




tacaacatggtgcctagtgacccatcttatgaagacatgcgtgaggtcgtgtgtgtg




aaacgcttgcggccaatcgtctctaaccgctggaacagtgatgaatgtcttcgagcc




gttttgaagctgatgtcagaatgctgggcccataatccagcatccagactcacagct




ttgagaatcaagaagacgctcgcaaagatggttgaatcccaggatgtaaagatttga




caaacagttttgagaaagaatttagactgcaagaaattcacccgaggaagggtggag




ttagcatggactaggatgtcggcttggtttccagactctctcctctacatcttcaca




ggctgctaacagtaaactttcaggactctgcagaatgcagggttggagcttcagaca




taggacttcagacatgctgttctttgcgtatggacagctttgttttaaatgtgggct




tttgatgcctttttggtttttatgaattgcatcaagactccaatcctgataagaagt




ctctggtcaaactctggttactcactatcctgtccataaagtggtgctttctgtgaa




agccttaaggaaattagtgagctcagcagagatggagaaaggcatatttgccctcta




cagagaaaatatctgtctgtgttctgtctctgtaaacagcctggactatgatctctt




tgggatgctgcctggttgatgatggtgcatcatgcctctgatatgcataccagactt




cctctgctgccatgggcttacaagacaagaatgtgaaggttgcacaggacggtattt




gtggccagtggtttaaatatgcaatatctaatcgacattcgccaatctcataaaagc




catctaccttgtaactgaagtaacttctctaccaactttatttttagcataatagtt




gtaaaggccaaactatgtataaagtgtccatagactcgaactgttttcctccagtca




ccattttgttttccttttggtaattatttttgttatataatccctcctatccagaat




tggcgctcactgtcttgaaccatactttgaaagaaatgcctcttcctggagtctgcc




ttactgcatctgatcaccatgtgcatacctctgatcaaattctggagtctttgttct




cggtacctcttaaaaagggaaattgtgtatcatgtgtagtgtgcttttattttcaaa




atcttcatagcctttattctagccatttttacctacatactcattctgtacaaaaca




gctcactcggtctcacggctgatcctcagtggaaatgatttaaagtagagctgtgta




cgaatttcagaattcatgtatttaaaaacttcacactaacactttactaagatattg




tctcatatcttttatgaggatgtcagctgattttcaatgactataaatgtatcttag




ctatctaaatcttttgaaatttggttttataatttctggtccctaacttgtgaagac




aaagaggcagaagtacccagtctaccacatttacactgtacattattaaataaaaaa




atgtatattttaaaaaaaaaaaaaaaaaaaaa


 


20
Human SMAD4 -
atgctcagtggcttctcgacaagttggcagcaacaacacggccctggtcgtcgtcgc



NM_005359.5
cgctgcggtaacggagcggtttgggtggcggagcctgcgttcgcgccttcccgctct




cctcgggaggcccttcctgctctcccctaggctccgcggccgcccagggggtgggag




cgggtgaggggagccaggcgcccagcgagagaggccccccgccgcagggcggcccgg




gagctcgaggcggtccggcccgcgcgggcagcggcgcggcgctgaggaggggcggcc




tggccgggacgcctcggggcgggggccgaggagctctccgggccgccggggaaagct




acgggcccggtgcgtccgcggaccagcagcgcgggagagcggactcccctcgccacc




gcccgagcccaggttatcctgaatacatgtctaacaattttccttgcaacgttagct




gttgtttttcactgtttccaaaggatcaaaattgcttcagaaattggagacatattt




gatttaaaaggaaaaacttgaacaaatggacaatatgtctattacgaatacaccaac




aagtaatgatgcctgtctgagcattgtgcatagtttgatgtgccatagacaaggtgg




agagagtgaaacatttgcaaaaagagcaattgaaagtttggtaaagaagctgaagga




gaaaaaagatgaattggattctttaataacagctataactacaaatggagctcatcc




tagtaaatgtgttaccatacagagaacattggatgggaggcttcaggtggctggtcg




gaaaggatttcctcatgtgatctatgcccgtctctggaggtggcctgatcttcacaa




aaatgaactaaaacatgttaaatattgtcagtatgcgtttgacttaaaatgtgatag




tgtctgtgtgaatccatatcactacgaacgagttgtatcacctggaattgatctctc




aggattaacactgcagagtaatgctccatcaagtatgatggtgaaggatgaatatgt




gcatgactttgagggacagccatcgttgtccactgaaggacattcaattcaaaccat




ccagcatccaccaagtaatcgtgcatcgacagagacatacagcaccccagctctgtt




agccccatctgagtctaatgctaccagcactgccaactttcccaacattcctgtggc




ttccacaagtcagcctgccagtatactggggggcagccatagtgaaggactgttgca




gatagcatcagggcctcagccaggacagcagcagaatggatttactggtcagccagc




tacttaccatcataacagcactaccacctggactggaagtaggactgcaccatacac




acctaatttgcctcaccaccaaaacggccatcttcagcaccacccgcctatgccgcc




ccatcccggacattactggcctgttcacaatgagcttgcattccagcctcccatttc




caatcatcctgctcctgagtattggtgttccattgcttactttgaaatggatgttca




ggtaggagagacatttaaggttccttcaagctgccctattgttactgttgatggata




cgtggacccttctggaggagatcgcttttgtttgggtcaactctccaatgtccacag




gacagaagccattgagagagcaaggttgcacataggcaaaggtgtgcagttggaatg




taaaggtgaaggtgatgtttgggtcaggtgccttagtgaccacgcggtctttgtaca




gagttactacttagacagagaagctgggcgtgcacctggagatgctgttcataagat




ctacccaagtgcatatataaaggtctttgatttgcgtcagtgtcatcgacagatgca




gcagcaggcggctactgcacaagctgcagcagctgcccaggcagcagccgtggcagg




aaacatccctggcccaggatcagtaggtggaatagctccagctatcagtctgtcagc




tgctgctggaattggtgttgatgaccttcgtcgcttatgcatactcaggatgagttt




tgtgaaaggctggggaccggattacccaagacagagcatcaaagaaacaccttgctg




gattgaaattcacttacaccgggccctccagctcctagacgaagtacttcataccat




gccgattgcagacccacaacctttagactgaggtcttttaccgttggggcccttaac




cttatcaggatggtggactacaaaatacaatcctgtttataatctgaagatatattt




cacttttgttctgctttatcttttcataaagggttgaaaatgtgtttgctgccttgc




tcctagcagacagaaactggattaaaacaattttttttttcctcttcagaacttgtc




aggcatggctcagagcttgaagattaggagaaacacattcttattaattcttcacct




gttatgtatgaaggaatcattccagtgctagaaaatttagccctttaaaacgtctta




gagccttttatctgcagaacatcgatatgtatatcattctacagaataatccagtat




tgctgattttaaaggcagagaagttctcaaagttaattcacctatgttattttgtgt




acaagttgttattgttgaacatacttcaaaaataatgtgccatgtgggtgagttaat




tttaccaagagtaactttactctgtgtttaaaaagtaagttaataatgtattgtaat




ctttcatccaaaatattttttgcaagttatattagtgaagatggtttcaattcagat




tgtcttgcaacttcagttttatttttgccaaggcaaaaaactcttaatctgtgtgta




tattgagaatcccttaaaattaccagacaaaaaaatttaaaattacgtttgttattc




ctagtggatgactgttgatgaagtatacttttcccctgttaaacagtagttgtattc




ttctgtatttctaggcacaaggttggttgctaagaagcctataagaggaatttcttt




tccttcattcatagggaaaggttttgtattttttaaaacactaaaagcagcgtcact




ctacctaatgtctcactgttctgcaaaggtggcaatgcttaaactaaataatgaata




aactgaatattttggaaactgctaaattctatgttaaatactgtgcagaataatgga




aacatcacagttcataataggtagtttggatatttttgtacttgatttgatgtgact




ttttttggtataatgtttaaatcatgtatgttatgatattgtttaaaattcagtttt




tgtatcttggggcaagactgcaaacttttttatatcttttggttattctaagccctt




tgccatcaatgatcatatcaattggcagtgactttgtatagagaatttaagtagaaa




agttgcagatgtattgactgtaccacagacacaatatgtatgctttttacctagctg




gtagcataaataaaactgaatctcaacatacaaagttgaattctaggtttgattttt




aagattttttttttcttttgcacttttgagtccaatctcagtgatgaggtaccttct




actaaatgacaggcaacagccagttctattgggcagctttgtttttttccctcacac




tctaccgggacttccccatggacattgtgtatcatgtgtagagttggtttttttttt




ttttaatttttattttactatagcagaaatagacctgattatctacaagatgataaa




tagattgtctacaggataaatagtatgaaataaaatcaaggattatctttcagatgt




gtttacttttgcctggagaacttttagctatagaaacacttgtgtgatgatagtcct




ccttatatcacctggaatgaacacagcttctactgccttgctcagaaggtcttttaa




atagaccatcctagaaaccactgagtttgcttatttctgtgatttaaacatagatct




tgatccaagctacatgacttttgtctttaaataacttatctaccacctcatttgtac




tcttgattacttacaaattctttcagtaaacacctaattttcttctgtaaaagtttg




gtgatttaagttttattggcagttttataaaaagacatcttctctagaaattgctaa




ctttaggtccattttactgtgaatgaggaataggagtgagttttagaataacagatt




tttaaaaatccagatgatttgattaaaaccttaatcatacattgacataattcattg




cttcttttttttgagatatggagtcttgctgtgctgcccaggcaggagtgcagtggt




atgatctcagctcactgcaacctctgcctcccgggttcaactgattctcctgcctca




gcctccctggtagctaggattacaggtgcccgccaccatgcctggctaacttttgta




gttttagtagagacggggttttgcctgttggccaggctggtcttgaactcctgacct




caagtgatccatccaccttggcctcccaaagtgctgggattacgggcgtgagccact




gtccctggcctcattgttcccttttctactttaaggaaagttttcatgtttaatcat




ctggggaaagtatgtgaaaaatatttgttaagaagtatctctttggagccaagccac




ctgtcttggtttctttctactaagagccataaagtatagaaatacttctagttgtta




agtgcttatatttgtacctagatttagtcacacgcttttgagaaaacatctagtatg




ttatgatcagctattcctgagagcttggttgttaatctatatttctatttcttagtg




gtagtcatctttgatgaataagactaaagattctcacaggtttaaaattttatgtct




actttaagggtaaaattatgaggttatggttctgggtgggttttctctagctaattc




atatctcaaagagtctcaaaatgttgaatttcagtgcaagctgaatgagagatgagc




catgtacacccaccgtaagacctcattccatgtttgtccagtgcctttcagtgcatt




atcaaagggaatccttcatggtgttgcctttattttccggggagtagatcgtgggat




atagtctatctcatttttaatagtttaccgcccctggtatacaaagataatgacaat




aaatcactgccatataaccttgctttttccagaaacatggctgttttgtattgctgt




aaccactaaataggttgcctataccattcctcctgtgaacagtgcagatttacaggt




tgcatggtctggcttaaggagagccatacttgagacatgtgagtaaactgaactcat




attagctgtgctgcatttcagacttaaaatccatttttgtggggcagggtgtggtgt




gtaaaggggggtgtttgtaatacaagttgaaggcaaaataaaatgtcctgtctccca




gatgatatacatcttattatttttaaagtttattgctaattgtaggaaggtgagttg




caggtatctttgactatggtcatctggggaaggaaaattttacattttactattaat




gctccttaagtgtctatggaggttaaagaataaaatggtaaatgtttctgtgcctgg




tttgatggtaactggttaatagttactcaccattttatgcagagtcacattagttca




caccctttctgagagccttttgggagaagcagttttattctctgagtggaacagagt




tctttttgttgataatttctagtttgctcccttcgttattgccaactttactggcat




tttatttaatgatagcagattgggaaaatggcaaatttaggttacggaggtaaatga




gtatatgaaagcaattacctctaaagccagttaacaattattttgtaggtggggtac




actcagcttaaagtaatgcatttttttttcccgtaaaggcagaatccatcttgttgc




agatagctatctaaataatctcatatcctcttttgcaaagactacagagaataggct




atgacaatcttgttcaagcctttccatttttttccctgataactaagtaatttcttt




gaacataccaagaagtatgtaaaaagtccatggccttattcatccacaaagtggcat




cctaggcccagccttatccctagcagttgtcccagtgctgctaggttgcttatcttg




tttatctggaatcactgtggagtgaaattttccacatcatccagaattgccttattt




aagaagtaaaacgttttaatttttagcctttttttggtggagttatttaatatgtat




atcagaggatatactagatggtaacatttctttctgtgcttggctatctttgtggac




ttcaggggcttctaaaacagacaggactgtgttgcctttactaaatggtctgagaca




gctatggttttgaatttttagttttttttttttaacccacttcccctcctggtctct




tccctctctgataattaccattcatatgtgagtgttagtgtgcctccttttagcatt




ttcttcttctctttctgattcttcatttctgactgcctaggcaaggaaaccagataa




ccaaacttactagaacgttctttaaaacacaagtacaaactctgggacaggacccaa




gacactttcctgtgaagtgctgaaaaagacctcattgtattggcatttgatatcagt




ttgatgtagcttagagtgcttcctgattcttgctgagtttcaggtagttgagataga




gagaagtgagtcatattcatattttcccccttagaataatattttgaaaggtttcat




tgcttccacttgaatgctgctcttacaaaaactggggttacaagggttactaaatta




gcatcagtagccagaggcaataccgttgtctggaggacaccagcaaacaacacacaa




caaagcaaaacaaaccttgggaaactaaggccatttgttttgttttggtgtcccctt




tgaagccctgccttctggccttactcctgtacagatatttttgacctataggtgcct




ttatgagaattgagggtctgacatcctgccccaaggagtagctaaagtaattgctag




tgttttcagggattttaacatcagactggaatgaatgaatgaaactttttgtccttt




ttttttctgtttttttttttctaatgtagtaaggactaaggaaaacctttggtgaag




acaatcatttctctctgttgatgtggatacttttcacaccgtttatttaaatgcttt




ctcaataggtccagagccagtgttcttgttcaacctgaaagtaatggctctgggttg




ggccagacagttgeactetctagtttgccctctgccacaaatttgatgtgtgacctt




tgggcaagtcatttatcttctctgggccttagttgcctcatctgtaaaatgagggag




ttggagtagattaattattccagctctgaaattctaagtgaccttggctaccttgca




gcagttttggatttcttccttatctttgttctgctgtttgagggggctttttactta




tttccatgttattcaaaggagactaggcttgatattttattactgttcttttatgga




caaaaggttacatagtatgcccttaagacttaattttaaccaaaggcctagcaccac




cttaggggctgcaataaacacttaacgcgcgtgcgcacgcgcgcgcgcacacacaca




cacacacacacacacacacacaggtcagagtttaaggctttcgagtcatgacattct




agcttttgaattgcgtgcacacacacacgcacgcacacactctggtcagagtttatt




aaggctttcgagtcatgacattatagcttttgagttggtgtgtgtgacaccaccctc




ctaagtggtgtgtgcttgtaattttttttttcagtgaaaatggattgaaaacctgtt




gttaatgcttagtgatattatgctcaaaacaaggaaattcccttgaaccgtgtcaat




taaactggttcatatgactcaagaaaacaataccagtagatgattattaactttatt




cttggctctttttaggtccattttgattaagtgacttttggctggatcattcagagc




tctcttctagcctacccttggatgagtacaattaatgaaattcatattttcaaggac




ctgggagccttccttggggctgggttgagggtggggggttggggagtcctggtagag




gccagctttgtggtagctggagaggaagggatgaaaccagctgctgttgcaaaggct




gcttgtcattgatagaaggactcacgggcttggattgattaagactaaacatggagt




tggcaaactttcttcaagtattgagttctgttcaatgcattggacatgtgatttaag




ggaaaagtgtgaatgcttatagatgatgaaaacctggtgggctgcagagcccagttt




agaagaagtgagttgggggttggggacagatttggtggtggtatttcccaactgttt




cctcccctaaattcagaggaatgcagctatgccagaagccagagaagagccactcgt




agcttctgctttggggacaactggtcagttgaaagtcccaggagttcctttgtggct




ttctgtatacttttgcctggttaaagtctgtggctaaaaaatagtcgaacctttctt




gagaactctgtaacaaagtatgtttttgattaaaagagaaagccaactaaaaaaaaa




aaaaaaaaaaa


 


21
Mouse SMAD4 -
ccgctgcggtaacggagcggctcgggcggcggagcccgtgttcgcgtccgtccgccc



NM_008540.2
gcccgcccgccgtcctccggaggcccttcccgcgccgcgctccgctccgcggccgtc




cccggggcgggagcgcgtgaccggagccggcgcccgcgagcgaggccccccgcagcg




gggcggctccggagctccagcggcccggccggccggcgcggtccgcggcgcggcggg




gagagggggccgcctgggccggacgccgcgggcggggcccgggaagcgacagcgagg




cgaggcgcggtgcggcgcggagcccaggtcatcctgctcaccagatgtcttgacagt




ttttcttgcaacattggccattggttttcactgccttcaaaagatcaaaattactcc




agaaatcggagagttggatttaaaagaaaaaacttgaacaaatggacaatatgtcta




taacaaatacaccaacaagtaacgatgcctgtctgagcattgtacatagtttgatgt




gccatagacaaggtggggaaagtgaaacctttgcaaaaagagcaattgagagtttgg




taaagaagctgaaagagaaaaaagatgaattggattctttaataacagctataacta




caaatggagctcatcctagcaagtgtgtcaccatacagagaacattggatggacgac




ttcaggtggctggtcggaaaggatttcctcatgtgatctatgcccgtctgtggaggt




ggcctgatctacacaagaatgaactaaagcatgttaaatattgtcagtatgcgtttg




acttaaaatgtgacagtgtctgtgtgaatccatatcactatgagcgggttgtctcac




ctggaattgatctctcaggattaacactgcagagtaatgctccaagtatgttagtga




aggatgagtacgttcacgactttgaaggacagccgtccttacccactgaaggacatt




cgattcaaaccatccaacacccgccaagtaatcgcgcatcaacggagacgtacagcg




ccccggctctgttagccccggcagagtctaacgccaccagcaccaccaacttcccca




acattcctgtggcttccacaagtcagccggccagtattctggcgggcagccatagtg




aaggactgttgcagatagcttcagggcctcagccaggacagcagcagaatggattta




ctgctcagccagctacttaccatcataacagcactaccacctggactggaagtagga




ctgcaccatacacacctaatttgcctcaccaccaaaacggccatcttcagcaccacc




cgcctatgccgccccatcctggacattactggccagttcacaatgagcttgcattcc




agcctcccatttccaatcatcctgctcctgagtactggtgctccattgcttactttg




aaatggacgttcaggtaggagagacgtttaaggtcccttcaagctgccctgttgcga




ctgtggatggctatgtggatccttcgggaggagatcgcttttgcttgggtcaactct




ccaatgtccacaggacagaagcgattgagagagcgaggttgcacataggcaaaggag




tgcagttggaatgtaaaggtgaaggtgacgtttgggtcaggtgccttagtgaccacg




cggtctttgtacagagttactacctggacagagaagctggccgagcacctggcgacg




ctgttcataagatctacccaagcgcgtatataaaggtctttgatctgcggcagtgtc




accggcagatgcagcaacaggcggccactgcgcaagctgcagctgctgctcaggcgg




cggccgtggcagggaacatccctggccctgggtccgtgggtggaatagctccagcca




tcagtctgtctgctgctgctggcatcggtgtggatgacctccggcgattgtgcattc




tcaggatgagctttgtgaagggctggggcccagactaccccaggcagagcatcaagg




aacccccgtgctggattgagattcaccttcaccgagctctgcagctcttggatgaag




tcctgcacaccatgcccattgcggacccacagcctttagactgagatctcacaccac




ggacgccctaaccatttccaggatggtggactatgaaatatactcgtgtttataatc




tgaagatctattgcattttgttctgctctgtcttttcctaaagggttgagagatgtg




tttgctgccttgctcttagcagacagaaactgaattaaaacttcttttctattttag




aactttcaggtgtggctcagtgcttgaagatcagaaagatgcagttcttgctgagtc




ttccctgctggttctgtatggaggagtcggccagtgctgggcgctcagccctttagt




gtgtgcgagcgccttgcatgccgaggagagtcagagctgctgattgtaaggctgaga




agttctcacagttaagccacctgccccttagtgggcgagttattaaacgcactgtgc




tcacgtggcgctgggccagccagctctaccaagagcaactttactctcctttaaaaa




ccttttagcaacctttgattcacaatggtttttgcaagttaaacagtgaaggtgaat




taaattcatactgtcttgcagacttcagggtttcttccccaagacaaaacactaatc




tgtgtgcatattgacaattccttacaattatcagtcaaagaaatgccatttaaaatt




acaatttttttaatccctaatggatgaccactatcaagatgtatactttgccctgtt




aaacagtaaatgaattcttctatatttctaggcacaaggttagttatttaaaaaaaa




aaaaaaaagcctaggggagggatttttcccttaattcctagggagaaggttttgtat




aaaacactaaaagcagcgtcactctgcctgctgcttcactgttctgcaaggtggcag




tacttcaactgaaataatgaatattttggaaactgctaaattctatgttaaatactg




tgcagaataatggaaacagtgcagttggtaacaggtggtttggatatttttgtactt




gatttgatgtgtgacttcttttcatatactgttaaaatcatgtatgttttgacattg




tttaaaattcagtttttgtatcttagggcaagactgcagacttttttataccttttg




gttataagccctgtgtttgccatccttgatcacttggcggtgactttgtagagattg




aagtggaggagttaagacacattgactgtaccacagacacacatgcatactttctac




ctagttactagcgtaaataaaactgagtcactataaaaaaaaaaaaaaaaaaaaa


 


22
Mouse IL6R -
gcagtgcgagctgagtgtggagcccgaggccgagggcgactgctctcgctgccccag



NM_010559.2
tctgccggccgcccggccccggctgcggagccgctctgccgcccgccgtcccgcgta




gaaggaagcatgctgaccgtcggctgcacgctgttggtcgccctgctggccgcgccc




gcggtcgcgctggtcctcgggagctaccgcgcgctggaggtggcaaatggcacagtg




acaagcctgccaggggccaccgttaccctgatttgccccgggaaggaagcagcaggc




aatgttaccattcactgggtgtactctggctcacaaaacagagaatggactaccaca




ggaaacacactggttctgagggacgtgcagctcagcgacactggggactatttatgc




tccctgaatgatcacctggtggggactgtgcccttgctggtggatgttcccccagag




gagcccaagctctcctgcttccggaagaacccccttgtcaacgccatctgtgagtgg




cgtccgagcagcaccccctctccaaccacgaaggctgtgctgcttgcaaagaaaatc




aacaccaccaacgggaagagtgacttccaggtgccctgtcagtattctcagcagctg




aaaagcttctcctgccaggtggagatcctggagggtgacaaagtataccacatagtg




tcactgtgcgttgcaaacagtgtgggaagcaagtccagccacaacgaagcgtttcac




agcttaaaaatggtgcagccggatccacctgccaaccttgtggtatcagccatacct




ggaaggccgcgctggctcaaagtcagctggcagcaccctgagacctgggacccgagt




tactacttgctgcagttccagcttcgataccgacctgtatgttcaaaggagttcacg




gtgttgctgctcccggtggcccagtaccaatgcgtcatccatgatgccttgcgagga




gtgaagcacgtggtccaggtccgtgggaaggaggagcttgaccttggccagtggagc




gaatggtccccagaggtcacgggcactccttggatagcagagcccaggaccaccccg




gcaggaatcctctggaaccccacacaggtctctgttgaagactctgccaaccacgag




gatcagtacgaaagttctacagaagcaacgagtgtcctcgccccagtgcaagaatcc




tcgtccatgtccctgcccacattcctggtagctggaggaagcttggcgtttgggttg




cttctctgtgtcttcatcatcctgagactcaagcagaaatggaagtcagaggctgag




aaggaaagcaagacgacctctcctccacccccaccgtattccttgggcccactgaag




ccgaccttccttctggttcctctcctcaccccacacagctctgggtctgacaatacc




gtaaaccacagctgcctgggtgtcagggacgcacagagcccttatgacaacagcaac




agagactacttattccccagacaatcatctggatggtacctggcagctggcagggca




ccacgagatcagcacacaagtttctcatgcgggtcccatccacctggggtggggtgg




ggcgggcggggctgcagcttcactaacccacaagagctctgcacaggttctgagtag




gtgcagctggtgctgcataggctctgaaggaaggaaggggctgtgaggaacacaggc




cattgcgaagacagcttgtgatgactgaatagagatgcccgtcagctccacatctga




tagtggctcacaagctgcaccctcaggaggcctcagaaaggggctccaaaggctgcc




ccagctgcctcgctctgcctcactgccccaagccaccttttagctctcgaactccta




aagtccaagcactttgccattctctttccgaggccactgaggccgggtggaagcttg




gttccgatttccttctcaacatctggaaagcagctgggcccggtggtggtgactaat




atctcagggcctgatggtttacgcgagtgacaatttcccacaagcagtttttaaatg




tgaatgatgaccccaggcactgctggctgcggaggcttcattttcctcttcgatctc




aggacttcaggcgaaaagcggagtggaagtagagagcggatgggtgtccaccgtcct




catggtacttgcgggaggtacagcctggaaaacacgtttcctgtccccctactctcc




caggagagggatgatggtagggggtgcctcttccagggcggagagaactactttacc




ccagccttgcccattctgatttcaactggactggagctactaggaaagtcgacattc




atgcaaaaagaaaaaacgttaactagcaagaatgcactttcattttggtttttagag




aactgttgcctgtttctctcaagagtctggaagaggccgctcactgcacactactgt




atgaaccctcactgcccaccctggaggaccaagtgcagtaacggtagcccaaacacc




aagtcaagtgaaaatcgagggaaaaaaaaaacaaacaagcaacaaaaaaaaaaaacc




aaaactaaactaaaaaacaaatcacccccccaaaaaaaaacaaaaccaaaaaccaaa




aaaaacaaaaaaacaaaacaacaacaacaaaaaaaacccaaaccaacccgctgtttc




ctataacagaaaagcctttggtttcattttttattttgatttttttgtcttaaaaag




tataaaaatagcctgtccatgctctgcttcagggaatgagcctgtgaacactcccag




gcgcaggcaggaagggtgtctgcttcctgctacacctcactgccaccttggccttcc




ttgctttacgtttgactgagtggcctcagacgctttcccctggggccttgaggaatc




cagtgatgttagtggtcaccgaggagaccacagagccacagtgtggtgcttagatta




aagtgacttctgcaaccacagcaccccacacctgccgtcttactgaactacgccagt




aacttgccttttctgccaccaccacgagacgagacgggcagagctcggaagctgtca




ccccatgccctctgcttgtccgctctaggggccactgacctaagcattagttatttt




attttattttatttttttgtgggttttgtacattttaggtcctgttgctgtcttaga




aaaggctctgtaggttgacagaaaatcaggccaagtattcatgttttgttttttttt




tttttccttctttcctcctttgctaagtttttgggactcaagggtagcaaaactgct




gtgaaagggaaatttattaaaaatgttacagatcgtg


 


23
Rat IL6R -
gccccacgtagaaggaaccatgctggccgtcggctgcaccctgctggtcgccctgct



NM_017020.3
ggccgcgcccgcagtcgcgctggtccttgggagctgccgcgcgctggaggcggcaaa




tggtacggtgacgagcctgccaggggccactgttaccctgatctgccctgggaagga




agcagcaggcaatgctaccattcactgggtgtactcaggctcacagagcagagaatg




gactaccacgggaaacacactggttctgagggccgtgcaggtcaacgacactgggca




ctatttgtgcttcctggatgatcatctggttgggactgtgcccttgctggtggatgt




tcccccagaggagcccaagctctcctgcttccggaagaacccccttgtaaatgcctt




ttgtgagtggcatccaagcagcactccctctccaaccacgaaggctgtgatgtttgc




aaagaaaatcaacaccaccaatgggaagagtgacttccaggtgccttgccagtattc




tcagcagctgaaaagcttctcctgcgaggtggagatcctggagggtgacaaagtgta




ccacatagtgtcactgtgcgttgcaaacagtgtcggaagcaggtccagccacaatgt




agtatttcagagtttaaaaatggtgcagccggatccacctgccaaccttgtggtatc




agccatacctggaaggcctcgttggctcaaagtcagttggcaagaccctgagtcctg




ggacccaagttactacttgttgcaattcgagcttcgataccgacctgtatggtcaaa




gacgttcacggtgtggccgctccaggtggcccagcatcaatgtgtcatccatgatgc




cttgcgaggagtaaagcatgtggtgcaggtccgagggaaggaggagtttgacattgg




ccagtggagcaaatggcccccggaggtcacaggcactccttggctagcagagcccag




gaccactccggcagggatcccggggaaccccacacaggtctctgttgaagactatga




caaccacgaggatcagtacggaagttctacagaagcaacgagtgtcctcgccccagt




gcaaggatcctcgcctatacccctgcccacattcctggtagctggaggaagcctggc




gtttggattgcttctctgtgtcttcatcatcttgagactcaagaagaaatggaagtc




acaggctgagaaggaaagcaagacgacttctcccccaccgtatcccttgggaccgct




gaagccgaccttcctcctggttcctctccctaccccatcagggtcccataacagctc




tgggactgacaacaccggaagccacagctgcctgggtgtcagggacccacagtgccc




taatgacaacagcaacagagactacttattccccagataattgtctggagggtacct




ggcagctggcacgcaagtttctcactgccggccccgtccaccagggctgggggcggg




gtgggcggggctgcagcttcacgatcccacaggagccttgcaaaggttctgagtggg




agaagactggtgtgctgcacgggcttcgaaagaaggggctgtgaggagcacgagcca




tcatgaagagagcccgtgatgactctgaatagagacgcccgcccatcagctacacac




ctgatggtggctctcaagctatcctctcaggaagcctctgggaggggcgacaaaggc




tgccccagttgcctagctctggctcactggcccaagctgccttttagcttgaactcc




taaaatccaagcaccttggccattctcttcctaggccaccgaggccgcggggaagct




tggttctactttccttctcaacacctggagaagcagctgcccggtggtggtgactaa




cgtatcagggcctgatggcttatgaggaatgacaattaattcctcataagcagtttt




taaatgtgaatagtaatcctaggcactgctgacttgaggttttattttcttcaatct




caggacttcaggagagaagcagagcagaagtagagagaggatgggtgtccattgtcc




gtgtggtacttgagggggatacagcctggaaaacacgtttcctgcccccctactctc




ccagaagaggtagggggtggcgcctcttccagggcagagagtataactactttacct




ggccttgcccatactggtttcaactggacttgagctactaggaaaaatgacattcat




gcaaaaagaaaactttaactagcaagaatgcacttccactttggtttctagaggact




gttgctcctcttgagacgctggaagaggccgctcactgtaccctggtgtatgagccc




tcaccccccaccccagggtaagtgcagtaactttagtctaaacaccgagtcaggtaa




aaatcgaggaaaaaacaaccccgtttcctgtaacagaaaagcctttggtttcgtttt




gtattttgattttttttttgtcttaaaaagtgtaaaaatagtctgtccatactctgc




ttcagggaatgacctgtgaatactccccaggcgtgggcaggaagggtgtctgcttcc




tgctacacctcactgccacctcggccttccttgctttacattcaactgagttgcctc




agctgctttcccctggggcgctgaaaaagccagtgatgttggtggtcaccgagaaga




ccacagagccacagagtaatgctgtgattgaagcgagctacgcaaccacagcacccc




acatttgctgtattatagaactatgctaggagcttgccctttcacaaaataccacca




ccacgagacgtggcagagctcggaagctgtcaccttgtgccatctgcttgccagctc




caaggggccactgacttaagcagttattttctttgtgggctttgttcatttcagggc




ctgttgctgtcttagaaaaagctctgtcggttgacaaaaacatcagacaggtagtca




tgtttatttattttttttcctcttttgctaagtctttgggactcaagggtagtaaaa




aatgctgtgaaaagggaaacattagaaacagcgatcttcggggaataggtgactgtg




cccacgcactgttcttcagtccctcacgtggctctgcccgagcgctgttccaagcca




ggcagagcaggctggcggaagattgaaatccagatagttcgttatctctgagagcta




aatagctttgatctccaagctgttattgctttcactattgtaacaggatagcctccc




ccccccatgtcaaaaggatgcttttcccttttgactttttataagctaagtcagtga




agtctgtttcatctgagctccagcttcgttcagttcgcacaggtgtatgccctcagc




tgcttcgggcctcagatctgtgctagttgaatggttgtcccatccttgggtcatcct




taccagagtttctgcagcccacaggtctgccttgtcaacagtaccacttaacaccag




cattcagtgcccaggcagccagatgtggagggtttacccagagatgatttaaacatg




accttaaacgtgtatggtagaacgaggggaacccataccagctcaggttctaaagag




atctttgattcttctggcattagtgaaatagctttaaactatttcaaggaagaagcc




ttggccacacccacgacatttggtgacaatcctttctctccatgagccttgtcttta




caccttctcacctggctgaaagctcacactgaatctttcctatgcccctggtgtctt




gggagaaaggaaactggtatgggcttcactgctggaattggcttggagccagcgtgt




ggcgcagcgcctggcagggtgggccaggcttagttatggtgtgctggtttaaggaat




gcctggcttgcctggttgcttgggttctgagctgcagagtttcctagcagttcttta




tggctgacctagttggggaagattcccacactcaactgcaggtggaggtggtgagaa




agctgttttcattcggagaggcaggatcagcccaagaagctttcagtgggagagcct




acagtgaggctgtacctcactgtgggaggaggcaggccagctggctcaggtcctggg




actggcactggggagggtctgccaaaggtccctccagcctgtagtcctagcatagtc




gggtgccagttccaggaagtttctacggcaaccttagtgctcattaaggaacattgt




cagttttgtgaacatatgctcagatggagatcttgctttcagagaaaggactggtac




agtgtgtaacaagctggagcagacagagagactttttggcaagagatcacatccgtt




aagcagaatacctcagtgctacatgtttttgtctttgagacaatgtttttaaggttt




ttatgctctgttacctgtaagctgatacctaaaactttctgcaaagtcagggttttt




caatgccttttttttttttttgccattgtttgctttaaagtgaagattgtaactgtt




tgaaataaataatttctaaaactgca


 


24
Human BMP6 -
caactgggggcgccccggacgaccatgagagataaggactgagggccaggaagggga



NM_001718.4
agcgagcccgccgagaggtggcggggactgctcacgccaagggccacagcggccgcg




ctccggcctcgctccgccgctccacgcctcgcgggatccgcgggggcagcccggccg




ggcggggatgccggggctggggcggagggcgcagtggctgtgctggtggcgggggct




gctgtgcagccgctgcgggcccccgccgctgcggccgcccttgcccgctgccgcggc




cgccgccgccggggggcagctgctgggggacggcgggagccccggccgcacggagca




gccgccgccgtcgccgcagtcctcctcgggcttcctgtaccggcggctcaagacgca




ggagaagcgggagatgcagaaggagatcttgtcggtgctggggctcccgcaccggcc




ccggcccctgcacggcctccaacagccgcagcccccggcgctccggcagcaggagga




gcagcagcagcagcagcagctgcctcgcggagagccccctcccgggcgactgaagtc




cgcgcccctcttcatgctggatctgtacaacgccctgtccgccgacaacgacgagga




cggggcgtcggagggggagaggcagcagtcctggccccacgaagcagccagctcgtc




ccagcgtcggcagccgcccccgggcgccgcgcacccgctcaaccgcaagagccttct




ggcccccggatctggcagcggcggcgcgtccccactgaccagcgcgcaggacagcgc




cttcctcaacgacgcggacatggtcatgagctttgtgaacctggtggagtacgacaa




ggagttctcccctcgtcagcgacaccacaaagagttcaagttcaacttatcccagat




tcctgagggtgaggtggtgacggctgcagaattccgcatctacaaggactgtgttat




ggggagttttaaaaaccaaacttttcttatcagcatttatcaagtcttacaggagca




tcagcacagagactctgacctgtttttgttggacacccgtgtagtatgggcctcaga




agaaggctggctggaatttgacatcacggccactagcaatctgtgggttgtgactcc




acagcataacatggggcttcagctgagcgtggtgacaagggatggagtccacgtcca




cccccgagccgcaggcctggtgggcagagacggcccttacgacaagcagcccttcat




ggtggctttcttcaaagtgagtgaggtgcacgtgcgcaccaccaggtcagcctccag




ccggcgccgacaacagagtcgtaatcgctctacccagtcccaggacgtggcgcgggt




ctccagtgcttcagattacaacagcagtgaattgaaaacagcctgcaggaagcatga




gctgtatgtgagtttccaagacctgggatggcaggactggatcattgcacccaaggg




ctatgctgccaattactgtgatggagaatgctccttcccactcaacgcacacatgaa




tgcaaccaaccacgcgattgtgcagaccttggttcaccttatgaaccccgagtatgt




ccccaaaccgtgctgtgcgccaactaagctaaatgccatctcggttctttactttga




tgacaactccaatgtcattctgaaaaaatacaggaatatggttgtaagagcttgtgg




atgccactaactcgaaaccagatgctggggacacacattctgccttggattcctaga




ttacatctgccttaaaaaaacacggaagcacagttggaggtgggacgatgagacttt




gaaactatctcatgccagtgccttattacccaggaagattttaaaggacctcattaa




taatttgctcacttggtaaatgacgtgagtagttgttggtctgtagcaagctgagtt




tggatgtctgtagcataaggtctggtaactgcagaaacataaccgtgaagctcttcc




taccctcctcccccaaaaacccaccaaaattagttttagctgtagatcaagctattt




ggggtgtttgttagtaaatagggaaaataatctcaaaggagttaaatgtattcttgg




ctaaaggatcagctggttcagtactgtctatcaaaggtagattttacagagaacaga




aatcggggaagtggggggaacgcctctgttcagttcattcccagaagtccacaggac




gcacagcccaggccacagccagggctccacggggcgcccttgtctcagtcattgctg




ttgtatgttcgtgctggagttttgttggtgtgaaaatacacttatttcagccaaaac




ataccatttctacacctcaatcctccatttgctgtactctttgctagtaccaaaagt




agactgattacactgaggtgaggctacaaggggtgtgtaaccgtgtaacacgtgaag




gcaatgctcacctcttctttaccagaacggttctttgaccagcacattaacttctgg




actgccggctctagtaccttttcagtaaagtggttctctgcctttttactatacagc




ataccacgccacagggttagaaccaacgaagaaaataaaatgagggtgcccagctta




taagaatggtgttagggggatgagcatgctgtttatgaacggaaatcatgatttccc




ttgtagaaagtgaggctcagattaaattttagaatattttctaaatgtctttttcac




aatcatgtactgggaaggcaatttcatactaaactgattaaataatacatttataat




ctacaactgtttgcacttacagctttttttgtaaatataaactataatttattgtct




attttatatctgttttgctgtaacattgaaggaaagaccagacttttaaaaaaaaag




agtttatttagaaagtatcatagtgtaaacaaacaaattgtaccactttgattttct




tggaatacaagactcgtgatgcaaagctgaagttgtgtgtacaagactcttgacagt




tgcgcttctctaggaggttgggtttttttaaaaaaagaattatctgtgaaccatacg




tgattaataaagatttcctttaaggca


 


25
Rhesus BMP6 -
agcgcagcccggactcggacgcacctggcctgtaccgcgcgcctctagagacctgcg



XM_001085364
cggggctgtggggctccccttcctcccctccaagcggttctcccggtgatcgcccct




tcgccacccctctatcctgggcaactgggggcgccccggacgaccatgagagataag




gactgagggccaggaaggggaagcgagcccgccgagaggtggcgggggctgctcacg




ccaagggccacagcggccgtgctccagcctcgctccgccgctccacgcctcgcggga




tccgcgggggcagcccggccgggcggggatgccggggctggggcggagggcgcagtg




gctgtgctggtggtgggggctgttgtgcagctgctgcgggcccccgccgctgcggcc




gcccctgcccgctgccgcggccgccgccgccgggggccagctgctgggggacggcgg




gagccccggccgcacggagcagccgccgccgtcgccgcaatcctccccgggcttcct




ctaccggcggctcaagacgcacgagaagcgggagatgcagaaggagatcttgtcggt




gctggggctcccacaccggccccggcccctgcacggcctccaacagccgcagccccc




ggcgctcccgcagcagcagcagcagcagcagcagccgcctcgcggagagccccctcc




cgggcagctgaagcccgcgcccctcttcatgctggatctgtacaacgccctgtccgc




cgacgacgaggaggacggggcgtcggagggggagaggcagcagccctggccccacga




aggagccagctcgtcccagcctcggcagccggccccgggcgccgcgcacccgctcaa




ccgcaagagcctcctggcccccggacctggcagcggcggcgcgtccccactgaccag




cgcgcaggacagcgccttcctcaacgacgcagacatggtcatgagctttgtgaacct




ggtggagtacgacaaggagttctcccctcgtcagcgacaccacaaagagttcaagtt




caacttatcccagattcctgagggtgaggcggtgacggctgcagaattccgcatcta




caaggactgtgttgtggggagttttaaaaaccaaacttttcttatcagcatttatca




agtcttacaggagcatcagcacagagactctgacctttttttgctggacacccgcgt




agtgtgggcctcagaagaaggctggctggaatttgacatcacggccactagcaatct




gtgggccgtgaccccgcagcataacatggggcttcagctgagtgtggtgacgcggga




tggagtccacatccatccccgagccgcgggcctggtgggcagagacggcccttacga




caagcagcccttcatggtggctttcttcaaagtgagtgaggtccacgtgcgcaccac




caggtcagcctctggctggcgccgacaacagagtcgtaatcgctctacccagtccca




ggacgtggcgcgggtctccagtgcttcagattacaacagcagtgaattaaaaacagc




ttgcaggaagcatgagctgtatgtgagtttccaagacctgggatggcaggactggat




cattgcacccaagggctacgctgccaattactgtgatggggaatgctccttcccact




caacgcacacatgaatgcaaccaaccacgcgatcgtgcagaccttggttcaccttat




gaaccctgagtatgtccccaaaccgtgctgtgcgccaactaaactaaatgccatctc




agttctttactttgatgacaattccaatgtcattctgaaaaaatacaggaatatggt




tgtaagagcttgtggatgccactaactcgaaaccagatgctggggacacacattctg




ccttggattcctagattacatctgccttaaaaaacacagaagcacagttggaggtgg




gacgatgagacttggaaactatctcatgccagtgccttattacccaagaagatttta




aaggacctcattaataatttgctcacttggtaaatgacgtgagtagttgttggtctg




tagcaagctgagtttggatgtctgtagtgcaaggtccggtaactgcagaaagcaccg




tgaagctcttcctcccctcctcccccaaaaacccaccaaaattagttttagctgtag




atcaagctatttggggtgttagtaagtagggaaaataatctcaaaggagttaaatgt




attcttggttaaagtatcagcctgttcagtactgtctatcaaaggtagattttacag




agaacagaaattggggaagttgggggaacgcctctgttcagatttcattcccaggaa




gttcaacttcatacatgacccacagcccaggccacagccagggctccatggggcgcc




tttgtctcagtcattgctgttatgtgttcatgctggagttttgttg


 


26
Mouse BMP6 -
gatcctggccgtcgccccgtcgtctcttctccacccgggcttctgggggcgccgcgg



NM_007556.2
atgaccatgagagataaggactgagtgccaggaccgggaagagagcccgccgagagg




tggcgggggctgcccactccgagggccacagcctccgcgctccggcctcgctccgcc




gctcgacgcctcgcgggccccgcgggggcagccgggctgggcggcgatgcccgggct




ggggcggagggcgcagtggctgtgctggtggtgggggttgctgtgcagctgcggccc




cccgccactgcggccccctctgccggtagccgcggccgccgccggggggcagctgct




gggagccggcgggagcccggtgcgcgctgagcagccaccgccacagtcctcttcttc




gggcttcctctatcggcggctcaagacccacgagaagcgggagatgcaaaaggagat




cctgtcggtgctggggctcccgcacaggccgcggcccctgcacggtctccagcagcc




tcagcccccggtactcccgccacagcagcagcagcagcagcagcagcagcagacggc




ccgcgaggagccccctccagggcggctgaagtccgctccactcttcatgctggatct




ctacaacgccctgtccaatgacgacgaagaggatggggcatcggagggtgtggggca




agagcctgggtcccacggaggggccagctcgtcccagctcaggcagccgtctcccgg




cgctgcacactccttgaaccgcaagagtctcctggccccgggacccggtggcggtgc




gtccccactgactagcgcgcaggacagcgctttcctcaacgacgcggacatggtcat




gagctttgtgaacctggtggagtacgacaaggagttctccccacatcaacgacacca




caaagagttcaagttcaacctatcccagattcctgagggtgaggcggtgacggctgc




tgagttccgcgtctacaaggactgtgtggtggggagttttaaaaaccaaacctttct




tatcagcatttaccaagtcttgcaggagcatcagcacagagactctgacctattttt




gttggacacccgggtggtgtgggcctcagaagaaggttggctggaatttgacatcac




agcaactagcaatctgtgggtggtgacaccgcagcacaacatggggctccagctgag




tgtggtgactcgggatggactccacgtcaacccccgtgcggcgggcctggtgggcag




agacggcccttacgacaagcagcccttcatggtggccttcttcaaggtgagcgaggt




ccacgtgcgcaccaccaggtcagcctccagtcggcggcggcagcagagtcgcaaccg




gtccacccagtcgcaggacgtgtcccggggctccggttcttcagactacaacggcag




tgagttaaaaacagcttgcaagaagcatgagctctatgtgagcttccaggacctggg




atggcaggactggatcattgcacccaaaggctacgctgccaactactgtgatggaga




gtgttccttcccactcaacgcacacatgaatgccaccaaccacgccattgtacagac




cttggtccaccttatgaatcccgagtacgtccccaaaccatgctgcgcaccaaccaa




actgaatgccatctcggttctttacttcgatgataactccaatgtcatcttgaaaaa




gtacaggaatatggtcgtgagagcttgtggttgccattaagttgaagctggtgtgtg




tgtgtgggtgggggcatggttctgccttggattcctaacaacaacatctgccttaaa




ccacgaacaacagcacagcgaagcgggatggtgacacacagagggatcgtgacacgc




agacacatctcccgctggtgccttacccacggaggcttttatgaggaccttgtcaag




ggctttcccagttcctaactgagcagttgctggtctgcaggaagctggaaggcttgt




agtacaggcctggaaactgcagttacctaatgttcgcctcccccaaccccgcccgga




gtagttttagcttttagatctagctgcttgtggtgtaagtagagagtaaacttgaag




gaatattaaatatccctgggttgaaagacccggtggtggctctacagcaeccatccc




agggagatttttgcagacatccgaatggaggggagaagggcactctttcaggttcca




ttcccagcaagggcagctcacacaggacctgcagcctggccatcagcaggctctgtg




gaggtgccttctgtctactgttgtagttacgtgttttgtgttgactctcggtggtgt




gagaatgtactaatctctgtcaagacaaactgtagcatttccaccccatcctcctcc




ctccctcacagaattc


 


27
Rat BMP6 -
atgcccgggctggggcggagggcgcagtggctgtgctggtggtgggggtcactgtgc



NM_013107.1
agctgcggccccccgccactgcggccccctctgccggtagccgcggccgccgccggg




gggcagctgctgggagccggcgggagccctgtgcgcgccgagcagccaccgccgcaa




tcctcctcttcgggcttcctctatcggcggctcaagacccacgagaagcgggagatg




caaaaggagatcctgtcggtgttggggctgcctcacaggccgcggcccctgcacggt




ctccagcagcctcaatcccccgtgctcccgcagcagcaacaatcgcaacagacggcc




cgcgaggagccccctccagggcggctgaagtccgctccgctcttcatgctggatctc




tacaactccctgtccaaggacgacgaagaggatggggtgtcagagggagagggactg




gagcccgagtcccacggaagggccagctcgtcccagctcaaacagccatctcccggg




gctgcacactccctgaaccgcaagagtctcctggccccgggacccggcggcagtgcg




tccccactgaccagcgcgcaggacagcgctttcctcaacgacgcggacatggtcatg




agctttgtgaacctggtggagtacgacaaggagttctccccacgccagcgacaccac




aaggagttcaagttcaacttatcccagattcccgagggtgaggcagtgacggctgca




gagttccgcgtctacaaggactgtgtggtggggagttttaaaaaccaaacttttctt




atcagcatttaccaagtcttacaggagcatcagcacagagactctgacctatttttg




ttggacacccgggtggtgtgggcctccgaagaaggctggctggaattcgacatcaca




gcaactagcaatctgtgggtggtgacaccgcagcacaacatgggactccagctgagt




gtggtgactcgggacggactccacatcaacccccgtgcggcgggcctggtgggcaga




gacggcccttacgacaagcagcccttcatggtggccttcttcaaggcgagcgaggtc




cacgtgcgcaccaccaggtcagcctccagtcggcgtcgacagcagagtcgcaatcgg




tccacccagtcgcaggacgtgtcccggggctccagtgcttcagactacaacagcagt




gagttaaaaacagcttgcaagaagcatgagctttacgtgagcttccaggacctggga




tggcaggactggatcatcgcacccaaaggctacgctgccaactattgtgacggagag




tgttccttccctctcaatgcacacatgaatgccaccaaccacgccattgtacagacc




ttggtccaccttatgaatcccgagtacgtccccaaaccatgctgcgcaccaaccaaa




ctgaatgccatctcggttctttacttcgacgacaactccaatgtcatcttgaaaaaa




tacaggaacatggttgtgagagcttgtggatgtcattga


 


28
Human NEO1 -
gggccgggccgggctgggctggagcagcggcggccgcgggagccgagcttgcagcga



NM_002499.2
gggaccggctgaggcgcgcgggagggaaggaggcaagggctccgcggcgctgtcgcc




gccgctgccgctcactctcggggaagagatggcggcggagcggggagcccggcgact




cctcagcaccccctccttctggctctactgcctgctgctgctcgggcgccgggcgcc




gggcgccgcggccgccaggagcggctccgcgccgcagtccccaggagccagcattcg




aacgttcactccattttattttctggtggagccggtggatacactctcagttagagg




ctcttctgttatattaaactgttcagcatattctgagccttctccaaaaattgaatg




gaaaaaagatggaacttttttaaacttagtatcagatgatcgacgccagcttctccc




ggatggatctttatttatcagcaatgtggtgcattccaaacacaataaacctgatga




aggttattatcagtgtgtggccactgttgagagtcttggaactattatcagtagaac




agcgaagctcatagtagcaggtcttccaagatttaccagccaaccataaccttcctc




agtttatgctgggaacaatgcaattctgaattgcgaagttaatgcagatttggtccc




atttgtgaggtgggaacagaacagacaaccccttcttctggatgatagagttatcaa




acttccaagtggaatgctggttatcagcaatgcaactgaaggagatggcgggcttta




tcgctgcgtagtggaaagtggtgggccaccaaagtatagtgatgaagttgaattgaa




ggttcttccagatcctgaggtgatatcagacttggtatttttgaaacagccttctcc




cttagtcagagtcattggtcaggatgtagtgttgccatgtgttgcttcaggacttcc




tactccaaccattaaatggatgaaaaatgaggaggcacttgacacagaaagctctga




aagattggtattgctggcaggcggtagcctggagatcagtgatgttactgaggatga




tgctgggacttatttttgtatagctgataatggaaatgagacaattgaagctcaagc




agagcttacagtgcaagctcaacctgaattcctgaagcagcctactaatatatatgc




tcacgaatctatggatattgtatttgaatgtgaagtgactggaaaaccaactccaac




tgtgaagtgggtcaaaaatggggatatggttatcccaagtgattattttaagattgt




aaaggaacataatcttcaagttttgggtctggtgaaaccagatgaagggttctatca




gtgcattgctgaaaatgatgttggaaatgcacaagctggagcccaactgataatcct




tgaacatgcaccagccacaacgggaccactgccttcagctcctcgggatgtcgtggc




ctccctggtctctacccgcttcatcaaattgacgtggcggacacctgcatcagatcc




tcacggagacaaccttacctactctgtgttctacaccaaggaagggattgctaggga




acgtgttgagaataccagtcacccaggagagatgcaagtaaccattcaaaacctaat




gccagcgaccgtgtacatctttagagttatggctcaaaataagcatggctcaggaga




gagttcagctccactgcgagtagaaacacaacctgaggttcagctccctggcccagc




acctaaccttcgtgcatatgcagcttcgcctacctccatcactgttacgtgggaaac




accagtgtctggcaatggggaaattcagaattataaattgtactacatggaaaaggg




gactgacaaagaacaggatgttgatgtttcaagtcacccttacaccattaatgggtt




gaaaaaatatacagagtatagtttccgagtggtggcctacaataaacatggtcctgg




agtttccacaccagatgttgctgttcgaacattgtcagatgctcccagtgctgctcc




tcagaatctgtccttggaagtgagaaattcaaagagtattatgattcactggcagcc




acctgctccagccacacaaaatgggcagattactggctacaagattcgctaccgaaa




ggcctcccgaaagagtgatgtcactgagaccttggtaagcgggacacagctgtctca




gctgattgaaggtcttgatcgggggactgagtataatttccgagtggctgctctaac




aatcaatggtacaggcccggcaactgactggctgtctgctgaaacttttgaaagtga




cctagatgaaactcgtgttcctgaagtgcctagctctcttcacgtacgcccgctcgt




tactagcatcgtagtgagctggactcctccagagaatcagaacattgtggtcagagg




ttacgccattggttatggcattggcagccctcatgcccagaccatcaaagtggacta




taaacagcgctattacaccattgaaaatctggatcccagctctcactatgtgattac




cctgaaagcatttaataacgtgggtgaaggcatccccctgtatgagagtgctgtgac




caggcctcacacagacacttctgaagttgatttatttgttattaatgctccatacac




tccagtgccagatcccactcccatgatgccaccagtgggagttcaggcttccattct




gagtcatgacaccatcaggattacgtgggcagacaactcgctgcccaagcaccagaa




gattacagactcccgatactacaccgtccgatggaaaaccaacatcccagcaaacac




caagtacaagaatgcaaatgcaaccactttgagttatttggtgactggtttaaagcc




gaatacactctatgaattctctgtgatggtgaccaaaggtcgaagatcaagtacatg




gagtatgacagcccatgggaccacctttgaattagttccgacttctccacccaagga




tgtgactgttgtgagtaaagaggggaaacctaagaccataattgtgaattggcagcc




tccctccgaagccaatggcaaaattacaggttacatcatatattacagtacagatgt




gaatgcagagatacatgactgggttattgagcctgttgtgggaaacagactgactca




ccagatacaagagttaactcttgacacaccatactacttcaaaatccaggcacggaa




ctcaaagggcatgggacccatgtctgaagctgtccaattcagaacacctaaagcgga




ctcctctgataaaatgcctaatgatcaagcctcagggtctggagggaaaggaagccg




gctgccagacctaggatccgactacaaacctccaatgagcggcagtaacagccctca




tgggagccccacctctcctctggacagtaatatgctgctggtcataattgtttctgt




tggcgtcatcaccatcgtggtggttgtgattatcgctgtcttttgtacccgtcgtac




cacctctcaccagaaaaagaaacgagctgcctgcaaatcagtgaatggctctcataa




gtacaaagggaattccaaagatgtgaaacctccagatctctggatccatcatgagag




actggagctgaaacccattgataagtctccagacccaaaccccatcatgactgatac




tccaattcctcgcaactctcaagatatcacaccagttgacaactccatggacagcaa




tatccatcaaaggcgaaattcatacagagggcatgagtcagaggacagcatgtctac




actggctggaaggcgaggaatgagaccaaaaatgatgatgccctttgactcccagcc




accccagcctgtgattagtgcccatcccatccattccctcgacaaccctcaccatca




tttccactccagcagcctcgcttctccagctcgcagtcatctctaccacccgggcag




cccatggcccattggcacacccatgcccctttcagacagggccaattccacagaatc




cgttcgaaatacccccagcactgacaccatgccagcctcttcgtctcaaacatgctg




cactgatcaccaggaccctgaaggtgctaccagctcctcttacttggccagctccca




agaggaagactcaggccagagtcttcccactgcccatgttcgcccttcccacccatt




gaagagcttcgccgtgccagcaatcccgcctccaggacctcccacctatgatcctgc




attgccaagcacaccattactgtcccagcaagctctgaaccatcacattcactcagt




gaagacagcctccaccgggactctaggaaggagccggcctcctatgccagtggttgt




tcccagtgcccctgaagtgcaggagaccacaaggatgttggaagactccgagagtag




ctatgaaccagatgagctgaccaaagagatggcccacctggaaggactaatgaagga




cctaaacgctatcacaacagcatgacgaccttcaccaggacctgacttcaaacctga




gtctggaagtcttggaacttacccttgaaaacaaggaattgtacagagtacgagagg




acagcacttgagaacacagaatgagccagcagactggccagcgcctctgtgtagggc




tggctccaggcatggccacctgccttcccctggtcagcctggaagaagcctgtgtcg




aggcagcttccctttgcctgctgatattctgcaggactgggcaccatgggccaaaat




tttgtgtccagggaagaggcgagaagtgcaacctgcatttcactttgtggtcaggcc




gtgtctttgtgctgtgactgcatcacctttatggagtgtagacattggcatttatgt




acaattttatttgtgtcttattttattttaccttcaaaaacaaaaacgccatccaaa




accaaggaagtccttggtgttctccacaagtggttgacatttgactgctcgttccaa




ttatgtatggaaagtctttgacagcgtgggtcgttcctggggttggcttgttttttg




gtttcatttttattttttaattctgagtcattgcatcctctaccagctgttaatcca




tcactctgagggggaggaaatgttgcattgctgtttgtaagctttttttattatttt




tttattataattattaaaggcctgactctttcctctcatcactgtgagattacagat




ctatttgaattgaatgaaatgtaacattgaaaagacttgtttgttgctttctgtgca




gtttcagtattggggcgggtggggggctgggggttggtaataggaaatggaggggct




gctgaggtcctgtgaatgtttctgtcattgtactttcttccagaagcctgcagagaa




tggaagcatcttctttattgtcctttcctggcatgtccatccttattgtcactacgt




tgcaactggagtttgatttggatctggttttaaaattcttctgtgcaatagatgggt




ttgaggatttagcggccctgatgtcttggtcatagcctggtaagaatgtccatgctg




aggagccagatgttgtatttctaactgcctgagtcacacagaatagggtaagagcct




gaccccattctgtaaatcagaaagcaaggatggagaccctttcctgctgctattatt




ggctctctttgaggaagttggaggttaaggaaggaacttgtttgtttccgtatacga




ctccttcttctctctagttcagtcttcagccagtccagcgctctcttccacacttca




gagccccttcagagaaagcattagcaggaatgagacaaggcagagctgcagtgcccc




ctgaggcttccacacatctttctgaatattatttttcaagtaacaagggcagggaca




gcggaaacagctgcccaccccccccatcccagcagctcagctaagccctgatgagaa




tgaagccacaggagttgtctgaggtgaacccagccgctcagccacacatggaagcca




ttgcctttgcacatagttcttgggttctttttcctaaaaaggtaaggagctgaggtg




tgtggttttttaatattaagaatatataatggaaaacacacgactgacgctcaggca




tcttcccctactccccaacagatccccagaagacagcgtggaaggcagtgtagacag




taaatcgggcttcagttctatagccaagaagagatcagctgctgaaaccaccagtgg




gtaccccaggccacctgcctttgaacttggggatttgccatgtttgatcttgtcaca




tacttgcttttttacaagatgaactctttgtatttatgatttggggggcaatgaaag




gtgcaatgcaggaactgctgctgccgagctcgctggtcacatgggggtgccaggcgg




gattctggaaaaccagtgcacttaaactgatcctgaagagagctgtcccagcactct




ggccaccaggagggccagattccccagaaactaccttttgcccaaagaacatgctca




gtatttggggcatttcctcccacaaaccctgactgcttctgttacctcagggccttg




gtacctggatactgccacagaattggggcgggtgggggaggggcctatttttaaata




aaataactgttcaaagttgggggttttttaaaaaattaagaaaaaggaaagctattc




tgtattgcaccttttcacaatttaatacattttcttacattttcctgtgattttcga




aactaaaccattgtgtgtcctgtagtgtcctggttgagctgccgctcagcagcttcc




tcggggggatttggaacacctgtgtctgtcgtcgcactgcctgtgggaggggcccag




agggctgctgggactggcgtctgtacacacttgtttggccttttctgtagttgatgc




tgtaaactctatggctttttaaaaacgatttcatgtttttatttagtattggaaatc




caatacacttttttaatccaatcaaaaaaaaaaaaaaaaaaaaaaa


 


29
Mouse NEO1 -
gcccccctcgctctaccgtgaagagcccgagtcggcgacgggtggcggcgcctggaa



NM_001042752
cctggagagaccgagccaccccccggctctcggccggaatgtactgattctcctctg




ctctcctccccgccccgctgcaggagggaggcgcccggagtctttccccctgggcgc




gcgagggggccgcgcgggccgggccgggccgggctggagccgagccctgcggcgcag




agaccggctgaggcgcgctgagggaagggcgcgagcgctccgcggcgctatcgccgc




cgccgccgccgccactcgtggggtagagatggcggcggagcgcgaagccgggcgact




cctctgcacctcctcctcccggcgctgctgcccgccaccgccgctgctgctgttgct




gccgctgctgctgctgctcggacgcccggcgtccggcgccgcggccacgaagagcgg




ctccccgccgcagtccgcaggagccagtgttcgaacattcactccgttttattttct




ggtggagccagtagacaccctctcagttagaggctcttctgttatattaaattgctc




ggcatattctgagccctctccaaacattgaatggaagaaagatgggacttttttaaa




cttagaatcagatgatcgacgccagctactcccagatggatctttattcatcagcaa




cgtggtgcattccaaacacaataagcctgacgaaggtttctatcagtgtgtagccac




tgtggataatcttggaaccattgtcagcagaacagccaagctcacagtagcaggtct




tccaagatttaccagccaaccagaaccttcttcagtctatgttggaaacagtgcaat




tctgaattgtgaagttaatgcagatttggccccatttgttaggtgggaacagaatcg




acagccccttcttctagatgacaggattgtcaaacttccaagtggaacactggttat




cagcaatgctactgaaggagatgggggactctaccgctgcattgttgaaagtggtgg




gccaccaaagtttagtgacgaagctgaattgaaagttcttcaagatcctgaggaaat




tgtagacttggtatttctgatgcgaccatcttctatgatgaaagtcactggtcagag




tgcagtgttgccatgtgttgtctcagggcttcctgctccagttgttagatggatgaa




aaacgaagaagtgcttgacacagaaagctctggcaggttggtcttgctagcaggagg




ttgcttggagatcagtgatgtcactgaggatgatgctgggacttatttttgcatagc




tgataatggaaataagacagttgaagctcaggcggagcttactgtgcaagtgccacc




tggattcctgaaacaacctgctaacatatatgctcacgaatccatagacattgtatt




tgaatgtgaagtcactgggaagccaactccaactgtgaagtgggtcaagaatgggga




tgtggttatccccagtgattactttaaaattgtaaaggaacataatcttcaagtttt




gggtctggtgaaatcagatgaagggttctatcaatgcattgctgagaatgatgttgg




aaatgcacaagctggagcccagctgataatccttgagcatgatgttgccatcccaac




attacctcccacttcactgaccagtgccactactgaccatctagcaccagccacaac




gggaccattaccttcagctcctcgagacgtcgtggcctccctggtctctactcgctt




cattaaattgacatggcgtacacctgcatcagaccctcatggagacaatctcaccta




ctctgtgttctacaccaaggaaggggttgctagggagcgtgttgagaataccagcca




gccaggagagatgcaggtgactattcaaaacttgatgccagcaactgtgtacatctt




caaagttatggctcaaaataagcatggctctggagaaagttcagctcctcttcgagt




agagacacagcctgaggttcagctccctggcccagcacctaatatccgtgcttatgc




aacgtcacctacttctatcactgtcacctgggaaacaccgttatctggcaatgggga




aattcaaaattacaaattgtactacatggaaaaaggaactgataaagaacaggatat




tgatgtttcaagtcactcctacaccattaatggactgaagaaatacacagaatacag




tttccgagtggtggcctacaataaacatggtcctggagtttctacacaagatgttgc




tgttcgaacattatcagatgttcccagtgctgctcctcagaatctgtccttagaagt




gagaaattcaaagagtatagtgatccactggcagcccccttcctcaaccacacaaaa




tgggcagataactggctacaagattcgatatcgaaaggcctcccgaaaaagtgatgt




cactgagaccttggtaactgggacacagctgtctcagctgattgaaggtcttgatcg




ggggacagaatataacttccgagtcgctgctctcacagtcaatggtacaggtccagc




aactgattggctgtctgctgaaacttttgaaagcgacctagatgaaactcgtgttcc




tgaagtgcccagctctcttcatgtccgtccgctcgtcactagcattgtagtgagctg




gactcctccagagaaccagaacattgtggtccgaggttatgccatcggttacggcat




tggcagccctcatgcccagaccatcaaagtggactataaacaacgttattacaccat




cgaaaacttggatccaagctctcattacgtgattaccttgaaagcatttaacaatgt




tggcgaaggcatccccctttatgagagtgctgtgaccagacctcacacagtgccaga




tcccactcccatgatgccaccagtgggagttcaggcttccattctgagtcacgacac




cataaggattacctgggcagacaactccctgcccaaacaccagaagattacagactc




ccgctactacacagtccggtggaagaccaacatcccagcaaacacgaagtacaagaa




tgcaaatgcaacgacgttaagctatttggttactggtttaaagccaaatacgctcta




tgagttctctgtgatggtgaccaaaggcagaaggtcaagcacgtggagtatgacagc




tcatggcgctacctttgaattagttcctacttctccacctaaggatgtgacagttgt




gagtaaggaaggaaaacctagaaccatcatagtgaattggcagcctccctctgaagc




taacggcaagattacaggttacatcatctattacagcacggatgtgaatgcagagat




acatgactgggttattgaaccagttgtgggaaacagactgactcaccagattcaaga




gttaacacttgatacgccatactacttcaaaatccaggcccggaactcaaaggacat




ggggcccatgtctgaagctgtacagttcagaacacctaaagccttagggtcagcagg




aaaaggaagccgactaccagacctgggatctgactacaaacctccaatgagtggcag




caacagccctcacgggagccccacctcccctctggacagcaacatgctgctggtcat




cattgtctctgttggcgtcatcactatcgtggtggttgtggtcattgctgctttttg




tacccggcgcaccacctctcaccagaagaagaaacgagctgcgtgcaaatcagtgaa




tggctcccataagtacaagggcaattgcaaagatgtgaagcctccagacctatggat




ccatcacgagagactagagttgaagcctattgacaagtctccagatcctaaccctgt




catgactgatactccaatcccctgaaactctcaagatatcacaccagtggacaattc




catggatagcaatatccatcaaaggcggaattcatacagagggcatgagtcagagga




cagcatgtctacactggctggaaggaggggaatgagaccaaaaatgatgatgccctt




tgactctcagccacctcagcctgtgattagcgcccatcccatccattccctcgataa




ccctcaccatcatttccactccagcagcctcgcttctccagcccgcagtcatctcta




ccacccaagcagcccatggcccattggcacatccatgtccctttcagacagggccaa




ttccacagaatctgttcgaaatacccccagcacggacaccatgccagcgtcctcgtc




tcagacgtgctgcactgaccatcaggaccctgagggtgctactagctcctcttactt




ggccagctcccaagaggaagactcaggccagagtcttcccacagcccatgtccgccc




ttcccaccctctgaagagcttcgctgtgccagcaatcccacccccaggacctcctct




ctatgatcctgcactgccaagcacaccattactgtcccagcaagctctgaaccatca




cattcactcagtgaaaacagcctccatcgggacgttaggaaggagccggcctcctat




gccagtggttgttccgagtgcccctgaagtacaggagaccaccaggatgctggaaga




ctccgagagtagctatgaaccagatgagctgaccaaagagatggcccacctggaagg




actaatgaaggacctaaatgccatcacaacagcctgatgaccttcgcctggacatga




ctccaagcctgagtctacaagtctcggaacttaaccttgaaaacaaggaattgtaca




gagtacgagaggacagcacttgagagcaggagccagcaaaccagccagtgcctccat




gtggggttggctccaggcacagccacctgccttctcctggtcagcctggattacact




tgtgtggaggcagcttccctttgcctgctgagagcctgcaggactgggcactatggg




ccaaaattttgtgtccagggaagaggcaagaagtacgacctgccttttgctttgtgg




tcagtggcttgtgtctttgtgctgcaactgcatcacttttatggagtgtagacattg




gcatttatgtacaattttgtgtcctattttattttaccttaaaacactatcagaagc




caagggagtctgtgatgttctctcaagcagttgacacttgactgtggttccagttac




ttacggaaagtcatcaacagtgaggttgtttgacaccactgacaggcattggcttgt




tgtgggtttcatttttattcttaattctgagacattgcatcctctgccagctgttaa




tccatcactttgaggggaggacatgttgcattgctgtttgtaagcttttttattatt




tttttattataattattaaaggcctgactttctcctctcatcactgtgagattacag




atctatttgaatgaaatgtaacattgaaaagacttgtttgttgctttctgtgcagat




tcagtattggggtgggattggggattgggaataggaaatggaggggctgctgaggcc




ctgtgaatgtttctgtcaatgtactttgttccagaagcctgccgagaatgaaagcag




catctttagtgtcctttcctggcatttccatcttcgtgtcaccgcatagcaactgga




gttttgtttggatctggttataaattcttgtacagtggatgactttggtgatttagc




tgccctggtatcttggtcatttcctctttggagtgtccacactgaggtctctatcaa




tgtatgtttaattgcttgagagatgccaagtagaaccagagcctgactgtgctctga




gaagctacaaagcacagggtggagactccctttgtgttgctagtattggttctctct




ggaaggttaaaatctaaggcaggatcttggtttcctattccaaataggatgcctgct




tctctgggcaccagtcctcagccaggcagctctcgtggcattgcagaggctctcctg




aaaaacatcaaccagggtgagagccaagatggggtggcacccatgacgcttccccac




atgtttcttcaaggagcagaggacagagatagtggaaagagggtcagcagaagcagg




tgccttcatctatcccagcagctcagccaaaccccagttagaatgaggcagcaggag




attccaggtgtgctgagggttcagccacacgcagaagacgttgcagagtgttaaaga




ggtaagctgaggtgtgtatttggttggctttgttgttgttgttaatgtataatgaaa




agtataagactaaccctcaggcctcatgttctccaatagatccctggaagacagtat




agaaagtcagtcgggcttgggctccttagccagtgagactactcagaccaccagtgg




ctagcctagcctacctgtccttgaacatgggtgattttacccctttgaggtcttaac




cctttttttactttcaacaagatgagctctttgtatgattgcgggcgggggatatga




aaatgcaatgatctaactcctgttgctcttctagctggtcacatgacggcaccaggc




agggttctgggacacccggtgtgctttgactgttctacaaaaagctgccagagcgtt




ctggcctcctggaggctagattcctcagaaactgtctagcctttgcccacagagcat




gctatgtaattagagcactccttcccatgaaccccagcacttgtgttacctcagggc




cttggtacctggatactgccacagaatttccatggggcgggaagggatgtattttta




aataaagtaacttaaaagttggggaaattttttaaattcagaaaatgcaaagctatt




ctgtattacaccatttcacaatttaatatgtcttatattttcctgtgactctggaaa




ctaaaccattgtgtgtcttgtcgtgtcctagttgagctggggcctagcagcttcctt




ccagtgggtgtggagcaaacgtgtatgtcgcctcgctacctgcttgaggggtccgaa




gggctgctgggactgagttctgtacacacttgtttggccttttctgtagttgatgct




gtaaaactctatggctttttaaaaacaatttcatgtttttattttgtattggaagtc




caatacacttttttaatccaatcaaactggtctggtcaaaaagttctttcccttaaa




agttcaggggctcctacttccagcttccgatgacttctctgtggctctcactgctat




aaagcaggatttagaatggcaatctgggcagaggtaacaaaagaaatgtctgactgc




cagccccaaaa
















TABLE 2







siRNA targeting HAMP 3′UTR














SEQ

SEQ



Duplex name
Start
ID NO
Sense (5′-3′)
ID NO
Antisense (5′-3′)















307-325_s
307
53
GGAUGUGCUGCAAGACGUA
 96
UACGUCUUGCAGCACAUCC


 


309-327_s
309
54
AUGUGCUGCAAGACGUAGA
 97
UCUACGUCUUGCAGCACAU


 


310-328_s
310
55
UGUGCUGCAAGACGUAGAA
 98
UUCUACGUCUUGCAGCACA


 


313-331_s
313
56
GCUGCAAGACGUAGAACCU
 99
AGGUUCUACGUCUUGUAGC


 


AD-11439.1_31
314
57
CUGCAAGACGUAGAACCUA
100
UAGGUUCUACGUCUUGCAG


4-332_s







 


322-340_s
322
58
CGUAGAACCUACCUGCCCU
101
AGGGCAGGUAGGUUCUACG


 


347-365_s_G1A
347
59
GUCCCCUCCCUUCCUUAUU
102
AAUAAGGAAGGGAGGGGAC


 


348-366_s
348
60
UCCCCUCCCUUCCUUAUUU
103
AAAUAAGGAAGGGAGGGGA


 


349-367_s
349
61
CCCCUCCCUUCCUUAUUUA
104
UAAAUAAGGAAGGGAGGGG


 


350-368_s
350
62
CCCUCCCUUCCUUAUUUAU
105
AUAAAUAAGGAAGGGAGGG


 


351-369_s
351
63
CCUCCCUUCCUUAUUUAUU
106
AAUAAAUAAGGAAGGGAGG


 


352-370_s_C19A
352
64
CUCCCUUCCUUAUUUAUUA
107
UAAUAAAUAAGGAAGGGAG


 


352-370_s_C19U
352
65
CUCCCUUCCUUAUUUAUUU
108
AAAUAAAUAAGGAAGGGAG


 


354-372_s
354
66
CCCUUCCUUAUUUAUUCCU
109
AGGAAUAAAUAAGGAAGGG


 


355-373_s_G19A
355
67
CCUUCCUUAUUUAUUCCUA
110
UAGGAAUAAAUAAGGAAGG


 


355-373_s_G19U
355
68
CCUUCCUUAUUUAUUCCUU
111
AAGGAAUAAAUAAGGAAGG


 


356-374_s_C19A
356
69
CUUCCUUAUUUAUUCCUGA
112
UCAGGAAUAAAUAAGGAAG


 


356-374_s_C19U
356
70
CUUCCUUAUUUAUUCCUGU
113
ACAGGAAUAAAUAAGGAAG


 


357-375_s
357
71
UUCCUUAUUUAUUCCUGCU
114
AGCAGGAAUAAAUAAGGAA


 


358-376_s_G19A
358
72
UCCUUAUUUAUUCCUGCUA
115
UAGCAGGAAUAAAUAAGGA


 


358-376_s_G19U
358
73
UCCUUAUUUAUUCCUGCUU
116
AAGCAGGAAUAAAUAAGGA


 


359-377_s_C19A
359
74
CCUUAUUUAUUCCUGCUGA
117
UCAGCAGGAAUAAAUAAGG


 


359-377_s_C19U
359
75
CCUUAUUUAUUCCUGCUGU
118
ACAGCAGGAAUAAAUAAGG


 


363-381_s
363
76
AUUUAUUCCUGCUGCCCCA
119
UGGGGCAGCAGGAAUAAAU


 


365-383_s
365
77
UUAUUCCUGCUGCCCCAGA
120
UCUGGGGCAGCAGGAAUAA


 


366-384_s
366
78
UAUUCCUGCUGCCCCAGAA
121
UUCUGGGGCAGCAGGAAUA


 


369-387_s
369
79
UCCUGCUGCCCCAGAACAU
122
AUGUUCUGGGGCAGCAGGA


 


370-388_s
370
80
CCUGCUGCCCCAGAACAUA
123
UAUGUUCUGGGGCAGCAGG


 


373-391_s
373
81
GCUGCCCCAGAACAUAGGU
124
ACCUAUGUUCUGGGGCAGC


 


375-393_s
375
82
UGCCCCAGAACAUAGGUCU
125
AGACCUAUGUUCUGGGGCA


 


376-394_s
376
83
GCCCCAGAACAUAGGUCUU
126
AAGACCUAUGUUCUGGGGC


 


379-397_s
379
84
CCAGAACAUAGGUCUUGGA
127
UCCAAGACCUAUGUUCUGG


 


380-398_s
380
85
CAGAACAUAGGUCUUGGAA
128
UUCCAAGACCUAUGUUCUG


 


381-399_s
381
86
AGAACAUAGGUCUUGGAAU
129
AUUCCAAGACCUAUGUUCU


 


AD-11442.1_38
382
87
GAACAUAGGUCUUGGAAUA
130
UAUUCCAAGACCUAUGUUC


2-400_s







 


383-401_s
383
88
AACAUAGGUCUUGGAAUAA
131
UUAUUCCAAGACCUAUGUU


 


396-414_s
396
89
GAAUAAAAUGGCUGGUUCU
132
AGAACCAGCCAUUUUAUUC


 


398-416_s
398
90
AUAAAAUGGCUGGUUCUUU
133
AAAGAACCAGCCAUUUUAU


 


399-417_s
399
91
UAAAAUGGCUGGUUCUUUU
134
AAAAGAACCAGCCAUUUUA


 


402-420_s
402
92
AAUGGCUGGUUCUUUUGUU
135
AACAAAAGAACCAGCCAUU


 


403-421_s
403
93
AUGGCUGGUUCUUUUGUUU
136
AAACAAAAGAACCAGCCAU


 


407-425_s
407
94
CUGGUUCUUUUGUUUUCCA
137
UGGAAAACAAAAGAACCAG


 


AD-11436.1_29
291
95
CAUCGAUCAAAGUGUGGGA
138
UCCCACACUUUGAUCGAUG


1-309_s





Note


that an overhang (e.g. TT, dTsdT) can be added to the 3′end of any duplex.













TABLE 3







siRNA targeting HAMP CDS














SEQ

SEQ



Duplex name
Start
ID NO
sense (5′-3′)
ID NO
Antisense (5′-3′)





62-80_s_G19U
 62
139
AGACGGCACGAUGGCACUU
186
AAGUGCCAUCGUGCCGUCU


 


67-85_s_C19A
 67
140
GCACGAUGGCACUGAGCUA
187
UAGCUCAGUGCCAUCGUGC


 


67-85_s_C19U
 67
141
GCACGAUGGCACUGAGCUU
188
AAGCUCAGUGCCAUCGUGC


 


74-92_s_C19A
 74
142
GGCACUGAGCUCCCAGAUA
189
UAUCUGGGAGCUCAGUGCC


 


74-92_s_C19U
 74
143
GGCACUGAGCUCCCAGAUU
190
AAUCUGGGAGCUCAGUGCC


 


76-94_s_G19A
 76
144
CACUGAGCUCCCAGAUCUA
191
UAGAUCUGGGAGCUCAGUG


 


76-94_s_G19U
 76
145
CACUGAGCUCCCAGAUCUU
192
AAGAUCUGGGAGCUCAGUG


 


132-150_s
132
146
CUGACCAGUGGCUCUGUUU
193
AAACAGAGCCACUGGUCAG


 


140-158_s
140
147
UGGCUCUGUUUUCCCACAA
194
UUGUGGGAAAACAGAGCCA


 


146-164_s_hcU1C_G19A
146
148
UGUUUUCCCACAACAGACA
195
UGUCUGUUGUGGGAAAACA


 


146-164_s_hcU1C_G19U
146
149
UGUUUUCCCACAACAGACU
196
AGUCUGUUGUGGGAAAACA


 


155-173_s
155
150
ACAACAGACGGGACAACUU
197
AAGUUGUCCCGUCUGUUGU


 


157-175_s_C19A
157
151
AACAGACGGGACAACUUGA
198
UCAAGUUGUCCCGUCUGUU


 


157-175_s_C19U
157
152
AGACGGGACAACUUGCAGA
199
UCUGCAAGUUGUCCCGUCU


 


160-178_s
160
153
AGACGGGACAACUUGCAGA
200
UCUGCAAGUUGUCCCGUCU


 


161-179_s_G19A
161
154
GACGGGACAACUUGCAGAA
201
UUCUGCAAGUUGUCCCGUC


 


161-179_s_G19U
161
155
GACGGGACAACUUGCAGAU
202
AUCUGCAAGUUGUCCCGUC


 


162-180_s_C19A
162
156
ACGGGACAACUUGCAGAGA
203
UCUCUGCAAGUUGUCCCGU


 


162-180_s_C19U
162
157
ACGGGACAACUUGCAGAGU
204
ACUCUGCAAGUUGUCCCGU


 


242-260_s_C19A
242
158
GAGGCGAGACACCCACUUA
205
UAAGUGGGUGUCUCGCCUC


 


242-260_s_C19U
242
159
GAGGCGAGACACCCACUUU
206
AAAGUGGGUGUCUCGCCUC


 


253-271_s
253
160
CCCACUUCCCCAUCUGCAU
207
AUGCAGAUGGGGAAGUGGG


 


258-276_s
258
161
UUCCCCAUCUGCAUUUUCU
208
AGAAAAUGCAGAUGGGGAA


 


261-279_s
261
162
CCCAUCUGCAUUUUCUGCU
209
AGCAGAAAAUGCAGAUGGG


 


275-293_s
275
163
CUGCUGCGGCUGCUGUCAU
210
AUGACAGCAGCCGCAGCAG


 


276-294_s_C19A
276
164
UGCUGCGGCUGCUGUCAUA
211
UAUGACAGCAGCCGCAGCA


 


276-294_s_C19U
276
165
UGCUGCGGCUGCUGUCAUU
212
AAUGACAGCAGCCGCAGCA


 


278-296_s
278
166
CUGCGGCUGCUGUCAUCGA
213
UCGAUGACAGCAGCCGCAG


 


279-297_s
279
167
UGCGGCUGCUGUCAUCGAU
214
AUCGAUGACAGCAGCCGCA


 


280-298_s_C19A
280
168
GCGGCUGCUGUCAUCGAUA
215
UAUCGAUGACAGCAGCCGC


 


280-298_s_C19U
280
169
GCGGCUGCUGUCAUCGAUU
216
AAUCGAUGACAGCAGCCGC


 


281-299_s
281
170
CGGCUGCUGUCAUCGAUCA
217
UGAUCGAUGACAGCAGCCG


 


AD-11443.1_282-300_s
282
171
GGCUGCUGUCAUCGAUCAA
218
UUGAUCGAUGACAGCAGCC


 


AD-11432.1_283-301_s
283
172
GCUGCUGUCAUCGAUCAAA
219
UUUGAUCGAUGACAGCAGC


 


284-302_s_G19A
284
173
CUGCUGUCAUCGAUCAAAA
220
UUUUGAUCGAUGACAGCAG


 


284-302_s_G19U
284
174
CUGCUGUCAUCGAUCAAAU
221
AUUUGAUCGAUGACAGCAG


 


AD-11441.1_285-303_s
285
175
UGCUGUCAUCGAUCAAAGU
222
ACUUUGAUCGAUGACAGCA


 


286-304_s_G19A
286
176
GCUGUCAUCGAUCAAAGUA
223
UACUUUGAUCGAUGACAGC


 


286-304_s_G19U
286
177
GCUGUCAUCGAUCAAAGUU
224
AACUUUGAUCGAUGACAGC


 


AD-11447.1_297-305_s
287
178
CUGUCAUCGAUCAAAGUGU
225
ACACUUUGAUCGAUGACAG


 


288-306_s_G19A
288
179
UGUCAUCGAUCAAAGUGUA
226
UACACUUUGAUCGAUGACA


 


288-306_s_G19U
288
180
UGUCAUCGAUCAAAGUGUU
227
AACACUUUGAUCGAUGACA


 


290-308_s_G19A
290
181
UCAUCGAUCAAAGUGUGGA
228
UCCACACUUUGAUCGAUGA


 


290-308_s_G19U
290
182
UCAUCGAUCAAAGUGUGGU
229
ACCACACUUUGAUCGAUGA


 


295-313_s_G19A
295
183
GAUCAAAGUGUGGGAUGUA
230
UACAUCCCACACUUUGAUC


 


295-313_s_G19U
295
184
GAUCAAAGUGUGGGAUGUU
231
AACAUCCCACACUUUGAUC


 


299-317_s_C19U
299
185
AAAGUGUGGGAUGUGCUGU
232
ACAGCACAUCCCACACUUU





Note


that an overhang (e.g. TT, dTsdT) can be added to the 3′end of any duplex.













TABLE 4







HAMP modified sequences




















SEQ
Antisense

SEQ




Start
Sense

ID


ID


Target
Duplex ID
Position
Name
Sense Sequence
NO
Name
Antisense Sequence
NO


















HAMP
AD-45073
  2
A-94166.1
AcuGucAcucGG
233
A-94167.1
UCUGGGACCGAG
458






ucccAGAdTsdT


UGAcAGUdTsdT



 


HAMP
AD-45079
  7
A-94168.1
cAcucGGucccA
234
A-94169.1
UGGUGUCUGGGA
459






GAcAccAdTsdT


CCGAGUGdTsdT



 


HAMP
AD-45085
 16
A-94170.1
ccAGAcAccAGA
235
A-94171.1
AGCUUGCUCUGG
460






GcAAGcudTsdT


UGUCUGGdTsdT



 


HAMP
AD-29928
 43
A-66808.1
AGcAGuGGGAcA
236
A-66809.1
GUCUGGCUGUCC
461






GccAGAcdTsdT


cACUGCUdTsdT



 


HAMP
AD-45674
 43
A-95618.1
AGcAGuGGGAcA
237
A-95619.1
UUCUGGCUGUCC
462






GccAGAAdTsdT


cACUGCUdTsdT



 


HAMP
AD-45680
 43
A-95620.1
AGcAGuGGGAcA
238
A-95621.1
AUCUGGCUGUCC
463






GccAGAudTsdT


cACUGCUdTsdT



 


HAMP
AD-45686
 48
A-95622.1
uGGGAcAGccAG
239
A-95623.1
CGUCUGUCUGGC
464






AcAGAcGdTsdT


UGUCCcAdTsdT



 


HAMP
AD-45698
 48
A-95626.1
uGGGAcAGccAG
240
A-95627.1
AGUCUGUCUGGC
465






AcAGAcudTsdT


UGUCCcAdTsdT



 


HAMP
AD-45692
 48
A-95624.1
uGGGAcAGccAG
241
A-95625.1
UGUCUGUCUGGC
466






AcAGAcAdTsdT


UGUCCcAdTsdT



 


HAMP
AD-45354
 51
A-94701.1
GAcAGccAGAcA
242
A-94702.1
UGCCGUCUGUCU
467






GAcGGcAdTsdT


GGCUGUCdTsdT



 


HAMP
AD-29929
 54
A-66810.1
AGccAGAcAGAc
243
A-66811.1
UCGUGCCGUCUG
468






GGcAcGAdTsdT


UCUGGCUdTsdT



 


HAMP
AD-45091
 55
A-94172.1
GccAGAcAGAcG
244
A-94173.1
AUCGUGCCGUCU
469






GcAcGAudTsdT


GUCUGGCdTsdT



 


HAMP
AD-29930
 59
A-66812.1
GAcAGAcGGcAc
245
A-66813.1
UGCcAUCGUGCC
470






GAuGGcAdTsdT


GUCUGUCdTsdT



 


HAMP
AD-29931
 60
A-66814.1
AcAGAcGGcAcG
246
A-66815.1
GUGCcAUCGUGC
471






AuGGcAcdTsdT


CGUCUGUdTsdT



 


HAMP
AD-45704
 60
A-95628.1
AcAGAcGGcAcG
247
A-95629.1
UUGCcAUCGUGC
472






AuGGcAAdTsdT


CGUCUGUdTsdT



 


HAMP
AD-45710
 60
A-95630.1
AcAGAcGGcAcG
248
A-95631.1
AUGCcAUCGUGC
473






AuGGcAudTsdT


CGUCUGUdTsdT



 


HAMP
AD-29932
 61
A-66816.1
cAGAcGGcAcGA
249
A-66817.1
AGUGCcAUCGUG
474






uGGcAcudTsdT


CCGUCUGdTsdT



 


HAMP
AD-47030
 62
A-98344.1
AGACfGGCfACf
250
A-98345.1
AAGUGCCfAUCG
475






GAUfGGCfACfU


UGCCGUCUdTsd







fUfdTsdT


T



 


HAMP
AD-29933
 62
A-66818.1
AGAcGGcAcGAu
251
A-66819.1
cAGUGCcAUCGU
476






GGcAcuGdTsdT


GCCGUCUdTsdT



 


HAMP
AD-45675
 62
A-95634.1
AGAcGGcAcGAu
250
A-95635.1
AAGUGCcAUCGU
475






GGcAcuudTsdT


GCCGUCUdTsdT



 


HAMP
AD-45716
 62
A-95632.1
AGAcGGcAcGAu
252
A-95633.1
uAGUGCcAUCGU
477






GGcAcuAdTsdT


GCCGUCUdTsdT



 


HAMP
AD-29934
 63
A-66820.1
GAcGGcAcGAuG
253
A-66821.1
UcAGUGCcAUCG
478






GcAcuGAdTsdT


UGCCGUCdTsdT



 


HAMP
AD-29935
 64
A-66822.1
AcGGcAcGAuGG
254
A-66823.1
CUcAGUGCcAUC
479






cAcuGAGdTsdT


GUGCCGUdTsdT



 


HAMP
AD-45687
 64
A-95638.1
AcGGcAcGAuGG
255
A-95639.1
AUcAGUGCcAUC
480






cAcuGAudTsdT


GUGCCGUdTsdT



 


HAMP
AD-45681
 64
A-95636.1
AcGGcAcGAuGG
256
A-95637.1
UUcAGUGCcAUC
481






cAcuGAAdTsdT


GUGCCGUdTsdT



 


HAMP
AD-29936
 66
A-66824.1
GGcAcGAuGGcA
257
A-66825.1
AGCUcAGUGCcA
482






cuGAGcudTsdT


UCGUGCCdTsdT



 


HAMP
AD-47043
 67
A-98348.1
GCfACfGAUfGG
258
A-98349.1
AAGCUCfAGUGC
483






CfACfUfGAGCf


CfAUCGUGCdTs







UfUfdTsdT


dT



 


HAMP
AD-47037
 67
A-98346.1
GCfACfGAUfGG
259
A-98347.1
CfAGCUCfAGUG
484






CfACfUfGAGCf


CCfAUCGUGCdT







UfAdTsdT


sdT



 


HAMP
AD-29937
 67
A-66826.1
GcAcGAuGGcAc
260
A-66827.1
GAGCUcAGUGCc
485






uGAGcucdTsdT


AUCGUGCdTsdT



 


HAMP
AD-45699
 67
A-95642.1
GcAcGAuGGcAc
258
A-95643.1
AAGCUcAGUGCc
483






uGAGcuudTsdT


AUCGUGCdTsdT



 


HAMP
AD-45693
 67
A-95640.1
GcAcGAuGGcAc
259
A-95641.1
uAGCUcAGUGCc
486






uGAGcuAdTsdT


AUCGUGCdTsdT



 


HAMP
AD-45711
 68
A-95646.1
cAcGAuGGcAcu
261
A-95647.1
UGAGCUcAGUGC
487






GAGcucAdTsdT


cAUCGUGdTsdT



 


HAMP
AD-45717
 68
A-95648.1
cAcGAuGGcAcu
262
A-95649.1
AGAGCUcAGUGC
488






GAGcucudTsdT


cAUCGUGdTsdT



 


HAMP
AD-45705
 68
A-95644.1
cAcGAuGGcAcu
263
A-95645.1
GGAGCUcAGUGC
489






GAGcuccdTsdT


cAUCGUGdTsdT



 


HAMP
AD-45682
 69
A-95652.1
AcGAuGGcAcuG
264
A-95653.1
UGGAGCUcAGUG
490






AGcuccAdTsdT


CcAUCGUdTsdT



 


HAMP
AD-45688
 69
A-95654.1
AcGAuGGcAcuG
265
A-95655.1
AGGAGCUcAGUG
491






AGcuccudTsdT


CcAUCGUdTsdT



 


HAMP
AD-45676
 69
A-95650.1
AcGAuGGcAcuG
266
A-95651.1
GGGAGCUcAGUG
492






AGcucccdTsdT


CcAUCGUdTsdT



 


HAMP
AD-45360
 70
A-94703.1
cGAuGGcAcuGA
267
A-94704.1
UGGGAGCUcAGU
493






GcucccAdTsdT


GCcAUCGdTsdT



 


HAMP
AD-45366
 71
A-94705.1
GAuGGcAcuGAG
268
A-94706.1
CUGGGAGCUcAG
494






cucccAGdTsdT


UGCcAUCdTsdT



 


HAMP
AD-29938
 72
A-66828.1
AuGGcAcuGAGc
269
A-66829.1
UCUGGGAGCUcA
495






ucccAGAdTsdT


GUGCcAUdTsdT



 


HAMP
AD-45372
 73
A-94707.1
uGGcAcuGAGcu
270
A-94708.1
AUCUGGGAGCUc
496






cccAGAudTsdT


AGUGCcAdTsdT



 


HAMP
AD-47055
 74
A-98352.1
GGCfACfUfGAG
271
A-98353.1
AAUCUGGGAGCU
497






CfUfCfCfCfAG


CfAGUGCCdTsd







AUfUfdTsdT


T



 


HAMP
AD-47049
 74
A-98350.1
GGCfACfUfGAG
272
A-98351.1
CfAUCUGGGAGC
498






CfUfCfCfCfAG


UCfAGUGCCdTs







AUfAdTsdT


dT



 


HAMP
AD-45700
 74
A-95658.1
GGcAcuGAGcuc
271
A-95659.1
AAUCUGGGAGCU
497






ccAGAuudTsdT


cAGUGCCdTsdT



 


HAMP
AD-29939
 74
A-66830.1
GGcAcuGAGcuc
273
A-66831.1
GAUCUGGGAGCU
499






ccAGAucdTsdT


cAGUGCCdTsdT



 


HAMP
AD-45694
 74
A-95656.1
GGcAcuGAGcuc
272
A-95657.1
uAUCUGGGAGCU
500






ccAGAuAdTsdT


cAGUGCCdTsdT



 


HAMP
AD-29940
 75
A-66832.1
GcAcuGAGcucc
274
A-66833.1
AGAUCUGGGAGC
501






cAGAucudTsdT


UcAGUGCdTsdT



 


HAMP
AD-47067
 76
A-98356.1
CfACfUfGAGCf
275
A-98357.1
AAGAUCUGGGAG
502






UfCfCfCfAGAU


CUCfAGUGdTsd







fCfUfUfdTsdT


T



 


HAMP
AD-47061
 76
A-98354.1
CfACfUfGAGCf
276
A-98355.1
CfAGAUCUGGGA
503






UfCfCfCfAGAU


GCUCfAGUGdTs







fCfUfAdTsdT


dT



 


HAMP
AD-45712
 76
A-95662.1
cAcuGAGcuccc
275
A-95663.1
AAGAUCUGGGAG
502






AGAucuudTsdT


CUcAGUGdTsdT



 


HAMP
AD-29941
 76
A-66834.1
cAcuGAGcuccc
277
A-66835.1
cAGAUCUGGGAG
503






AGAucuGdTsdT


CUcAGUGdTsdT



 


HAMP
AD-45706
 76
A-95660.1
cAcuGAGcuccc
276
A-95661.1
uAGAUCUGGGAG
504






AGAucuAdTsdT


CUcAGUGdTsdT



 


HAMP
AD-45097
 88
A-94174.1
AGAucuGGGccG
278
A-94175.1
AGGcAAGCGGCC
505






cuuGccudTsdT


cAGAUCUdTsdT



 


HAMP
AD-45103
 91
A-94176.1
ucuGGGccGcuu
279
A-94177.1
AGGAGGcAAGCG
506






GccuccudTsdT


GCCcAGAdTsdT



 


HAMP
AD-45378
116
A-94709.1
ccuccuccucGc
280
A-94710.1
cAGGCUGGCGAG
507






cAGccuGdTsdT


GAGGAGGdTsdT



 


HAMP
AD-45383
117
A-94711.1
cuccuccucGcc
281
A-94712.1
UcAGGCUGGCGA
508






AGccuGAdTsdT


GGAGGAGdTsdT



 


HAMP
AD-45388
118
A-94713.1
uccuccucGccA
282
A-94714.1
GUcAGGCUGGCG
509






GccuGAcdTsdT


AGGAGGAdTsdT



 


HAMP
AD-45393
120
A-94715.1
cuccucGccAGc
283
A-94716.1
UGGUcAGGCUGG
510






cuGAccAdTsdT


CGAGGAGdTsdT



 


HAMP
AD-45355
121
A-94717.1
uccucGccAGcc
284
A-94718.1
CUGGUcAGGCUG
511






uGAccAGdTsdT


GCGAGGAdTsdT



 


HAMP
AD-45361
122
A-94719.1
ccucGccAGccu
285
A-94720.1
ACUGGUcAGGCU
512






GAccAGudTsdT


GGCGAGGdTsdT



 


HAMP
AD-45367
123
A-94721.1
cucGccAGccuG
286
A-94722.1
cACUGGUcAGGC
513






AaccAGuGdTsd


UGGCGAGdTsdT







T






 


HAMP
AD-45373
126
A-94723.1
GccAGccuGAcc
287
A-94724.1
AGCcACUGGUcA
514






AGuGGcudTsdT


GGCUGGCdTsdT



 


HAMP
AD-45109
132
A-94178.1
cuGAccAGuGGc
288
A-94179.1
AAAcAGAGCcAC
515






ucuGuuudTsdT


UGGUcAGdTsdT



 


HAMP
AD-47032
140
A-98360.1
UfGGCfUfCfUfG
289
A-98361.1
UUGUGGGAAAAC
516






UfUfUfUfCfCfC


fAGAGCCfAdTs







fACfAAdTsdT


dT



 


HAMP
AD-45115
140
A-94180.1
uGGcucuGuuuu
289
A-94181.1
UUGUGGGAAAAc
516






cccAcAAdTsdT


AGAGCcAdTsdT



 


HAMP
AD-45074
142
A-94182.1
GcucuGuuuucc
290
A-94183.1
UGUUGUGGGAAA
517






cAcAAcAdTsdT


AcAGAGCdTsdT



 


HAMP
AD-47038
146
A-98362.1
UfGUfUfUfUfC
291
A-98363.1
UGUCUGUUGUGG
518






fCfCfACfAACf


GAAAACfAdTsd







AGACfAdTsdT


T



 


HAMP
AD-47044
146
A-98364.1
UfGUfUfUfUfC
292
A-98365.1
AGUCUGUUGUGG
519






fCfCfACfAACf


GAAAACfAdTsd







AGACfUfdTsdT


T



 


HAMP
AD-45677
146
A-95666.1
uGuuuucccAcA
291
A-95667.1
UGUCUGUUGUGG
518






AcAGAcAdTsdT


GAAAAcAdTsdT



 


HAMP
AD-45683
146
A-95668.1
uGuuuucccAcA
292
A-95669.1
AGUCUGUUGUGG
519






AcAGAcudTsdT


GAAAAcAdTsdT



 


HAMP
AD-45718
146
A-95664.1
uGuuuucccAcA
293
A-95665.1
CGUCUGUUGUGG
520






AcAGAcGdTsdT


GAAAAcAdTsdT



 


HAMP
AD-45080
149
A-94184.1
uuucccAcAAcA
294
A-94185.1
UCCCGUCUGUUG
521






GAcGGGAdTsdT


UGGGAAAdTsdT



 


HAMP
AD-45379
150
A-94725.1
uucccAcAAcAG
295
A-94726.1
GUCCCGUCUGUU
522






AcGGGAcdTsdT


GUGGGAAdTsdT



 


HAMP
AD-29942
151
A-66836.1
ucccAcAAcAGA
296
A-66837.1
UGUCCCGUCUGU
523






cGGGAcAdTsdT


UGUGGGAdTsdT



 


HAMP
AD-29943
152
A-66838.1
cccAcAAcAGAc
297
A-66839.1
UUGUCCCGUCUG
524






GGGAcAAdTsdT


UUGUGGGdTsdT



 


HAMP
AD-29944
153
A-66840.1
ccAcAAcAGAcG
298
A-15142.2
GUUGUCCCGUCU
525






GGAcAAcdTsdT


GUUGUGGdTsdT



 


HAMP
AD-45695
153
A-95672.1
ccAcAAcAGAcG
299
A-95673.1
AUUGUCCCGUCU
526






GGAcAAudTsdT


GUUGUGGdTsdT



 


HAMP
AD-45689
153
A-95670.1
ccAcAAcAGAcG
300
A-95671.1
UUUGUCCCGUCU
527






GGAcAAAdTsdT


GUUGUGGdTsdT



 


HAMP
AD-29945
154
A-66841.1
cAcAAcAGAcGG
301
A-15116.1
AGUUGUCCCGUC
528






GAcAAcudTsdT


UGUUGUGdTsdT



 


HAMP
AD-47050
155
A-98366.1
ACfAACfAGACf
302
A-15182.3
AAGUUGUCCCGU
529






GGGACfAACfUf


CUGUUGUdTsdT







UfdTsdT






 


HAMP
AD-29946
155
A-66842.1
AcAAcAGAcGGG
302
A-15182.1
AAGUUGUCCCGU
529






AcAAcuudTsdT


CUGUUGUdTsdT



 


HAMP
AD-47062
157
A-98369.1
AACfAGACfGGG
303
A-98370.1
ACfAAGUUGUCC
530






ACfAACfUfUfG


CGUCUGUUdTsd







UfdTsdT


T



 


HAMP
AD-47056
157
A-98367.1
AACfAGACfGGG
304
A-98368.1
UCfAAGUUGUCC
531






ACfAACfUfUfG


CGUCUGUUdTsd







AdTsdT


T



 


HAMP
AD-45713
157
A-95678.1
AAcAGAcGGGAc
303
A-95679.1
AcAAGUUGUCCC
530






AAcuuGudTsdT


GUCUGUUdTsdT



 


HAMP
AD-45707
157
A-95676.1
AAcAGAcGGGAc
304
A-95677.1
UcAAGUUGUCCC
531






AAcuuGAdTsdT


GUCUGUUdTsdT



 


HAMP
AD-45701
157
A-95674.1
AAcAGAcGGGAc
305
A-95675.1
GcAAGUUGUCCC
532






AAcuuGcdTsdT


GUCUGUUdTsdT



 


HAMP
AD-45394
159
A-94727.1
cAGAcGGGAcAA
306
A-94728.1
CUGcAAGUUGUC
533






cuuGcAGdTsdT


CCGUCUGdTsdT



 


HAMP
AD-47068
160
A-98371.1
AGACfGGGACfA
307
A-98372.1
UCUGCfAAGUUG
534






ACfUfUfGCfAG


UCCCGUCUdTsd







AdTsdT


T



 


HAMP
AD-45389
160
A-94729.1
AGAcGGGAcAAc
307
A-94730.1
UCUGcAAGUUGU
534






uuGcAGAdTsdT


CCCGUCUdTsdT



 


HAMP
AD-47033
161
A-98375.1
GACfGGGACfAA
308
A-98376.1
AUCUGCfAAGUU
535






CfUfUfGCfAGA


GUCCCGUCdTsd







UfdTsdT


T



 


HAMP
AD-47074
161
A-98373.1
GACfGGGACfAA
309
A-98374.1
UUCUGCfAAGUU
536






CfUfUfGCfAGA


GUCCCGUCdTsd







AdTsdT


T



 


HAMP
AD-45678
161
A-95682.1
GAcGGGAcAAcu
308
A-95683.1
AUCUGcAAGUUG
535






uGcAGAudTsdT


UCCCGUCdTsdT



 


HAMP
AD-45719
161
A-95680.1
GAcGGGAcAAcu
309
A-95681.1
UUCUGcAAGUUG
536






uGcAGAAdTsdT


UCCCGUCdTsdT



 


HAMP
AD-29947
161
A-66843.1
GAcGGGAcAAcu
310
A-66844.1
CUCUGcAAGUUG
537






uGcAGAGdTsdT


UCCCGUCdTsdT



 


HAMP
AD-47039
162
A-98377.1
ACfGGGACfAAC
311
A-98378.1
UCUCUGCfAAGU
538






fUfUfGCfAGAG


UGUCCCGUdTsd







AdTsdT


T



 


HAMP
AD-47045
162
A-98379.1
ACfGGGACfAAC
312
A-98380.1
ACUCUGCfAAGU
539






fUfUfGCfAGAG


UGUCCCGUdTsd







UfdTsdT


T



 


HAMP
AD-45690
162
A-95686.1
AcGGGAcAAcuu
311
A-95687.1
UCUCUGcAAGUU
538






GcAGAGAdTsdT


GUCCCGUdTsdT



 


HAMP
AD-45696
162
A-95688.1
AcGGGAcAAcuu
312
A-95689.1
ACUCUGcAAGUU
539






GcAGAGudTsdT


GUCCCGUdTsdT



 


HAMP
AD-45684
162
A-95684.1
AcGGGAcAAcuu
313
A-95685.1
GCUCUGcAAGUU
540






GcAGAGcdTsdT


GUCCCGUdTsdT



 


HAMP
AD-30016
163
A-66845.1
cGGGAcAAcuuG
314
A-66846.1
AGCUCUGcAAGU
541






cAGAGcudTsdT


UGUCCCGdTsdT



 


HAMP
AD-45394
164
A-94731.1
GGGAcAAcuuGc
315
A-94732.1
cAGCUCUGcAAG
542






AGAGcuGdTsdT


UUGUCCCdTsdT



 


HAMP
AD-45702
165
A-95690.1
GGAcAAcuuGcA
316
A-95691.1
GcAGCUCUGcAA
543






GAGcuGcdTsdT


GUUGUCCdTsdT



 


HAMP
AD-45708
165
A-95692.1
GGAcAAcuuGcA
317
A-95693.1
UcAGCUCUGcAA
544






GAGcUGAdTsdT


GUUGUCCdTsdT



 


HAMP
AD-45714
165
A-95694.1
GGAcAAcuuGcA
318
A-95695.1
AcAGCUCUGcAA
545






GAGcuGudTsdT


GUUGUCCdTsdT



 


HAMP
AD-29949
166
A-66847.1
GAcAAcuuGcAG
319
A-66848.1
UGcAGCUCUGcA
546






AGcuGcAdTsdT


AGUUGUCdTsdT



 


HAMP
AD-45086
167
A-94186.1
AcAAcuuGcAGA
320
A-94187.1
UUGcAGCUCUGc
547






GcuGcAAdTsdT


AAGUUGUdTsdT



 


HAMP
AD-45356
168
A-94733.1
cAAcuuGcAGAG
321
A-94734.1
GUUGcAGCUCUG
548






cuGcAAcdTsdT


cAAGUUGdTsdT



 


HAMP
AD-45685
169
A-95700.1
AAcuuGcAGAGc
322
A-95701.1
AGUUGcAGCUCU
549






uGcAAcudTsdT


GcAAGUUdTsdT



 


HAMP
AD-45679
169
A-95698.1
AAcuuGcAGAGc
323
A-95699.1
UGUUGcAGCUCU
550






uGcAAcAdTsdT


GcAAGUUdTsdT



 


HAMP
AD-45720
169
A-95696.1
AAcuuGcAGAGc
324
A-95697.1
GGUUGcAGCUCU
551






uGcAAccdTsdT


GcAAGUUdTsdT



 


HAMP
AD-45703
170
A-95706.1
AcuuGcAGAGcu
325
A-95707.1
AGGUUGcAGCUC
552






GcAAccudTsdT


UGcAAGUdTsdT



 


HAMP
AD-45697
170
A-95704.1
AcuuGcAGAGcu
326
A-95705.1
UGGUUGcAGCUC
553






GcAAccAdTsdT


UGcAAGUdTsdT



 


HAMP
AD-45691
170
A-95702.1
AcuuGcAGAGcu
327
A-95703.1
GGGUUGcAGCUC
554






GcAAcccdTsdT


UGcAAGUdTsdT



 


HAMP
AD-45362
189
A-94735.1
cAGGAcAGAGcu
328
A-94736.1
UGGCUCcAGCUC
555






GGAGccAdTsdT


UGUCCUGdTsdT



 


HAMP
AD-45368
190
A-94737.1
AGGAcAGAGcuG
329
A-94738.1
CUGGCUCcAGCU
556






GAGccAGdTsdT


CUGUCCUdTsdT



 


HAMP
AD-45374
199
A-94739.1
cuGGAGccAGGG
330
A-94740.1
cAGCUGGCCCUG
557






ccAGcuGdTsdT


GCUCcAGdTsdT



 


HAMP
AD-45092
222
A-94188.1
cccAuGuuccAG
331
A-94189.1
UUCGCCUCUGGA
558






AGGcGAAdTsdT


AcAUGGGdTsdT



 


HAMP
AD-45721
228
A-95712.1
uuccAGAGGcGA
332
A-95713.1
ACCUCCUUCGCC
559






AGGAGGudTsdT


UCUGGAAdTsdT



 


HAMP
AD-45715
228
A-95710.1
uuccAGAGGcGA
333
A-95711.1
UCCUCCUUCGCC
560






AGGAGGAdTsdT


UCUGGAAdTsdT



 


HAMP
AD-45709
228
A-95708.1
uuccAGAGGcGA
334
A-95709.1
GCCUCCUUCGCC
561






AGGAGGcdTsdT


UCUGGAAdTsdT



 


HAMP
AD-45380
230
A-94741.1
ccAGAGGcGAAG
335
A-94742.1
UCGCCUCCUUCG
562






GAGGcGAdTsdT


CCUCUGGdTsdT



 


HAMP
AD-45385
231
A-94743.1
cAGAGGcGAAGG
336
A-94744.1
CUCGCCUCCUUC
563






AGGcGAGdTsdT


GCCUCUGdTsdT



 


HAMP
AD-29950
232
A-66849.1
AGAGGcGAAGGA
337
A-66850.1
UCUCGCCUCCUU
564






GGcGAGAdTsdT


CGCCUCUdTsdT



 


HAMP
AD-45390
233
A-94745.1
GAGGcGAAGGAG
338
A-94746.1
GUCUCGCCUCCU
565






GcGAGAcdTsdT


UCGCCUCdTsdT



 


HAMP
AD-29951
234
A-66851.1
AGGcGAAGGAGG
339
A-66852.1
UGUCUCGCCUCC
566






cGAGAcAdTsdT


UUCGCCUdTsdT



 


HAMP
AD-45395
235
A-94747.1
GGcGAAGGAGGc
340
A-94748.1
GUGUCUCGCCUC
567






GAGAcAcdTsdT


CUUCGCCdTsdT



 


HAMP
AD-45727
239
A-95714.1
AAGGAGGcGAGA
341
A-95715.1
UUGGGUGUCUCG
568






cAcccAAdTsdT


CCUCCUUdTsdT



 


HAMP
AD-45732
239
A-95716.1
AAGGAGGcGAGA
342
A-95717.1
AUGGGUGUCUCG
569






cAcccAudTsdT


CCUCCUUdTsdT



 


HAMP
AD-29952
239
A-66853.1
AAGGAGGcGAGA
343
A-66854.1
GUGGGUGUCUCG
570






cAcccAcdTsdT


CCUCCUUdTsdT



 


HAMP
AD-29953
240
A-66855.1
AGGAGGcGAGAc
344
A-66856.1
AGUGGGUGUCUC
571






AcccAcudTsdT


GCCUCCUdTsdT



 


HAMP
AD-30017
241
A-66857.1
GGAGGcGAGAcA
345
A-66858.1
AAGUGGGUGUCU
572






cccAcuudTsdT


CGCCUCCdTsdT



 


HAMP
AD-47057
242
A-98383.1
GAGGCfGAGACf
346
A-95721.2
AAAGUGGGUGUC
573






ACfCfCfACfUf


UCGCCUCdTsdT







UfUfdTsdT






 


HAMP
AD-47051
242
A-98381.1
GAGGCfGAGACf
347
A-98382.1
CfAAGUGGGUGU
574






ACfCfCfACfUf


CUCGCCUCdTsd







UfAdTsdT


T



 


HAMP
AD-30018
242
A-66859.1
GAGGcGAGAcAc
348
A-66860.1
GAAGUGGGUUCG
575






ccAcuucdTsdT


CCUCGUCdTsdT



 


HAMP
AD-45737
242
A-95718.1
GAGGcGAGAcAc
347
A-95719.1
uAAGUGGGUGUC
576






ccAcuuAdTsdT


UCGCCUCdTsdT



 


HAMP
AD-29956
246
A-66861.1
cGAGAcAcccAc
349
A-66862.1
UGGGGAAGUGGG
577






uuccccAdTsdT


UGUCUCGdTsdT



 


HAMP
AD-45357
247
A-94749.1
GAGAcAcccAcu
350
A-94750.1
AUGGGGAAGUGG
578






uccccAudTsdT


GUGUCUCdTsdT



 


HAMP
AD-45363
248
A-94751.1
AGAcAcccAcuu
351
A-94752.1
GAUGGGGAAGUG
579






ccccAucdTsdT


GGUGUCUdTsdT



 


HAMP
AD-45747
251
A-95722.1
cAcccAcuuccc
352
A-95723.1
GcAGAUGGGGAA
580






cAucuGcdTsdT


GUGGGUGdTsdT



 


HAMP
AD-45752
251
A-95724.1
cAcccAcuuccc
353
A-95725.1
UcAGAUGGGAAG
581






cAucuGAdTsdT


UGGGUGdTsdT



 


HAMP
AD-45757
251
A-95726.1
cAcccAcuuccc
354
A-95727.1
AcAGAUGGGGAA
582






cAucuGudTsdT


GUGGGUGdTsdT



 


HAMP
AD-29957
252
A-66863.1
AcccAcuucccc
355
A-66864.1
UGcAGAUGGGGA
583






AucuGcAdTsdT


AGUGGGUdTsdT



 


HAMP
AD-47063
253
A-98384.1
CfCfCfACfUfU
356
A-98385.1
AUGCfAGAUGGG
584






fCfCfCfCfAUf


GAAGUGGGdTsd







CfUfGCfAUfdT


T







sdT






 


HAMP
AD-45399
253
A-94753.1
cccAcuuccccA
356
A-94754.1
AUGcAGAUGGGG
584






ucuGcAudTsdT


AAGUGGGdTsdT



 


HAMP
AD-45098
255
A-94190.1
cAcuuccccAuc
357
A-94191.1
AAAUGcAGAUGG
585






uGcAuuudTsdT


GGAAGUGdTsdT



 


HAMP
AD-45400
256
A-94755.1
AcuuccccAucu
358
A-94756.1
AAAAUGcAGAUG
586






GcAuuuudTsdT


GGGAAGUdTsdT



 


HAMP
AD-45381
257
A-94757.1
cuuccccAucuG
359
A-94758.1
GAAAAUGcAGAU
587






cAuuuucdTsdT


GGGGAAGdTsdT



 


HAMP
AD-47069
258
A-98386.1
UfUfCfCfCfCf
360
A-98387.1
AGAAAAUGCfAG
588






AUfCfUfGCfAU


AUGGGGAAdTsd







fUfUfUfCfUfd


T







TsdT






 


HAMP
AD-45401
258
A-94759.1
uuccccAucuGc
360
A-94760.1
AGAAAAUGcAGA
588






AuuuucudTsdT


UGGGGAAdTsdT



 


HAMP
AD-47075
261
A-98388.1
CfCfCfAUfCfU
361
A-98389.1
AGCfAGAAAAUG
589






fGCfAUfUfUfU


CfAGAUGGGdTs







fCfUfGCfUfdT


dT







sdT






 


HAMP
AD-29958
261
A-66865.1
cccAucuGcAuu
361
A-66866.1
AGcAGAAAAUGc
589






uucuGcudTsdT


AGAUGGGdTsdT



 


HAMP
AD-45391
262
A-94761.1
ccAucuGcAuuu
362
A-94762.1
cAGcAGAAAAUG
590






ucuGcuGdTsdT


cAGAUGGdTsdT



 


HAMP
AD-29959
267
A-66867.1
uGcAuuuucuGc
363
A-66868.1
AGCCGcAGcAGA
591






uGcGGcudTsdT


AAAUGcAdTsdT



 


HAMP
AD-29960
268
A-66869.1
GcAuuuucuGcu
364
A-66870.1
cAGCCGcAGcAG
592






GcGGcuGdTsdT


AAAAUGCdTsdT



 


HAMP
AD-30019
270
A-66871.1
AuuuucuGcuGc
365
A-66872.1
AGcAGCCGcAGc
593






GGcuGcudTsdT


AGAAAAUdTsdT



 


HAMP
AD-45396
271
A-94763.1
uuuucuGcuGcG
366
A-94764.1
cAGcAGCCGcAG
594






GcuGcuGdTsdT


cAGAAAAdTsdT



 


HAMP
AD-45358
272
A-94765.1
uuucuGcuGcGG
367
A-94766.1
AcAGcAGCCGcA
595






cuGcuGudTsdT


GcAGAAAdTsdT



 


HAMP
AD-45364
273
A-94767.1
uucuGcuGcGGc
368
A-94768.1
GAcAGcAGCCGc
596






uGcuGucdTsdT


AGcAGAAdTsdT



 


HAMP
AD-29962
274
A-66873.1
ucuGcuGcGGcu
369
A-66874.1
UGAcAGcAGCCG
597






GcuGucAdTsdT


cAGcAGAdTsdT



 


HAMP
AD-47034
275
A-98390.1
CfUfGCfUfGCf
370
A-98391.1
AUGACfFGCfAG
598






GGCfUfGCfUfG


CCGCfAGCfAGd







UfCfAUfdTsdT


TsdT



 


HAMP
AD-45370
275
A-94769.1
cuGcuGcGGcuG
370
A-94770.1
AUGAcAGcAGCC
598






cuGucAudTsdT


GcAGcAGdTsdT



 


HAMP
AD-47046
276
A-98394.1
UfGCfUfGCfGG
371
A-98395.1
AAUGACfAGCfA
599






CfUfGCfUfGUf


GCCGCfAGCfAd







CfAUfUfdTsdT


TsdT



 


HAMP
AD-47040
276
A-98392.1
UfGCfUfGCfGG
372
A-98393.1
CfAUGACfAGCf
600






CfUfGCfUfGUf


AGCCGCfAGCfA







CfAUfAdTsdT


dTsdT



 


HAMP
AD-45728
276
A-95730.1
uGcuGcGGcuGc
371
A-95731.1
AAUGAcAGcAGC
599






uGucAuudTsdT


CGcAGcAdTsdT



 


HAMP
AD-45722
276
A-95728.1
uGcuGcGGcuGc
372
A-95729.1
uAUGAcAGcAGC
601






uGucAuAdTsdT


CGcAGcAdTsdT



 


HAMP
AD-29963
276
A-66875.1
uGcuGcGGcuGc
373
A-66876.1
GAUGAcAGcAGC
602






uGucAucdTsdT


CGcAGcAdTsdT



 


HAMP
AD-45104
278
A-94192.1
cuGcGGcuGcuG
374
A-94193.1
UCGAUGAcAGcA
603






ucAucGAdTsdT


GCCGcAGdTsdT



 


HAMP
AD-47058
279
A-98398.1
UfGCfGGCfUfG
375
A-98399.1
AUCGAUGACfAG
604






CfUfGUfCfAUf


CfAGCCGCfAdT







CfGAUfdTsdT


sdT



 


HAMP
AD-29964
279
A-66877.1
uGcGGcuGcuGu
375
A-66878.1
AUCGAUGAcAGc
604






cAucGAudTsdT


AGCCGcAdTsdT



 


HAMP
AD-47070
280
A-98402.1
GCfGGCfUfGCf
376
A-98403.1
AAUCGAUGACfA
605






UfGUfCfAUfCf


GCfAGCCGCdTs







GAUfUfdTsdT


dT



 


HAMP
AD-47064
280
A-98400.1
GCfGGCfUfGCf
377
A-98401.1
CfAUCGAUGACf
606






UfGUfCfAUfCf


AGCfAGCCGCdT







GAUfFdTsdT


sdT



 


HAMP
AD-45738
280
A-95734.1
GcGGcuGcuGuc
376
A-95735.1
AAUCGAUGAcAG
605






AucGAuudTsdT


cAGCCGCdTsdT



 


HAMP
AD-45733
280
A-95732.1
GcGGcuGcuGuc
377
A-95733.1
uAUCGAUGAcAG
607






AucGAuAdTsdT


cAGCCGCdTsdT



 


HAMP
AD-47076
281
A-98404.1
CfGGCfUfGCfU
378
A-98405.1
UGAUCGAUGACf
608






fGUfCfAUfCfG


AGCfAGCCGdTs







AUfCfAdTsdT


dT



 


HAMP
AD-29965
281
A-66879.1
cGGcuGcuGucA
378
A-66880.1
UGAUCGAUGAcA
608






ucGAucAdTsdT


GcAGCCGdTsdT



 


HAMP
AD-47035
282
A-98406.1
GGCfUfGCfUfG
379
A-98407.1
UUGAUCGAUGAC
609






UfCfAUfCfGAU


fAGCfAGCCdTs







fCfAAdTsdT


dT



 


HAMP
AD-47041
283
A-98408.1
GCfUfGCfUfGU
380
A-98409.1
UUUGAUCGAUGA
610






fCfAUfCfGAUf


CfAGCfAGCdTs







CfAAAdTsdT


dT



 


HAMP
AD-30020
283
A-18260.1
GcuGcuGucAuc
380
A-18261.1
UUUGAUCGAUGA
610






GAucAAAdTsdT


cAGcAGCdTsdT



 


HAMP
AD-47053
284
A-98412.1
CfUfGCfUfGUf
381
A-98413.1
AUUUGAUCGAUG
611






CfAUfCfGAUfC


ACfAGCfAGdTs







fAAAUfdTsdT


dT



 


HAMP
AD-47047
284
A-98410.1
CfUfGCfUfGUf
382
A-98411.1
UUUUGAUCGAUG
612






CfAUfCfGAUfC


ACfAGCfAGdTs







fAAAAdTsdT


dT



 


HAMP
AD-45748
284
A-95738.1
cuGcuGucAucG
381
A-95739.1
AUUUGAUCGAUG
611






AucAAAudTsdT


AcAGcAGdTsdT



 


HAMP
AD-45743
284
A-95736.1
cuGcuGucAucG
382
A-95737.1
UUUUGAUCGAUG
612






AucAAAAdTsdT


AcAGcAGdTsdT



 


HAMP
AD-30021
284
A-18284.1
cuGcuGucAucG
383
A-18285.1
CUUUGAUCGAUG
613






AucAAAGdTsdT


AcAGcAGdTsdT



 


HAMP
AD-47059
285
A-98414.1
UfGCfUfGUfCf
384
A-98415.1
ACUUUGAUCGAU
614






AUfCfGAUfCfA


GACfAGCfAdTs







AAGUfdTsdT


dT



 


HAMP
AD-11441
285
A-18278.3
uGcuGucAucGA
384
A-18279.2
ACUUUGAUCGAU
614






ucAAAGudTsdT


GAcAGcAdTsdT



 


HAMP
AD-47071
286
A-98418.1
GCfUfGUfCfAU
385
A-98419.1
AACUUUGAUCGA
615






fCfGAUfCfAAA


UGACfAGCdTsd







GUfUfdTsdT


T



 


HAMP
AD-47065
286
A-98416.1
GCfUfGUfCfAU
386
A-98417.1
CfACUUUGAUCG
616






fCfGAUfCfAAA


AUGACfAGCdTs







GUfAdTsdT


dT



 


HAMP
AD-45758
286
A-95742.1
GcuGucAcuGAc
385
A-95743.1
AACUUUGAUCGA
615






uAAAGuudTsdT


UGAcAGCdTsdT



 


HAMP
AD-45753
286
A-95740.1
GcuGucAucGAu
386
A-95741.1
uACUUUGAUCGA
617






cAAAGuAdTsdT


UGAcAGCdTsdT



 


HAMP
AD-29968
286
A-18288.1
GcuGucAucGAu
387
A-18289.1
cACUUUGAUCGA
616






cAAAGuGdTsdT


UGAcAGCdTsdT



 


HAMP
AD-47077
287
A-98420.1
CfUfGUfCfAUf
388
A-98421.1
ACfACUUUGAUC
618






CfGAUfCfAAAG


GAUGACfAGdTs







UfGUfdTsdT


dT



 


HAMP
AD-29969
287
A-18290.3
cuGucAucGAuc
388
A-18291.1
AcACUUUGAUCG
618






AAAgUgUdTsdT


AUGAcAGdTsdT



 


HAMP
AD-48208
288
A-100241.2
uGucAucGAucA
389
A-100243.1
AACACUUUgAuC
619






AAGuGuudTsdT


gAuGaCadTsdT



 


HAMP
AD-47042
288
A-98424.1
UfGUfCfAUfCf
389
A-98425.1
AACfACUUUGAU
619






FAUfCfAAAGUf


CGAUGACfAdTs







GUfUfdTsdT


dT



 


HAMP
AD-48202
288
A-100241.1
uGucAcuGAucA
389
A-100242.1
AACACuuUGauC
619






AAGuGuudTsdT


GAuGacadTsdT



 


HAMP
AD-47036
288
A-98422.1
UfGUfCfAUfCf
390
A-98423.1
CfACfACUUUGA
620






GAUfCfAAAGUf


UCGAUGACfAdT







GUfAdTsdT


sdT



 


HAMP
AD-45729
288
A-95746.1
uGucAucGAucA
389
A-95747.1
AAcACUUUGAUC
619






AAGuGuudTsdT


GAUGAcAdTsdT



 


HAMP
AD-45723
288
A-95744.1
uGucAucGAucA
390
A-95745.1
uAcACUUUGAUC
621






AAGuGuAdTsdT


GAUGAcAdTsdT



 


HAMP
AD-29970
288
A-66881.1
uGucAucGAucA
391
A-66882.1
cAcACUUUGAUC
620






AAGuGuGdTsdT


GAUGAcAdTsdT



 


HAMP
AD-47048
290
A-98426.1
UfCfAUfCfGAU
392
A-98427.1
UUCfACfACUUU
622






fCfAAAGUfGUf


GAUCGAUGAdTs







GGAdTsdT


dT



 


HAMP
AD-47054
290
A-98428.1
UfCfAUfCfGAU
393
A-98429.1
ACCfACfACUUU
623






fCfAAAGUfGUf


GAUCGAUGAdTs







GGUfdTsdT


dT



 


HAMP
AD-45744
290
A-95752.1
ucAucGAucAAA
393
A-95753.1
ACcAcACUUUGA
623






GuGuGGudTsdT


UCGAUGAdTsdT



 


HAMP
AD-45739
290
A-95750.1
ucAucGAucAAA
392
A-95751.1
UCcAcACUUUGA
622






GuGuGGAdTsdT


UCGAUGAdTsdT



 


HAMP
AD-45734
290
A-95748.1
ucAucGAucAAA
397
A-95749.1
CCcAcACUUUGA
624






GuGuGGGdTsdT


UCGAUGAdTsdT



 


HAMP
AD-47005
291
A-98342.1
CfAUfCfGAUfC
395
A-98343.1
UCCCfACfACUU
625






fAAAGUfGUfGG


UGAUCGAUGdTs







GAdTsdT


dT



 


HAMP
AD-11436
291
A-18268.1
cAucGAucAAAG
395
A-18269.1
UCCcAcACUUUG
625






uGuGGGAdTsdT


AUCGAUGdTsdT



 


HAMP
AD-11436
291
A-18268.1
cAucGAucAAAG
395
A-18269.1
UCCcAcACUUUG
625






uGuGGGAdTsdT


AUCGAUGdTsdT



 


HAMP
AD-29971
291
A-18268.1
cAucGAucAAAG
395
A-18269.1
UCCcAcACUUUG
625






uGuGGGAdTsdT


AUCGAUGdTsdT



 


HAMP
AD-45376
292
A-94771.1
AucGAucAAAGu
396
A-94772.1
AGAUCGAUUCCc
626






GuGGGAudTsdT


AcACUUUdTsdT



 


HAMP
AD-45382
293
A-94773.1
ucGAucAAAGuG
397
A-94774.1
cAUCCcAcACUU
627






uGGGAuGdTsdT


UGAUCGAdTsdT



 


HAMP
AD-29972
294
A-66883.1
cGAucAAAGuGu
398
A-66884.1
AcAUCCcAcACU
628






GGGAuGudTsdT


UUGAUCGdTsdT



 


HAMP
AD-47066
295
A-98432.1
GAUfCfAAAGUf
399
A-98433.1
AACfAUCCCfAC
629






GUfGGGAUfFUf


fACUUUGAUdTs







UfdTsdT


dT



 


HAMP
AD-47060
295
A-98430.1
GAUfCfAAAGUf
400
A-98431.1
CfACfAUCCCfA
630






GUfGGGAUfGUf


CfACUUUGAUCd







AdTsdT


TsdT



 


HAMP
AD-45754
295
A-95756.1
GAucAAAGuGuG
399
A-95757.1
AAcAUCCcAcAC
629






GGAuGuudTsdT


UUUGAUCdTsdT



 


HAMP
AD-45749
295
A-95754.1
GAucAAAGuGuG
400
A-95755.1
uAcAUCCcAcAC
631






GGAuGuAdTsdT


UUUGAUCdTsdT



 


HAMP
AD-29973
295
A-66885.1
GAucAAAGuGuG
401
A-66886.1
cAcAUCCcAcAC
630






GGAuGuGdTsdT


UUUGAUCdTsdT



 


HAMP
AD-45730
296
A-95762.1
AucAAAGuGuGG
402
A-95763.1
AcAcAUCCcAcA
632






GAuGuGudTsdT


CUUUGAUdTsdT



 


HAMP
AD-45724
296
A-95760.1
AucAAAGuGuGG
403
A-95761.1
UcAcAUCCcAcA
633






GAuGuGAdTsdT


CUUUGAUdTsdT



 


HAMP
AD-45759
296
A-95758.1
AucAAAGuGuGG
404
A-95759.1
GcAcAUCCcAcA
634






GAuGuGcdTsdT


CUUUGAUdTsdT



 


HAMP
AD-45110
297
A-94194.1
ucAAAGuGuGGG
405
A-94195.1
AGcAcAUCCcAc
635






AuGuGcudTsdT


ACUUUGAdTsdT



 


HAMP
AD-45387
298
A-94775.1
cAAAGuGuGGGA
406
A-94776.1
cAGcAcAUCCcA
636






uGuGcuGdTsdT


cACUUUGdTsdT



 


HAMP
AD-47072
299
A-98434.1
AAAGUfGUfGGG
407
A-98435.1
ACfAGCfACfAU
637






AUfGUfGCfUfG


CCCfACfACUUU







UfdTsdT


dTsdT



 


HAMP
AD-45740
299
A-95766.1
AAAGuGuGGGAu
408
A-95767.1
UcAGcAcAUCCc
638






GuGcuGAdTsdT


AcACUUUdTsdT



 


HAMP
AD-45745
299
A-95768.1
AAAGuGuGGGAu
407
A-95769.1
AcAGcAcAUCCc
637






GuGcuGudTsdT


AcACUUUdTsdT



 


HAMP
AD-45735
299
A-95764.1
AAAGuGuGGGAu
409
A-95765.1
GcAGcAcAUCCc
639






GuGcuGcdTsdT


dAcACUUUTsdT



 


HAMP
AD-29974
300
A-66887.1
AAGuGuGGGAuG
410
A-66888.1
UGcAGcAcAUCC
640






uGcuGcAdTsdT


cAcACUUdTsdT



 


HAMP
AD-29975
301
A-66889.1
AGuGuGGGAuGu
411
A-66890.1
UUGcAGcAcAUC
641






GcuGcAAdTsdT


CcAcACUdTsdT



 


HAMP
AD-45116
306
A-94196.1
GGGAuGuGcuGc
412
A-94197.1
ACGUCUUGcAGc
642






AAGAcGudTsdT


AcAUCCCdTsdT



 


HAMP
AD-46988
307
A-98258.1
GGAUfGUfGCfU
413
A-98259.1
CfACGUCUUGCf
643






fGCfAAGACfGU


AGCfACfAUCCd







fAdTsdT


TsdT



 


HAMP
AD-45075
307
A-94198.1
GGAuGuGcuGcA
413
A-94199.1
uACGUCUUGcAG
644






AGAcGuAdTsdT


cAcAUCCdTsdT



 


HAMP
AD-46994
309
A-98260.1
AUfGUfGCfUfG
414
A-98261.1
UCCfACGUCUUG
645






CfAAGACfGUfA


CfAGCfACfAUd







GAdTsdT


TsdT



 


HAMP
AD-45081
309
A-94200.1
AuGuGcuGcAAG
414
A-94201.1
UCuACGUCUUGc
646






AcGuAGAdTsdT


AGcAcAUdTsdT



 


HAMP
AD-47000
310
A-98262.1
UfGUfGCfUfGC
415
A-98263.1
UUCCfACGUCUU
647






fAAGACfGUfAG


GCfAGCfACfAd







AAdTsdT


TsdT



 


HAMP
AD-45087
310
A-94202.1
uGuGcuGcAAGA
415
A-94203.1
UUCuACGUCUUG
648






cGuAGAAdTsdT


cAGcAcAdTsdT



 


HAMP
AD-47006
313
A-98264.1
GCfUfGCfAAGA
416
A-98265.1
AGGUUCCfACGU
649






CfGUfAGAACfC


CUUGCfAGCdTs







fUfdTsdT


dT



 


HAMP
AD-45093
313
A-94204.1
GcuGcAAGAcGu
416
A-94205.1
AGGUUCuACGUC
650






AGAAccudTsdT


UUGcAGCdTsdT



 


HAMP
AD-47011
314
A-98266.1
CfUfGCfAAGAC
417
A-98267.1
CfAGGUUCCfAC
651






fGUfAGAACfCf


GUCUUGCfAGdT







UfAdTsdT


sdT



 


HAMP
AD-47016
322
A-98268.1
CfGUfAGAACfC
418
A-98269.1
AGGGCfAAGCfA
652






fUfACfCfUfGC


GGUUCCfACGdT







fCfCfUfdTsdT


sdT



 


HAMP
AD-45099
322
A-94206.1
cGuAGAAccuAc
418
A-94207.1
AGGGcAGGuAGG
653






cuGcccudTsdT


UUCuACGdTsdT



 


HAMP
AD-47021
347
A-98270.1
GUfCfCfCfCfU
419
A-98271.1
AACfAAGGAAGG
654






fCfCfCfUfUfC


GAGGGGACdTsd







fCfUfUfAUfUf


T







dTsdT






 


HAMP
AD-47026
348
A-98272.1
UfCfCfCfCfUf
420
A-98273.1
AAACfAAGGAAG
655






CfCfCfUfUfCf


GGAGGGGAdTsd







CfUfUfAUfUfd


T







TsdT






 


HAMP
AD-46989
349
A-98274.1
CfCfCfCfUfCf
421
A-98275.1
CfAAACfAAGGA
656






CfCfUfUFCfCf


AGGGAGGGGdTs







UfUfAUfUfUfA


dT







dTsdT






 


HAMP
AD-46995
350
A-98276.1
CfCfCfUfCfCf
422
A-98277.1
ACfAAACfAAGG
657






CfUfUfCfCfUf


AAGGGAGGGdTs







UfAUfUfUfAUf


dT







dTsdT






 


HAMP
AD-47001
351
A-98278.1
CfCfUfCfCfCf
423
A-98279.1
AACfAAACfAAG
658






UfUfCfCfUfUf


GAAGGGAGGdTs







AUfUfUfAUfUf


dT







dTsdT






 


HAMP
AD-47012
352
A-98282.1
CfUfCfCfCfUf
424
A-98283.1
AAACfAAACfAA
659






UfCfCfUfUfAU


GGAAGGGAGdTs







fUfUfAUfUfUf


dT







dTsdT






 


HAMP
AD-47007
352
A-98280.1
CfUfCfCfCfUf
425
A-98281.1
CfAACfAAACfA
660






UfCfCfUfUfAU


AGGAAGGGAGdT







fUfUfAUfUfAd


sdT







TsdT






 


HAMP
AD-47017
354
A-98284.1
CfCfCfUfUfCf
426
A-98285.1
AGGAACfAAACf
661






CfUfUfAUfUfU


AAGGAAGGGdTs







fAUfUfCfCfUf


dT







dTsdT






 


HAMP
AD-47022
355
A-98286.1
CfCfUfUfCfCf
427
A-98287.1
CfAGGAACfAAA
662






UfUfAUfUfUfA


CfAAGGAAGGdT







UfUfCfCfUfAd


sdT







TsdT






 


HAMP
AD-47027
355
A-98288.1
CfCfUfUfCfCf
428
A-98289.1
AAGGAACfAAAC
663






UfUfAUfUfUfA


fAAGGAAGGdTs







UfUfCfCfUfUf


dT







dTsdT






 


HAMP
AD-46996
356
A-98292.1
CfUfUfCfCfUf
429
A-98293.1
ACfAGGAACfAA
664






UfAUfUfUfAUf


ACfAAGGAAGdT







UfCfCfUfGUfd


sdT







TsdT






 


HAMP
AD-46990
356
A-98290.1
CfUfUfCfCfUf
430
A-98291.1
UCfAGGAACfAA
665






UfAUfUfUfAUf


ACfAAGGAAGdT







UfCfCfUfGAdT


sdT







sdT






 


HAMP
AD-47002
357
A-98294.1
UfUfCfCfUfUf
431
A-98295.1
AGCfAGGAACfA
666






AUfUfUfAUfUf


AACfAAGGAAdT







CfCfUfGCfUfd


sdT







TsdT






 


HAMP
AD-47013
358
A-98298.1
UfCfCfUfUfAU
432
A-98299.1
AAGCfAGGAACf
667






fUfUfAUfUfCf


AAACfAAGGAdT







CfUfGCfUfUfd


sdT







TsdT






 


HAMP
AD-47008
358
A-98296.1
UfCfCfUfUfAU
433
A-98297.1
CfAGCfAGGAAC
668






fUfUfAUfUfCf


fAAACfAAGGAd







CfUfGCfUfAdT


TsdT







sdT






 


HAMP
AD-47023
359
A-98302.1
CfCfUfUfAUfU
434
A-98303.1
ACfAGCfAGGAA
669






fUfAUfUfCfCf


CfAAACfAAGGd







UfGCfUfGUfdT


TsdT







sdT






 


HAMP
AD-47018
359
A-98300.1
CfCfUfUfAUfU
435
A-98301.1
UCfAGCfAGGAA
670






fUfAUfUUCfCf


CfAAACfAAGGd







UfGCfUfGAdTs


TsdT







dT






 


HAMP
AD-47028
363
A-98304.1
AUfUfUfAUfUf
436
A-98305.1
UGGGGCfAGCfA
671






CfCfCfGCfUfG


GGAACfAAAUdT







CfCfCfCfAdTs


sdT







dT






 


HAMP
AD-46991
365
A-98306.1
UfUfAUfUfCfC
437
A-98307.1
UCUGGGGCfAGC
672






fUfGCfUfGCfC


fAGGAACfAAdT







fCfCfAGAdTsd


sdT







T






 


HAMP
AD-46997
366
A-98308.1
UfAUfUfCfCfU
438
A-98309.1
UUCUGGGGCfAG
673






fGCfUfGCfCfC


CfAGGAACfAdT







fCfAGAAdTsdT


sdT



 


HAMP
AD-47003
369
A-98310.1
UfCfCfUfGCfU
439
A-98311.1
AUGUUCUGGGGC
674






fGCfCfCfCfAG


fAGCfAGGAdTs







AACfAUfdTsdT


dT



 


HAMP
AD-45105
369
A-94208.1
uccuGcuGcccc
439
A-94209.1
AUGUUCUGGGGc
674






AGAAcAudTsdT


AGcAGGAdTsdT



 


HAMP
AD-47009
370
A-98312.1
CfCfUfGCfUfG
440
A-98313.1
CfAUGUUCUGGG
675






CfCfCfCfAGAA


GCfAGCfAGGdT







CfAUfAdTsdT


sdT



 


HAMP
AD-45111
370
A-94210.1
ccuGcuGccccA
440
A-94211.1
uAUGUUCUGGGG
676






GAAcAuAdTsdT


cAGcAGGdTsdT



 


HAMP
AD-47014
373
A-98314.1
GCfUfGCfCfCf
441
A-98315.1
ACCCfAUGUUCU
677






CfAGAACfAUfA


GGGGCfAGCdTs







GGUfdTsdT


dT



 


HAMP
AD-45117
373
A-94212.1
GcuGccccAGAA
441
A-94213.1
ACCuAUGUUCUG
678






cAuAGGudTsdT


GGGcAGCdTsdT



 


HAMP
AD-47019
375
A-98316.1
UfGCfCfCfCfA
442
A-98317.1
AGACCCfAUGUU
679






GAACfAUfAGGU


CUGGGGCfAdTs







fCfUfdTsdT


dT



 


HAMP
AD-45076
375
A-94214.1
uGccccAGAAcA
442
A-94215.1
AGACCuAUGUUC
680






uAGGucudTsdT


UGGGGcAdTsdT



 


HAMP
AD-48214
376
A-100244.1
GccccAGAAcAu
443
A-100245.1
AAGACCuAUGUU
681






AGGucuudTdT


CUGGGGCdTdT



 


HAMP
AD-48219
376
A-100246.1
GcCCCAGAAcAu
443
A-100247.1
AAGACCuaUGuu
681






AGGucuudTdT


CUGGGGcdTdT



 


HAMP
AD-47024
376
A-98318.1
GCfCfCfCfAGA
443
A-98319.1
AAGACCCfAUGU
682






ACfAUfAGGUfC


UCUGGGGCdTsd







fUfUfdTsdT


T



 


HAMP
AD-45082
376
A-94216.1
GccccAGAAcAu
443
A-94217.1
AAGACCuAUGUU
681






AGGucuudTsdT


CUGGGGCdTsdT



 


HAMP
AD-48224
379
A-100248.1
ccAGAAcAuAGG
444
A-100249.1
UCcAAGACCuAU
683






ucuuGGAdTdT


GUUCUCGdTdT



 


HAMP
AD-48187
379
A-100248.2
ccAGAAcAuAGG
444
A-100250.1
uCCAAGACCuAU
684






ucuuGGAdTdT


GuUCuggdTdT



 


HAMP
AD-47029
379
A-98320.1
CfCfAGAACfAU
444
A-98321.1
UCCfAAGACCCf
685






fAGGUfCfUfUf


AUGUUCUGGdTs







GGAdTsdT


dT



 


HAMP
AD-48192
379
A-100248.3
ccAGAAcAuAGG
444
A-100251.1
uCCAAGACCUaU
684






ucuuGGAdTdT


gUuCuGgdTdT



 


HAMP
AD-45088
379
A-94218.1
ccAGAAcAuAGG
444
A-94219.1
UCcAAGACCuAU
684






ucuuGGAdTsdT


GUUCUGGdTsdT



 


HAMP
AD-46992
380
A-98322.1
CfAGAACfAUfA
445
A-98323.1
UUCCfAAGACCC
686






GGUfCfUfUfGG


fAUGUUCUGdTs







AAdTsdT


dT



 


HAMP
AD-45094
380
A-94220.1
cAGAAcAuAGGu
445
A-94221.1
UUCcAAGACCuA
687






cuuGGAAdTsdT


UGUUCUGdTsdT



 


HAMP
AD-46998
381
A-98324.1
AGAACfAUfAGG
446
A-98325.1
AUUCCfAAGACC
688






UfCfUfUfGGAA


CfAUGUUCUdTs







UfdTsdT


dT



 


HAMP
AD-45100
381
A-94222.1
AGAAcAuAGGuc
446
A-94223.1
AUUCcAAGACCu
689






uuGGAAudTsdT


AUGUUCUdTsdT



 


HAMP
AD-48137
382
A-100195.1
GAAcAuAGGUCU
 30
A-98136.7
UAUUCCAAGACC
 44






UGGAAUAdTdT


UAUGUUCdTdT



 


HAMP
AD-48196
382
A-100179.22
GAAcAuAGGucu
 30
A-100228.1
UAuUCCAAGaCC
 44






uGGAAuAdTdT


uAuGuucdTdT



 


HAMP
AD-48195
382
A-98135.8
GAACAUAGGUCU
 30
A-100218.1
UAUUcCaAgAcC
 44






UGGAAUAdTdT


uAuGuUcdTdT



 


HAMP
AD-48201
382
A-100179.23
GAAcAuAGGucu
 30
A-100229.1
UAUUCCAAGaCC
 44






uGGAAuAdTdT


uAuGuucdTdT



 


HAMP
AD-48207
382
A-100179.24
GAAcAuAGGucu
 30
A-100230.1
UAUUCCAAgAcC
 44






uGGAAuAdTdT


uAuGuucdTdT



 


HAMP
AD-48159
382
A-100179.5
GAAcAuAGGucu
 30
A-100184.1
UAUUCCAAGACC
 44






uGGAAuAdTdT


UAuGuUCdTdT



 


HAMP
AD-48147
382
A-100179.3
GAAcAuAGGucu
 30
A-100182.1
UAUUCcAAGACC
 44






uGGAAuAdTdT


uAuGuUCdTdT



 


HAMP
AD-48161
382
A-98135.4
GAACAUAGGUCU
 30
A-100188.2
UAUUCCAAGACC
 44






UGGAAUAdTdT


UAuGuUcdTdT



 


HAMP
AD-48172
382
A-100193.1
GAAcAuAGGucU
 30
A-98136.5
UAUUCCAAGACC
 44






UGGAAUAdTdT


UAUGUUCdTdT



 


HAMP
AD-48156
382
A-100194.4
GAAcAuAGGuCU
 30
A-100188.3
UAUUCCAAGACC
 44






UGGAAUAdTdT


UAuGuUcdTdT



 


HAMP
AD-48195
382
A-98135.8
GAACAUAGGUCU
 30
A-100218.1
UAUUcCaAgAcC
 44






UGGAAUAdTdT


uAuGuUcdTdT



 


HAMP
AD-48136
382
A-100179.9
GAAcAuAGGucu
 30
A-100187.1
UAUUCCAAGACC
 44






uGGAAuAdTdT


UAUGuUcdTdT



 


HAMP
AD-48166
382
A-100192.1
GAAcAuAGGucu
 30
A-98136.1
UAUUCCAAGACC
 44






UGGAAUAdTdT


UAUGUUCdTdT



 


HAMP
AD-48213
382
A-100179.25
GAAcAuAGGucu
 30
A-100231.1
UAuUCCAAgAcC
 44






uGGAAuAdTdT


uAuGuucdTdT



 


HAMP
AD-48173
382
A-98135.6
GAACAUAGGUCU
 30
A-100190.2
UAUUCCAAGACc
 44






UGGAAUAdTdT


uAuGuUcdTdT



 


HAMP
AD-48154
382
A-100179.12
GAAcAuAGGucu
 30
A-100190.1
UAUUCCAAGACc
 44






uGGAAuAdTdT


uAuGuUcdTdT



 


HAMP
AD-48141
382
A-100179.2
GAAcAuAGGuCU
 30
A-100181.1
UAuUCcAAGACC
 44






UGGAAUAdTdT


uAuGuUCdTdT



 


HAMP
AD-48216
382
A-100217.3
GAAcAuAGGuCU
 30
A-100215.3
UAUUCCAAGACc
 44






UGGAAUAdTsdT


uAuGuUcdTsdT



 


HAMP
AD-48180
382
A-100194.8
GAAcAuAGGuCU
 30
A-100183.2
UAUUCCAAGACC
 44






UGGAAUAdTdT


uAuGuUCdTdT



 


HAMP
AD-48143
382
A-100196.1
GAAcAUAGGUCU
 30
A-98136.8
UAUUCCAAGACC
 44






UGGAAUAdTdT


UAUGUUCdTdT



 


HAMP
AD-48142
382
A-100179.10
GAAcAuAGGucu
 30
A-100188.1
UAUUCCAAGACC
 44






duGGAAuATdT


UAuGuUcdTdT



 


HAMP
AD-48221
382
A-18280.13
GAAcAuAGGucu
 30
A-15168.3
UAUUCCAAGACC
 44






uGGAAuAdTsdT


UAUGUUCdTsdT



 


HAMP
AD-48171
382
A-100179.7
GAAcAUAGGucu
 30
A-98136.2
UAUUCCAAGACC
 44






uGGAAuAdTdT


UAUGUUCdTdT



 


HAMP
AD-48145
382
A-100195.4
GAAcAuAGGUCU
 30
A-100183.3
UAUUCCAAGACC
 44






UGGAAUAdTdT


uAuGuUCdTdT



 


HAMP
AD-48160
382
A-100191.1
GAAcAuAGGucu
 30
A-98136.3
UAUUCCAAGACC
 44






uGGAAUAdTdT


UAUGUUCdTdT



 


HAMP
AD-48144
382
A-100194.2
GAAcAuAGGuCU
 30
A-100190.5
UAUUCCAAGACc
 44






UGGAAUAdTdT


uAuGuUcdTdT



 


HAMP
AD-48167
382
A-98135.5
GAACAUAGGUCU
 30
A-100189.2
UAUUCCAAGACC
 44






UGGAAUAdTdT


uAuGuUcdTdT



 


HAMP
AD-48177
382
A-100179.8
GAAcAuAGGucu
 30
A-100186.1
UAUUCCAAGACC
 44






uGGAAuAdTdT


UAUGUUcdTdT



 


HAMP
AD-48153
382
A-100179.4
GAAcAuAGGucu
 30
A-100183.1
UAUUCCAAGACC
 44






uGGAAuAdTdT


uAuGuUCdTdT



 


HAMP
AD-48178
382
A-100194.1
GAAcAuAGGuCU
 30
A-98136.6
UAUUCCAAGACC
 44






UGGAAUAdTdT


UAUGUUCdTdT



 


HAMP
AD-48155
382
A-98135.3
GAACAUAGGUCU
 30
A-100187.2
UAUUCCAAGACC
 44






UGGAAUAdTdT


UAUGuUcdTdT



 


HAMP
AD-48174
382
A-100194.7
GAAcAuAGGuCU
 30
A-100197.1
UAUUCCAAGACc
 44






UGGAAUAdTdT


uAuGuUCdTdT



 


HAMP
AD-48205
382
A-15167.2
GAACAUAGGUCU
 30
A-100215.2
UAUUCCAAGACc
 44






UGGAAUAdTsdT


uAuGuUcdTsdT



 


HAMP
AD-48179
382
A-100196.2
GAAcAUAGGUCU
 30
A-100190.3
UAUUCCAAGACc
 44






UGGAAUAdTdT


uAuGuUcdTdT



 


HAMP
AD-48168
382
A-100194.6
GAAcAuAGGuCU
 30
A-100186.3
UAUUCCAAGACC
 44






UGGAAUAdTdT


UAUGUUcdTdT



 


HAMP
AD-48149
382
A-98135.2
GAACAUAGGUCU
 30
A-100186.2
UAUUCCAAGACC
 44






UGGAAUAdTdT


UAUGUUcdTdT



 


HAMP
AD-48211
382
A-100217.2
GAAcAuAGGuCU
 30
A-100214.2
UAUUCCAAGACC
 44






UGGAAUAdTsdT


uAuGuUCdTsdT



 


HAMP
AD-48200
382
A-100217.1
GAAcAuAGGuCU
 30
A-15168.2
UAUUCCAAGACC
 44






UGGAAUAdTsdT


UAUGUUCdTsdT



 


HAMP
AD-48188
382
A-100179.20
GAAcAuAGGucu
 30
A-100205.1
UAuUCcAAGACC
 44






uGGAAuAdTdT


uAuGuUcdTdT



 


HAMP
AD-48183
382
A-18280.10
GAAcAuAGGucu
 30
A-100214.1
UAUUCCAAGACC
 44






uGGAAuAdTsdT


uAuGuUCdTsdT



 


HAMP
AD-48150
382
A-100194.3
GAAcAuAGGuCU
 30
A-100189.3
UAUUCCAAGACC
 44






UGGAAUAdTdT


uAuGuUcdTdT



 


HAMP
AD-48162
382
A-100194.5
GAAcAuAGGuCU
 30
A-100187.3
UAUUCCAAGACC
 44






UGGAAUAdTdT


UAUGuUcdTdT



 


HAMP
AD-48139
382
A-100195.3
GAAcAuAGGUCU
 30
A-100197.2
UAUUCCAAGACc
 44






UGGAAUAdTdT


uAuGuUCdTdT



 


HAMP
AD-9940
382
A-15167.1
GAACAUAGGUCU
 30
A-15168.2
UAUUCCAAGACC
 44






UGGAAUAdTsdT


UAUGUUCdTsdT



 


HAMP
AD-48138
382
A-100195.2
GAAcAuAGGUCU
 30
A-100190.4
UAUUCCAAGACc
 44






UGGAAUAdTdT


uAuGuUcdTdT



 


HAMP
AD-11459
382
A-18280.2
GAAcAuAGGucu
 30
A-18304.1
uAuUCcAAGACC
 44






uGGAAuAdTsdT


uAuGuUCdTsdT



 


HAMP
AD-48189
382
A-18280.11
GAAcAuAGGucu
 30
A-100215.1
UAUUCCAAGACc
 44






uGGAAuAdTsdT


uAuGuUcdTsdT



 


HAMP
AD-48148
382
A-100179.11
GAAcAuAGGucu
 30
A-100189.1
UAUUCCAAGACC
 44






uGGAAuAdTdT


uAuGuUcdTdT



 


HAMP
AD-48215
382
A-18280.8
GAAcAuAGGucu
 30
A-100212.1
UAuUCcAAGACC
 44






uGGAAuAdTsdT


uAuGuUCdTsdT



 


HAMP
AD-48218
382
A-100179.26
GAAcAuAGGucu
 30
A-100232.1
uAuUCCAAgAcC
 44






uGGAAuAdTdT


uAuGuucdTdT



 


HAMP
AD-48135
382
A-100179.1
GAAcAuAGGucu
 30
A-100180.1
uAuUCcAAGACC
 44






uGGAAuAdTdT


uAuGuUCdTdT



 


HAMP
AD-47004
382
A-98326.1
GAACfAUfAGGU
 30
A-98327.1
CfAUUCCfAAGA
690






fCfUfUfGGAAU


CCCfAUGUUCdT







fAdTsdT


sdT



 


HAMP
AD-48194
382
A-18280.12
GAAcAuAGGucu
 30
A-100216.1
uAUUCCAAGACC
 44






uGGAAuAdTsdT


uAuGuUCdTsdT



 


HAMP
AD-48197
382
A-100239.1
GAAcAuAGGuCd
447
A-100240.1
dTAdTUdCCdAA
691






TUdGGdAAdTAd


dGACCuAuGuuc







TdT


dTdT



 


HAMP
AD-11459
382
A-18280.2
GAAcAuAGGucu
 30
A-18304.1
uAuUCcAAGACC
 44






uGGAAuAdTsdT


uAuGuUCdTsdT



 


HAMP
AD-48164
382
A-100179.16
GAAcAuAGGucu
 30
A-100201.1
uAUUCcAAGACC
 44






uGGAAuAdTdT


uAuGuUCdTdT



 


HAMP
AD-48158
382
A-100179.15
GAAcAuAGGucu
 30
A-100200.1
uAUUCCAAGACC
 44






uGGAAuAdTdT


uAuGuUCdTdT



 


HAMP
AD-48204
382
A-100208.1
GAAcAcAGGucu
448
A-100209.1
uAuUCcAAGACC
692






uGGAAuAdTdT


uGuGuUCdTdT



 


HAMP
AD-48181
382
A-100192.2
GAAcAuAGGucu
 30
A-100180.7
uAuUCcAAGACC
 44






UGGAAUAdTdT


uAuGuUCdTdT



 


HAMP
AD-48223
382
A-100233.1
GAAcAuAGGucu
449
A-100234.1
uAuUCcAAGACC
706






uGGAAuAuu


uAuGuUCuu



 


HAMP
AD-48190
382
A-100179.21
GAAcAuAGGucu
 30
A-100227.1
uAuUCCAAGaCC
 44






uGGAAuAdTdT


uAuGuucdTdT



 


HAMP
AD-48163
382
A-100195.5
GAAcAuAGGUCU
 30
A-100180.4
uAuUCcAAGACC
 44






UGGAAUAdTdT


uAuGuUCdTdT



 


HAMP
AD-48140
382
A-100191.2
GAAcAuAGGucu
 30
A-100180.8
uAuUCcAAGACC
 44






uGGAAUAdTdT


uAuGuUCdTdT



 


HAMP
AD-48169
382
A-100194.9
GAAcAuAGGuCU
 30
A-100180.5
uAuUCcAAGACC
 44






UGGAAUAdTdT


uAuGuUCdTdT



 


HAMP
AD-48220
382
A-18280.9
GAAcAuAGGucu
 30
A-100213.1
uAuUCcAAGACC
 44






uGGAAuAdTsdT


uAuGuUcdTsdT



 


HAMP
AD-48184
382
A-15167.3
GAACAUAGGUCU
 30
A-18304.6
uAuUCcAAGACC
 44






UGGAAUAdTsdT


uAuGuUCdTsdT



 


HAMP
AD-48176
382
A-100179.18
GAAcAuAGGucu
 30
A-100203.1
uAUUCCAAGACC
 44






uGGAAuAdTdT


uAuGuUcdTdT



 


HAMP
AD-48175
382
A-100193.2
GAAcAuAGGucU
 30
A-100180.6
uAuUCcAAGACC
 44






UGGAAUAdTdT


uAuGuUCdTdT



 


HAMP
AD-48146
382
A-100179.13
GAAcAuAGGucu
 30
A-100198.1
uAUUCCAAGACC
 44






uGGAAuAdTdT


UAUGUUcdTdT



 


HAMP
AD-48182
382
A-100179.19
GAAcAuAGGucu
 30
A-100204.1
uAuUCcAAGACC
 44






uGGAAuAdTdT


uAuGuUcdTdT



 


HAMP
AD-48199
382
A-100207.1
GAAcAuAGGUCU
 30
A-100180.10
uAuUCcAAGACC
 44






UGGAAuAdTdT


uAuGuUCdTdT



 


HAMP
AD-48157
382
A-100196.3
GAAcAUAGGUCU
 30
A-100180.3
uAuUCcAGACCu
 44






UGGAAUAdTdT


AuGuUCdTdT



 


HAMP
AD-48206
382
A-100219.1
GAAcAcAGGucu
448
A-100220.1
uAuUCcAAGACC
692






uGGAAuAdTsdT


uGuGuUCdTsdT



 


HAMP
AD-48193
382
A-100206.1
GAAcAuAGGuCu
 30
A-100180.9
uAuUCcAAGACC
 44






uGGAAuAdTdT


uAuGuUCdTdT



 


HAMP
AD-48152
382
A-100179.14
GAAcAuAGGucu
 30
A-100199.1
uAUUCCAAGACC
 44






uGGAAuAdTdT


UAUGUUCdTdT



 


HAMP
AD-48151
382
A-98135.7
GAACAUAGGUCU
 30
A-100180.2
uAuUCcAAGACC
 44






UGGAAUAdTdT


uAuGuUCdTdT



 


HAMP
AD-48170
382
A-100179.17
GAAcAuAGGucu
 30
A-100202.1
uAuUCUAAGACC
693






uGGAAuAdTdT


uAuGuUCdTdT



 


HAMP
AD-47010
383
A-98328.1
AACfAUfAGGUf
450
A-98329.1
UCfAUUCCfAAG
694






CfUfUfGGAAUf


ACCCfAUGUUdT







AAdTdT


dT



 


HAMP
AD-45106
383
A-94224.1
AAcAuAGGucuu
450
A-94225.1
UuAUUCcAAGAC
695






GGAAuAAdTsdT


CuAUGUUdTsdT



 


HAMP
AD-48222
385
A-100224.1
AAcAuAGGucuu
451
A-100225.1
UuAUUCcAAGAC
696






GGAAuAAdTsdT


CuAuGUUdTsdT



 


HAMP
AD-48217
385
A-100221.2
cAuAGGucuuGG
451
A-100223.1
UUUuAUUCcAAG
696






AAuAAAAdTdT


ACCuAUdTdT



 


HAMP
AD-48185
385
A-100221.3
cAuAGGucuuGG
451
A-100226.1
uUUUAuuCCaaG
696






AAuAAAAdTdT


ACCUaugdTdT



 


HAMP
AD-48212
385
A-100221.1
cAuAGGucuuGG
451
A-100222.1
UuUuAuUCcAAG
696






AAuAAAAdTdT


ACCuAuGdTdT



 


HAMP
AD-48198
396
A-100252.1
GAAuAAAAuGGc
452
A-100253.1
AGAACcAGCcAU
697






uGGuucudTdT


UUuAUUCdTdT



 


HAMP
AD-48209
396
A-100252.3
GAAuAAAAuGGc
452
A-100255.1
AGAACCAGcCaU
697






uGGuucudTdT


uUuAuUcdTdT



 


HAMP
AD-48203
396
A-100252.2
GAAuAAAAuGGc
452
A-100254.1
AGAACcAGCCAu
697






uGGuucudTdT


uUUAuucdTdT



 


HAMP
AD-47015
396
A-98330.1
GAAUfAAAAUfG
452
A-98331.1
AGAACCfAGCCf
698






GCfUfGGUfUfC


AUUUCfAUUCdT







fUfdTsdT


sdT



 


HAMP
AD-45112
396
A-94226.1
GAAuAAAAuGGc
452
A-94227.1
AGAACcAGCcAU
697






uGGuucudTsdT


UUuAUUCdTsdT



 


HAMP
AD-47020
398
A-98332.1
AUfAAAAUfGGC
453
A-98333.1
AAAGAACCfAGC
699






fUfGGUfUfCfU


CfAUUUCfAUdT







fUfUfdTsdT


sdT



 


HAMP
AD-45118
398
A-94228.1
AuAAAAuGGcuG
453
A-94229.1
AAAGAACcAGCc
700






GuucuuudTsdT


AUUUuAUdTsdT



 


HAMP
AD-47025
399
A-98334.1
UfAAAAUfGGCf
454
A-98335.1
AAAAGAACCfAG
701






UfGGUfUfCfUf


CCfAUUUCfAdT







UfUfUfdTsdT


sdT



 


HAMP
AD-45077
399
A-94230.1
uAAAAuGGcuGG
454
A-94231.1
AAAAGAACcAGC
702






uucuuuudTsdT


cAUUUuAdTsdT



 


HAMP
AD-47030
402
A-98336.1
AAUfGGCfUfGG
455
A-98337.1
AACfAAAAGAAC
703






UfUfCfUfUfUf


CfAGCCfAUUdT







UfGUfUfdTsdT


sdT



 


HAMP
AD-45083
402
A-94232.1
AAuGGcuGGuuc
455
A-94233.1
AAcAAAAGAACc
703






uuuuGuudTsdT


AGCcAUUdTsdT



 


HAMP
AD-46993
403
A-98338.1
AUfGGCfUfGGU
456
A-98339.1
AAACfAAAAGAA
704






fUfCfUfUfUfU


CCfAGCCfAUdT







fGUfUfUfdTsd


sdT







T






 


HAMP
AD-45089
403
A-94234.1
AuGGcuGGuucu
456
A-94235.1
AAAcAAAAGAAC
704






uuuGuuudTsdT


cAGCcAUdTsdT



 


HAMP
AD-46999
407
A-98340.1
CfUfGGUfUfCf
457
A-98341.1
UGGAAAACfAAA
705






UfUfUfUfGUfU


AGAACCfAGdTs







fUfUfCfCfAdT


dT







sdT





It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention.













TABLE 5







HAMP unmodified seqeunces

















Start
Antisense

SEQ ID
Sense

SEQ ID


Target
Duplex ID
Position
Name
Antisense Sequence
NO
Name
Sense Sequence
NO


















HAMP
AD-47121
62
A-98153.1
AGACGGCACGAUGG
250
A-98154.1
AAGUGCCAUCGU
 475






CACUUdTdT


GCCGUCUdTdT



 


HAMP
AD-47133
67
A-98157.1
GCACGAUGGCACUG
258
A-98158.1
AAGCUCAGUGCCA 
 483






AGCUUdTdT


UCGUGCdTdT



 


HAMP
AD-47127
67
A-98155.1
GCACGAUGGCACUG
259
A-98156.1
UAGCUCAGUGCC
 486






AGCUAdTdT


AUCGUGCdTdT



 


HAMP
AD-47145
74
A-98161.1
GGCACUGAGCUCCCA
271
A-98162.1
AAUCUGGGAGCU
 497






GAUUdTdT


CAGUGCCdTdT



 


HAMP
AD-47139
74
A-98159.1
GGCACUGAGCUCCCA
272
A-98160.1
UAUCUGGGAGCU
 500






GAUAdTdT


CAGUGCCdTdT



 


HAMP
AD-47157
76
A-98165.1
CACUGAGCUCCCAGA
275
A-98166.1
AAGAUCUGGGAG
 502






UCUUdTdT


CUCAGUGdTdT



 


HAMP
AD-47151
76
A-98163.1
CACUGAGCUCCCAGA
276
A-98164.1
UAGAUCUGGGAG
 504






UCUAdTdT


CUCAGUGdTdT



 


HAMP
AD-47163
132
A-98167.1
CUGACCAGUGGCUC
288
A-98168.1
AAACAGAGCCACU
 515






UGUUUdTdT


GGUCAGdTdT



 


HAMP
AD-47122
140
A-98169.1
UGGCUCUGUUUUCC
289
A-98170.1
UUGUGGGAAAAC
 516






CACAAdTdT


AGAGCCAdTdT



 


HAMP
AD-47128
146
A-98171.1
UGUUUUCCCACAAC
291
A-98172.1
UGUCUGUUGUGG
 518






AGACAdTdT


GAAAACAdTdT



 


HAMP
AD-47134
146
A-98173.1
UGUUUUCCCACAAC
292
A-98174.1
AGUCUGUUGUGG
 519






AGACUdTdT


GAAAACAdTdT



 


HAMP
AD-47140
155
A-98175.1
ACAACAGACGGGACA
302
A-98176.1
AAGUUGUCCCGU
 529






ACUUdTdT


CUGUUGUdTdT



 


HAMP
AD-47152
157
A-98179.1
AACAGACGGGACAAC
303
A-98180.1
ACAAGUUGUCCC
 530






UUGUdTdT


GUCUGUUdTdT



 


HAMP
AD-47146
157
A-98177.1
AACAGACGGGACAAC
304
A-98178.1
UCAAGUUGUCCC
 531






UUGAdTdT


GUCUGUUdTdT



 


HAMP
AD-47158
160
A-98181.1
AGACGGGACAACUU
307
A-98182.1
UCUGCAAGUUGU
 534






GCAGAdTdT


CCCGUCUdTdT



 


HAMP
AD-47164
161
A-98183.1
GACGGGACAACUUG
309
A-98184.1
UUCUGCAAGUUG
 536






CAGAAdTdT


UCCCGUCdTdT



 


HAMP
AD-47123
161
A-98185.1
GACGGGACAACUUG
308
A-98186.1
AUCUGCAAGUUG
 535






CAGAUdTdT


UCCCGUCdTdT



 


HAMP
AD-47135
162
A-98189.1
ACGGGACAACUUGC
312
A-98190.1
ACUCUGCAAGUU
 539






AGAGUdTdT


GUCCCGUdTdT



 


HAMP
AD-47129
162
A-98187.1
ACGGGACAACUUGC
311
A-98188.1
UCUCUGCAAGUU
 538






AGAGAdTdT


GUCCCGUdTdT



 


HAMP
AD-47141
242
A-98191.1
GAGGCGAGACACCCA
347
A-98192.1
UAAGUGGGUGUC
 576






CUUAdTdT


UCGCCUCdTdT



 


HAMP
AD-47147
242
A-98193.1
GAGGCGAGACACCCA
346
A-98194.1
AAAGUGGGUGUC
 573






CUUUdTdT


UCGCCUCdTdT



 


HAMP
AD-47153
253
A-98195.1
CCCACUUCCCCAUCU
356
A-98196.1
AUGCAGAUGGGG
 584






GCAUdTdT


AAGUGGGdTdT



 


HAMP
AD-47159
258
N-98197.1
UUCCCCAUCUGCAU
360
A-98198.1
AGAAAAUGCAGA
 588






UUUCUdTdT


UGGGGAAdTdT



 


HAMP
AD-47165
261
A-98199.1
CCCAUCUGCAUUUU
361
A-98200.1
AGCAGAAAAUGC
 589






CUGCUdTdT


AGAUGGGdTdT



 


HAMP
AD-47124
275
A-98201.1
CUGCUGCGGCUGCU
370
A-98202.1
AUGACAGCAGCCG
 598






GUCAUdTdT


CAGCAGdTdT



 


HAMP
AD-47136
276
A-98205.1
UGCUGCGGCUGCUG
371
A-98206.1
AAUGACAGCAGCC
 599






UCAUUdTdT


GCAGCAdTdT



 


HAMP
AD-47130
276
A-98203.1
UGCUGCGGCUGCUG
372
A-98204.1
UAUGACAGCAGCC
 601






UCAUAdTdT


GCAGCAdTdT



 


HAMP
AD-47142
278
A-98207.1
CUGCGGCUGCUGUC
374
A-98208.1
UCGAUGACAGCA
 603






AUCGAdTdT


GCCGCAGdTdT



 


HAMP
AD-47148
279
A-98209.1
UGCGGCUGCUGUCA
375
A-98210.1
AUCGAUGACAGC
 604






UCGAUdTdT


AGCCGCAdTdT



 


HAMP
AD-47160
280
A-98213.1
GCGGCUGCUGUCAU
376
A-98214.1
AAUCGAUGACAG
 605






CGAUUdTdT


CAGCCGCdTdT



 


HAMP
AD-47154
280
A-98211.1
GCGGCUGCUGUCAU
377
A-98212.1
UAUCGAUGACAG
 607






CGAUAdTdT


CAGCCGCdTdT



 


HAMP
AD-47166
281
4-98215.1
CGGCUGCUGUCAUC
378
A-98216.1
UGAUCGAUGACA
 608






GAUCAdTdT


GCAGCCGdTdT



 


HAMP
AD-47125
282
A-98217.1
GGCUGCUGUCAUCG
379
A-98218.1
UUGAUCGAUGAC
 609






AUCAAdTdT


AGCAGCCdTdT



 


HAMP
AD-47131
283
A-98219.1
GCUGCUGUCAUCGA
380
A-98220.1
UUUGAUCGAUGA
 610






UCAAAdTdT


CAGCAGCdTdT



 


HAMP
AD-47137
284
A-98221.1
CUGCUGUCAUCGAU
382
A-98222.1
UUUUGAUCGAUG
 612






CAAAAdTdT


ACAGCAGdTdT



 


HAMP
AD-47143
284
A-98223.1
CUGCUGUCAUCGAU
381
A-98224.1
AUUUGAUCGAUG
 611






CAAAUdTdT


ACAGCAGdTdT



 


HAMP
AD-47149
285
A-98225.1
UGCUGUCAUCGAUC
384
A-98226.1
ACUUUGAUCGAU
 614






AAAGUdTdT


GACAGCAdTdT



 


HAMP
AD-47161
286
A-98229.1
GCUGUCAUCGAUCA
385
A-98230.1
AACUUUGAUCGA
 615






AAGUUdTdT


UGACAGCdTdT



 


HAMP
AD-47155
286
4-98227.1
GCUGUCAUCGAUCA
386
A-98228.1
UACUUUGAUCGA
 617






AAGUAdTdT


UGACAGCdTdT



 


HAMP
AD-47167
287
A-98231.1
CUGUCAUCGAUCAA
388
A-98232.1
ACACUUUGAUCG
 618






AGUGUdTdT


AUGACAGdTdT



 


HAMP
AD-47132
288
A-98235.1
UGUCAUCGAUCAAA
389
A-98236.1
AACACUUUGAUC
 619






GUGUUdTdT


GAUGACAdTdT



 


HAMP
AD-47126
288
A-98233.1
UGUCAUCGAUCAAA
390
A-98234.1
UACACUUUGAUC
 621






GUGUAdTdT


GAUGACAdTdT



 


HAMP
AD-47138
290
A-98237.1
UCAUCGAUCAAAGU
392
A-98238.1
UCCACACUUUGA
 622






GUGGAdTdT


UCGAUGAdTdT



 


HAMP
AD-47144
290
A-98239.1
UCAUCGAUCAAAGU
393
A-98240.1
ACCACACUUUGAU
 623






GUGGUdTdT


CGAUGAdTdT



 


HAMP
AD-47095
291
A-98151.1
CAUCGAUCAAAGUG
395
A-98152.1
UCCCACACUUUGA
 625






UGGGAdTdT


UCGAUGdTdT



 


HAMP
AD-47156
295
A-98243.1
GAUCAAAGUGUGGG
399
A-98244.1
AACAUCCCACACU
 629






AUGUUdTdT


UUGAUCdTdT



 


HAMP
AD-47150
295
A-98241.1
GAUCAAAGUGUGGG
400
A-98242.1
UACAUCCCACACU
 631






AUGUAdTdT


UUGAUCdTdT



 


HAMP
AD-47162
299
A-98245.1
AAAGUGUGGGAUGU
407
A-98246.1
ACAGCACAUCCCA
 637






GCUGUdTdT


CACUUUdTdT



 


HAMP
AD-47078
307
A-98067.1
GGAUGUGCUGCAAG
413
A-98068.1
UACGUCUUGCAG
 644






ACGUAdTdT


CACAUCCdTdT



 


HAMP
AD-47084
309
A-98069.1
AUGUGCUGCAAGAC
414
A-98070.1
UCUACGUCUUGC
 646






GUAGAdTdT


AGCACAUdTdT



 


HAMP
AD-47090
310
A-98071.1
UGUGCUGCAAGACG
415
A-98072.1
UUCUACGUCUUG
 648






UAGAAdTdT


CAGCACAdTdT



 


HAMP
AD-47096
313
A-98073.1
GCUGCAAGACGUAG
416
A-98074.1
AGGUUCUACGUC
 650






AACCUdTdT


UUGCAGCdTdT



 


HAMP
AD-47101
314
A-98075.1
CUGCAAGACGUAGA
417
A-98076.1
UAGGUUCUACGU
 707






ACCUAdTdT


CUUGCAGdTdT



 


HAMP
AD-47106
322
A-98077.1
CGUAGAACCUACCU
418
A-98078.1
AGGGCAGGUAGG
 653






GCCCUdTdT


UUCUACGdTdT



 


HAMP
AD-47111
347
A-98079.1
GUCCCCUCCCUUCCU
419
A-98080.1
AAUAAGGAAGGG
1358






UAUUdTdT


AGGGGACdTdT



 


HAMP
AD-47116
348
A-98081.1
UCCCCUCCCUUCCUU
420
A-98082.1
AAAUAAGGAAGG
1359






AUUUdTdT


GAGGGGAdTdT



 


HAMP
AD-47079
349
A-98083.1
CCCCUCCCUUCCUUA
421
A-98084.1
UAAAUAAGGAAG
1360






UUUAdTdT


GGAGGGGdTdT



 


HAMP
AD-47085
350
A-98085.1
CCCUCCCUUCCUUAU
422
A-98086.1
AUAAAUAAGGAA
1361






UUAUdTdT


GGGAGGGdTdT



 


HAMP
AD-47091
351
A-98087.1
CCUCCCUUCCUUAU
423
A-98088.1
AAUAAAUAAGGA
1362






UUAUUdTdT


AGGGAGGdTdT



 


HAMP
AD-47097
352
A-98089.1
CUCCCUUCCUUAUU
425
A-98090.1
UAAUAAAUAAGG
1363






UAUUAdTdT


AAGGGAGdTdT



 


HAMP
AD-47102
352
A-98091.1
CUCCCUUCCUUAUU
424
A-98092.1
AAAUAAAUAAGG
1364






UAUUUdTdT


AAGGGAGdTdT



 


HAMP
AD-47107
354
A-98093.1
CCCUUCCUUAUUUA
426
A-98094.1
AGGAAUAAAUAA
1365






UUCCUdTdT


GGAAGGGdTdT



 


HAMP
AD-47112
355
A-98095.1
CCUUCCUUAUUUAU
427
A-98096.1
UAGGAAUAAAUA
1366






UCCUAdTdT


AGGAAGGdTdT



 


HAMP
AD-47117
355
A-98097.1
CCUUCCUUAUUUAU
428
A-98098.1
AAGGAAUAAAUA
1367






UCCUUdTdT


AGGAAGGdTdT



 


HAMP
AD-47086
356
A-98101.1
CUUCCUUAUUUAU
429
A-98102.1
ACAGGAAUAAAU
1368






CCUGUdTdt


AAGGAAGdTdT



 


HAMP
AD-47080
356
N-98099.1
CUUCCUUAUUUAUU
430
A-98100.1
UCAGGAAUAAAU
1369






CCUGAdTdT


AAGGAAGdTdT



 


HAMP
AD-47092
357
A-98103.1
UUCCUUAUUUAUUC
431
A-98104.1
AGCAGGAAUAAA
1370






CUGCUdTdT


UAAGGAAdTdT



 


HAMP
AD-47103
358
A-98107.1
UCCUUAUUUAUUCC
432
A-98108.1
AAGCAGGAAUAA
1371






UGCUUdTdT


AUAAGGAdTdT



 


HAMP
AD-47098
358
A-98105.1
UCCUUAUUUAUUCC
433
A-98106.1
UAGCAGGAAUAA
1372






UGCUAdTdT


AUAAGGAdTdT



 


HAMP
AD-47113
359
A-98111.1
CCUUAUUUAUUCCU
434
A-98112.1
ACAGCAGGAAUA
1373






GCUGUdTdT


AAUAAGGdTdT



 


HAMP
AD-47108
359
A-98109.1
CCUUAUUUAUUCCU
435
A-98110.1
UCAGCAGGAAUA
1374






GCUGAdTdT


AAUAAGGdTdT



 


HAMP
AD-47118
363
A-98113.1
AUUUAUUCCUGCUG
436
A-98114.1
UGGGGCAGCAGG
1375






CCCCAdTdT


AAUAAAUdTdT



 


HAMP
AD-47081
365
N-98115.1
UUAUUCCUGCUGCC
437
A-98116.1
UCUGGGGCAGCA
1376






CCAGAdTdT


GGAAUAAdTdT



 


HAMP
AD-47087
366
A-98117.1
UAUUCCUGCUGCCC
438
A-98118.1
UUCUGGGGCAGC
1377






CAGAAdTdT


AGGAAUAdTdT



 


HAMP
AD-47093
369
A-98119.1
UCCUGCUGCCCCAGA
439
A-98120.1
AUGUUCUGGGGC
 674






ACAUdTdT


AGCAGGAdTdT



 


HAMP
AD-47099
370
A-98121.1
CCUGCUGCCCCAGAA
440
A-98122.1
UAUGUUCUGGGG
 676






CAUAdTdT


CAGCAGGdTdT



 


HAMP
AD-47104
373
A-98123.1
GCUGCCCCAGAACAU
441
A-98124.1
ACCUAUGUUCUG
 678






AGGUdTdT


GGGCAGCdTdT



 


HAMP
AD-47109
375
A-98125.1
UGCCCCAGAACAUAG
442
A-98126.1
AGACCUAUGUUC
 680






GUCUdTdT


UGGGGCAdTdT



 


HAMP
AD-47114
376
A-93127.1
GCCCCAGAACAUAGG
443
A-98128.1
AAGACCUAUGUU
 681






UCUUdTdT


CUGGGGCdTdT



 


HAMP
AD-47119
379
A-98129.1
CCAGAACAUAGGUC
444
A-98130.1
UCCAAGACCUAUG
 684






UUGGAdTdT


UUCUGGdTdT



 


HAMP
AD-47082
380
A-98131.1
CAGAACAUAGGUCU
445
A-98132.1
UUCCAAGACCUAU
 687






UGGAAdTdT


GUUCUGdTdT



 


HAMP
AD-47088
381
A-98133.1
AGAACAUAGGUCUU
446
A-98134.1
AUUCCAAGACCUA
 689






GGAAUdTdT


UGUUCUdTdT



 


HAMP
AD-47094
382
A-98135.1
GAACAUAGGUCUUG
 30
A-98136.1
UAUUCCAAGACCU
  44






GAAUAdTdT


AUGUUCdTdT








  















HAMP
AD-47094
382
N-98135.1
GAACAUAGGUCUUG
 30
A-98136.1
UAUUCCAAGACCU
  44






GAAUAdTdT


AUGUUCdTdT








 















HAMP
AD-48210
382
A-100210.1
GAACACAGGUCUUG
448
A-100211.1
UAUUCCAAGACCU
 692






GAAUAdTdT


GUGUUCdTdT



 


HAMP
AD-47100
383
A-98137.1
AACAUAGGUCUUGG
450
A-98138.1
UUAUUCCAAGACC
 695






AAUAAdTdT


UAUGUUdTdT



 


HAMP
AD-47105
396
A-98139.1
GAAUAAAAUGGCUG
452
A-98140.1
AGAACCAGCCAUU
 697






GUUCUdTdT


UUAUUCdTdT



 


HAMP
AD-47110
398
A-98141.3
AUAAAAUGGCUGGU
453
A-98142.1
AAAGAACCAGCCA
 700






UCUUUdTdT


UUUUAUdTdT



 


HAMP
AD-47115
399
A-98143.1
UAAAAUGGCUGGUU
454
A-98144.1
AAAAGAACCAGCC
 702






CUUUUdTdT


AUUUUAdTdT



 


HAMP
AD-47120
402
A-98145.1
AAUGGCUGGUUCUU
455
A-98146.1
AACAAAAGAACCA
 703






UUGUUdTdT


GCCAUUdTdT



 


HAMP
AD-47083
403
A-98147.1
AUGGCUGGUUCUUU
456
A-98148.1
AAACAAAAGAACC
 704






UGUUUdTdT


AGCCAUdTdT



 


HAMP
AD-47089
407
A-98149.1
CUGGUUCUUUUGUU
457
A-98150.1
UGGAAAACAAAA
 705






UUCCAdTdT


GAACCAGdTdT
















TABLE 6







HAMP single dose screen (Modified Duplexes, Dual Luciferase Assay)













10 nM
0.1 nM
0.01 nM















Target
Duplex ID
Start Position
Avg
SD
Avg
SD
Avg
SD


















HAMP
AD-45073
2
107.73
0.50
92.94
7.41




HAMP
AD-45079
7
110.26
7.14
101.78
5.79




HAMP
AD-45085
16
90.81
0.48
96.06
2.19




HAMP
AD-29928
43
102.01
15.80
96.01
1.70




HAMP
AD-45674
43
94.81
4.68
108.44
7.69




HAMP
AD-45680
43
109.80
2.04
111.06
5.64




HAMP
AD-45686
48
89.78
15.04
110.29
0.29




HAMP
AD-45698
48
103.33
8.83
112.53
1.57




HAMP
AD-45692
48
110.03
6.99
115.05
0.14




HAMP
AD-45354
51
111.45
7.56
105.64
4.49




HAMP
AD-29929
54
99.33
11.26
104.08
6.92




HAMP
AD-45091
55
116.71
3.20
102.27
0.81




HAMP
AD-29930
59
88.47
0.38
102.18
7.79




HAMP
AD-29931
60
104.54
3.36
104.80
3.55




HAMP
AD-45704
60
142.74
0.80
122.02
1.37




HAMP
AD-45710
60
135.87
3.55
129.05
1.72




HAMP
AD-29932
61
103.48
6.29
108.36
1.31




HAMP
AD-29933
62
110.13
1.03
104.36
6.68




HAMP
AD-45675
62
113.15
1.01
107.56
0.54




HAMP
AD-45716
62
111.06
12.39
113.09
8.16




HAMP
AD-29934
63
101.68
3.60
96.37
6.01




HAMP
AD-29935
64
100.63
8.13
93.98
8.75




HAMP
AD-45687
64
103.09
8.83
105.61
3.09




HAMP
AD-45681
64
117.87
2.59
111.72
1.69




HAMP
AD-29936
66
98.38
12.53
98.56
13.20




HAMP
AD-29937
67
93.41
2.34
97.50
10.28




HAMP
AD-45699
67
47.01
9.59
98.55
3.80




HAMP
AD-45693
67
84.68
3.15
113.79
5.11




HAMP
AD-45711
68
113.03
9.72
108.10
3.83




HAMP
AD-45717
68
99.40
12.84
110.38
0.04




HAMP
AD-45705
68
110.22
3.84
117.90
9.96




HAMP
AD-45682
69
96.60
3.60
103.41
1.06




HAMP
AD-45688
69
100.44
9.14
104.93
5.18




HAMP
AD-45676
69
106.83
9.15
106.73
1.89




HAMP
AD-45360
70
92.88
0.12
93.73
2.85




HAMP
AD-45366
71
92.46
2.58
99.04
0.39




HAMP
AD-29938
72
62.08
21.83
75.55
6.85




HAMP
AD-45372
73
59.85
2.76
96.31
5.86




HAMP
AD-45700
74
12.85
5.11
63.97
14.79




HAMP
AD-29939
74
36.40
19.57
67.18
9.10




HAMP
AD-45694
74
17.85
4.97
90.43
0.13




HAMP
AD-29940
75
49.83
6.31
76.05
7.08




HAMP
AD-45712
76
32.07
2.85
63.27
3.48




HAMP
AD-29941
76
81.10
0.03
97.49
9.32




HAMP
AD-45706
76
43.48
6.67
97.60
1.61




HAMP
AD-45097
88
50.62
0.50
71.18
1.94




HAMP
AD-45103
91
53.20
9.02
96.52
7.45




HAMP
AD-45378
116
95.96
1.21
103.17
3.99




HAMP
AD-45383
117
99.99
2.44
104.79
5.38




HAMP
AD-45388
118
98.52
4.10
105.96
3.21




HAMP
AD-45393
120
103.62
5.17
102.44
6.38




HAMP
AD-45355
121
73.28
0.51
96.56
1.06




HAMP
AD-45361
122
98.67
0.23
99.82
4.47




HAMP
AD-45367
123
90.48
1.28
102.75
4.07




HAMP
AD-45373
126
106.01
8.36
99.38
4.05




HAMP
AD-45109
132
85.86
5.55
95.06
3.75




HAMP
AD-45115
140
100.97
1.25
90.90
9.28




HAMP
AD-45074
142
95.53
2.12
95.37
2.74




HAMP
AD-45677
146
58.20
5.06
80.37
5.46




HAMP
AD-45683
146
67.80
1.12
88.08
7.96




HAMP
AD-45718
146
76.16
3.48
100.37
3.35




HAMP
AD-45080
149
52.89
1.28
84.16
0.02




HAMP
AD-45379
150
48.97
3.64
103.43
1.19




HAMP
AD-29942
151
88.17
4.85
97.49
7.95




HAMP
AD-29943
152
43.37
9.93
73.15
23.90




HAMP
AD-29944
153
80.38
9.90
92.54
7.85




HAMP
AD-45695
153
65.57
2.52
92.72
5.87




HAMP
AD-45689
153
72.67
0.78
93.00
2.67




HAMP
AD-29945
154
69.81
13.13
76.49
17.11




HAMP
AD-29946
155
75.80
1.18
80.39
14.15




HAMP
AD-45713
157
70.69
1.76
94.45
0.39




HAMP
AD-45707
157
71.62
6.17
94.94
2.22




HAMP
AD-45701
157
79.39
1.97
101.46
0.70




HAMP
AD-45384
159
89.86
1.67
102.53
2.37




HAMP
AD-45389
160
41.14
0.44
90.29
3.94




HAMP
AD-45678
161
55.04
0.96
76.03
3.63




HAMP
AD-45719
161
55.02
5.94
84.45
2.01




HAMP
AD-29947
161
81.45
6.55
89.78
7.92




HAMP
AD-45690
162
105.99
3.29
97.21
2.61




HAMP
AD-45696
162
105.48
0.08
99.13
0.28




HAMP
AD-45684
162
96.14
6.48
104.09
2.52




HAMP
AD-30016
163
57.89
8.28
90.60
14.06




HAMP
AD-45394
164
87.68
5.01
108.27
2.32




HAMP
AD-45702
165
70.60
2.02
93.12
5.12




HAMP
AD-45708
165
74.75
3.73
98.51
2.26




HAMP
AD-45714
165
73.26
2.24
102.34
12.47




HAMP
AD-29949
166
102.90
8.09
91.62
0.16




HAMP
AD-45086
167
120.81
3.27
106.79
7.19




HAMP
AD-45356
168
81.17
4.40
93.13
0.76




HAMP
AD-45685
169
114.45
9.16
98.53
0.41




HAMP
AD-45679
169
105.22
9.07
101.56
5.80




HAMP
AD-45720
169
121.03
5.25
110.57
1.75




HAMP
AD-45703
170
44.33
1.60
79.12
4.45




HAMP
AD-45697
170
46.91
2.65
87.12
1.48




HAMP
AD-45691
170
54.15
1.94
92.73
6.86




HAMP
AD-45362
189
40.88
0.51
88.62
2.54




HAMP
AD-45368
190
31.23
1.19
95.59
2.85




HAMP
AD-45374
199
101.82
3.03
101.10
0.65




HAMP
AD-45092
222
87.17
5.48
98.58
1.36




HAMP
AD-45721
228
46.67
6.31
81.09
9.13




HAMP
AD-45715
228
49.86
3.40
88.14
4.98




HAMP
AD-45709
228
77.17
5.09
98.27
3.31




HAMP
AD-45380
230
62.83
3.09
103.76
1.31




HAMP
AD-45385
231
98.28
0.86
102.61
0.12




HAMP
AD-29950
232
55.13
8.89
67.22
10.51




HAMP
AD-45390
233
43.22
3.42
94.61
0.86




HAMP
AD-29951
234
37.28
9.48
53.43
13.93




HAMP
AD-45395
235
60.56
0.93
96.88
1.63




HAMP
AD-45727
239
41.79
5.36
73.07
5.68




HAMP
AD-45732
239
40.15
8.90
73.60
14.88




HAMP
AD-29952
239
97.66
18.17
104.87
4.70




HAMP
AD-29953
240
86.68
10.48
88.35
7.38




HAMP
AD-30017
241
33.76
16.25
60.73
30.76




HAMP
AD-30018
242
41.44
14.83
70.97
23.75




HAMP
AD-45737
242
17.97
4.49
71.13
9.76




HAMP
AD-29956
246
89.56
4.13
97.82
5.97




HAMP
AD-45357
247
82.69
2.17
93.22
4.53




HAMP
AD-45363
248
93.32
5.91
91.24
0.20




HAMP
AD-45747
251
70.65
8.85
97.73
2.26




HAMP
AD-45752
251
89.51
3.39
98.35
2.19




HAMP
AD-45757
251
82.94
6.75
102.34
3.76




HAMP
AD-29957
252
81.99
11.58
93.00
9.35




HAMP
AD-45399
253
64.38
0.64
97.91
2.54




HAMP
AD-45098
255
82.48
2.01
76.84
2.07




HAMP
AD-45400
256
41.85
0.69
73.87
3.09




HAMP
AD-45381
257
33.48
1.75
76.90
0.24




HAMP
AD-45401
258
20.19
1.67
47.65
3.20




HAMP
AD-29958
261
56.65
14.92
84.66
28.04




HAMP
AD-45391
262
24.94
0.82
89.45
2.12




HAMP
AD-29959
267
31.48
11.63
63.85
30.99




HAMP
AD-29960
268
79.91
12.47
93.49
6.68




HAMP
AD-30019
270
63.27
10.61
74.99
18.30




HAMP
AD-45396
271
119.24
2.30
111.41
3.10




HAMP
AD-45358
272
94.71
8.18
101.07
2.21




HAMP
AD-45364
273
84.76
0.19
96.53
5.62




HAMP
AD-29962
274
76.15
9.81
86.71
13.14




HAMP
AD-45370
275
72.03
3.07
96.93
2.90




HAMP
AD-45728
276
16.69
2.26
33.05
13.53




HAMP
AD-45722
276
14.19
2.15
36.38
8.98




HAMP
AD-29963
276
33.66
10.79
68.80
33.38




HAMP
AD-45104
278
68.72
2.74
87.22
0.49




HAMP
AD-29964
279
71.02
18.87
76.03
27.29




HAMP
AD-45738
280
50.02
8.64
70.44
7.26




HAMP
AD-45733
280
57.29
5.28
84.47
5.76




HAMP
AD-29965
281
55.85
8.35
72.34
23.30




HAMP
AD-30020
283
68.86
8.88
66.02
23.24




HAMP
AD-45748
284
21.85
1.77
35.95
6.98




HAMP
AD-45743
284
29.01
1.73
42.99
5.69




HAMP
AD-30021
284
42.30
7.75
66.28
27.47




HAMP
AD-11441
285
15.04
8.59
34.60
10.87
63.42
16.67


HAMP
AD-45758
286
17.08
0.43
33.34
3.43




HAMP
AD-45753
286
25.19
4.02
80.83
6.73




HAMP
AD-29968
286
57.05
12.26
85.22
13.56




HAMP
AD-29969
287
81.97
16.19
102.53
21.58




HAMP
AD-45729
288
9.67
1.06
32.83
13.93




HAMP
AD-45723
288
20.87
3.89
66.57
4.73




HAMP
AD-29970
288
65.21
1.72
84.12
5.99




HAMP
AD-45744
290
40.34
1.92
56.56
3.09




HAMP
AD-45739
290
29.46
2.77
67.24
9.80




HAMP
AD-45734
290
53.39
2.32
83.49
1.19




HAMP
AD-11436
291
19.18
8.22
42.74
14.18
76.43
23.00


HAMP
AD-29971
291
29.02
9.90
52.08
14.09




HAMP
AD-45376
292
47.54
0.51
87.50
1.26




HAMP
AD-45382
293
37.05
0.44
93.25
4.03




HAMP
AD-29972
294
32.08
7.08
53.51
17.12




HAMP
AD-45754
295
30.78
4.04
62.74
0.50




HAMP
AD-45749
295
48.49
11.81
92.97
1.08




HAMP
AD-29973
295
101.69
8.46
97.65
8.24




HAMP
AD-45730
296
78.23
2.00
86.13
5.69




HAMP
AD-45724
296
82.07
4.46
86.67
2.23




HAMP
AD-45759
296
92.10
6.68
97.71
2.40




HAMP
AD-45110
297
69.77
2.74
90.01
2.11




HAMP
AD-45387
298
98.34
5.75
108.20
1.85




HAMP
AD-45740
299
124.12
4.66
101.03
6.78




HAMP
AD-45745
299
131.69
10.22
103.23
1.76




HAMP
AD-45735
299
111.96
3.19
103.86
6.46




HAMP
AD-29974
300
34.04
6.78
53.98
24.23




HAMP
AD-29975
301
53.80
12.05
67.73
22.08




HAMP
AD-45116
306
25.93
2.21
55.49
5.82




HAMP
AD-45075
307
19.84
2.98
63.83
1.45




HAMP
AD-45081
309
14.66
0.55
38.67
3.25




HAMP
AD-45087
310
11.95
0.17
29.23
1.61




HAMP
AD-45093
313
14.07
0.68
45.44
2.30




HAMP
AD-45099
322
108.37
1.23
107.51
2.82




HAMP
AD-45105
369
102.93
6.40
101.31
4.27




HAMP
AD-45111
370
117.00
3.72
104.04
2.33




HAMP
AD-45117
373
99.33
0.61
102.95
0.52




HAMP
AD-45076
375
62.84
4.39
90.32
1.71




HAMP
AD-45082
376
75.58
1.59
95.17
0.21




HAMP
AD-45088
379
83.27
12.84
101.84
1.83




HAMP
AD-45094
380
99.51
2.23
102.51
2.51




HAMP
AD-45100
381
112.68
6.11
107.46
7.75




HAMP
AD-45106
383
138.19
1.98
112.49
0.89




HAMP
AD-45112
396
128.11
6.65
106.21
3.64




HAMP
AD-45118
398
116.86
12.51
103.78
4.53




HAMP
AD-45077
399
98.64
1.22
103.30
2.55




HAMP
AD-45083
402
114.82
2.26
104.50
0.17




HAMP
AD-45089
403
107.59
7.70
103.43
1.69





Data are expressed as percent of mock or AD-1955.













TABLE 7







HAMP single dose screen (Unmodified Duplexes, Human Endogenous)















10 nM
0.1 nM
0.01 nM















Target
Duplex ID
Start Position
Avg
SD
Avg
SD
Avg
SD


















HAMP
AD-47121
62
22.18
1.49
60.31
16.26




HAMP
AD-47133
67
27.53
1.12
53.58
2.92




HAMP
AD-47127
67
20.45
4.63
54.16
7.57




HAMP
AD-47145
74
19.51
6.65
54.67
10.88




HAMP
AD-47139
74
20.72
0.17
58.12
2.90




HAMP
AD-47157
76
10.07
0.35
28.24
6.49




HAMP
AD-47151
76
12.08
1.83
33.95
2.14




HAMP
AD-47163
132
8.58
0.51
44.65
17.97




HAMP
AD-47122
140
25.66
0.45
72.63
2.29




HAMP
AD-47128
146
30.88
3.04
64.36
2.04




HAMP
AD-47134
146
48.07
0.91
72.33
12.48




HAMP
AD-47140
155
15.20
1.25
34.69
0.30




HAMP
AD-47152
157
13.21
6.55
28.17
1.17




HAMP
AD-47146
157
14.77
0.68
32.02
1.58




HAMP
AD-47158
160
9.73
1.66
32.92
1.87




HAMP
AD-47164
161
5.71
0.44
32.90
2.89




HAMP
AD-47123
161
7.88
3.02
39.31
19.09




HAMP
AD-47135
162
26.84
0.87
74.06
15.78




HAMP
AD-47129
162
27.18
1.57
83.62
13.59




HAMP
AD-47141
242
110.80
16.98
127.17
42.39




HAMP
AD-47147
242
116.01
9.55
132.52
28.61




HAMP
AD-47153
253
34.69
7.47
66.88
2.08




HAMP
AD-47159
258
33.41
1.23
57.26
7.97




HAMP
AD-47165
261
25.12
0.71
85.70
6.86




HAMP
AD-47124
275
36.35
7.66
87.65
11.44




HAMP
AD-47136
276
6.06
0.70
40.72
13.94




HAMP
AD-47130
276
8.76
0.58
46.31
13.29




HAMP
AD-47142
278
24.10
2.89
56.75
18.44




HAMP
AD-47148
279
19.36
1.09
57.95
18.21




HAMP
AD-47160
280
5.75
0.73
35.24
4.98




HAMP
AD-47154
280
15.01
3.91
36.32
0.45




HAMP
AD-47166
281
11.98
0.47
51.40
12.88




HAMP
AD-47125
282
14.62
1.15
54.37
11.47




HAMP
AD-47131
283
8.74
0.45
42.66
12.21




HAMP
AD-47137
284
9.97
0.73
36.35
5.96




HAMP
AD-47143
284
9.66
0.84
39.72
8.37




HAMP
AD-47149
285
13.85
0.47
47.21
9.13




HAMP
AD-47161
286
7.28
0.89
31.75
8.03




HAMP
AD-47155
286
8.27
0.74
36.03
14.37




HAMP
AD-47167
287
8.98
0.14
41.61
6.60




HAMP
AD-47132
288
9.08
0.17
38.01
4.01




HAMP
AD-47126
288
8.59
3.66
40.28
10.49




HAMP
AD-47138
290
41.75
1.27
81.70
14.64




HAMP
AD-47144
290
60.81
13.34
107.58
15.10




HAMP
AD-47095
291
34.79
5.48
58.98
7.36




HAMP
AD-47156
295
39.09
4.26
90.08
4.38




HAMP
AD-47150
295
53.01
9.58
99.42
9.61




HAMP
AD-47162
299
122.90
8.44
123.74
15.71




HAMP
AD-47078
307
26.81
9.00
59.79
15.73




HAMP
AD-47084
309
31.16
5.91
59.33
19.95




HAMP
AD-47090
310
15.07
5.19
49.74
11.70




HAMP
AD-47096
313
49.34
9.86
68.64
0.71




HAMP
AD-47101
314
13.36
5.68
38.13
10.64




HAMP
AD-47106
322
29.91
1.99
61.30
1.25




HAMP
AD-47111
347
21.57
4.45
46.65
10.06




HAMP
AD-47116
348
32.95
7.34
65.00
10.06




HAMP
AD-47079
349
10.10
2.16
24.36
4.46




HAMP
AD-47085
350
8.08
5.13
20.39
9.47




HAMP
AD-47091
351
20.73
6.86
42.28
5.15




HAMP
AD-47097
352
10.57
2.97
24.58
3.18




HAMP
AD-47102
352
15.48
7.81
25.60
8.37




HAMP
AD-47107
354
50.89
12.39
61.80
6.52




HAMP
AD-47112
355
42.93
6.42
53.00
3.93




HAMP
AD-47117
355
33.82
2.18
60.78
7.57




HAMP
AD-47086
356
16.50
3.69
34.88
9.79




HAMP
AD-47080
356
13.76
3.39
38.95
7.09




HAMP
AD-47092
357
35.01
5.39
48.61
6.81




HAMP
AD-47103
356
45.09
9.10
66.18
7.81




HAMP
AD-47098
358
63.20
11.74
70.69
1.23




HAMP
AD-47113
359
27.42
9.95
49.88
7.22




HAMP
AD-47108
359
30.30
9.89
52.33
12.30




HAMP
AD-47118
363
7.45
0.35
19.20
1.31




HAMP
AD-47081
365
4.25
1.97
22.94
6.70




HAMP
AD-47087
366
9.49
2.18
37.51
12.04




HAMP
AD-47093
369
4.75
1.38
23.36
3.30




HAMP
AD-47099
370
5.05
0.01
17.71
7.66




HAMP
AD-47104
373
32.32
8.82
37.72
8.15




HAMP
AD-47109
375
25.45
4.46
35.56
1.25




HAMP
AD-47114
376
10.65
4.55
17.30
6.01




HAMP
AD-47119
379
7.99
0.50
17.44
6.45




HAMP
AD-47082
380
13.13
1.08
27.19
8.88




HAMP
AD-47088
381
5.80
2.75
12.26
7.13




HAMP
AD-47094
382
5.59
2.35
11.95
7.39




HAMP
AD-47094
382
8.63
3.05
14.02
2.30
22.83
0.56


HAMP
AD-48210
382
7.43
7.88
17.06
4.17
30.21
0.63


HAMP
AD-47100
383
3.80
2.75
8.41
3.86




HAMP
AD-47105
396
6.56
2.25
12.11
4.90




HAMP
AD-47110
398
10.42
5.14
21.44
0.24




HAMP
AD-47115
399
4.86
0.27
9.25
1.57




HAMP
AD-47120
402
5.78
0.12
15.68
0.67




HAMP
AD-47083
403
4.36
1.88
14.49
5.26




HAMP
AD-47089
407
17.68
1.22
21.61
6.91





Data are expressed as percent of mock.













TABLE 8







HAMP single dose screen (Modified Duplexes, Human Endogenous)















10 nM
0.1 nM
0.01 nM















Target
Duplex ID
Start Position
Avg
SD
Avg
SD
Avg
SD


















HAMP
AD-47031
62
15.53
6.23
42.68
4.24




HAMP
AD-47043
67
21.87
1.62
50.05
4.67




HAMP
AD-47037
67
23.85
4.92
53.84
0.43




HAMP
AD-47055
74
31.38
2.06
59.08
4.48




HAMP
AD-47049
74
30.11
3.10
64.35
3.66




HAMP
AD-47067
76
8.71
1.38
28.60
2.87




HAMP
AD-47061
76
11.78
3.07
29.53
0.18




HAMP
AD-47032
140
37.89
8.04
60.90
3.74




HAMP
AD-47038
146
33.92
2.79
53.24
7.14




HAMP
AD-47044
146
39.99
7.45
60.74
5.99




HAMP
AD-47050
155
14.55
1.46
35.51
0.33




HAMP
AD-47062
157
13.42
1.10
45.34
3.82




HAMP
AD-47056
157
23.31
0.44
46.02
0.80




HAMP
AD-47068
160
24.68
0.67
56.12
4.66




HAMP
AD-47033
161
11.56
4.54
36.94
0.19




HAMP
AD-47074
161
9.99
1.07
44.47
0.95




HAMP
AD-47039
162
63.29
2.38
80.39
15.70




HAMP
AD-47045
162
86.89
5.22
96.60
14.33




HAMP
AD-47057
242
66.74
2.06
82.90
10.89




HAMP
AD-47051
242
72.68
0.12
86.34
3.07




HAMP
AD-47063
253
26.21
0.40
58.94
7.25




HAMP
AD-47069
258
30.01
3.26
41.02
3.03




HAMP
AD-47075
261
30.80
2.74
75.66
1.48




HAMP
AD-47034
275
54.15
10.01
75.48
16.26




HAMP
AD-47046
276
13.55
0.80
30.18
6.37




HAMP
AD-47040
276
18.09
3.87
40.15
14.54




HAMP
AD-47058
279
36.00
4.98
64.23
1.93




HAMP
AD-47070
280
12.74
1.13
34.84
9.02




HAMP
AD-47064
280
17.08
0.13
49.50
0.21




HAMP
AD-47076
281
12.07
1.81
36.35
5.58




HAMP
AD-47035
282
31.01
7.32
61.60
1.15




HAMP
AD-47041
283
16.92
0.64
39.03
10.75




HAMP
AD-47053
284
10.31
0.77
23.40
7.24




HAMP
AD-47047
284
12.12
0.18
30.96
7.74




HAMP
AD-47059
285
20.79
0.79
45.23
6.52




HAMP
AD-47071
286
15.36
1.48
36.67
8.67




HAMP
AD-47065
286
19.45
0.16
53.77
19.91




HAMP
AD-47077
287
9.85
0.40
45.43
2.39




HAMP
AD-48208
288
9.71
4.88
14.16
3.25
40.26
4.14


HAMP
AD-47042
288
9.47
2.61
24.02
11.39




HAMP
AD-48202
288
11.49
3.71
27.05
3.20
69.29
1.70


HAMP
AD-47036
288
10.22
1.87
38.40
8.79




HAMP
AD-47048
290
38.00
2.44
80.14
9.40




HAMP
AD-47054
290
46.82
5.24
87.19
6.81




HAMP
AD-47005
291
34.54
2.08
63.87
11.34




HAMP
AD-11436
291
43.37
7.53
74.23
14.15




HAMP
AD-47066
295
37.84
6.67
66.36
3.03




HAMP
AD-47060
295
52.68
4.93
83.68
16.47




HAMP
AD-47072
299
74.58
22.86
117.51
7.68




HAMP
AD-46988
307
39.46
7.63
78.38
1.82




HAMP
AD-46994
309
91.00
6.12
100.96
9.30




HAMP
AD-47000
310
42.88
7.35
65.34
6.09




HAMP
AD-47006
313
27.81
0.36
71.03
9.01




HAMP
AD-47011
314
24.50
4.38
63.98
14.14




HAMP
AD-47016
322
65.73
3.26
90.84
9.34




HAMP
AD-47021
347
80.76
0.51
86.40
9.24




HAMP
AD-47026
348
71.64
5.09
81.58
5.61




HAMP
AD-46989
349
90.45
10.46
99.05
11.53




HAMP
AD-46995
350
20.68
3.39
75.89
9.25




HAMP
AD-47001
351
74.47
1.50
80.49
21.50




HAMP
AD-47012
352
71.82
13.01
84.03
5.63




HAMP
AD-47007
352
82.28
15.46
89.63
9.85




HAMP
AD-47017
354
66.26
8.83
100.80
21.89




HAMP
AD-47022
355
63.73
4.49
87.39
9.12




HAMP
AD-47027
355
68.87
6.64
108.08
32.59




HAMP
AD-46996
355
37.91
1.83
48.04
6.32




HAMP
AD-46990
356
41.87
4.92
54.43
6.70




HAMP
AD-47002
357
16.19
0.33
42.98
3.19




HAMP
AD-47013
358
22.95
0.97
44.27
7.76




HAMP
AD-47008
358
20.38
2.71
50.46
16.50




HAMP
AD-47023
359
77.40
10.19
95.51
9.29




HAMP
AD-47018
359
95.24
14.37
97.19
8.72




HAMP
AD-47028
363
28.25
2.86
62.93
4.48




HAMP
AD-46991
365
15.53
2.49
29.41
0.30




HAMP
AD-46997
366
31.51
3.85
48.07
6.21




HAMP
AD-47003
369
9.85
2.64
34.31
6.01




HAMP
AD-47009
370
6.69
1.11
24.11
3.57




HAMP
AD-47014
373
55.85
3.19
60.89
10.51




HAMP
AD-47019
375
28.54
1.87
49.45
14.83




HAMP
AD-48214
376
12.63
3.25
16.69
1.38
29.21
0.40


HAMP
AD-48219
376
15.92
0.02
18.92
0.48
33.17
2.16


HAMP
AD-47024
376
19.61
1.81
42.20
5.93




HAMP
AD-48224
379
22.20
5.60
33.45
1.62
52.72
1.81


HAMP
AD-48187
379
25.57
5.25
46.92
0.04
73.94
0.20


HAMP
AD-47029
379
24.31
0.26
47.37
6.54




HAMP
AD-48192
379
19.69
0.78
55.32
4.62
88.04
0.76


HAMP
AD-46992
380
23.41
3.32
37.52
4.13




HAMP
AD-46998
381
26.55
2.19
49.95
0.87




HAMP
AD-48137
382
8.66
0.33
11.24
1.02
26.89
1.08


HAMP
AD-48196
382
6.92
3.59
11.81
1.33
22.33
0.98


HAMP
AD-48195
382
6.10
2.66
12.50
3.26
26.60
1.17


HAMP
AD-48201
382
12.95
1.61
13.01
2.07
25.66
6.95


HAMP
AD-48207
382
7.91
2.77
13.17
0.62
21.22
1.42


HAMP
AD-48159
382
15.26
0.13
13.59
1.45
28.89
4.67


HAMP
AD-48147
382
14.28
0.17
13.65
0.38
25.68
3.81


HAMP
AD-48161
382
9.61
3.69
13.77
1.67
22.95
0.59


HAMP
AD-48172
382
11.67
0.41
13.98
1.39
27.28
5.22


HAMP
AD-48156
382
12.14
0.96
14.06
1.75
28.85
2.18


HAMP
AD-48195
382
6.81
0.62
14.14
0.37
27.99
0.61


HAMP
AD-48136
382
10.81
4.42
14.16
1.41
29.89
1.13


HAMP
AD-48166
382
8.74
2.41
14.22
0.42
25.21
2.09


HAMP
AD-48213
382
8.35
1.61
14.49
3.71
22.38
0.33


HAMP
AD-48173
382
12.84
3.32
14.51
0.96
27.87
5.30


HAMP
AD-48154
382
9.93
2.07
14.80
0.75
23.47
0.94


HAMP
AD-48141
382
12.73
2.32
14.92
0.32
26.97
5.76


HAMP
AD-48216
382
10.39
0.95
15.18
2.11
22.38
1.02


HAMP
AD-48180
382
7.71
0.47
15.20
1.30
29.01
1.11


HAMP
AD-48143
382
7.00
2.73
15.44
1.88
24.35
2.09


HAMP
AD-48142
382
10.10
3.42
15.50
0.90
25.84
2.08


HAMP
AD-48221
382
9.54
1.43
15.56
0.24
23.59
1.06


HAMP
AD-48171
382
13.46
5.06
15.67
0.63
26.44
6.58


HAMP
AD-48145
382
10.87
0.68
15.70
2.69
28.65
2.93


HAMP
AD-48160
382
10.77
0.26
15.74
1.29
28.12
4.11


HAMP
AD-48144
382
9.88
0.46
15.75
0.94
33.60
0.84


HAMP
AD-48167
382
10.83
1.48
15.87
3.03
24.90
2.15


HAMP
AD-48177
382
15.05
2.15
15.87
0.86
28.68
8.05


HAMP
AD-48153
382
12.07
5.83
15.92
1.04
31.19
3.85


HAMP
AD-48178
382
11.02
0.05
16.06
0.45
27.12
2.27


HAMP
AD-48155
382
12.92
0.25
16.32
4.70
27.64
0.32


HAMP
AD-48174
382
11.50
2.09
16.39
0.74
27.90
4.46


HAMP
AD-48205
382
7.46
7.62
16.39
2.29
24.17
1.56


HAMP
AD-48179
382
10.80
3.42
16.50
0.71
27.82
3.57


HAMP
AD-48168
382
12.14
4.14
16.63
1.58
27.25
1.24


HAMP
AD-48149
382
10.42
0.41
16.71
3.88
28.30
1.91


HAMP
AD-48211
382
9.46
4.30
16.80
1.44
25.08
0.20


HAMP
AD-48200
382
9.05
1.30
16.97
2.20
28.99
0.38


HAMP
AD-48188
382
11.09
3.14
16.99
2.41
32.42
1.58


HAMP
AD-48183
382
9.79
11.87
17.20
0.54
42.02
0.63


HAMP
AD-48150
382
9.99
8.19
17.30
2.27
35.68
0.32


HAMP
AD-48162
382
8.48
2.96
17.38
1.26
29.63
0.85


HAMP
AD-48139
382
10.35
1.92
17.78
0.51
36.00
2.79


HAMP
AD-9942
382
8.96
1.21
18.03
1.85
30.09
0.13


HAMP
AD-48138
382
10.06
2.32
18.04
1.88
26.97
1.56


HAMP
AD-11459
382
7.07
0.09
18.93
3.13




HAMP
AD-48189
382
8.39
0.33
19.16
0.02
26.30
0.33


HAMP
AD-48148
382
10.68
1.22
19.23
3.07
32.33
1.11


HAMP
AD-48215
382
12.87
3.87
19.50
0.69
35.59
3.70


HAMP
AD-48218
382
12.77
1.57
22.32
2.42
48.01
5.44


HAMP
AD-48135
382
13.43
1.45
26.06
4.80
57.92
1.12


HAMP
AD-47004
382
10.04
1.61
26.68
3.90




HAMP
AD-48194
382
13.40
1.06
27.15
1.41
44.44
2.80


HAMP
AD-48197
382
13.40
6.22
27.28
1.85
47.77
5.73


HAMP
AD-11459
382
14.32
0.61
28.20
2.18
50.13
0.65


HAMP
AD-48164
382
16.52
2.52
28.92
3.09
55.94
0.41


HAMP
AD-48158
382
11.63
2.57
29.78
1.47
51.55
0.47


HAMP
AD-48204
382
11.53
13.16
29.90
1.81
68.66
2.16


HAMP
AD-48181
382
11.31
3.03
30.04
2.07
65.39
3.38


HAMP
AD-48223
382
12.35
5.46
30.39
3.74
60.92
1.91


HAMP
AD-48190
382
10.09
1.16
30.78
0.29
61.44
0.86


HAMP
AD-48163
382
13.43
2.71
31.64
4.03
60.73
1.22


HAMP
AD-48140
382
11.77
2.88
31.73
2.21
57.59
0.57


HAMP
AD-48169
382
12.50
0.13
32.00
4.43
60.20
3.22


HAMP
AD-48220
382
15.52
3.73
32.05
1.48
58.71
2.57


HAMP
AD-48184
382
13.23
0.16
33.25
3.63
56.78
2.43


HAMP
AD-48176
382
16.68
3.88
34.04
1.12
68.51
2.16


HAMP
AD-48175
382
13.60
9.49
34.34
2.07
63.89
2.17


HAMP
AD-48146
382
13.16
4.10
35.07
0.23
61.14
1.09


HAMP
AD-48182
382
13.71
8.96
36.24
0.98
71.73
0.19


HAMP
AD-48199
382
11.16
0.69
36.33
1.26
66.19
0.94


HAMP
AD-48157
382
11.27
0.12
36.54
2.61
71.05
0.88


HAMP
AD-48206
382
10.51
2.71
36.79
4.47
61.74
0.21


HAMP
AD-48193
382
13.00
3.25
37.58
2.12
73.07
0.26


HAMP
AD-48152
382
21.49
5.83
39.17
6.15
68.81
10.01


HAMP
AD-48151
382
13.62
10.83
39.31
6.55
66.68
1.47


HAMP
AD-48170
382
14.54
2.27
47.27
4.01
70.43
2.22


HAMP
AD-47010
383
41.47
1.29
64.00
4.87




HAMP
AD-48222
385
9.49
0.13
10.82
1.20
18.78
2.86


HAMP
AD-48217
385
14.39
3.84
14.26
0.55
19.91
0.71


HAMP
AD-48185
385
14.22
4.44
18.35
1.29
37.50
0.03


HAMP
AD-48212
385
20.56
4.86
22.61
3.43
26.13
0.41


HAMP
AD-48198
396
8.31
23.06
13.10
3.95
50.72
1.39


HAMP
AD-48209
396
8.76
5.65
14.85
3.44
33.81
2.06


HAMP
AD-48203
396
9.38
2.78
15.08
2.34
35.67
2.38


HAMP
AD-47015
396
16.43
2.26
30.67
1.14




HAMP
AD-47020
398
50.18
1.59
68.91
17.54




HAMP
AD-47025
399
13.04
0.87
19.74
1.31




HAMP
AD-47030
402
5.12
0.55
12.72
0.67




HAMP
AD-46993
403
5.82
2.21
12.55
1.15




HAMP
AD-46999
407
11.34
1.35
15.21
1.41





Data are expressed as percent of mock.













TABLE 9







HAMP dose-response (Dual Luciferase, HepG2, Cyno primary


hepatocytes; Unmodified & Modified duplexes)











Start
Modification
IC50 (nM)













Target
Duplex ID
position
status
Luc
HepG2
Cyno
















HAMP
AD-29939
74
Modified
0.288




HAMP
AD-45700
74
Modified

0.752



HAMP
AD-29940
75
Modified
0.929




HAMP
AD-29943
152
Modified
0.567




HAMP
AD-29950
232
Modified
1.527




HAMP
AD-29951
234
Modified
0.408




HAMP
AD-30017
241
Modified
0.163




HAMP
AD-30018
242
Modified
0.517




HAMP
AD-29959
267
Modified
0.147




HAMP
AD-29963
276
Modified
0.155




HAMP
AD-45722
276
Modified

0.299



HAMP
AD-29965
281
Modified
1.149




HAMP
AD-30020
283
Modified
39.122




HAMP
AD-30021
284
Modified
0.308




HAMP
AD-11441
285
Modified
0.042
0.135
0.027


HAMP
AD-11458
285
Modified
0.358




HAMP
AD-45729
288
Modified

0.068
0.016


HAMP
AD-48208
288
Modified

0.012
0.016


HAMP
AD-11436
291
Modified
0.054
>10 nM



HAMP
AD-11453
291
Modified
0.134




HAMP
AD-29971
291
Modified
0.108




HAMP
AD-29972
294
Modified
0.154




HAMP
AD-29974
300
Modified
0.137




HAMP
AD-29975
301
Modified
1.392




HAMP
AD-45081
309
Modified

>10 nM



HAMP
AD-45087
310
Modified

>10 nM



HAMP
AD-45093
313
Modified

>10 nM



HAMP
AD-29979
352
Modified

>10 nM



HAMP
AD-45750
352
Modified

1.558



HAMP
AD-45755
352
Modified

0.296



HAMP
AD-45725
355
Modified

>10 nM



HAMP
AD-29981
357
Modified

>10 nM



HAMP
AD-45761
359
Modified

>10 nM



HAMP
AD-45377
364
Modified

>10 nM



HAMP
AD-29982
365
Modified

1.723



HAMP
AD-29983
366
Modified

>10 nM



HAMP
AD-47099
370
Unmodified

0.017
0.081


HAMP
AD-47114
376
Unmodified

0.008
0.036


HAMP
AD-48214
376
Modified

0.008
1.575


HAMP
AD-47119
379
Unmodified

0.004
0.040


HAMP
AD-47088
381
Unmodified

0.007
>10 nM


HAMP
AD-11442
382
Modified

0.028
0.010


HAMP
AD-11459
382
Unmodified

0.038
0.045


HAMP
AD-45062
382
Modified

0.088
0.030


HAMP
AD-47094
382
Unmodified

0.005
0.039


HAMP
AD-48141
382
Modified

0.004
0.023


HAMP
AD-48147
382
Modified

0.007
0.008


HAMP
AD-48154
382
Modified

0.006
0.019


HAMP
AD-48189
382
Modified

0.005
0.145


HAMP
AD-48195
382
Modified

0.011
0.009


HAMP
AD-48196
382
Modified

0.017
0.031


HAMP
AD-48201
382
Modified

0.007
0.009


HAMP
AD-48205
382
Modified

0.014
0.022


HAMP
AD-48207
382
Modified

0.007
0.017


HAMP
AD-48213
382
Modified

0.009
0.027


HAMP
AD-48216
382
Modified

0.014
0.035


HAMP
AD-47100
383
Unmodified

0.007
0.172


HAMP
AD-48217
385
Modified

0.028
0.021


HAMP
AD-47105
396
Unmodified

0.005
>10 nM


HAMP
AD-48209
396
Modified

0.013
>10 nM


HAMP
AD-47115
399
Unmodified

0.007
>10 nM


HAMP
AD-47030
402
Modified

0.015
>10 nM


HAMP
AD-47120
402
Unmodified

0.006
>10 nM


HAMP
AD-46993
403
Modified

0.015
>10 nM


HAMP
AD-47083
403
Unmodified

0.007
>10 nM


HAMP
AD-46999
407
Modified

0.011
>10 nM
















TABLE 10A







Tables 10A and 10B: Secondary Target sequences

















Start


SEQ
Antisense

SEQ


Target
Duplex ID
Position
Sense Name
Sense Sequence
ID NO
Name
Antisense Sequence
ID NO





HFE2
AD-47391
 177
A-98855.1
AGAGuAGGGAAucAu
 31
A-98856.1
AGCcAUGAUUCCCuACUC
 33






GGcudTsdT


UdTsdT



 


HFE2
AD-47397
 193
A-98857.1
GcuGGAGAAuuGGAu
708
A-98858.1
UGCuAUCcAAUUCUCcAG
753






AGcAdTsdT


CdTsdT



 


HFE2
AD-47403
 195
A-98859.1
uGGAGAAuuGGAuAG
709
A-98860.1
UCUGCuAUCcAAUUCUCc
754






cAGAdTsdT


AdTsdT



 


HFE2
AD-47409
 199
A-98861.1
GAAuuGGAuAGcAGA
710
A-98862.1
UuACUCUGCuAUCcAAUU
755






GuAAdTsdT


CdTsdT



 


HFE2
AD-47415
 200
A-98863.1
AAuuGGAuAGcAGAG
711
A-98864.1
AUuACUCUGCuAUCcAAU
756






uAAudTsdT


UdTsdT



 


HFE2
AD-47421
 206
A-98865.1
AuAGcAGAGuAAuGu
712
A-98866.1
UcAAAcAUuACUCUGCuA
757






uuGAdTsdT


UdTsdT



 


HFE2
AD-47427
 211
A-98867.1
AGAGuAAuGuuuGAc
713
A-98868.1
AGAGGUcAAAcAUuACUC
758






cucudTsdT


UdTsdT



 


HFE2
AD-47433
 244
A-98869.1
ucAuAuuuAAGAAcA
714
A-98870.1
UGcAUGUUCUuAAAuAUG
759






uGcAdTsdT


AdTsdT



 


HFE2
AD-47392
 257
A-98871.1
cAuGcAGGAAuGcAu
715
A-98872.1
AUcAAUGcAUUCCUGcAU
760






uGAudTsdT


GdTsdT



 


HFE2
AD-47398
 261
A-98873.1
cAGGAAuGcAuuGAu
716
A-98874.1
UCUGAUcAAUGcAUUCCU
761






cAGAdTsdT


GdTsdT



 


HFE2
AD-47404
 290
A-98875.1
GGcuGAGGuGGAuAA
717
A-98876.1
AAGAUuAUCcACCUcAGC
762






ucuudTsdT


CdTsdT



 


HFE2
AD-47410
 360
A-98877.1
uccAGuuuGucGAuu
718
A-98878.1
UUUGAAUCGAcAAACUGG
763






cAAAdTsdT


AdTsdT



 


HFE2
AD-47416
 367
A-98879.1
uGucGAuucAAAcuG
719
A-98880.1
UuAGcAGUUUGAAUCGAc
764






cuAAdTsdT


AdTsdT



 


HFE2
AD-47422
 404
A-98881.1
GAuccAAGcuGccuA
720
A-98882.1
AAUGuAGGcAGCUUGGAU
765






cAuudTsdT


CdTsdT



 


HFE2
AD-47428
 415
A-98883.1
ccuAcAuuGGcAcAA
721
A-98884.1
AuAGUUGUGCcAAUGuAG
766






cuAudTsdT


GdTsdT



 


HFE2
AD-47434
 417
A-98885.1
uAcAuuGGcAcAAcu
722
A-98886.1
UuAuAGUUGUGCcAAUGu
767






AuAAdTsdT


AdTsdT



 


HFE2
AD-47393
 472
A-98887.1
ucAAGGuAGcAGAGG
723
A-98888.1
AcAUCCUCUGCuACCUUG
768






AuGudTsdT


AdTsdT



 


HFE2
AD-47399
 585
A-98889.1
GGAGcuAuAAccAuu
724
A-98890.1
uAUcAAUGGUuAuAGCUC
769






GAuAdTsdT


CdTsdT



 


HFE2
AD-47405
 587
A-98891.1
AGcuAuAAccAuuGA
725
A-98892.1
AGuAUcAAUGGUuAuAGC
770






uAcudTsdT


UdTsdT



 


HFE2
AD-47417
 638
A-98895.1
GGAAGAuGcuuAcuu
726
A-98896.1
AUGGAAGuAAGcAUCUUC
771






ccAudTsdT


CdTsdT



 


HFE2
AD-47423
 642
A-98897.1
GAuGcuuAcuuccAu
727
A-98898.1
AGGAAUGGAAGuAAGcAU
772






uccudTsdT


CdTsdT



 


HFE2
AD-47429
 646
A-98899.1
cuuAcuuccAuuccu
728
A-98900.1
AcAcAGGAAUGGAAGuAA
773






GuGudTsdT


GdTsdT



 


HFE2
AD-47435
 656
A-98901.1
uuccuGuGucuuuGA
729
A-98902.1
AAcAUcAAAGAcAcAGGA
774






uGuudTsdT


AdTsdT



 


HFE2
AD-47394
 657
A-98903.1
uccuGuGucuuuGAu
730
A-98904.1
AAAcAUcAAAGAcAcAGG
775






GuuudTsdT


AdTsdT



 


HFE2
AD-47400
 678
A-98905.1
AuuucuGGuGAuccc
731
A-98906.1
AGUUGGGAUcACcAGAAA
776






AAcudTsdT


UdTsdT



 


HFE2
AD-47406
1121
A-98907.1
ccAuuuAcuGcAGAu
732
A-98908.1
UGAAAUCUGcAGuAAAUG
777






uucAdTsdT


GdTsdT



 


HFE2
AD-47412
1151
A-98909.1
uuAGAGGucAuGAAG
733
A-98910.1
AAACCUUcAUGACCUCuA
778






GuuudTsdT


AdTsdT



 


HFE2
AD-47418
1152
A-98911.1
uAGAGGucAuGAAGG
734
A-98912.1
AAAACCUUcAUGACCUCu
779






uuuudTsdT


AdTsdT



 


HFE2
AD-47424
1203
A-98913.1
uuAAGAGGcAAGAGc
735
A-98914.1
UUcAGCUCUUGCCUCUuA
780






uGAAdTsdT


AdTsdT



 


HFE2
AD-47430
1228
A-98915.1
AGAcAuGAucAuuAG
736
A-98916.1
AUGGCuAAUGAUcAUGUC
781






ccAudTsdT


UdTsdT



 


HFE2
AD-47436
1230
A-98917.1
AcAuGAucAuuAGcc
737
A-98918.1
UuAUGGCuAAUGAUcAUG
782






AuAAdTsdT


UdTsdT



 


HFE2
AD-47395
1233
A-98919.1
uGAucAuuAGccAuA
738
A-98920.1
UUCUuAUGGCuAAUGAUc
783






AGAAdTsdT


AdTsdT



 


HFE2
AD-47401
1272
A-98921.1
AuuAGGGAAAGAAGu
739
A-98922.1
AuAGACUUCUUUCCCuAA
784






cuAudTsdT


UdTsdT



 


HFE2
AD-47407
1273
A-98923.1
uuAGGGAAAGAAGuc
740
A-98924.1
AAuAGACUUCUUUCCCuA
785






uAuudTsdT


AdTsdT



 


HFE2
AD-51740
1273
A-107281.4
uuAGGGAAAGAAGuC
740
A-107275.3
AAuAGACUUCUUUCCCuA
785






uAuUdTsdT


adTsdT



 


HFE2
AD-51747
1273
A-107280.6
uuAGGGAAAGAAGuc
740
A-107277.2
AAuAGACUuCUuUCcCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51744
1273
A-107281.5
uuAGGGAAAGAAGuC
740
A-107276.3
AAuAGACUuCUuUCCCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51731
1273
A-107280.2
uuAGGGAAAGAAGuc
740
A-107273.2
AAuAGACUUCUuUCCCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51736
1273
A-107281.3
uuAGGGAAAGAAGuC
740
A-107274.3
AAuAGACUUCUUUCcCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51732
1273
A-107281.2
uuAGGGAAAGAAGuC
740
A-107273.3
AAuAGACUUCUuUCCCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51734
1273
A-98923.4
uuAGGGAAAGAAGuc
740
A-107274.1
AAuAGACUUCUUUCcCuA
785






uAuudTsdT


AdTsdT



 


HFE2
AD-51748
1273
A-107281.6
uuAGGGAAAGAAGuC
740
A-107277.3
AAuAGACUuCUuUCcCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51735
1273
A-107280.3
uuAGGGAAAGAAGuc
740
A-107274.2
AAuAGACUUCUUUCcCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51749
1273
A-107282.6
uuAgGGAAAGAAGuC
740
A-107277.4
AAuAGACUuCUuUCcCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51752
1273
A-107281.7
uuAGGGAAAGAAGuC
740
A-107278.3
AAuAGACUuCUuUCcCuA
785






uAuUdTsdT


adTsdT



 


HFE2
AD-51738
1273
A-98923.5
uuAGGGAAAGAAGuc
740
A-107275.1
AAuAGACUUCUUUCCCuA
785






uAuudTsdT


adTsdT



 


HFE2
AD-51730
1273
A-98923.3
uuAGGGAAAGAAGuc
740
A-107273.1
AAuAGACUUCUuUCCCuA
785






uAuudTsdT


AdTsdT



 


HFE2
AD-51745
1273
A-107282.5
uuAgGGAAAGAAGuC
740
A-107276.4
AAuAGACUuCUuUCCCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51737
1273
A-107282.3
uuAgGGAAAGAAGuC
740
A-107274.4
AAuAGACUUCUUUCcCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51743
1273
A-107280.5
uuAGGGAAAGAAGuc
740
A-107276.2
AAuAGACUuCUuUCCCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51751
1273
A-107280.7
uuAGGGAAAGAAGuc
740
A-107278.2
AAuAGACUuCUuUCcCUA
785






uAuUdTsdT


adTsdT



 


HFE2
AD-51750
1273
A-98923.8
uuAGGGAAAGAAGuc
740
A-107278.1
AAuAGACUuCUuUCcCuA
785






uAuudTsdT


adTsdT



 


HFE2
AD-51741
1273
A-107282.4
uuAgGGAAAGAAGuC
740
A-107275.4
AAuAGACUUCUUUCCCuA
785






uAuUdTsdT


adTsdT



 


HFE2
AD-51742
1273
A-98923.6
uuAGGGAAAGAAGuc
740
A-107276.1
AAuAGACUuCUuUCCCuA
785






uAuudTsdT


AdTsdT



 


HFE2
AD-51733
1273
A-107082.2
uuAgGGAAAGAAGuC
740
A-107273.4
AAuAGACUUCUuUCCCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51755
1273
A-107280.8
uuAGGGAAAGAAGuc
740
A-107279.2
AAUAGACUuCUuUCcCuA
785






uAuUdTsdT


adTsdT



 


HFE2
AD-51756
1273
A-107281.8
uuAGGGAAAGAAGuC
740
A-107279.3
AAUAGACUuCUuUCcCuA
785






uAuUdTsdT


adTsdT



 


HFE2
AD-51728
1273
A-107281.1
uuAGGGAAAGAAGuC
740
A-107272.3
AAuAGACUuCUUUCCCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51729
1273
A-107282.1
uuAgGGAAAGAAGuC
740
A-107272.4
AAuAGACUuCUUUCCCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51726
1273
A-98923.2
uuAGGGAAAGAAGuc
740
A-107272.1
AAuAGACUuCUUUCCCuA
785






uAuudTsdT


AdTsdT



 


HFE2
AD-51746
1273
A-98923.7
uuAGGGAAAGAAGuc
740
A-107277.1
AAuAGACUuCUuUCcCuA
785






uAuudTsdT


AdTsdT



 


HFE2
AD-51757
1273
A-107282.8
uuAgGGAAAGAAGuC
740
A-107279.4
AAUAGACUuCUuUCcCuA
785






uAuUdTsdT


adTsdT



 


HFE2
AD-51727
1273
A-107280.1
uuAGGGAAAGAAGuc
740
A-107272.2
AAuAGACUuCUUUCCCuA
785






uAuUdTsdT


AdTsdT



 


HFE2
AD-51753
1273
A-107282.7
uuAgGGAAAGAAGuC
740
A-107278.4
AAuAGACUuCUuUCcCuA
785






uAuUdTsdT


adTsdT



 


HFE2
AD-51754
1273
A-98923.9
uuAGGGAAAGAAGuc
740
A-107279.1
AAUAGACUuCUuUCcCuA
785






uAuudTsdT


adTsdT



 


HFE2
AD-51739
1273
A-107280.4
uuAGGGAAAGAAGuc
740
A-107275.2
AAuAGACUUCUUUCCCuA
785






uAuUdTsdT


adTsdT



 


HFE2
AD-47413
1274
A-98925.1
uAGGGAAAGAAGucu
741
A-98926.1
AAAuAGACUUCUUUCCCu
786






AuuudTsdT


AdTsdT



 


HFE2
AD-47419
1279
A-98927.1
AAAGAAGucuAuuuG
742
A-98928.1
UcAUcAAAuAGACUUCUU
787






AuGAdTsdT


UdTsdT



 


HFE2
AD-47425
1280
A-98929.1
AAGAAGucuAuuuGA
743
A-98930.1
UUcAUcAAAuAGACUUCU
788






uGAAdTsdT


UdTsdT



 


HFE2
AD-47431
1303
A-98931.1
uGuGuGuAAGGuAuG
744
A-98932.1
AGAAcAuACCUuAcAcAc
789






uucudTsdT


AdTsdT



 


HFE2
AD-47437
1366
A-98933.1
GuGAAGGGAGucucu
745
A-98934.1
AAGcAGAGACUCCCUUcA
790






GcuudTsdT


CdTsdT



 


HFE2
AD-47396
1367
A-98935.1
uGAAGGGAGucucuG
746
A-98936.1
AAAGcAGAGACUCCCUUc
791






cuuudTsdT


AdTsdT



 


HFE2
AD-47402
1396
A-98937.1
cAcAGGuAGGAcAGA
747
A-98938.1
uACUUCUGUCCuACCUGU
792






AGuAdTsdT


GdTsdT



 


HFE2
AD-47408
1397
A-98939.1
AcAGGuAGGAcAGAA
748
A-98940.1
AuACUUCUGUCCuACCUG
793






GuAudTsdT


UdTsdT



 


HFE2
AD-47414
1399
A-98941.1
AGGuAGGAcAGAAGu
749
A-98942.1
UGAuACUUCUGUCCuACC
794






AucAdTsdT


UdTsdT



 


HFE2
AD-47420
1400
A-98943.1
GGuAGGAcAGAAGuA
750
A-98944.1
AUGAuACUUCUGUCCuAC
795






ucAudTsdT


CdTsdT



 


HFE2
AD-47426
1404
A-98945.1
GGAcAGAAGuAucAu
751
A-98946.1
AGGGAUGAuACUUCUGUC
796






cccudTsdT


CdTsdT



 


HFE2
AD-47432
1441
A-98947.1
uAuuAAAGcuAcAAA
752
A-98948.1
AGAAUUUGuAGCUUuAAu
797






uucudTsdT


AdTsdT






It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention.





















TABLE 10B







Start
Sense

SEQ
Antisense

SEQ


Target
Duplex ID
Position
Name
Sense Sequence
ID NO:
Name
Antisense Sequence
ID NO:







TFR2
AD-47814
  64
A-99594.1
uccAGAGAGcGc
798
A-99595.1
AGUUGUUGCGCU
841






AAcAAcudTsdT


CUCUGGAdTsdT



 


TFR2
AD-47820
  66
A-99596.1
cAGAGAGcGcAA
799
A-99597.1
AcAGUUGUUGCG
842






cAAcuGudTsdT


CUCUCUGdTsdT



 


TFR2
AD-47826
 239
A-99598.1
cAGGcAGccAAA
 35
A-99599.1
AAUGAGGUUUGG
 38






ccucAuudTsdT


CUGCCUGdTsdT



 


TFR2
AD-47819
 772
A-99674.1
AGcuGGuGuAcG
800
A-99675.1
uAGUGGGCGuAc
843






cccAcuAdTsdT


ACcAGCUdTsdT



 


TFR2
AD-47832
 884
A-99600.1
ccAGAAGGuGAc
801
A-99601.1
AGcAUUGGUcAC
844






cAAuGcudTsdT


CUUCUGGdTsdT



 


TFR2
AD-47838
 886
A-99602.1
AGAAGGuGAccA
802
A-99603.1
UGAGcAUUGGUc
845






AuGcucAdTsdT


ACCUUCUdTsdT



 


TFR2
AD-47844
 915
A-99604.1
GcucAAGGAGuG
803
A-99605.1
AuAUGAGcACUC
856






cucAuAudTsdT


CUUGAGCdTsdT



 


TFR2
AD-47849
 916
A-99606.1
cucAAGGAGuGc
804
A-99607.1
uAuAUGAGcACU
847






ucAuAuAdTsdT


CCUUGAGdTsdT



 


TFR2
AD-47854
 920
A-99608.1
AGGAGuGcucAu
805
A-99609.1
UGGGuAuAUGAG
848






AuAcccAdTsdT


cACUCCUdTsdT



 


TFR2
AD-47815
 922
A-99610.1
GAGuGcucAuAu
806
A-99611.1
UCUGGGuAuAUG
849






AcccAGAdTsdT


AGcACUCdTsdT



 


TFR2
AD-47825
1004
A-99676.1
AcAuGuGcAccu
807
A-99677.1
AGUUCCcAGGUG
850






GGGAAcudTsdT


cAcAUGUdTsdT



 


TFR2
AD-47821
1048
A-99612.1
cuuccuucAAuc
808
A-99613.1
UGGGUUUGAUUG
851






AAAcccAdTsdT


AAGGAAGdTsdT



 


TFR2
AD-47827
1050
A-99614.1
uccuucAAucAA
809
A-99615.1
ACUGGGUUUGAU
852






AcccAGudTsdT


UGAAGGAdTsdT



 


TFR2
AD-47833
1051
A-99616.1
ccuucAAucAAA
 47
A-99617.1
AACUGGGUUUGA
 48






cccAGuudTsdT


UUGAAGGdTsdT



 


TFR2
AD-51696
1051
A-107271.3
ccuucAAucAAA
 47
A-107257.2
AACUGGGUUUGA
 48






cccAGuUdTsdT


uUGAAGGdTsdT



 


TFR2
AD-51708
1051
A-107271.5
ccuucAAucAAA
 47
A-107259.2
AACUGGGUuUGA
 48






cccAGuUdTsdT


uUGAAGGdTsdT



 


TFR2
AD-51700
1051
A-99616.5
ccuucAAucAAA
 47
A-107258.1
AACUGGGUuUGA
 48






cccAGuudTsdT


UUGAAGGdTsdT



 


TFR2
AD-51701
1051
A-99616.13
ccuucAAucAAA
 47
A-107266.1
AACuGGGUuUGA
 48






cccAGuudTsdT


UUGAAGGdTsdT



 


TFR2
AD-51702
1051
A-107271.4
ccuucAAucAAA
 47
A-107258.2
AACUGGGUuUGA
 48






cccAGuUdTsdT


UUGAAGGdTsdT



 


TFR2
AD-51707
1051
A-99616.14
ccuucAAucAAA
 47
A-107267.1
AACuGGGUuUGA
 48






cccAGuudTsdT


uUGAAGGdTsdT



 


TFR2
AD-51694
1051
A-99616.4
ccuucAAucAAA
 47
A-107257.1
AACUGGGUUUGA
 48






cccAGuudTsdT


uUGAAGGdTsdT



 


TFR2
AD-51706
1051
A-99616.6
ccuucAAucAAA
 47
A-107259.1
AACUGGGUuUGA
 48






cccAGuudTsdT


uUGAAGGdTsdT



 


TFR2
AD-51695
1051
A-99616.12
ccuucAAucAAA
 47
A-107265.1
AACuGGGUUUGA
 48






cccAGuudTsdT


uUGAAGGdTsdT



 


TFR2
AD-51713
1051
A-99616.15
ccuucAAucAAA
 47
A-107268.1
AACuGGGUuUGA
 48






cccAGuudTsdT


uuGAAGGdTsdT



 


TFR2
AD-51714
1051
A-107271.6
ccuucAAucAAA
 47
A-107260.1
AACUGGGUuUGA
 48






cccAGuUdTsdT


uuGAAGGdTsdT



 


TFR2
AD-51683
1051
A-99616.10
ccuucAAucAAA
 47
A-107263.1
AACuGGGUUUGA
 48






cccAGuudTsdT


UUGAAGgdTsdT



 


TFR2
AD-51712
1051
A-99616.7
ccuucAAucAAA
 47
A-107260.1
AACUGGGUuUGA
 48






cccAGuudTsdT


uuGAAGGdTsdT



 


TFR2
AD-51720
1051
A-107271.7
ccuucAAucAAA
 47
A-107261.2
AACUGGGUuUGA
 48






cccAGuUdTsdT


uuGAAGgdTsdT



 


TFR2
AD-51719
1051
A-99616.16
ccuucAAucAAA
 47
A-107269.1
AACuGGGUuUGA
 48






cccAGuudTsdT


uuGAAGgdTsdT



 


TFR2
AD-51684
1051
A-107271.1
ccuucAAucAAA
 47
A-107255.2
AACUGGGUUUGA
 48






cccAGuUdTsdT


UUGAAGgdTsdT



 


TFR2
AD-51690
1051
A-107271.2
ccuucAAucAAA
 47
A-107256.2
AACUGGGUUUGA
 48






cccAGuUdTsdT


UuGAAGGdTsdT



 


TFR2
AD-51689
1051
A-99616.11
ccuucAAucAAA
 47
A-107264.1
AACuGGGUUUGA
 48






cccAGuudTsdT


UuGAAGGdTsdT



 


TFR2
AD-51682
1051
A-99616.2
ccuucAAucAAA
 47
A-107255.1
AACUGGGUUUGA
 48






cccAGuudTsdT


UUGAAGgdTsdT



 


TFR2
AD-51688
1051
A-99616.3
ccuucAAucAAA
 47
A-107256.1
AACUGGGUUUGA
 48






cccAGuudTsdT


UuGAAGGdTsdT



 


TFR2
AD-51718
1051
A-99616.8
ccuucAAucAAA
 47
A-107261.1
AACUGGGUuUGA
 48






cccAGuudTsdT


uuGAAGgdTsdT



 


TFR2
AD-51725
1051
A-99616.17
ccuucAAucAAA
 47
A-107270.1
AACuGGGUuUGA
 48






cccAGuudTsdT


UuGAAGgdTsdT



 


TFR2
AD-51724
1051
A-99616.9
ccuucAAucAAA
 47
A-107262.1
AACUGGGUuUGA
 48






cccAGuudTsdT


UuGAAGgdTsdT



 


TFR2
AD-47839
1067
A-99618.1
GuucccuccAGu
810
A-99619.1
UGAUGcAACUGG
853






uGcAucAdTsdT


AGGGAACdTsdT



 


TFR2
AD-47845
1068
A-99620.1
uucccuccAGuu
811
A-99621.1
AUGAUGcAACUG
854






GcAucAudTsdT


GAGGGAAdTsdT



 


TFR2
AD-47850
1299
A-99622.1
cGcucAGAGccA
812
A-99623.1
AGUGAUCUGGCU
855






GAucAcudTsdT


CUGAGCGdTsdT



 


TFR2
AD-47855
1355
A-99624.1
AGGAGcAGcuAA
813
A-99625.1
AGCGGAUUuAGC
856






AuccGcudTsdT


UGCUCCUdTsdT



 


TFR2
AD-47816
1441
A-99626.1
cccGcAGAAGuc
814
A-99267.1
AAGAGGAGACUU
857






uccucuudTsdT


CUGCGGGdTsdT



 


TFR2
AD-47831
1548
A-99678.1
GuGuAcGuGAGc
815
A-99679.1
UGUCcAGGCUcA
858






cuGGAcAdTsdT


CGuAcACdTsdT



 


TFR2
AD-47822
1584
A-99628.1
GAcAAGuuucAu
816
A-99629.1
UCUUGGcAUGAA
859






GccAAGAdTsdT


ACUUGUCdTsdT



 


TFR2
AD-47828
1612
A-99630.1
uucuGAcAAGuc
817
A-99631.1
UcAAUGAGACUU
860






ucAuuGAdTsdT


GUcAGAAdTsdT



 


TFR2
AD-47834
1614
A-99632.1
cuGAcAAGucuc
818
A-99633.1
UCUcAAUGAGAC
861






AuuGAGAdTsdT


UUGUcAGdTsdT



 


TFR2
AD-47840
1616
A-99634.1
GAcAAGucucAu
819
A-99635.1
ACUCUcAAUGAG
862






uGAGAGudTsdT


ACUUGUCdTsdT



 


TFR2
AD-47846
1618
A-99636.1
cAAGucucAuuG
820
A-99637.1
AcACUCUcAAUG
863






AGAGuGudTsdT


AGACUUGdTsdT



 


TFR2
AD-47851
2140
A-99638.1
AGcGAcuGAcAc
821
A-99639.1
uAcAUGCGUGUc
864






GcAuGuAdTsdT


AGUCGCUdTsdT



 


TFR2
AD-47856
2142
A-99640.1
cGAcuGAcAcGc
822
A-99641.1
UGuAcAUGCGUG
865






AuGuAcAdTsdT


UcAGUCGdTsdT



 


TFR2
AD-47817
2143
A-99642.1
GAcuGAcAcGcA
823
A-99643.1
UUGuAcAUGCGU
866






uGuAcAAdTsdT


GUcAGUCdTsdT



 


TFR2
AD-47823
2146
A-99644.1
uGAcAcGcAuGu
824
A-99645.1
ACGUUGuAcAUG
867






AcAAcGudTsdT


CGUGUcAdTsdT



 


TFR2
AD-47837
2151
A-99680.1
cGcAuGuAcAAc
825
A-99681.1
UGCGcACGUUGu
868






GuGcGcAdTsdT


AcAUGCGdTsdT



 


TFR2
AD-47843
2152
A-99682.1
GcAuGuAcAAcG
826
A-99683.1
AUGCGcACGUUG
869






uGcGcAudTsdT


uAcAUGCdTsdT



 


TFR2
AD-47829
2154
A-99646.1
AuGuAcAAcGuG
827
A-99647.1
UuAUGCGcACGU
870






cGcAuAAdTsdT


UGuAcAUdTsdT



 


TFR2
AD-47835
2155
A-99648.1
uGuAcAAcGuGc
828
A-99649.1
AUuAUGCGcACG
871






GcAuAAudTsdT


UUGuAcAdTsdT



 


TFR2
AD-47841
2170
A-99650.1
uAAuGcGGGuGG
829
A-99651.1
uAGAACUCcACC
872






AGuucuAdTsdT


CGcAUuAdTsdT



 


TFR2
AD-51703
2170
A-99650.6
uAAuGcGGGuGG
829
A-107249.1
UAGAACuCcACC
872






AGuucuAdTsdT


CGcAUuAdTsdT



 


TFR2
AD-51710
2170
A-107254.2
uAAuGcGGGuGG
829
A-107246.3
UAGAACUCcACC
872






AGuUCuAdTsdT


CGcAUUAdTsdT



 


TFR2
AD-51697
2170
A-99650.5
uAAuGcGGGuGG
829
A-107248.1
UAGAACUCcACC
872






AGuucuAdTsdT


CGcAuuadTsdT



 


TFR2
AD-51692
2170
A-107253.3
uAAuGcGGGuGG
829
A-107247.2
UAGAACUCcACC
872






AGuuCuAdTsdT


CGcAUuadTsdT



 


TFR2
AD-51685
2170
A-99650.3
uAAuGcGGGuGG
829
A-107246.1
UAGAACUCcACC
872






AGuucuAdTsdT


CGcAUUAdTsdT



 


TFR2
AD-51691
2170
A-99650.4
uAAuGcGGGuGG
829
A-107247.1
UAGAACUCcACC
872






AGuucuAdTsdT


CGcAUuadTsdT



 


TFR2
AD-51698
2170
A-107253.4
uAAuGcGGGuGG
829
A-107248.2
UAGAACUCcACC
872






AGuuCuAdTsdT


CGcAuuadTsdT



 


TFR2
AD-51686
2170
A-107253.2
uAAuGcGGGuGG
829
A-107246.2
UAGAACUCcACC
872






AGuuCuAdTsdT


CGcAuuAdTsdT



 


TFR2
AD-51709
2170
A-99650.7
uAAuGcGGGuGG
829
A-107250.1
UAGAACuCcACC
872






AGuucuAdTsdT


CGcAuuAdTsdT



 


TFR2
AD-51679
2170
A-99650.2
uAAuGcGGGuGG
829
A-107245.1
UAGAACUCcACC
872






AGuucuAdTsdT


CGcAUuAdTsdT



 


TFR2
AD-51705
2170
A-107254.5
uAAuGcGGGuGG
829
A-107249.3
UAGAACuCcACC
872






AGuUCuAdTsdT


CGcAUuAdTsdT



 


TFR2
AD-51704
2170
A-107254.1
uAAuGcGGGuGG
829
A-107245.3
UAGAACUCcACC
872






AGuUCuAdTsdT


CGcAUuAdTsdT



 


TFR2
AD-51687
2170
A-107253.6
uAAuGcGGGuGG
829
A-107250.2
UAGAACuCcACC
872






AGuuCuAdTsdT


CGcAuuAdTsdT



 


TFR2
AD-51681
2170
A-107253.5
uAAuGcGGGuGG
829
A-107249.2
UAGAACuCcACC
872






AGuuCuAdTsdT


CGcAUuAdTsdT



 


TFR2
AD-51716
2170
A-107254.3
uAAuGcGGGuGG
829
A-107247.3
UAGAACUCcACC
872






AGuUCuAdTsdT


CGcAUuadTsdT



 


TFR2
AD-51693
2170
A-107253.7
uAAuGcGGGuGG
829
A-107251.2
UAGAACuCcACC
872






AGuuCuAdTsdT


CGcUAuadTsdT



 


TFR2
AD-51711
2170
A-107254.6
uAAuGcGGGuGG
829
A-107250.3
UAGAACuCcACC
872






AGuUCuAdTsdT


CGcAuuAdTsdT



 


TFR2
AD-51699
2170
A-107253.8
uAAuGcGGGuGG
829
A-107252.2
UAGAACuCcACC
872






AGuuCuAdTsdT


CGcAuuadTsdT



 


TFR2
AD-51722
2170
A-107254.4
uAAuGcGGGuGG
829
A-107248.3
UAGAACUCcACC
872






AGuUCuAdTsdT


CGcAuuadTsdT



 


TFR2
AD-51715
2170
A-99650.8
uAAuGcGGGuGG
829
A-107251.1
UAGAACuCcACC
872






AGuucuAdTsdT


CGcAUuadTsdT



 


TFR2
AD-51680
2170
A-107253.1
uAAuGcGGGuGG
829
A-107245.2
UAGAACUCcACC
872






AGuuCuAdTsdT


CGcAUuAdTsdT



 


TFR2
AD-51717
2170
A-107254.7
uAAuGcGGGuGG
829
A-107251.3
UAGAACuCcACC
872






AGuUCuAdTsdT


CGcAUuadTsdT



 


TFR2
AD-51723
2170
A-107254.8
uAAuGcGGGuGG
829
A-107252.3
UAGAACuCcACC
872






AGuUCuAdTsdT


CGcAuuadTsdT



 


TFR2
AD-51721
2170
A-99650.9
uAAuGcGGGuGG
829
A-107252.1
UAGAACuCcACC
872






AGuucuAdTsdT


CGcAuuadTsdT



 


TFR2
AD-47847
2178
A-99652.1
GuGGAGuucuAc
830
A-99653.1
AAAGGAAGuAGA
873






uuccuuudTsdT


ACUCcACdTsdT



 


TFR2
AD-47852
2224
A-99654.1
cGuuccGccAcA
831
A-99655.1
AUGAAGAUGUGG
874






ucuucAudTsdT


CGGAACGdTsdT



 


TFR2
AD-47857
2425
A-99656.1
GGAAcAuuGAuA
832
A-99657.1
AAGUUGUuAUcA
875






AcAAcuudTsdT


AUGUUCCdTsdT



 


TFR2
AD-47818
2602
A-99658.1
cAGcAcAGAuAu
833
A-99659.1
UGUGUGGAuAUC
876






ccAcAcAdTsdT


UGUGCUGdTsdT



 


TFR2
AD-47824
2656
A-99660.1
GGucAuAcuGuc
834
A-99661.1
AUuAACCGAcAG
877






GGuuAAudTsdT


uAUGACCdTsdT



 


TFR2
AD-47830
2658
A-99662.1
ucAuAcuGucGG
835
A-99663.1
UGAUuAACCGAc
878






uuAAucAdTsdT


AGuAUGAdTsdT



 


TFR2
AD-47836
2660
A-99664.1
AuAcuGucGGuu
836
A-99665.1
UCUGAUuAACCG
879






AAucAGAdTsdT


AcAGuAUdTsdT



 


TFR2
AD-47842
2662
A-99666.1
AcuGucGGuuAA
837
A-99667.1
UCUCUGAUuAAC
880






ucAGAGAdTsdT


CGAcAGUdTsdT



 


TFR2
AD-47848
2719
A-99668.1
GGuccuccAuAc
838
A-99669.1
UCUCuAGGuAUG
881






cuAGAGAdTsdT


GAGGACCdTsdT



 


TFR2
AD-47853
2795
A-99670.1
ucGcuGGcAccA
839
A-99671.1
AAGGCuAUGGUG
882






uAGccuudTsdT


CcAGCGAdTsdT



 


TFR2
AD-47858
2802
A-99672.1
cAccAuAGccuu
840
A-99673.1
UGGCcAuAAGGC
883






AuGGccAdTsdT


uAUGGUGdTsdT





It should be noted that unmodified versions of each on the modified sequences shown are included within the scope of the invention.













TABLE 11







Secondary Target single-dose












Start
10 nM overall
0.1 nM overall
0.01 nM overall
















Target
Reactivity
Duplex Name
Position
Avg
SD
Avg
SD
Avg
SD



















HFE2
Human
AD-47391
177
97.5
10.8
111.9
21.2




HFE2
Human
AD-47397
193
27.3
4.2
36.9
3.3




HFE2
Human
AD-47403
195
31.2
10.0
48.6
7.6




HFE2
Human
AD-47409
199
82.3
15.8
89.5
11.4




HFE2
Human
AD-47415
200
44.8
5.9
51.1
7.0




HFE2
Human
AD-47421
206
27.8
8.1
28.8
0.9




HFE2
Human
AD-47427
211
96.4
25.8
79.8
18.2




HFE2
Human
AD-47433
244
7.5
1.3
21.2
4.0




HFE2
Human
AD-47392
257
8.6
2.0
20.5
8.1




HFE2
Human
AD-47398
261
30.0
6.5
45.9
6.4




HFE2
Human
AD-47404
290
9.3
2.8
20.5
0.5




HFE2
Human
AD-47410
360
28.7
9.8
36.7
1.8




HFE2
Human
AD-47416
367
72.3
32.5
79.2
19.3




HFE2
Human
AD-47422
404
20.4
2.5
35.4
2.5




HFE2
Human
AD-47428
415
66.8
22.5
80.6
11.4




HFE2
Human
AD-47434
417
34.7
5.9
28.6
3.4




HFE2
Human
AD-47393
472
96.3
9.7
99.8
31.3




HFE2
Human
AD-47399
585
10.0
6.6
16.3
0.6




HFE2
Human
AD-47405
587
11.3
2.1
14.0
0.4




HFE2
Human
AD-47417
638
39.3
2.0
62.6
7.6




HFE2
Human
AD-47423
642
109.4
4.1
58.5
0.9




HFE2
Human
AD-47429
646
56.0
13.0
76.3
21.8




HFE2
Human
AD-47435
656
17.7
1.4
29.3
9.4




HFE2
Human
AD-47394
657
8.8
7.3
9.8
6.3




HFE2
Human
AD-47400
678
21.2
2.8
25.1
8.4




HFE2
Human
AD-47406
1121
12.9
1.4
20.5
1.3




HFE2
Human
AD-47412
1151
16.5
5.2
11.8
3.2




HFE2
Human
AD-47418
1152
16.0
1.6
8.4
2.2




HFE2
Human
AD-47424
1203
9.2
1.6
14.0
2.4




HFE2
Human
AD-47430
1228
14.8
2.7
19.2
0.9




HFE2
Human
AD-47436
1230
17.9
9.6
19.7
1.4




HFE2
Human
AD-47395
1233
15.3
2.1
12.7
2.2




HFE2
Human
AD-47401
1272
6.3
1.2
10.5
0.9




HFE2
Human
AD-47407
1273
5.6
1.8
5.6
0.8




HFE2
Human
AD-51740
1273
5.7
0.0
6.5
0.7
6.3
0.4


HFE2
Human
AD-51747
1273
7.1
1.6
6.0
0.1
7.0
0.1


HFE2
Human
AD-51744
1273
11.8
5.8
18.4
14.1
7.7
0.0


HFE2
Human
AD-51731
1273
6.2
0.7
7.1
0.2
8.1
3.2


HFE2
Human
AD-51736
1273
6.3
0.3
7.2
0.7
8.2
0.5


HFE2
Human
AD-51732
1273
6.0
1.0
8.2
0.5
8.3
0.6


HFE2
Human
AD-51734
1273
6.9
0.3
14.5
13.3
8.4
1.4


HFE2
Human
AD-51748
1273
6.6
0.2
7.7
0.9
8.5
1.3


HFE2
Human
AD-51735
1273
6.4
1.5
6.3
0.3
8.5
0.7


HFE2
Human
AD-51749
1273
6.8
1.0
8.3
0.4
8.7
2.2


HFE2
Human
AD-51752
1273
12.7
6.4
10.3
3.6
8.8
1.0


HFE2
Human
AD-51738
1273
5.8
0.6
9.2
3.0
8.9
1.4


HFE2
Human
AD-51730
1273
7.6
1.7
7.8
1.0
9.3
0.5


HFE2
Human
AD-51745
1273
5.8
0.4
6.5
1.6
9.5
1.2


HFE2
Human
AD-51737
1273
5.9
0.1
19.8
18.4
9.6
1.3


HFE2
Human
AD-51743
1273
6.5
1.6
7.0
1.5
9.9
2.0


HFE2
Human
AD-51751
1273
6.4
1.4
7.5
1.6
10.3
1.6


HFE2
Human
AD-51750
1273
6.9
0.2
8.8
0.3
10.7
1.0


HFE2
Human
AD-51741
1273
6.0
2.1
8.5
1.1
10.8
4.0


HFE2
Human
AD-51742
1273
7.0
1.0
6.1
0.2
11.0
0.9


HFE2
Human
AD-51733
1273
6.7
1.1
7.2
0.1
11.0
1.3


HFE2
Human
AD-51755
1273
6.1
0.8
13.4
6.9
11.2
2.8


HFE2
Human
AD-51756
1273
9.8
0.3
8.9
0.4
11.6
0.3


HFE2
Human
AD-51728
1273
7.1
0.8
8.2
0.2
11.6
0.6


HFE2
Human
AD-51729
1273
6.8
1.2
8.9
0.4
11.7
0.5


HFE2
Human
AD-51726
1273
7.1
0.6
9.0
1.0
12.6
2.4


HFE2
Human
AD-51746
1273
7.3
1.4
14.9
6.0
12.6
5.5


HFE2
Human
AD-51757
1273
9.1
2.0
10.4
0.6
13.1
1.9


HFE2
Human
AD-51727
1273
6.7
1.0
8.5
1.1
13.8
0.4


HFE2
Human
AD-51753
1273
7.2
0.3
13.6
8.4
14.2
7.8


HFE2
Human
AD-51754
1273
6.9
0.4
10.1
1.0
14.7
2.8


HFE2
Human
AD-51739
1273
6.1
0.1
8.2
0.1
14.8
8.9


HFE2
Human
AD-47413
1274
7.2
0.2
6.4
0.9




HFE2
Human
AD-47419
1279
8.6
2.3
10.0
2.2




HFE2
Human
AD-47425
1280
14.5
1.0
14.1
0.8




HFE2
Human
AD-47431
1303
49.5
0.6
72.2
0.7




HFE2
Human
AD-47437
1366
6.4
4.2
11.4
2.4




HFE2
Human
AD-47396
1367
4.6
0.1
10.0
0.2




HFE2
Human
AD-47402
1396
11.8
0.2
19.9
4.4




HFE2
Human
AD-47408
1397
12.0
3.4
13.7
0.2




HFE2
Human
AD-47414
1399
5.6
1.5
8.2
0.1




HFE2
Human
AD-47420
1400
3.6
1.0
5.7
0.8




HFE2
Human
AD-47426
1404
13.7
3.8
27.1
3.1




HFE2
Human
AD-47432
1441
3.8
0.0
5.6
1.0




TFR2
Human
AD-47814
64
7.8
0.4
16.3
0.1




TFR2
Human
AD-47820
66
13.7
2.5
25.1
3.7




TFR2
Human
AD-47826
239
13.5
1.8
25.4
4.3




TFR2
Human
AD-47819
772
112.4
2.9
102.9
3.8




TFR2
Human
AD-47832
884
24.2
1.8
52.4
2.7




TFR2
Human
AD-47838
886
23.6
0.4
39.0
1.6




TFR2
Human
AD-47844
915
19.5
3.9
40.9
4.5




TFR2
Human
AD-47849
916
14.2
6.9
22.8
0.5




TFR2
Human
AD-47854
920
69.4
4.2
88.3
0.8




TFR2
Human
AD-47815
922
66.3
6.7
71.2
8.8




TFR2
Human
AD-47825
1004
23.9
2.9
46.2
3.8




TFR2
Human
AD-47821
1048
57.4
15.9
78.5
5.0




TFR2
Human
AD-47827
1050
18.9
8.3
37.9
2.9




TFR2
Human
AD-47833
1051
8.3
4.3
19.7
5.4




TFR2
Human
AD-51696
1051
8.0
2.1
21.1
2.1
27.2
0.5


TFR2
Human
AD-51708
1051
8.8
1.2
17.7
0.8
28.5
3.7


TFR2
Human
AD-51700
1051
9.3
1.2
19.8
3.7
30.1
5.0


TFR2
Human
AD-51701
1051
9.4
0.6
22.3
8.1
30.8
2.7


TFR2
Human
AD-51702
1051
8.7
2.1
19.7
0.1
30.9
1.4


TFR2
Human
AD-51707
1051
8.1
2.5
19.1
2.6
32.2
8.2


TFR2
Human
AD-51694
1051
9.3
1.9
19.3
2.5
38.8
0.0


TFR2
Human
AD-51706
1051
8.4
0.3
19.5
1.5
39.9
6.9


TFR2
Human
AD-51695
1051
10.1
1.6
19.9
2.4
40.1
4.2


TFR2
Human
AD-51713
1051
14.6
1.8
45.3
2.5
59.0
1.6


TFR2
Human
AD-51714
1051
22.1
0.1
44.2
1.5
62.8
1.7


TFR2
Human
AD-51683
1051
9.7
0.6
36.5
2.4
66.0
2.4


TFR2
Human
AD-51712
1051
21.2
2.6
44.1
4.5
67.1
5.9


TFR2
Human
AD-51720
1051
34.5
6.1
58.3
10.4
67.4
0.0


TFR2
Human
AD-51719
1051
38.6
1.2
57.7
1.2
68.8
3.2


TFR2
Human
AD-51684
1051
14.7
3.5
48.3
1.9
69.3
3.5


TFR2
Human
AD-51690
1051
19.7
0.0
49.8
0.5
74.1
12.6


TFR2
Human
AD-51689
1051
40.5
2.4
53.1
9.7
75.0
6.4


TFR2
Human
AD-51682
1051
12.7
1.1
42.3
10.1
75.7
6.0


TFR2
Human
AD-51688
1051
34.9
2.9
62.2
6.9
78.1
3.2


TFR2
Human
AD-51718
1051
31.6
6.6
53.1
6.5
80.2
1.3


TFR2
Human
AD-51725
1051
47.9
5.0
76.1
1.8
83.7
3.2


TFR2
Human
AD-51724
1051
52.0
1.2
66.1
32.9
87.8
14.9


TFR2
Human
AD-47839
1067
54.0
3.1
71.5
8.4




TFR2
Human
AD-47845
1068
105.7
20.1
98.0
3.0




TFR2
Human
AD-47850
1299
16.7
4.8
21.3
3.2




TFR2
Human
AD-47855
1355
64.6
0.5
66.1
8.0




TFR2
Human
AD-47816
1441
10.6
2.6
30.6
6.9




TFR2
Human
AD-47831
1548
22.8
0.2
36.6
9.5




TFR2
Human
AD-47822
1584
57.2
7.0
72.6
1.6




TFR2
Human
AD-47828
1612
38.2
5.9
61.2
9.9




TFR2
Human
AD-47834
1614
9.2
3.6
20.1
3.0




TFR2
Human
AD-47840
1616
50.1
3.7
55.6
3.8




TFR2
Human
AD-47846
1618
75.0
7.9
94.6
4.3




TFR2
Human
AD-47851
2140
94.1
0.4
101.3
10.6




TFR2
Human
AD-47856
2142
63.3
4.1
60.7
3.1




TFR2
Human
AD-47817
2143
50.2
2.7
50.3
6.5




TFR2
Human
AD-47823
2146
26.1
2.3
40.9
3.3




TFR2
Human
AD-47837
2151
119.5
21.7
89.5
6.9




TFR2
Human
AD-47843
2152
20.6
1.7
34.9
7.8




TFR2
Human
AD-47829
2154
53.4
4.1
60.3
0.5




TFR2
Human
AD-47835
2155
15.5
1.8
18.3
2.4




TFR2
Human
AD-47841
2170
26.6
1.5
24.7
2.0




TFR2
Human
AD-51703
2170
25.2
2.8
27.9
3.5
23.2
1.1


TFR2
Human
AD-51710
2170
22.1
3.4
23.1
0.5
24.0
0.6


TFR2
Human
AD-51697
2170
30.9
3.6
25.3
0.9
24.5
0.8


TFR2
Human
AD-51692
2170
23.1
1.3
24.6
1.2
24.9
6.4


TFR2
Human
AD-51685
2170
24.6
2.2
23.9
0.6
25.6
1.7


TFR2
Human
AD-51691
2170
29.1
3.2
21.3
0.2
26.4
3.7


TFR2
Human
AD-51698
2170
23.1
2.3
25.8
3.0
26.8
2.8


TFR2
Human
AD-51686
2170
20.7
2.5
24.7
0.7
27.5
1.4


TFR2
Human
AD-51709
2170
23.1
1.3
25.1
2.7
27.7
2.1


TFR2
Human
AD-51679
2170
27.4
2.2
26.4
4.3
28.3
5.1


TFR2
Human
AD-51705
2170
27.8
5.3
24.6
2.0
28.8
2.4


TFR2
Human
AD-51704
2170
23.9
2.1
26.1
0.5
29.2
4.6


TFR2
Human
AD-51687
2170
20.8
3.9
27.7
2.8
29.4
1.0


TFR2
Human
AD-51681
2170
30.0
1.8
31.2
1.8
29.5
4.7


TFR2
Human
AD-51716
2170
20.0
1.7
25.9
2.2
30.2
1.1


TFR2
Human
AD-51693
2170
26.2
0.8
26.1
1.0
30.6
0.6


TFR2
Human
AD-51711
2170
20.8
0.5
24.8
3.2
31.3
3.0


TFR2
Human
AD-51699
2170
20.9
0.7
27.3
1.5
31.7
5.1


TFR2
Human
AD-51722
2170
28.3
3.7
30.0
0.5
32.1
1.2


TFR2
Human
AD-51715
2170
22.2
6.1
30.4
0.6
34.6
1.3


TFR2
Human
AD-51680
2170
26.4
2.5
26.7
5.4
36.6
2.6


TFR2
Human
AD-51717
2170
28.2
6.2
24.6
0.2
37.2
7.7


TFR2
Human
AD-53723
2170
25.9
4.0
30.7
4.0
40.7
3.1


TFR2
Human
AD-51721
2170
30.7
1.6
28.1
0.9
40.8
0.3


TFR2
Human
AD-47847
2178
21.7
2.1
25.1
3.5




TFR2
Human
AD-47852
2224
71.4
2.2
66.7
7.1




TFR2
Human
AD-47857
2425
37.4
4.8
29.5
5.4




TFR2
Human
AD-47818
2602
48.3
4.8
50.8
4.3




TFR2
Human
AD-47824
2656
19.9
3.3
25.7
0.1




TFR2
Human
AD-47830
2658
25.8
7.7
25.8
6.4




TFR2
Human
AD-47836
2660
34.6
0.1
37.4
6.1




TFR2
Human
AD-47842
2662
39.2
6.8
26.3
1.1




TFR2
Human
AD-47848
2719
76.8
2.2
90.1
9.7




TFR2
Human
AD-47853
2795
28.1
6.3
43.7
3.8




TFR2
Human
AD-47858
2802
66.9
8.2
73.6
3.4





Data are expressed as percent of control (Mock transfected or 1955).













TABLE 12







Secondary Target does-response











Target
Reactivity
Duplex Name
Start Position
IC50 (nM)














HFE2
HumaWn
AD-47394
657
0.004


HFE2
Human
AD-47395
1233
0.011


HFE2
Human
AD-47407
1273
0.002


HFE2
Human
AD-51747
1273
0.001


HFE2
Human
AD-51736
1273
0.001


HFE2
Human
AD-51734
1273
0.001


HFE2
Human
AD-51732
1273
0.002


HFE2
Human
AD-51731
1273
0.002


HFE2
Human
AD-51744
1273
0.002


HFE2
Human
AD-51748
1273
0.002


HFE2
Human
AD-51735
1273
0.002


HFE2
Human
AD-47407
1273
0.002


HFE2
Human
AD-51740
1273
0.003


HFE2
Human
AD-47413
1274
0.003


HFE2
Human
AD-47425
1280
0.021


HFE2
Human
AD-47437
1366
0.015


HFE2
Human
AD-47396
1367
0.013


HFE2
Human
AD-47414
1399
0.005


HFE2
Human
AD-47420
1400
0.010


HFE2
Human
AD-47432
1441
0.004


TFR2
Human
AD-47814
64
0.012


TFR2
Human
AD-47820
66
0.011


TFR2
Human
AD-47826
239
0.014


TFR2
Human
AD-47849
916
0.067


TFR2
Human
AD-47833
1051
0.013


TFR2
Human
AD-51701
1051
0.015


TFR2
Human
AD-51708
1051
0.017


TFR2
Human
AD-51700
1051
0.017


TFR2
Human
AD-47833
1051
0.023


TFR2
Human
AD-51696
1051
0.024


TFR2
Human
AD-47850
1299
0.011


TFR2
Human
AD-47834
1614
0.014


TFR2
Human
AD-47835
2155
0.023


TFR2
Human
AD-47841
2170
0.009


TFR2
Human
AD-51710
2170
0.003


TFR2
Human
AD-51703
2170
0.005


TFR2
Human
AD-51697
2170
0.006


TFR2
Human
AD-51692
2170
0.010


TFR2
Human
AD-47841
2170
0.024


TFR2
Human
AD-47847
2178
0.013
















TABLE 13







TFR2 Duplex Sequences

















Start
Sense

SEQ
Antisense

SEQ


Target
Duplex ID
Position
Name
Sense Sequence
ID NO:
Name
Antisense Sequence
ID NO:





TFR2
AD-52549
 64
A-108802.1
uccAGAGAGcGc
798
A-108798.2
AGUUGUUGCGCU
841






AAcAAcUdTsdT


CUCuGGAdTsdT



 


TFR2
AD-52550
 64
A-108802.5
uccAGAGAGcGc
798
A-108803.2
AGUUGUUGCGCU
841






AAcAAcUdTsdT


CUCuGGadTsdT



 


TFR2
AD-52555
 64
A-108802.2
UccAGAGAGcGc
798
A-108799.2
AGUUGUUGCGCU
841






AAcAAcUdTsdT


CuCuGGAdTsdT



 


TFR2
AD-52556
 64
A-108802.6
uccAGAGAGcGc
798
A-108804.2
AGUUGUUGCGCU
841






AAcAAcUdTsdT


CuCuGGadTsdT



 


TFR2
AD-52561
 64
A-108802.3
uccAGAGAGcGc
798
A-108800.2
AGUUGUUGCGCu
841






AAcAAcUdTsdT


CuCuGGAdTsdT



 


TFR2
AD-52562
 64
A-108802.7
uccAGAGAGcGc
798
A-108805.2
AGUUGUUGCGCu
841






AAcAAcUdTsdT


CuCuGGadTsdT



 


TFR2
AD-52567
 64
A-108802.4
uccAGAGAGcGc
798
A-108801.2
AGUUGuUGCGCu
841






AAcAAcUdTsdT


CuCuGGAdTsdT



 


TFR2
AD-52568
 64
A-108802.8
uccAGAGAGcGc
798
A-108806.2
AGUUGuUGCGCu
841






AAcAAcUdTsdT


CuCuGGadTsdT



 


TFR2
AD-52572
 64
A-99594.2
uccAGAGAGcGc
798
A-108798.1
AGUUGUUGCGCU
841






AAcAAcudTsdT


CUCuGGAdTsdT



 


TFR2
AD-52573
 64
A-99594.6
uccAGAGAGcGc
798
A-108803.1
AGUUGUUGCGCU
841






AAcAAcudTsdT


CUCuGGadTsdT



 


TFR2
AD-52577
 64
A-99594.3
uccAGAGAGcGc
798
A-108799.1
AGUUGUUGCGCU
841






AAcAAcudTsdT


CuCuGGAdTsdT



 


TFR2
AD-52578
 64
A-99594.7
uccAGAGAGcGc
798
A-108804.1
AGUUGUUGCGCU
841






AAcAAcudTsdT


CuCuGGadTsdT



 


TFR2
AD-52582
 64
A-99594.4
uccAGAGAGcGc
798
A-108800.1
AGUUGUUGCGCu
841






AAcAAcudTsdT


CuCuGGAdTsdT



 


TFR2
AD-52583
 64
A-99594.8
uccAGAGAGcGc
798
A-108805.1
AGUUGUUGCGCu
841






AAcAAcudTsdT


CuCuGGadTsdT



 


TFR2
AD-52587
 64
A-99594.5
uccAGAGAGcGc
798
A-108801.1
AGUUGuUGCGCu
841






AAcAAcudTsdT


CuCuGGAdTsdT



 


TFR2
AD-52588
 64
A-99594.9
uccAGAGAGcGc
798
A-108806.1
AGUUGuUGCGCu
841






AAcAAcudTsdT


CuCuGGadTsdT



 


TFR2
AD-52551
239
A-108811.1
cAGGcAGccAAA
 35
A-108810.2
AAUGAGGUuUGG
 38






ccucAuUdTsdT


CuGcCuGdTsdT



 


TFR2
AD-52552
239
A-108811.3
cAGGcAGccAAA
 35
A-108816.1
AAuGAGGUuUGG
 38






ccucAuUdTsdT


CUGCcugdTsdT



 


TFR2
AD-52557
239
A-108812.1
cAGGcAGccAAA
 35
A-108810.3
AAUGAGGUuUGG
 38






ccuCAuUdTsdT


CuGcCuGdTsdT



 


TFR2
AD-52558
239
A-108812.3
cAGGcAGccAAA
 35
A-108816.2
AAuGAGGUuUGG
 38






ccuCAuUdTsdT


CUGcCugdTsdT



 


TFR2
AD-52563
239
A-108813.1
cAGGcAGccAAA
 35
A-108810.4
AAUGAGGUuUGG
 38






cCuCAuUdTsdT


CuGcCuGdTsdT



 


TFR2
AD-52564
239
A-108813.3
cAGGcAGccAAA
 35
A-108816.3
AAuGAGGUuUGG
 38






cCuCAuUdTsdT


CUGcCugdTsdT



 


TFR2
AD-52569
239
A-108814.1
cAGGcAGcCAAA
 35
A-108810.5
AAUGAGGUuUGG
 38






cCuCAuUdTsdT


CuGcCuGdTsdT



 


TFR2
AD-52570
239
A-108814.3
cAGGcAGcCAAA
 35
A-108816.4
AAuGAGGUuUGG
 38






cCuCAuUdTsdT


CUGcCugdTsdT



 


TFR2
AD-52574
239
A-99598.2
cAGGcAGccAAA
 35
A-108807.1
AAUGAGGUUUGG
 38






ccucAuudTsdT


CUGCCuGdTsdT



 


TFR2
AD-52575
239
A-108811.2
cAGGcAGccAAA
 35
A-108815.1
AAUGAGGUuUGG
 38






ccucAuUdTsdT


CUGcCugdTsdT



 


TFR2
AD-52579
239
A-99598.3
cAGGcAGccAAA
 35
A-108808.1
AAUGAGGUUUGG
 38






ccucAuudTsdT


CUGcCuGdTsdT



 


TFR2
AD-52580
239
A-108812.2
cAGGcAGccAAA
 35
A-108815.2
AAUGAGGUuUGG
 38






ccuCAuUdTsdT


CUGcCugdTsdT



 


TFR2
AD-52584
239
A-99598.4
cAGGcAGccAAA
 35
A-108809.1
AAUGAGGUUUGG
 38






ccucAuudTsdT


CuGcCuGdTsdT



 


TFR2
AD-52585
239
A-108813.2
cAGGcAGccAAA
 35
A-108815.3
AAUGAGGUuUGG
 38






cCuCAuUdTsdT


CUGcCugdTsdT



 


TFR2
AD-52589
239
A-99598.5
cAGGcAGccAAA
 35
A-108810.1
AAUGAGGUuUGG
 38






ccucAuudTsdT


CuGcCuGdTsdT



 


TFR2
AD-52590
239
A-108814.2
cAGGcAGcCAAA
 35
A-108815.4
AAUGAGGUuUGG
 38






cCuCAuUdTsdT


CUGcCugdTsdT





It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention.













TABLE 14







TFR2 Dose Response











Target
Reactivity
Duplex Name
Start Position
IC50 (nM)














TFR2
Human
AD-47814
64
0.019


TFR2
Human
AD-52549
64
0.034


TFR2
Human
AD-52572
64
0.059


TFR2
Human
AD-52550
64
0.062


TFR2
Human
AD-52573
64
0.102


TFR2
Human
AD-52570
239
0.035


TFR2
Human
AD-47826
239
0.036


TFR2
Human
AD-52590
239
0.038


TFR2
Human
AD-52574
239
0.065


TFR2
Human
AD-52558
239
0.236
















TABLE 15







SMAD4 Unmodified Duplexes


















Sense

SEQ
Antis

SEQ


Duplex Name
Start
Target
Oligo Name
Trans Seq
ID NO:
Oligo Name
Trans Seq
ID NO:


















AD-48090.1
 481
SMAD4
A-100350.1
AUGCCUGUCU
884
A-100351.1
ACAAUGCUCA
929






GAGCAUUGU


GACAGGCAU



 


AD-48091.1
 772
SMAD4
A-100366.1
AUGUUAAAUA
885
A-100367.1
UACUGACAAU
930






UUGUCAGUA


AUUUAACAU



 


AD-48092.1
 817
SMAD4
A-100382.1
UCUGUGUGAA
886
A-100383.1
UGAUAUGGAU
931






UCCAUAUCA


UCACACAGA



 


AD-48093.1
1212
SMAD4
A-100398.1
ACUUACCAUC
887
A-100399.1
UGCUGUUAUG
932






AUAACAGCA


AUGGUAAGU



 


AD-48094.1
1351
SMAD4
A-100414.1
ACAAUGAGCU
888
A-100415.1
UGGAAUGCAA
933






UGCAUUCCA


GCUCAUUGU



 


AD-48095.1
1712
SMAD4
A-100430.1
UGUUCAUAAG
889
A-100431.1
UGGGUAGAUC
934






AUCUACCCA


UUAUGAACA



 


AD-48096.1
 590
SMAD4
A-100352.1
AAAAGAUGAA
890
A-100353.1
AGAAUCCAAU
935






UUGGAUUCU


UCAUCUUUU



 


AD-48097.1
 773
SMAD4
A-100368.1
UGUUAAAUAU
891
A-100369.1
AUACUGACAA
936






UGUCAGUAU


UAUUUAACA



 


AD-48098.1
 819
SMAD4
A-100384.1
UGUGUGAAUC
892
A-100385.1
AGUGAUAUGG
937






CAUAUCACU


AUUCACACA



 


AD-48099.1
1232
SMAD4
A-100400.1
UACCACCUGG
893
A-100401.1
ACUUCCAGUC
938






ACUGGAAGU


CAGGUGGUA



 


AD-48100.1
1362
SMAD4
A-100416.1
GCAUUCCAGC
894
A-100417.1
AAAUGGGAGG
939






CUCCCAUUU


CUGGAAUGC



 


AD-48101.1
1713
SMAD4
A-100432.1
GUUCAUAAGA
895
A-100433.1
UUGGGUAGAU
940






UCUACCCAA


CUUAUGAAC



 


AD-48102.1
 602
SMAD4
A-100354.1
GGAUUCUUUA
896
A-100355.1
AGCUGUUAUU
941






AUAACAGCU


AAAGAAUCC



 


AD-48103.1
 777
SMAD4
A-100370.1
AAAUAUUGUC
897
A-100371.1
ACGCAUACUG
942






AGUAUGCGU


ACAAUAUUU



 


AD-48104.1
 820
SMAD4
A-100386.1
GUGUGAAUCC
898
A-100387.1
UAGUGAUAUG
943






AUAUCACUA


GAUUCACAC



 


AD-48105.1
1238
SMAD4
A-100402.1
CUGGACUGGA
899
A-100403.1
AGUCCUACUU
944






AGUAGGACU


CCAGUCCAG



 


AD-48106.1
1367
SMAD4
A-100418.1
CCAGCCUCCC
900
A-100419.1
AUUGGAAAUG
945






AUUUCCAAU


GGAGGCUGG



 


AD-48107.1
2816
SMAD4
A-100434.1
UAUUUCUAGG
901
A-100435.1
AACCUUGUGC
946






CACAAGGUU


CUAGAAAUA



 


AD-48108.1
 608
SMAD4
A-100356.1
UUUAAUAACA
902
A-100357.1
AGUUAUAGCU
947






GCUAUAACU


GUUAUUAAA



 


AD-48109.1
 778
SMAD4
A-100372.1
AAUAUUGUCA
903
A-100373.1
AACGCAUACU
948






GUAUGCGUU


GACAAUAUU



 


AD-48110.1
 861
SMAD4
A-100388.1
AUUGAUCUCU
904
A-100389.1
UUAAUCCUGA
949






CAGGAUUAA


GAGAUCAAU



 


AD-48111.1
1250
SMAD4
A-100404.1
UAGGACUGCA
905
A-100405.1
UGUGUAUGGU
950






CCAUACACA


GCAGUCCUA



 


AD-48112.1
1370
SMAD4
A-100420.1
GCCUCCCAUU
906
A-100421.1
AUGAUUGGAA
951






UCCAAUCAU


AUGGGAGGC



 


AD-48113.1
2984
SMAD4
A-100436.1
AAUAUUUUGG
907
A-100437.1
UAGCAGUUUC
952






AAACUGCUA


CAAAAUAUU



 


AD-48114.1
 611
SMAD4
A-100358.1
AAUAACAGCU
908
A-100359.1
UGUAGUUAUA
953






AUAACUACA


GCUGUUAUU



 


AD-48115.1
 781
SMAD4
A-100374.1
AUUGUCAGUA
909
A-100375.1
UCAAACGCAU
954






UGCGUUUGA


ACUGACAAU



 


AD-48116.1
1090
SMAD4
A-100390.1
CUGUGGCUUC
910
A-100391.1
UGACUUGUGG
955






CACAAGUCA


AAGCCACAG



 


AD-48117.1
1257
SMAD4
A-100406.1
GCACCAUACA
911
A-100407.1
AAUUAGGUGU
956






CACCUAAUU


GUAUGGUGC



 


AD-48118.1
1601
SMAD4
A-100422.1
GUUGGAAUGU
912
A-100423.1
UUCACCUUUA
957






AAAGGUGAA


CAUUCCAAC



 


AD-48119.1
3013
SMAD4
A-100438.1
UAAAUACUGU
913
A-100439.1
UUAUUCUGCA
958






GCAGAAUAA


CAGUAUUUA



 


AD-48120.1
 659
SMAD4
A-100360.1
CAUACAGAGA
914
A-100361.1
AUCCAAUGUU
959






ACAUUGGAU


CUCUGUAUG



 


AD-48121.1
 783
SMAD4
A-100376.1
UGUCAGUAUG
915
A-100377.1
AGUCAAACGC
960






CGUUUGACU


AUACUGACA



 


AD-48122.1
1137
SMAD4
A-100392.1
AGUGAAGGAC
916
A-100393.1
UCUGCAACAG
961






UGUUGCAGA


UCCUUCACU



 


AD-48123.1
1262
SMAD4
A-100408.1
AUACACACCU
917
A-100409.1
AGGCAAAUUA
962






AAUUUGCCU


GGUGUGUAU



 


AD-48124.1
1633
SMAD4
A-100424.1
UCAGGUGCCU
918
A-100425.1
UGGUCACUAA
963






UAGUGACCA


GGCACCUGA



 


AD-48125.1
 698
SMAD4
A-100362.1
UCGGAAAGGA
919
A-100363.1
AUGAGGAAAU
964






UUUCCUCAU


CCUUUCCGA



 


AD-48126.1
 784
SMAD4
A-100378.1
GUCAGUAUGC
920
A-100379.1
AAGUCAAACG
965






GUUUGACUU


CAUACUGAC



 


AD-48126.2
 784
SMAD4
A-100378.2
GUCAGUAUGC
920
A-100379.2
AAGUCAAACG
965






GUUUGACUU


CAUACUGAC



 


AD-48127.1
1207
SMAD4
A-100394.1
CAGCUACUUA
921
A-100395.1
UUAUGAUGGU
966






CCAUCAUAA


AAGUAGCUG



 


AD-48128.1
1272
SMAD4
A-100410.1
AAUUUGCCUC
922
A-100411.1
UUUGGUGGUG
967






ACCACCAAA


AGGCAAAUU



 


AD-48129.1
1650
SMAD4
A-100426.1
CACGCGGUCU
923
A-100427.1
UCUGUACAAA
968






UUGUCAAGA


GACCGCGUG



 


AD-48130.1
 771
SMAD4
A-100364.1
CAUGUUAAAU
924
A-100365.1
AUCGACAAUA
969






AUUGUCAGU


UUUAACAUG



 


AD-48131.1
 791
SMAD4
A-100380.1
UGCGUUUGAC
925
A-100381.1
ACAUUUUAAG
970






UUAAAAUGU


UCAAACGCA



 


AD-48132.1
1209
SMAD4
A-100396.1
GCUACUUACC
926
A-100397.1
UGUUAUGAUG
971






AUCAUAACA


GUAAGUAGC



 


AD-48133.1
1273
SMAD4
A-100412.1
AUUUGCCUCA
927
A-100413.1
UUUUGGUGGU
972






CCACCAAAA


GAGGCAAAU



 


AD-48134.1
1652
SMAD4
A-100428.1
CGCGGUCUUU
928
A-100429.1
ACUCUGUACA
973






GUACAGAGU


AAGACCGCG





Note


that an overhang (e.g. TT, dTsdT) can be added to the 3′end of any duplex.













TABLE 16







SMAD4 Modified Duplexes


















Sense

SEQ
Antis

SEQ


Target
Duplex Name
Start
Oligo Name
Oligo Seq
ID NO:
Oligo Name
Oligo Seq
ID NO:


















SMAD4
AD-48090.1
 481
A-100350.1
AuGccuGucuGA
 974
A-100351.1
AcAAUGCUcAGA
1020






GcAuuGudTsdT


cAGGcAUdTsdT



 


SMAD4
AD-48091.1
 772
A-100366.1
AuGuuAAAuAuu
 975
A-100367.1
uACUGAcAAuAU
1021






GucAGuAdTsdT


UuAAcAUdTsdT



 


SMAD4
AD-48092.1
 817
A-100382.1
ucuGuGuGAAuc
 976
A-100383.1
UGAuAUGGAUUc
1022






cAuAucAdTsdT


AcAcAGAdTsdT



 


SMAD4
AD-48093.1
1212
A-100398.1
AcuuAccAucAu
 977
A-100399.1
UGCUGUuAUGAU
1023






AAcAGcAdTsdT


GGuAAGUdTsdT



 


SMAD4
AD-48094.1
1351
A-100414.1
AcAAuGAGcuuG
 978
A-100415.1
UGGAAUGcAAGC
1024






cAuuccAdTsdT


UcAUUGUdTsdT



 


SMAD4
AD-48095.1
1712
A-100430.1
uGuucAuAAGAu
 979
A-100431.1
UGGGuAGAUCUu
1025






cuAcccAdTsdT


AUGAAcAdTsdT



 


SMAD4
AD-48096.1
 590
A-100352.1
AAAAGAuGAAuu
 980
A-100353.1
AGAAUCcAAUUc
1026






GGAuucudTsdT


AUCUUUUdTsdT



 


SMAD4
AD-48097.1
 773
A-100368.1
uGuuAAAuAuuG
 981
A-100369.1
AuACUGAcAAuA
1027






ucAGuAudTsdT


UUuAAcAdTsdT



 


SMAD4
AD-48098.1
 819
A-100384.1
uGuGuGAAuccA
 982
A-100385.1
AGUGAuAUGGAU
1028






uAucAcudTsdT


UcAcAcAdTsdT



 


SMAD4
AD-48099.1
1232
A-100400.1
uAccAccuGGAc
 983
A-100401.1
ACUUCcAGUCcA
1029






uGGAAGudTsdT


GGUGGuAdTsdT



 


SMAD4
AD-48100.1
1362
A-100416.1
GcAuuccAGccu
 984
A-100417.1
AAAUGGGAGGCU
1030






cccAuuudTsdT


GGAAUGCdTsdT



 


SMAD4
AD-48101.1
1713
A-100432.1
GuucAuAAGAuc
 985
A-100433.1
UUGGGuAGAUCU
1031






uAcccAAdTsdT


uAUGAACdTsdT



 


SMAD4
AD-48102.1
 602
A-100354.1
GGAuucuuuAAu
 986
A-100355.1
AGCUGUuAUuAA
1032






AAcAGcudTsdT


AGAAUCCdTsdT



 


SMAD4
AD-48103.1
 777
A-100370.1
AAAuAuuGucAG
 987
A-100371.1
ACGcAuACUGAc
1033






uAuGcGudTsdT


AAuAUUUdTsdT



 


SMAD4
AD-48104.1
 820
A-100386.1
GuGuGAAuccAu
 988
A-100387.1
uAGUGAuAUGGA
1034






AucAcuAdTsdT


UUcAcACdTsdT



 


SMAD4
AD-48105.1
1238
A-100402.1
cuGGAcuGGAAG
 989
A-100403.1
AGUCCuACUUCc
1035






uAGGAcudTsdT


AGUCcAGdTsdT



 


SMAD4
AD-48106.1
1367
A-100418.1
ccAGccucccAu
 990
A-100419.1
AUUGGAAAUGGG
1036






uuccAAudTsdT


AGGCUGGdTsdT



 


SMAD4
AD-48107.1
2816
A-100434.1
uAuuucuAGGcA
 991
A-100435.1
AACCUUGUGCCu
1037






cAAGGuudTsdT


AGAAAuAdTsdT



 


SMAD4
AD-48108.1
 608
A-100356.1
uuuAAuAAcAGc
 992
A-100357.1
AGUuAuAGCUGU
1038






uAuAAcudTsdT


uAUuAAAdTsdT



 


SMAD4
AD-48109.1
 778
A-100372.1
AAuAuuGucAGu
 993
A-100373.1
AACGcAuACUGA
1039






AuGcGuudTsdT


cAAuAUUdTsdT



 


SMAD4
AD-48110.1
 861
A-100388.1
AuuGAucucucA
 994
A-100389.1
UuAAUCCUGAGA
1040






GGAuuAAdTsdT


GAUcAAUdTsdT



 


SMAD4
AD-48111.1
1250
A-100404.1
uAGGAcuGcAcc
 995
A-100405.1
UGUGuAUGGUGc
1041






AuAcAcAdTsdT


AGUCCuAdTsdT



 


SMAD4
AD-48112.1
1370
A-100420.1
GccucccAuuuc
 996
A-100421.1
AUGAUUGGAAAU
1042






cAAucAudTsdT


GGGAGGCdTsdT



 


SMAD4
AD-48113.1
2984
A-100436.1
AAuAuuuuGGAA 
 997
A-100437.1
uAGcAGUCCCcA
1043






AcuGcuAdTsdT


AAAuAUUdTsdT



 


SMAD4
AD-48114.1
 611
A-100358.1
AAuAAcAGcuAu
 998
A-100359.1
UGuAGUuAuAGC
1044






AAcuAcAdTsdT 


UGUuAUUdTsdT



 


SMAD4
AD-48115.1
 781
A-100374.1
AuuGucAGuAuG
 999
A-100375.1
UcAAACGcAuAC
1045






cGuuuGAdTsdT


UGAcAAUdTsdT



 


SMAD4
AD-48116.1
1090
A-100390.1
cuGuGGcuuccA
1000
A-100391.1
UGACUUGUGGAA
1046






cAAGucAdTsdT


GCcAcAGdTsdT



 


SMAD4
AD-48117.1
1257
A-100406.1
GcAccAUaCaCa
1001
A-100407.1
AAUuAGGUGUGu
1047






ccuAAuudTsdT


AUGGUGCdTsdT



 


SMAD4
AD-48118.1
1601
A-100422.1
GuuGGAAuGuAA
1002
A-100423.1
UUcACCUUuAcA
1048






AGGuGAAdTsdT


UUCcAACdTsdT



 


SMAD4
AD-48119.1
3013
A-100438.1
uAAAuAcuGuGc
1003
A-100439.1
UuAUUCUGcAcA
1049






AGAAuAAdTsdT


GuAUUuAdTsdT



 


SMAD4
AD-48120.1
 659
A-100360.1
cAuAcAGAGAAc
1004
A-100361.1
AUCcAAUGUUCU
1050






AuuGGAudTsdT


CUGuAUGdTsdT



 


SMAD4
AD-48121.1
 783
A-100376.1
uGucAGuAuGcG
1005
A-100377.1
AGUcAAACGcAu
1051






uuuGAcudTsdT


ACUGAcAdTsdT



 


SMAD4
AD-48122.1
1137
A-100392.1
AGuGAAGGAcuG
1006
A-100393.1
UCUGcAAcAGUC
1052






uuGcAGAdTsdT


CUUcACUdTsdT



 


SMAD4
AD-48123.1
1262
A-100408.1
AuAcAcAccuAA
1007
A-100409.1
AGGcAAAUuAGG
1053






uuuGccudTsdT


UGUGuAUdTsdT



 


SMAD4
AD-48124.1
1633
A-100424.1
ucAGGuGccuuA
1008
A-100425.1
UGGUcACuAAGG
1054






GuGAccAdTsdT


cACCUGAdTsdT



 


SMAD4
AD-48125.1
 698
A-100362.1
ucGGAAAGGAuu
1009
A-100363.1
AUGAGGAAAUCC
1055






uccucAudTsdT


UUUCCGAdTsdT



 


SMAD4
AD-48126.1
 784
A-100378.1
GucAGuAuGcGu
1010
A-100379.1
AAGUcAAACGcA
1056






uuGAcuudTsdT


uACUGACdTsdT



 


SMAD4
AD-48126.2
 784
A-100378.2
GucAGuAuGcGu
1011
A-100379.2
AAGUcAAACGcA
1057






uuGAcuudTsdT


uACUGACdTsdT



 


SMAD4
AD-48127.1
1207
A-100394.1
cAGcuAcuuAcc
1012
A-100395.1
UuAUGAUGGuAA
1058






AucAuAAdTsdT


GuAGCUGdTsdT



 


SMAD4
AD-48128.1
1272
A-100410.1
AAuuuGccucAc
1013
A-100411.1
UUUGGUGGUGAG
1059






cAccAAAdTsdT


GcAAAUUdTsdT



 


SMAD4
AD-48120.1
1650
A-100426.1
cAcGcGGucuuu
1014
A-100427.1
UCUGuAcAAAGA
1060






GuAcAGAdTsdT


CCGCGUGdTsdT



 


SMAD4
AD-48130.1
 771
A-100364.1
cAuGuuAAAuAu
1015
A-100365.1
ACUGAcAAuAUU
1061






uGucAGudTsdT


uAAcAUGdTsdT



 


SMAD4
AD-48131.1
 791
A-100380.1
uGcGuuuGAcuu
1016
A-100381.1
AcAUUUuAAGUc
1062






AAAAuGudTsdT


AAACGcAdTsdT



 


SMAD4
AD-48132.1
1209
A-100396.1
GcuAcuuAccAu
1017
A-100397.1
UGUuAUGAUGGu
1063






cAuAAcAdTsdT


AAGuAGCdTsdT



 


SMAD4
AD-48133.1
1273
A-100412.1
AuuuGccucAcc
1018
A-100413.1
UUUUGGUGGUGA
1064






AccAAAAdTsdT


GGcAAAUdTsdT



 


SMAD4
AD-48134.1
1652
A-100428.1
cGcGGucuuuGu
1019
A-100429.1
ACUCUGuAcAAA
1065






AcAGAGudTsdT


GACCGCGdTsdT





It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention.













TABLE 17







NEO1 Unmodified Duplexes


















Sense

SEQ
Antis

SEQ


Target
Duplex Name
Start
OligoName
Trans Seq
ID NO:
Oligo Name
Trans Seq
ID NO:


















NEO1
AD-48273.1
4618
A-100622.1
CUCCGAGAGU
1066
A-100623.1
UUCAUAGCUA
1110






AGCUAUGAA


CUCUCGGAG



 


NEO1
AD-48287.1
 546
A-100564.1
GCUCUUCUGU
1067
A-100565.1
UUUAAUAUAA
1111






UAUAUUAAA


CAGAAGAGC



 


NEO1
AD-48274.1
5060
A-100638.1
GAGUGUAGAC
1068
A-100639.1
AAUGCCAAUG
1112






AUUGGCAUU


UCUACACUC



 


NEO1
AD-48309.1
4778
A-100634.1
GGAAUUGUAC
1069
A-100635.1
UCGUACUCUG
1113






AGAGUACGA


UACAAUUCC



 


NEO1
AD-48309.2
4778
A-100634.2
GGAAUUGUAC
1070
A-100635.2
UCGUACUCUG
1114






AGAGUACGA


UACAAUUCC



 


NEO1
AD-48297.1
4674
A-100630.1
GACUAAUGAA
1071
A-100631.1
UUUAGGUCCU
1115






GGACCUAAA


UCAUUAGUC



 


NEO1
AD-48296.1
4495
A-100614.1
GAACCAUCAC
1072
A-100615.1
UGAGUGAAUG
1116






AUUCACUCA


UGAUGGUUC



 


NEO1
AD-48280.1
5062
A-100640.1
GUGUAGACAU
1073
A-100641.1
UAAAUGCCAA
1117






UGGCAUUUA


UGUCUACAC



 


NEO1
AD-48275.1
 535
A-100560.1
CUCAGUUAGA
1074
A-100561.1
AGAAGAGCCU
1118






GGCUCUUCU


CUAACUGAG



 


NEO1
AD-48276.1
1283
A-100576.1
GAUGAUGCUG
1075
A-100577.1
AAUAAGUCCC
1119






GGACUUAUU


AGCAUCAUC



 


NEO1
AD-48269.1
 533
A-100558.1
CUCUCAGUUA
1075
A-100559.1
AAGAGCCUCU
1120






GAGGCUCUU


AACUGAGAG



 


NEO1
AD-48286.1
5069
A-100642.1
CAUUGGCAUU
1077
A-100643.1
UUGUACAUAA
1121






UAUGUACAA


AUGCCAAUG



 


NEO1
AD-48299.1
 791
A-100568.1
GCAGGUCUUC
1078
A-100569.1
UAAAUCUUGG
1122






CAAGAUUUA


AAGACCUGC



 


NEO1
AD-48295.1
2602
A-100598.1
CCUAGAUGAA
1079
A-100599.1
AACACGAGUU
1123






ACUCGUGUU


UCAUCUAGG



 


NEO1
AD-48292.1
5329
A-100644.1
GCAUUGCUGU
1080
A-100645.1
AGCUUACAAA
1124






UUGUAAGCU


CAGCAAUGC



 


NEO1
AD-48293.1
 686
A-100566.1
GUGGUGCAUU
1081
A-100567.1
UGUGUUUGGA
1125






CCAAACACA


AUGCACCAC



 


NEO1
AD-48288.1
1535
A-100580.1
GUUUUGGGUC
1082
A-100581.1
AUUUCACCAG
1126






UGGUGAAAU


ACCCAAAAC



 


NEO1
AD-48307.1
4066
A-100602.1
GCCUGUGAUU
1083
A-100603.1
AUGGGCACUA
1127






AGUGCCCAU


AUCACAGGC



 


NEO1
AD-48270.1
1282
A-100574.1
GGAUGAUGCU
1084
A-100575.1
AUAAGUCCCA
1128






GGGACUUAU


GCAUCAUCC



 


NEO1
AD-48300.1
1949
A-100584.1
GCUCAAAAUA
1085
A-100585.1
AGCCAUGCUU
1129






AGCAUGGCU


AUUUUGAGC



 


NEO1
AD-48306.1
2227
A-100586.1
CCGAGUGGUG
1086
A-100578.1
AUUGUAGGCC
1130






GCCUACAAU


ACCACUCGG



 


NEO1
AD-48315.1
5059
A-100636.1
GGAGUGUAGA
1087
A-100637.1
AUGCCAAUGU
1131






CAUUGGCAU


CUACACUCC



 


NEO1
AD-48291.1
4673
A-100628.1
GGACUAAUGA
1088
A-100629.1
UUAGGUCCUU
1132






AGGACCUAA


CAUUAGUCC



 


NEO1
AD-48272.1
4096
A-100606.1
CCUCGAUAAC
1089
A-100607.1
AUGGUGAGGG
1133






CCUCACCAU


UUAUCGAGG



 


NEO1
AD-48271.1
2273
A-100590.1
GAUGUUGCUG
1090
A-100591.1
AUGUUCGAAC
1134






UUCGAACAU


AGCAACAUC



 


NEO1
AD-48294.1
1540
A-100582.1
GGGUCUGGUG
1091
A-100583.1
AUCUGAUUUC
1135






AAAUCAGAU


ACCAGACCC



 


NEO1
AD-48278.1
4123
A-100608.1
CUCCAGCAGC
1092
A-100609.1
AGAAGCGAGG
1136






CUCGCUUCU


CUGCUGGAG



 


NEO1
AD-48277.1
2312
A-100592.1
GCUCCUCAGA
1093
A-100593.1
AGGACAGAUU
1137






AUCUGUCCU


CUGAGGAGC



 


NEO1
AD-48313.1
4086
A-100604.1
CCAUCCAUUC
1094
A-100605.1
UUAUCGAGGG
1138






CCUCGAUAA


AAUGGAUGG



 


NEO1
AD-48289.1
2484
A-100596.1
CUCAGCUGAU
1095
A-100597.1
AGACCUUCAA
1139






UGAAGGUCU


UCAGCUGAG



 


NEO1
AD-48290.1
4179
A-100612.1
GGCCCAUUGG
1096
A-100613.1
AUGGAUGUGC
1140






CACAUCCAU


CAAUGGGCC



 


NEO1
AD-48284.1
4174
A-100610.1
CCCAUGGCCC
1097
A-100611.1
UGUGCCAAUG
1141






AUUGGCACA


GGCCAUGGG



 


NEO1
AD-48298.1
6731
A-100646.1
GUACCUGGAU
1098
A-100647.1
UGUGGCAGUA
1142






ACUGCCACA


UCCAGGUAC



 


NEO1
AD-48311.1
 852
A-100572.1
CAAUUCUGAA
1099
A-100573.1
ACUUCACAAU
1143






UUGUGAAGU


UCAGAAUUG



 


NEO1
AD-48285.1
4664
A-100626.1
CACCUGGAAG
1100
A-100627.1
UCAUUAGUCC
1144






GACUAAUGA


UUCCAGGUG



 


NEO1
AD-48282.1
1448
A-100578.1
CCAACUCCAA
1101
A-100579.1
ACUUCACAGU
1145






CUGUGAAGU


UGGAGUUGG



 


NEO1
AD-48302.1
4542
A-100616.1
GAAGGAGCCG
1102
A-100617.1
AUAGGAGGCC
1146






GCCUCCUAU


GGCUCCUUC



 


NEO1
AD-48303.1
4767
A-100632.1
CUUGAAAACA
1103
A-100633.1
ACAAUUCCUU
1147






AGGAAUUGU


GUUUUCAAG



 


NEO1
AD-48279.1
4629
A-100624.1
GCUAUGAACC
1104
A-100625.1
AGCUCAUCUG
1148






AGAUGAGCU


GUUCAUAGC



 


NEO1
AD-48301.1
3361
A-100600.1
GAUACAUGAC
1105
A-100601.1
AAUAACCCAG
1149






UGGGUUAUU


UCAUGUAUC



 


NEO1
AD-48314.1
4613
A-100620.1
GAAGACUCCG
1106
A-100621.1
AGCUACUCUC
1150






AGAGUAGCU


GGAGUCUUC



 


NEO1
AD-48312.1
2236
A-100588.1
GGCCUACAAU
1107
A-100589.1
ACCAUGUUUA
1151






AAACAUGGU


UUGUAGGCC



 


NEO1
AD-48304.1
7033
A-100648.1
GUACACACUU
1108
A-100649.1
AGGCCAAACA
1152






GUUUGGCUU


AGUGUGUAC



 


NEO1
AD-48310.1
7043
A-100650.1
GUUUGGCCUU
1109
A-100651.1
ACUACAGAAA
1153






UUCUGUAGU


AGGCCAAAC





Note


that an overhang (e.g. TT, dTsdT) can be added to the 3′end of any duplex.













TABLE 18







NEO1 Modified Duplexes


















Sense

SEQ
Antis

SEQ


Duplex Name
Target
Start
Oligo Name
Oligo Seq
ID NO:
Oligo Name
Oligo Seq
ID NO:


















AD-48273.1
NEO1
4618
A-100622.1
cuccGAGAGuAG
1154
A-100623.1
UUcAuAGCuACU
1198






cuAuGAAdTsdT


CUCGGAGdTsdT



 


AD-48287.1
NEO1
 546
A-100564.1
GcucuucuGuuA
1155
A-100565.1
UUuAAuAuAAcA
1199






uAuuAAAdTsdT


GAAGAGCdTsdT



 


AD-48274.1
NEO1
5060
A-100638.1
GAGuGuAGAcAu
1156
A-100639.1
AAUGCcAAUGUC
1200






uGGcAuudTsdT


uAcACUCdTsdT



 


AD-48309.1
NEO1
4778
A-100634.1
GGAAuuGuAcAG
1157
A-100635.1
UCGuACUCUGuA
1201






AGuAcGAdTsdT


cAAUUCCdTsdT



 


AD-48309.2
NEO1
4778
A-100634.2
GGAAuuGuAcAG
1158
A-100635.2
UCGuACUCUGuA
1202






AGuAcGAdTsdT


cAAUUCCdTsdT



 


AD-48297.1
NEO1
4674
A-100630.1
GAcuAAuGAAGG
1159
A-100631.1
UUuAGGUCCUUc
1203






AccuAAAdTsdT


AUuAGUCdTsdT



 


AD-48296.1
NEO1
4495
A-100614.1
GAAccAucAcAu
1160
A-100615.1
UGAGUGAAUGUG
1204






ucAcucAdTsdT


AUGGUUCdTsdT



 


AD-48280.1
NEO1
5062
A-100640.1
GuGuAGAcAuuG
1161
A-100641.1
uAAAUGCcAAUG
1205






GcAuuuAdTsdT


UCuAcACdTsdT



 


AD-48275.1
NEO1
 535
A-100560.1
cucAGuuAGAGG
1162
A-100561.1
AGAAGAGCCUCu
1206






cucuucudTsdT


AACUGAGdTsdT



 


AD-48276.1
NEO1
1283
A-100576.1
GAuGAuGcuGGG
1163
A-100577.1
AAuAAGUCCcAG
1207






AcuuAuudTsdT


cAUcAUCdTsdT



 


AD-48269.1
NEO1
 533
A-100558.1
cucucAGuuAGA
1164
A-100559.1
AAGAGCCUCuAA
1208






GGcucuudTsdT


CUGAGAGdTsdT



 


AD-48286.1
NEO1
5069
A-100642.1
cAuuGGcAuuuA
1165
A-100643.1
UUGuAcAuAAAU
1209






uGuAcAAdTsdT


GCcAAUGdTsdT



 


AD-48299.1
NEO1
 791
A-100568.1
GcAGGucuuccA
1166
A-100569.1
uAAAUCUUGGAA
1210






AGAuuuAdTsdT


GACCUGCdTsdT



 


AD-48295.1
NEO1
2602
A-100598.1
ccuAGAuGAAAc
1167
A-100599.1
AAcACGAGUUUc
1211






ucGuGuudTsdT


AUCuAGGdTsdT



 


AD-48292.1
NEO1
5329
A-100644.1
GcAuuGcuGuuu
1168
A-100645.1
AGCUuAcAAAcA
1212






GuAAGcudTsdT


GcAAUGCdTsdT



 


AD-48293.1
NEO1
 686
A-100566.1
GuGGuGcAuucc
1169
A-100567.1
UGUGUUUGGAAU
1213






AAAcAcAdTsdT


GcACcACdTsdT



 


AD-48288.1
NEO1
1535
A-100580.1
GuuuuGGGucuG
1170
A-100581.1
AUUUcACcAGAC
1214






GuGAAAudTsdT


CcAAAACdTsdT



 


AD-48307.1
NEO1
4066
A-100602.1
GccuGuGAuuAG
1171
A-100603.1
AUGGGcACuAAU
1215






uGcccAudTsdT


cAcAGGCdTsdT



 


AD-48270.1
NEO1
1282
A-100574.1
GGAuGAuGcuGG
1172
A-100575.1
AuAAGUCCcAGc
1216






GAcuuAudTsdT


AUcAUCCdTsdT



 


AD-48300.1
NEO1
1949
A-100584.1
GcucAAAAuAAG
1173
A-100585.1
AGCcAUGCUuAU
1217






cAuGGcudTsdT


UUUGAGCdTsdT



 


AD-48306.1
NEO1
2227
A-100586.1
ccGAGuGGuGGc
1174
A-100587.1
AUUGuAGGCcAC
1218






cuAcAAudTsdT


cACUCGGdTsdT



 


AD-48315.1
NEO1
5059
A-100636.1
GGAGuGuAGAcA
1175
A-100637.1
AUGCcAAUGUCu
1219






uuGGcAudTsdT


AcACUCCdTsdT



 


AD-48291.1
NEO1
4673
A-100628.1
GGAcuAAuGAAG
1176
A-100629.1
UuAGGUCCUUcA
1220






GAccuAAdTsdT


UuAGUCCdTsdT



 


AD-48272.1
NEO1
4096
A-100606.1
ccucGAuAAccc
1177
A-100607.1
AUGGUGAGGGUu
1221






ucAccAudTsdT


AUCGAGGdTsdT



 


AD-48271.1
NEO1
2273
A-100590.1
GAuGuuGcuGuu
1178
A-100591.1
AUGUUCGAAcAG
1222






cGAAcAudTsdT


cAAcAUCdTsdT



 


AD-48294.1
NEO1
1540
A-100582.1
GGGucuGGuGAA
1179
A-100583.1
AUCUGAUUUcAC
1223






AucAGAudTsdT


cAGACCCdTsdT



 


AD-48278.1
NEO1
4123
A-100608.1
cuccAGcAGccu
1180
A-100609.1
AGAAGCGAGGCU
1224






cGcuucudTsdT


GCUGGAGdTsdT



 


AD-48277.1
NEO1
2312
A-100592.1
GcuccucAGAAu
1181
A-100593.1
AGGAcAGAUUCU
1225






cuGuccudTsdT


GAGGAGCdTsdT



 


AD-48313.1
NEO1
4086
A-100604.1
ccAuccAuuccc
1182
A-100605.1
UuAUCGAGGGAA
1226






ucGAuAAdTsdT


UGGAUGGdTsdT



 


AD-48289.1
NEO1
2484
A-100596.1
cucAGcuGAuuG
1183
A-100597.1
AGACCUUcAAUc
1227






AAGGucudTsdT


AGCUGAGdTsdT



 


AD-48290.1
NEO1
4179
A-100612.1
GGcccAuuGGcA
1184
A-100613.1
AUGGAUGUGCcA
1228






cAuccAudTsdT


AUGGGCCdTsdT



 


AD-48284.1
NEO1
4174
A-100610.1
cccAuGGcccAu
1185
A-100611.1
UGUGCcAAUGGG
1229






uGGcAcAdTsdT


CcAUGGGdTsdT



 


AD-48298.1
NEO1
6731
A-100646.1
GuAccuGGAuAc
1186
A-100647.1
UGUGGcAGuAUC
1230






uGccAcAdTsdT


cAGGuACdTsdT



 


AD-48311.1
NEO1
 852
A-100572.1
cAAuucuGAAuu
1187
A-100573.1
ACUUcAcAAUUc
1231






GuGAAGudTsdT


AGAAUUGdTsdT



 


AD-48285.1
NEO1
4664
A-100626.1
cAccuGGAAGGA
1188
A-100627.1
UcAUuAGUCCUU
1232






cuAAuGAdTsdT


CcAGGUGdTsdT



 


AD-48282.1
NEO1
1448
A-100578.1
ccAAcuccAAcu
1189
A-100579.1
ACUUcAcAGUUG
1233






GuGAAGudTsdT


GAGUUGGdTsdT



 


AD-48302.1
NEO1
4542
A-100616.1
GAAGGAGccGGc
1190
A-100617.1
AuAGGAGGCCGG
1234






cuccuAudTsdT


CUCCUUCdTsdT



 


AD-48303.1
NEO1
4767
A-100632.1
cuuGAAAAcAAG
1191
A-100633.1
AcAAUUCCUUGU
1235






GAAuuGudTsdT


UUUcAAGdTsdT



 


AD-48279.1
NEO1
4629
A-100624.1
GcuAuGAAccAG
1192
A-100625.1
AGCUcAUCUGGU
1236






AuGAGcudTsdT


UcAuAGCdTsdT



 


AD-48301.1
NEO1
3361
A-100600.1
GAuAcAuGAcuG
1193
A-100601.1
AAuAACCcAGUc
1237






GGuuAuudTsdT


AUGuAUCdTsdT



 


AD-48314.1
NEO1
4613
A-100620.1
GAAGAcuccGAG
1194
A-100621.1
AGCuACUCUCGG
1238






AGuAGcudTsdT


AGUCUUCdTsdT



 


AD-48312.1
NEO1
2236
A-100588.1
GGccuAcAAuAA
1195
A-100589.1
ACcAUGUUuAUU
1239






AcAuGGudTsdT


GuAGGCCdTsdT



 


AD-48304.1
NEO1
7033
A-100648.1
GuAcAcAcuuGu
1196
A-100649.1
AGGCcAAAcAAG
1240






uuGGccudTsdT


UGUGuACdTsdT



 


AD-48310.1
NEO1
7043
A-100650.1
GuuuGGccuuuu
1197
A-100651.1
ACuAcAGAAAAG
1241






cuGuAGudTsdT


GCcAAACdTsdT





It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention.













TABLE 19







SMAD4 Percent Inhibition












0.1 nM (%

10 nM (%




message

message




remaining)

remaning)















Target
ID
Avg
SD
Avg
SD

















SMAD4
AD-48090
93.6
4.6
54.6
5.6



SMAD4
AD-48091
98.0
5.0
60.8
3.3



SMAD4
AD-48092
64.6
0.2
47.8
12.0



SMAD4
AD-48093
96.4
3.5
45.0
8.0



SMAD4
AD-48094
41.3
0.4
16.3
5.4



SMAD4
AD-48095
64.4
9.1
30.0
0.5



SMAD4
AD-48096
70.5
1.8
44.3
0.7



SMAD4
AD-48097
19.6
2.5
10.0
1.6



SMAD4
AD-48098
60.6
2.1
29.9
1.8



SMAD4
AD-48099
83.1
5.5
57.2
2.5



SMAD4
AD-48100
73.4
1.6
50.4
1.2



SMAD4
AD-48101
34.8
3.7
23.3
0.9



SMAD4
AD-48102
66.9
3.2
35.5
4.0



SMAD4
AD-48103
43.4
8.9
20.5
1.0



SMAD4
AD-48104
53.5
6.2
20.5
1.5



SMAD4
AD-48105
59.4
0.6
23.8
3.0



SMAD4
AD-48106
68.4
0.3
40.7
0.5



SMAD4
AD-48107
40.9
3.0
26.9
6.6



SMAD4
AD-48108
21.4
4.3
15.2
4.3



SMAD4
AD-48109
19.2
4.1
12.1
5.2



SMAD4
AD-48110
46.1
6.4
28.4
8.1



SMAD4
AD-48111
75.9
5.1
68.4
12.1



SMAD4
AD-48112
75.8
2.0
72.0
10.4



SMAD4
AD-48113
87.4
11.1
72.0
2.7



SMAD4
AD-48114
36.7
3.2
19.2
0.6



SMAD4
AD-48115
35.8
2.8
18.6
1.9



SMAD4
AD-48116
37.1
0.2
13.6
0.9



SMAD4
AD-48117
32.1
0.8
21.1
1.4



SMAD4
AD-48118
26.3
1.1
16.4
5.5



SMAD4
AD-48119
52.1
4.7
38.8
4.5



SMAD4
AD-48120
32.1
1.0
13.9
1.4



SMAD4
AD-48121
24.3
2.3
10.0
0.7



SMAD4
AD-48122
31.4
5.7
14.6
1.7



SMAD4
AD-48123
27.4
1.5
14.6
2.2



SMAD4
AD-48124
76.8
7.0
55.8
1.0



SMAD4
AD-48125
28.7
2.6
12.6
0.9



SMAD4
AD-48126
18.9
1.9
7.4
0.2



SMAD4
AD-48127
67.5
3.7
39.6
4.0



SMAD4
AD-48128
69.8
4.0
44.5
6.1



SMAD4
AD-48129
73.1
3.4
42.6
2.0



SMAD4
AD-48130
18.1
0.1
12.5
0.9



SMAD4
AD-48131
44.4
0.5
17.1
4.1



SMAD4
AD-48132
47.7
0.1
22.6
5.4



SMAD4
AD-48133
57.1
1.8
30.4
10.0



SMAD4
AD-48134
86.3
18.0
42.4
9.2
















TABLE 20







NEO1 Percent Inhibition












0.1 nM (%

10 nM (%




message

message




remaining)

remaining)















Target
ID
Avg
SD
Avg
SD

















Neo1
AD-48273
8.4
0.7
9.3
3.6



Neo1
AD-48287
8.6
5.5
10.4
2.7



Neo1
AD-48274
11.0
4.3
6.5
2.2



Neo1
AD-48309
11.0
0.6
6.5
0.8



Neo1
AD-48297
12.9
1.6
8.7
2.4



Neo1
AD-48296
14.0
6.9
7.6
0.1



Neo1
AD-48280
15.6
3.7
10.8
7.1



Neo1
AD-48275
17.7
6.9
8.4
3.8



Neo1
AD-48276
17.8
9.8
6.8
2.0



Neo1
AD-48269
18.4
5.5
10.9
4.4



Neo1
AD-48286
21.4
3.8
11.7
2.1



Neo1
AD-48299
22.9
3.0
11.7
3.8



Neo1
AD-48295
36.2
16.3
12.0
0.4



Neo1
AD-48292
44.3
6.8
14.8
2.2



Neo1
AD-48293
44.7
14.1
30.7
1.8



Neo1
AD-48288
46.9
21.9
31.9
5.2



Neo1
AD-48307
50.2
10.1
16.8
3.9



Neo1
AD-48270
54.2
10.6
65.9
42.5



Neo1
AD-48300
54.6
0.1
18.6
1.9



Neo1
AD-48306
56.6
19.5
16.0
2.3



Neo1
AD-48315
57.7
3.5
17.6
8.0



Neo1
AD-48291
60.2
12.0
35.2
6.4



Neo1
AD-48272
61.9
4.1
25.2
3.2



Neo1
AD-48271
62.6
4.7
35.4
6.8



Neo1
AD-48294
62.6
2.1
22.7
11.0



Neo1
AD-48278
62.9
13.8
27.4
1.3



Neo1
AD-48277
63.2
20.4
26.1
2.6



Neo1
AD-48313
68.2
18.7
43.7
2.2



Neo1
AD-48289
70.6
15.3
53.6
12.3



Neo1
AD-48290
73.8
22.6
60.0
3.9



Neo1
AD-48284
74.0
19.2
106.9
43.7



Neo1
AD-48298
76.0
6.9
75.4
19.3



Neo1
AD-48311
77.9
22.6
23.5
11.1



Neo1
AD-48285
81.0
11.5
65.3
14.2



Neo1
AD-48282
82.7
16.3
47.0
15.3



Neo1
AD-48302
83.3
3.1
32.8
6.7



Neo1
AD-48303
85.0
16.3
29.2
7.7



Neo1
AD-48279
90.2
6.2
51.7
14.3



Neo1
AD-48301
91.8
8.5
88.2
11.1



Neo1
AD-48314
96.7
16.7
128.8
37.8



Neo1
AD-48312
107.9
30.0
94.0
27.8



Neo1
AD-48304
111.6
22.3
91.6
33.2



Neo1
AD-48310
118.0
36.4
118.8
29.0
















TABLE 21







BMP6 Duplexes














SEQ

SEQ



duplexName
sOligoSeq
ID NO
asOligoSeq
ID NO
Set





AD-47955.1
GcAGAAuuccGcAucuAcAdTsdT
1242
UGuAGAUGCGGAAUUCUGCdTsdT
1300
humanRhesus


 


AD-47957.1
GAAuAuGGuuGuAAGAGcudTsdT
1243
AGCUCUuAcAACcAuAUUCdTsdT
1301
humanRhesus


 


AD-47966.1
cucuucAuGcuGGAucuGudTsdT
1244
AcAGAUCcAGcAUGAAGAGdTsdT
1302
humanRhesus


 


AD-47989.1
GAGuucAAGuucAAcuuAudTsdT
1245
AuAAGUUGAACUUGAACUCdTsdT
1303
humanRhesus


 


AD-47993.1
cGuGAGuAGuuGuuGGucudTsdT
1246
AGACcAAcAACuACUcACGdTsdT
1304
humanRhesus


 


AD-47960.1
GGAcGAccAuGAGAGAuAAdTsdT
1247
UuAUCUCUcAUGGUCGUCCdTsdT
1305
humanRhesus


 


AD-47997.1
ccuAGAuuAcAucuGccuudTsdT
1248
AAGGcAGAUGuAAUCuAGGdTsdT
1306
humanRhesus


 


AD-47985.1
cAAcAGAGucGuAAucGcudTsdT
1249
AGCGAUuACGACUCUGUUGdTsdT
1307
humanRhesus


 


AD-47983.1
GucuAucAAAGGuAGAuuudTsdT
1250
AAAUCuACCUUUGAuAGACdTsdT
1308
humanRhesus


 


AD-47954.1
cccGGAcGAccAuGAGAGAdTsdT
1251
UCUCUcAUGGUCGUCCGGGdTsdT
1309
humanRhesus


 


AD-47972.1
cucGucAGcGAcAccAcAAdTsdT
1252
UUGUGGUGUCGCUGACGAGdTsdT
1310
humanRhesus


 


AD-47981.1
ccAcuAAcucGAAAccAGAdTsdT
1253
UCUGGUUUCGAGUuAGUGGdTsdT
1311
humanRhesus


 


AD-47982.1
GuAAAuGAcGuGAGuAGuudTsdT
1254
AACuACUcACGUcAUUuACdTsdT
1312
humanRhesus


 


AD-47987.1
GGGGAcAcAcAuucuGccudTsdT
1255
AGGcAGAAUGUGUGUCCCCdTsdT
1313
humanRhesus


 


AD-47994.1
cGGcuGcAGAAuuccGcAudTsdT
1256
AUGCGGAAUUCUGcAGCCGdTsdT
1314
humanRhesus


 


AD-47973.1
GccGAcAAcAGAGucGuAAdTsdT
1257
UuACGACUCUGUUGUCGGCdTsdT
1315
humanRhesus


 


AD-47975.1
GGAuGccAcuAAcucGAAAdTsdT
1258
UUUCGAGUuAGUGGcAUCCdTsdT
1316
humanRhesus


 


AD-47979.1
ccGAcAAcAGAGucGuAAudTsdT
1259
AUuACGACUCUGUUGUCGGdTsdT
1317
humanRhesus


 


AD-47996.1
cGuGcuGuGcGccAAcuAAdTsdT
1260
UuAGUUGGCGcAcAGcACGdTsdT
1318
humanRhesus


 


AD-47968.1
cAAcGcAcAcAuGAAuGcAdTsdT
1261
UGcAUUcAUGUGUGCGUUGdTsdT
1319
humanRhesus


 


AD-47977.1
cuGucuAucAAAGGuAGAudTsdT
1262
AUCuACCUUUGAuAGAcAGdTsdT
1320
humanRhesus


 


AD-47995.1
GcGGGucuccAGuGcuucAdTsdT
1263
UGAAGcACUGGAGACCCGCdTsdT
1321
humanRhesus


 


AD-47959.1
cuGAGuuuGGAuGucuGuAdTsdT
1264
uAcAGAcAUCcAAACUcAGdTsdT
1322
humanRhesus


 


AD-47962.1
cAGGAAGcAuGAGcuGuAudTsdT
1265
AuAcAGCUcAUGCUUCCUGdTsdT
1323
humanRhesus


 


AD-47967.1
GGcuGGcuGGAAuuuGAcAdTsdT
1266
UGUcAAAUUCcAGCcAGCCdTsdT
1324
humanRhesus


 


AD-47986.1
GcAGAccuuGGuucAccuudTsdT
1267
AAGGUGAACcAAGGUCUGCdTsdT
1325
humanRhesus


 


AD-47988.1
GAcGuGAGuAGuuGuuGGudTsdT
1268
ACcAAcAACuACUcACGUCdTsdT
1326
humanRhesus


 


AD-47990.1
cAGAGucGuAAucGcucuAdTsdT
1269
uAGAGCUGAuACGACUCUGdTsdT
1327
humanRhesus


 


AD-47991.1
cAGAccuuGGuucAccuuAdTsdT
1270
uAAGGUGAACcAAGGUCUGdTsdT
1328
humanRhesus


 


AD-47956.1
GGGucuccAGuGcuucAGAdTsdT
1271
UCUGAAGcACUGGAGACCCdTsdT
1329
humanRhesus


 


AD-47974.1
GcAcAcAuGAAuGcAAccAdTsdT
1272
UGGUUGcAUUcAUGUGUGCdTsdT
1330
humanRhesus


 


AD-47976.1
GGuAAAuGAcGuGAGuAGudTsdT
1273
ACuACUcACGUcAUUuACCdTsdT
1331
humanRhesus


 


AD-47980.1
cAcAcAuGAAuGcAAccAAdTsdT
1274
UUGGUUGcAUUcAUGUGUGdTsdT
1332
humanRhesus


 


AD-47984.1
cGAcAccAcAAAGAGuucAdTsdT
1275
UGAACUCUUUGUGGUGUCGdTsdT
1333
humanRhesus


 


AD-47964.1
cucAuuAAuAAuuuGcucAdTsdT
1276
UGAGcAAAUuAUuAAUGAGdTsdT
1334
humanRhesus


 


AD-47970.1
cAuuAAuAAuuuGcucAcudTsdT
1277
AGUGAGcAAAUuAUuAAUGdTsdT
1335
humanRhesus


 


AD-47971.1
GuAcuGucuAucAAAGGuAdTsdT
1278
uACCUUUGAuAGAcAGuACdTsdT
1336
humanRhesus


 


AD-47963.1
cuuGuGGAuGccAcuAAcudTsdT
1279
AGUuAGUGGcAUCcAcAAGdTsdT
1337
humanRhesus


 


AD-47965.1
GuucAGuAcuGucuAucAAdTsdT
1280
UUGAuAGAcAGuACUGAACdTsdT
1338
humanRhesus


 


AD-47992.1
cuuGGAuuccuAGAuuAcAdTsdT
1281
UGuAAUCuAGGAAUCcAAGdTsdT
1339
humanRhesus


 


AD-47998.1
GGucuGuAGcAAGcuGAGudTsdT
1282
ACUcAGCUUGCuAcAGACCdTsdT
1340
humanRhesus


 


AD-47958.1
GAuuuuAAAGGAccucAuudTsdT
1283
AAUGAGGUCCUUuAAAAUCdTsdT
1341
humanRhesus


 


AD-47961.1
cAAAcuuuucuuAucAGcAdTsdT
1284
UGCUGAuAAGAAAAGUUUGdTsdT
1342
humanRhesus


 


AD-47969.1
GuGGAuGccAcuAAcucGAdTsdT
1285
UCGAGUuAGUGGcAUCcACdTsdT
1343
humanRhesus


 


AD-47978.1
GucAGcGAcAccAcAAAGAdTsdT
1286
UCUUUGUGGUGUCGCUGACdTsdT
1344
humanRhesus


 


AD-47305.1
ucAuGAGcuuuGuGAAccudTsdT
1287
AGGUUcAcAAAGCUcAUGAdTsdT
1345
humanRhesus







Mouse


 


AD-47325.1
GAGAcGGcccuuAcGAcAAdTsdT
1288
UUGUCGuAAGGGCCGUCUCdTsdT
1346
humanRhesus







Mouse


 


AD-47329.1
AcGGcccuuAcGAcAAGcAdTsdT
1289
UGCUUGUCGuAAGGGCCGUdTsdT
1347
humanRhesus







Mouse


 


AD-47309.1
AAccuGGuGGAGuAcGAcAdTsdT
1290
UGUCGuACUCcACcAGGUUdTsdT
1348
humanRhesus







Mouse


 


AD-47317.1
GcAGAGAcGGcccuuAcGAdTsdT
1291
UCGuAAGGGCCGUCUCUGCdTsdT
1349
humanRhesus







Mouse


 


AD-47313.1
AccuGGuGGAGuAcGAcAAdTsdT
1292
UUGUCGuACUCcACcAGGUdTsdT
1350
humanRhesus







Mouse


 


AD-47321.1
AGAGAcGGcccuuAcGAcAdTsdT
1293
UGUCGuAAGGGCCGUCUCUdTsdT
1351
humanRhesus







Mouse


 


AD-47333.1
ucccAcucAAcGcAcAcAudTsdT
1294
AUGUGUGCGUUGAGUGGGAdTsdT
1352
humanRhesus







Mouse


 


AD-48038.1
ucAAcGAcGcGGAcAuGGudTsdT
1295
ACcAUGUCCGCGUCGUUGAdTsdT
1353
mouseRat


 


AD-48010.1
GccAucucGGuucuuuAcudTsdT
1296
AGuAAAGAACCGAGAUGGCdTsdT
1354
mouseRat


 


AD-48042.1
AAuGccAucucGGuucuuudTsdT
1297
AAAGAACCGAGAUGGcAUUdTsdT
1355
mouseRat


 


AD-48000.1
AAcGAcGcGGAcAuGGucAdTsdT
1298
UGACcAUGUCCGCGUCGUUdTsdT
1356
mouseRat


 


AD-48004.1
AuGccAucucGGuucuuuAdTsdT
1299
uAAAGAACCGAGAUGGcAUdTsdT
1357
mouseRat





It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention.

## Claims

1. A double-stranded ribonucleic acid (dsRNA) compound, comprising a sense strand and an antisense strand that are each equal to or less than 30 nucleotides in length, wherein the compound is targeted to a nucleic acid molecule encoding transferrin receptor 2(TRF2), wherein the compound specifically hybridizes with the nucleic acid molecule so that the abundance of TRF2 mRNA is inhibited by at least 20% relative to a PBS control, and wherein the antisense strand comprises at least 15 contiguous nucleotides from the nucleotide sequence of SEQ ID NO:38.

2. The dsRNA of claim 1, wherein the nucleotide sequence of the sense strand comprises 15 or more contiguous nucleotides of SEQ ID NO:35 with a start position of position 239 of the TFR2 mRNA transcript and the nucleotide sequence of the antisense strand comprises 15 or more contiguous nucleotides of SEQ ID NO:38 with a start position complementary to position 239 of the TFR2 mRNA transcript.

3. The dsRNA of claim 1, wherein the nucleotide sequence of the sense strand comprises 16, 17, 18, 19, 20, or 21 contiguous nucleotides of SEQ ID NO:35 with a start position of position 239 of the TFR2 mRNA transcript and the nucleotide sequence of the antisense strand comprises 16, 17, 18, 19, 20, or 21 contiguous nucleotides of SEQ ID NO:38 with a start position complementary to position 239 of the TFR2 mRNA transcript.

4. The dsRNA of claim 1, wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:35 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:38.

5. The dsRNA of claim 1, further comprising a phosphorothioate at the first internucleotide linkage at the 3′ end of the sense strand, the antisense strand or both the sense strand and the antisense strand.

6. The dsRNA of claim 1, wherein the sense strand, the antisense strand or both the sense and antisense strand further comprises at least one 3′n-overhang wherein the 3′-overhang comprises from 1 to 6 nucleotides.

7. The dsRNA of claim 1, wherein the dsRNA further comprises a non-nucleotide moiety.

8. The dsRNA of claim 1, wherein said dsRNA further comprises at least one modified nucleotide.

9. The dsRNA of claim 1, further comprising a 2′-modified nucleotide in the sense strand, r the antisense strand or both the sense and antisense strand.

10. The dsRNA of claim 8, wherein at least one of said modified nucleotides is selected from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.

11. The dsRNA of claim 8, wherein the modified nucleotide is selected from the group consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

12. The dsRNA of claim 1, wherein the dsRNA is formulated in a nucleic acid lipid particle formulation.

13. The dsRNA of claim 1, wherein the nucleic acid lipid particle formulation comprises a cationic lipid, a non-cationic lipid, and a cholesterol/ polyethyleneglycol-lipid conjugate.

14. The dsRNA of claim 1, wherein the dsRNA is selected from the group consisting of AD-52551, AD-52552, AD-52557, AD-52558, AD-52563, AD-52564, AD-52569, AD-52570, AD-52574, AD-52575, AD-52579, AD-52580, AD-52584, AD-52585, AD-52589, AD-52590, and AD-47826.

15. A cell comprising the dsRNA of claim 1.

16. A vector encoding at least one of the antisense strand and the sense strand of the dsRNA of claim 1.

17. A cell comprising the vector of claim 15.

18. A pharmaceutical composition comprising the dsRNA of claim 1 and a pharmaceutically acceptable carrier.

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

20. A method of inhibiting TFR2 expression in a cell, the method comprising:
(a) introducing into the cell the dsRNA of claim 1 and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a TFR2 gene, thereby inhibiting expression of the TFR2 gene in the cell.

21. A method of treating a disorder associated with TFR2 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of the dsRNA of claim 1.

22. The dsRNA of claim 1, wherein the sense strand consists of the nucleotide sequence set forth in SEQ ID NO:35 and the antisense strand consists of the nucleotide sequence set forth in SEQ ID NO:38.