阅读说明：本技术 用于抑制tmprss6基因表达的组合物和方法 (Compositions and methods for inhibiting expression of TMPRSS6 gene ) 是由 D·邦克罗特 布莱恩·贝当古 I·陶德贾斯卡 于 2012-03-28 设计创作，主要内容包括：本发明涉及用于抑制TMPRSS6基因表达的组合物和方法,具体而言,本发明涉及靶向TMPRSS6基因的双链核糖核酸(dsRNA)组合物,以及使用这类dsRNA组合物来抑制TMPRSS6表达的方法。(The present invention relates to compositions and methods for inhibiting expression of the TMPRSS6 gene, in particular, the invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the TMPRSS6 gene, and methods of using such dsRNA compositions to inhibit expression of TMPRSS 6.)

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

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

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

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

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

6. The dsRNA of claim 1, wherein the region of complementarity is between 19 and 21 nucleotides in length.

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

8. The dsRNA of claim 1, wherein each strand is no more than 30 nucleotides in length.

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

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

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

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

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

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

15. A cell containing the dsRNA of claim 1.

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

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

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

19. A method of inhibiting expression of TMPRSS6 in a cell, the method comprising:

a) Introducing the dsRNA of claim 1 into the cell; and is

b) Maintaining the cells resulting from step a) for a time sufficient to effect degradation of the mRNA transcript of the TMPRSS6 gene, thereby inhibiting expression of the TMPRSS6 gene in the cells.

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

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

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

23. The method of claim 21, wherein the human has β -thalassemia.

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

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

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

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

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

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

30. A cell comprising the vector of claim 27.

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

Technical Field

The present invention relates to the specific inhibition of expression of the TMPRSS6 gene.

Background

TMPRSS6 (transmembrane protease, serine 6) encodes a type II serine protease and is expressed predominantly in the liver. TMPRSS6 affects iron levels in the liver by binding to and proteolytically degrading the hepcidin (hepcidin) activator and the BMP co-receptor HJV (hemojuvelin), which causes down-regulation of hepcidin levels.

TMPRSS6 consists of: a short N-terminal intracytoplasmic tail, a type II transmembrane domain, a stem region consisting of two extracellular CUB (complement factor C1s/C1r, sea urchin embryo growth factor and BMP (bone morphogenetic protein)) domains, three LDLR (class a low density lipoprotein receptor) domains and a C-terminal trypsin-like serine protease domain. Consensus sites for N-glycosylation also exist in the extracellular domain and potential phosphorylation sites exist within the intracytoplasmic tail region.

Disclosure of Invention

Described herein are compositions and methods for effecting RNA-induced silencing complex (RISC) -mediated cleavage of an RNA transcript of TMPRSS6 gene, e.g., in a cell or mammal. Also described are compositions and methods for treating pathological conditions and diseases caused by expression of the TMPRSS6 gene, such as disorders characterized by iron overload (e.g., thalassemia, e.g., beta-thalassemia intermedia or alpha-thalassemia). Also described are compositions and methods for reducing or preventing iron absorption or mobilization, thereby alleviating iron excesses in certain pathological conditions. The methods and compositions described herein are generally useful for treating hemochromatosis (iron accumulation in the body).

As used herein, the term "iRNA" refers to a substance that contains an RNA as that term is defined herein and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein inhibits expression of TMPRSS6 in a cell or mammal. Alternatively, in another embodiment, the iRNA upregulates expression of TMPRSS6 in the cell or mammal.

iRNA included in the compositions characterized herein encompass double-stranded RNA (dsrna) having an RNA strand (antisense strand) with a region of 30 or fewer nucleotides in length, generally 19-24 nucleotides, that is substantially complementary to at least a portion of an mRNA transcript of the TMPRSS6 gene. In one embodiment, the dsRNA comprises a region of at least 15 contiguous nucleotides.

In one embodiment, the iRNA that inhibits expression of TMPRSS6 gene comprises at least two sequences complementary to each other. The iRNA includes a sense strand having a first sequence and an antisense strand having a second sequence. The antisense strand comprises a nucleotide sequence that is substantially complementary to at least a portion of the mRNA encoding TMPRSS6, and this region of complementarity is 30 nucleotides or less and at least 15 nucleotides in length. In general, the iRNA is 19 to 24, e.g., 19 to 21 nucleotides in length. In some embodiments, the iRNA is from about 15 to about 25 nucleotides in length, and in other embodiments, the iRNA is from about 25 to about 30 nucleotides in length. Once contacted with a cell expressing TMPRSS6, the iRNA inhibits expression of TMPRSS6 gene by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more as analyzed by the methods as described herein. In one embodiment, TMPRSS6 iRNA is formulated in Stable Nucleic Acid Lipid Particles (SNALP).

In one embodiment, the iRNA characterized herein comprises a first sequence of dsRNA selected from the sense sequences of table 2, 3 or 4 and a second sequence selected from the corresponding antisense sequences of table 2, 3 or 4. The iRNA molecules characterized herein can include naturally occurring nucleotides or can include at least one modified nucleotide including, but not limited to, 2 '-O-methyl modified nucleotides, a nucleotide group having a 5' -phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative. Alternatively, the modified nucleotide may be selected from: 2' -deoxy-2 ' -fluoro modified nucleotides, 2' -deoxy modified nucleotides, locked nucleotides, non-base nucleotides, 2' -amino modified nucleotides, 2' -alkyl modified nucleotides, morpholino nucleotides, phosphoramidates and nucleotides comprising non-natural bases. In general, such modified sequences will be based on a first sequence of the iRNA selected from the sense sequences of tables 2, 3 or 4, and a second sequence selected from the antisense sequences of tables 2, 3 or 4.

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

In another embodiment, a composition containing a dsRNA targeting TMPRSS6 is administered to a subject having an elevated iron level (e.g., an elevated iron level in the liver). Subjects with elevated iron levels can be identified as subjects who having elevated serum iron levels (e.g., above 350 μ g/dL, above 500 μ g/dL, or above 1000 μ g/dL or higher), elevated serum ferritin levels, or transferrin saturation levels greater than 40%, greater than 45%, greater than 50%, greater than 60%, or more.

Mild to moderate iron excesses are indicated by serum ferritin levels of 300-. Serum ferritin at > 1000 μ g/L has been shown to be associated with adverse consequences in primary and secondary iron excesses. Serum ferritin levels above 200 μ g/L in premenopausal women and above 300 μ g/L in men and postmenopausal women indicate primary iron overload due to hemochromatosis, and ferritin levels above 1000 μ g/L generally indicate liver damage due to iron overload. Subjects with serum ferritin levels above 300, 500, 1000, 1500, 2000, or 2500 μ g/L or higher are candidates for dsRNA therapy targeting TMPRSS 6.

In another embodiment, a composition containing a dsRNA targeting TMPRSS6 is administered to a subject having an elevated transferrin level (e.g., a transferrin level greater than 400mg/dL, greater than 500mg/dL, greater than 1000mg/dL, or higher).

Iron levels can also be measured by the TIBC (total iron binding capacity) assay. The TIBC test measures the amount of iron that blood will carry if transferrin is fully saturated. Since transferrin is produced by the liver, TIBCs can be used to monitor liver function and nutrition in a subject. Subjects with TIBC values greater than 400 μ g/dL, greater than 500 μ g/dL, or greater than 1000 μ g/dL or higher are candidates for receiving dsRNA therapy targeting TMPRSS 6.

In one embodiment, administration of the dsRNA reduces iron levels, e.g., in the liver or in serum, by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 60% or more. In some embodiments, one or more of serum ferritin levels, serum transferrin levels, transferrin saturation levels, or TIBC values are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 60% or more compared to pre-treatment levels. In another embodiment, the reduction in iron levels, the reduction in serum ferritin levels, the reduction in transferrin or transferrin saturation levels, or the reduction in TIBC values is maintained for at least 5, 10, 20, 30, or 40 days or longer.

In one embodiment, the subject is selected based at least in part on a need for a lower level of iron (as compared to selecting the patient based only on who happens to be needed).

In one embodiment, irnas as described herein target wild-type TMPRSS6 RNA transcripts, and in another embodiment, irnas target mutant transcripts (e.g., TMPRSS6 RNA carrying allelic variants). For example, irnas characterized in the present invention can target polymorphic variants of TMPRSS6, such as Single Nucleotide Polymorphisms (SNPs). In another embodiment, the iRNA targets both the wild-type and mutant TMPRSS6 transcripts. In yet another embodiment, the iRNA targets a transcript variant of TMPRSS 6.

In one embodiment, the iRNA characterized in the present invention targets a non-coding region, e.g., a 5 'or 3' untranslated region, of the TMPRSS6 RNA transcript.

In one embodiment, the iRNA characterized in the invention is delivered to the liver, e.g., hepatocytes or kupffer cells of the liver, e.g., hypertrophic kupffer cells.

In one aspect, embodiments characterized in the present invention provide a cell containing at least one iRNA characterized in the present invention. The cells are typically mammalian cells, such as human cells.

In another aspect, the embodiments characterized herein provide pharmaceutical compositions for inhibiting TMPRSS6 gene expression in an organism, typically a human subject. Such compositions generally include one or more irnas as described herein and a pharmaceutically acceptable carrier or delivery vehicle. In one embodiment, the composition is used to treat disorders that cause an increase in iron levels, such as hemochromatosis. For example, the composition can be used to treat thalassemia, such as beta-thalassemia intermedia.

In another embodiment, such pharmaceutical compositions are formulated for administration of a dosage regimen described herein, e.g., no more than 1 every two months, no more than 1 every month, no more than 2 every month, no more than 1 every 4 weeks, no more than 1 every 3 weeks, no more than 1 every 2 weeks, or no more than 1 every week. In another embodiment, administration of the pharmaceutical composition can be maintained for one month or more, e.g., 1, 2, 3, or 6 months, or 1 year, or 5 years, or 10 years or more, including the remainder of the subject's life.

In another embodiment, a composition containing an iRNA described herein (e.g., a dsRNA targeting TMPRSS 6) is administered with a non-iRNA therapeutic agent (e.g., a drug known to treat hemochromatosis or a disorder that causes hemochromatosis (such as thalassemia)). For example, irnas characterized in the present invention can be administered with a drug that treats beta thalassemia (e.g., beta-thalassemia intermedia) or another disorder associated with increased iron levels.

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

In another aspect, provided herein is a method of inhibiting expression of TMPRSS6 gene in a cell by performing the steps of:

(a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises at least two sequences that are complementary to each other. The dsRNA has a sense strand comprising a first sequence and an antisense strand comprising a second sequence; the antisense strand has a region of complementarity that is substantially complementary to at least a portion of an mRNA encoding TMPRSS6, and wherein the region of complementarity is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, and generally 19-24 nucleotides in length, and wherein upon contact with a cell expressing TMPRSS6, then the dsRNA inhibits expression of the TMPRSS6 gene by at least 10%, preferably at least 20%, at least 30%, at least 40% or more; and is

(b) Maintaining the cells produced in step (a) for a time sufficient to effect degradation of the mRNA transcript of the TMPRSS6 gene, thereby inhibiting expression of the TMPRSS6 gene in the cells.

In another aspect, the invention provides methods and compositions useful for activating TMPRSS6 gene expression in a cell or mammal.

In another aspect, the invention provides a method of modulating TMPRSS6 gene expression in a cell by performing the steps of:

(a) Introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises at least two sequences that are complementary to each other. The dsRNA has a sense strand comprising a first sequence and an antisense strand comprising a second sequence; the antisense strand has a region of complementarity that is substantially complementary to at least a portion of an mRNA encoding TMPRSS6, and wherein the region of complementarity is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, and generally 19-24 nucleotides in length, and wherein upon contact with a cell expressing TMPRSS6, then the dsRNA modulates expression of TMPRSS6 gene by at least 10%, preferably at least 20%, at least 30%, at least 40% or more; and is

(b) Maintaining the cells produced in step (a) for a time sufficient to obtain degradation or protection of the mRNA transcript of the TMPRSS6 gene, thereby modulating expression of the TMPRSS6 gene in the cells.

In one embodiment, the method is used to inhibit gene expression in a hepatocyte, such as a hepatocyte or kupffer cell. In another embodiment, the method is used to activate gene expression in a hepatocyte.

In other aspects, the invention provides methods for treating, preventing, reversing, or managing pathological processes mediated by TMPRSS6 expression (e.g., disorders associated with hemochromatosis). In one embodiment, the method comprises administering to a patient in need of such treatment, prevention, reversal or management a therapeutically or prophylactically effective amount of one or more irnas characterized in the present invention. In one embodiment, the patient has thalassemia, such as beta-thalassemia intermedia. In another embodiment, administration of iRNA targeting TMPRSS6 reduces or attenuates the severity of at least one symptom of a TMPRSS 6-mediated disorder (such as a symptom associated with an excess of iron, e.g., joint pain, abdominal pain, or weakness) in the patient.

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

In one aspect, the invention provides a cell comprising a vector for inhibiting expression of TMPRSS6 gene in the cell. The vector comprises at least one regulatory sequence operably linked to a nucleotide sequence encoding at least one strand of one of the irnas as described herein.

In yet another aspect, the invention provides a composition comprising TMPRSS6 iRNA in combination with a second iRNA targeting a second gene involved in a pathological disease and useful for treating a disease, e.g., beta-thalassemia. For example, the second iRNA can target a negative regulator of hepcidin, such as a hypoxia inducible factor, e.g., HIF-1a or HIF-2 a; GDF 15; or TWSG 1. In one embodiment, the second iRNA targets a gene involved in a second disorder resulting from β -thalassemia. For example, the second iRNA may target a gene involved in diabetes, thrombosis, or bone mass reduction.

The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Drawings

FIG. 1 is the sequence of human TMPRSS6 mRNA (reference sequence NM-153609.2, GI:56682967, date of registration 2011, 1, 23, SEQ ID NO: 1).

Figures 2A and 2B depict the efficacy of two chemically modified TMPRSS6 targeting sirnas in reducing TMPRSS6 gene expression in primary mouse hepatocytes.

FIGS. 3A and 3B depict the effect of LNP-TMPRSS6 siRNA-1(AD-46273) and LNP-TMPRSS6 siRNA-2(AD-46286) on TMPRSS6 and HAMP1 gene expression, respectively, in WT C57BL/6 mice.

Figure 4 depicts the duration of TMPRSS6 siRNA mediated effect on TMPRSS6 gene expression, HAMP1 gene expression and serum iron levels in WT C57BL/6 mice.

Figure 5 depicts TMPRSS6 siRNA mediated levels of TMPRSS6 silencing required to maintain TMPRSS6 siRNA mediated effects on HAMP1 gene expression and serum iron levels in WT C57BL/6 mice.

FIGS. 6A and 6B depict the effect of TMPRSS6 siRNA-mediated TMPRSS6 silencing on hematological parameters in WT C57BL/6 mice. Figure 6A depicts the effect of TMPRSS6 siRNA mediated silencing of TMPRSS6 on Hemoglobin (HBG) in WT C57BL/6 mice 6 hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days post-administration. Figure 6B depicts the effect of TMPRSS6 siRNA mediated silencing of TMPRSS6 on hematocrit in WT C57BL/6 mice 6 hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days post-administration.

Figure 7 depicts the effect of TMPRSS6 siRNA mediated silencing of TMPRSS6 in thalassemic mice (Th3/+) on serum iron parameters including serum iron levels, Unsaturated Iron Binding Capacity (UIBC) levels and transferrin saturation levels.

Figures 8A to 8C depict the effect of TMPRSS6 siRNA mediated silencing of TMPRSS6 on reticulocyte and erythrocyte parameters in thalassemia mice (Th 3/+). Fig. 8A depicts the effect on reticulocyte number (%), fig. 8B depicts the effect on hemoglobin content (CHr) of reticulocytes, and fig. 8C depicts the effect on mature red blood cell number.

Figures 9A to 9D depict the effect of TMPRSS6 siRNA mediated silencing of TMPRSS6 on hematological parameters in thalassemic mice (Th 3/+). Fig. 9A depicts the effect on Hematocrit (HCT) levels, fig. 9B depicts the effect on Hemoglobin (HGB), fig. 9C depicts the effect on Red Blood Cell (RBC) distribution width (RDW), and fig. 9D depicts the effect on mean red blood cell volume value (MCV).

FIGS. 10A to 10C depict the effect of TMPRSS6 siRNA-mediated TMPRSS6 silencing on spleen and liver iron content in thalassemic mice (Th 3/+). Fig. 10A depicts the effect on total spleen iron content, fig. 10B depicts the effect on spleen weight, and fig. 10C depicts the effect of TMPRSS6 siRNA mediated TMPRSS6 silencing on iron concentration in the liver.

Detailed Description

Described herein are methods and uses thereof for inhibiting iRNA expression from the TMPRSS6 gene in a cell or mammal in which the iRNA targets the TMPRSS6 gene. Also provided are compositions and methods for treating pathological conditions and diseases caused by gene expression, such as conditions associated with elevated iron levels. irnas direct sequence-specific degradation of mRNA by a process called RNA interference (RNAi). In an alternative embodiment, the iRNA activates expression of the TMPRSS6 gene in a cell or mammal in which the iRNA targets the TMPRSS6 gene.

TMPRSS6 plays an important role in iron homeostasis as an inhibitor of HAMP gene expression. The HAMP gene encodes the hepatic hormone, hepcidin, which is the core regulator of iron homeostasis. The hepcidin binds to the ferroportin-ferroportin (FPN1) which is predominantly localized on absorptive intestinal cells, hepatocytes and macrophages. Binding of hepcidin to the extracellular domain of ferroportin results in internalization and degradation of ferroportin, thus reducing dietary iron absorption from the intestine and iron release from macrophages and hepatocytes. HAMP gene expression may be stimulated in response to BMP-co-receptor Hemojuvelin (HJV) -mediated Bone Morphogenetic Protein (BMP)/progeny of the mother Against Decapentaplegic (SMAD) dependent signaling cascades. A key role of TMPRSS6 in central HAMP regulation is to inhibit BMP-mediated HAMP upregulation. TMPRSS6 inhibits BMP-mediated HAMP upregulation by cleaving the BMP co-receptor HJV, which is essential for BMP-mediated HAMP upregulation; thus preventing BMP signaling, SMAD transport to the nucleus and HAMP transcriptional activation.

Several human and mouse studies have demonstrated the role of TMPRSS6 in HAMP regulation and iron homeostasis (Du) et al, Science (Science) 2008, vol 320, p 1088-. Studies have shown that loss of functional mutations in TMPRSS6 may lead to upregulation of hepcidin expression, resulting in hereditary iron-deficiency anemia (IRIDA), known as refractory iron-deficiency anemia (Finberg. seninars in Hematology (Hematology corp.) 2009, volume 46, pages 378-86), characterized by elevated hepcidin levels, hypochromous minicell anemia, low mean erythrocyte volume (MCV), low transferrin saturation, poor absorption of oral iron, and inadequate response to parenteral iron. However, loss of functional mutations in HAMP positive regulators (e.g., BMP1, BMP4, and HFE) have been shown to down-regulate hepcidin expression and cause iron-excess dysregulation (Milet et al, Am J Hum Gen 2007, vol.81, p.799-. In primary iron-overabundance disorders (collectively referred to as Hereditary Hemochromatosis (HH)), hepcidin levels are low in anemias characterized by massive ineffective hematopoiesis and in iron excess (secondary hemochromatosis), such as beta-Thalassemia Intermedia (TI), despite elevated serum iron concentrations and iron stores. The intermediate beta-Thalassemia Mouse Model has shown that loss of expression of TMPRSS6 results in elevated Hepcidin levels (Finberg 2010 oral presentation: "TMPRSS 6, an inhibitor of Heapatic BMP/Smad Signaling, is required for Hepcidin Suppression and Iron Loading in a Mouse Model of beta-Thalassemia (" Hepatic BMP/Smad Sigss 6 is required for Hepcidin Suppression and Iron Loading in the beta-Thalassemia Mouse Model), American Society of health Annual Meeting 2010 (Annual Meeting of the Annual Society of American Society of Hematology 2010), Abstract number: 164).

The present invention describes methods and iRNA compositions for modulating expression of the TMPRSS6 gene. In certain embodiments, the use of TMPRSS 6-specific irnas reduces or inhibits expression of TMPRSS6, thereby resulting in increased HAMP expression and decreased serum iron levels. Thus, inhibition of TMPRSS6 gene expression or activity using the RNA compositions characterized in the present invention may be a useful method of therapy directed to reducing iron levels in a subject. Such inhibition may be useful for treating disorders associated with elevated iron levels, such as hemochromatosis or thalassemia, e.g., beta-thalassemia.

The iRNA of the compositions described herein includes an RNA strand (antisense strand) having a region that is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides, that is substantially complementary to at least a portion of an mRNA transcript of the TMPRSS6 gene. The use of these irnas makes it possible to target mRNA degrading genes involved in pathologies associated with expression of TMPRSS6 in mammals. Very low doses of TMPRSS6 iRNA may in particular and efficiently mediate RNAi, resulting in a significant inhibition of TMPRSS6 gene expression. Using cell-based assays, the inventors have shown that irnas targeting TMPRSS6 can mediate RNAi particularly and efficiently, resulting in significant inhibition of TMPRSS6 gene expression. Thus, methods and compositions comprising these irnas are useful for treating pathological processes that may be mediated by TMPRSS6, for example for treating disorders that cause elevated levels of iron, such as hemochromatosis, or β -thalassemia, such as β -thalassemia intermedia. The following detailed description discloses how to make and use iRNA-containing compositions to inhibit TMPRSS6 gene expression, as well as compositions and methods for treating diseases and conditions caused by expression of such genes.

Embodiments of the pharmaceutical compositions characterized herein also include irnas having an antisense strand, wherein the antisense strand comprises a region that is 30 or fewer nucleotides in length, generally 19-24 nucleotides, that is substantially complementary to at least a portion of an RNA transcript of the TMPRSS6 gene, along with a pharmaceutically acceptable carrier. Embodiments of the compositions characterized in this invention also include irnas having an antisense strand, wherein the antisense strand has a region of complementarity that is 30 or fewer nucleotides in length, generally 19-24 nucleotides, and that is substantially complementary to at least a portion of the RNA transcript of the TMPRSS6 gene.

Thus, in some aspects, pharmaceutical compositions containing TMPRSS6 iRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of TMPRSS6 gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of TMPRSS6 gene are characterized in the present invention.

I.Definition of

For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. Definitions in this section should prevail if there is a clear inconsistency between the use of a term in other sections of the description and its definition provided in this section.

"G", "C", "A", "T" and "U" each generally denote nucleotides containing guanine, cytosine, adenine, thymidine and uracil, respectively, as bases. However, it is to be understood that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide, as described in further detail below, or to a substitute part. It is well understood by the skilled artisan 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 a replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequence of the dsRNA characterized herein by nucleotides containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively, to form G-U wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for use in the compositions and methods described herein.

As used herein, "transmembrane serine protease 6" ("TMPSSR 6") refers to a particular polypeptide expressed in a cell. TMPRSS6 is also known as matriptase-2, IRIDA (refractory iron deficiency anemia), transmembrane protease serine 6, type II transmembrane serine protease 6, and membrane-bound mosaic serine protease matriptase-2. TMPRSS6 is a serine protease type II transmembrane protein of about 899 amino acids in length. TMPRSS6 contains multiple domains, for example, a short endo-domain a transmembrane domain, a echinospermin/enteropeptidase domain (SEA) domain, two complement factors/echinococcus embryo growth factor domains (CUBs), three LDL-R like domains (LDLa), and a trypsin-like serine protease domain with a conserved His-Asp-Ser triad (HDS). The sequence of the human TMPRSS6 mRNA transcript can be found at (NM-153609.2 (SEQ ID NO:1) (FIG. 1)).

As used herein, the term "iRNA" refers to a substance that contains an RNA as that term is defined herein and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. In one embodiment, iRNA as described herein effects inhibition of TMPRSS6 expression. Alternatively, in another embodiment, iRNA activates TMPRSS6 expression as described herein.

As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule (including messenger RNA (mRNA), which is the product of RNA processing of a primary transcript product) formed during transcription of the TMPRSS6 gene. The target portion of the sequence will be at least long enough to serve as a substrate for the iRNA to direct cleavage at or near this portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all subranges therebetween. As a non-limiting example, the target sequence may have from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

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

As used herein and unless otherwise specified, when used to describe a first nucleotide sequence relative to a second nucleotide sequence, the term "complementary" refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as will be understood by the skilled artisan. Such conditions may for example be stringent conditions, wherein stringent conditions may comprise: 400mM NaCl, 40mM PIPES pH 6.4, 1mM EDTA, 50 ℃ or 70 ℃ for 12-16 hours, followed by washing. Other conditions may be used, such as physiologically relevant conditions that may be encountered inside an organism, for example. The skilled person will be able to determine the set of conditions most suitable for checking the complementarity of the two sequences, depending on the final application of the hybridized nucleotides.

Complementary sequences within an iRNA (e.g., within a dsRNA as described herein) include base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, where the first sequence is referred to herein as "substantially complementary" with respect to the second sequence, the two sequences may be fully complementary, or when hybridized to a duplex up to 30 base pairs (bp), they may form one or more, but typically no more than 5, 4, 3 or 2 mismatched base pairs, while still retaining the ability to hybridize under conditions most relevant to their end use, e.g., to inhibit gene expression via the RISC pathway. However, where two oligonucleotides are designed to hybridize forming one or more single stranded overhangs, such overhangs should not be considered mismatches in terms of determining complementarity. For example, for the purposes described herein, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length may still be referred to as "fully complementary" wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide.

As used herein, "complementary" sequences may also include or be formed entirely of non-Watson-Crick base pairs and/or base pairs consisting of non-natural and modified nucleotides, provided that the above requirements for their hybridization capabilities are met. Such non-Watson-Crick base pairs include, but are not limited to, G: U wobble or Hoogstein base pairing.

The terms "complementary," "fully complementary," and "substantially complementary" may be used herein with respect to base matching between the sense strand and the antisense strand of a dsRNA or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context in which they are used.

As used herein, a polynucleotide that is "substantially complementary to at least a portion of messenger rna (mRNA)" refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding TMPRSS 6). For example, if a polynucleotide is substantially complementary to an uninterrupted portion of the mRNA encoding TMPRSS6, that sequence is complementary to at least a portion of the TMPRSS6 mRNA.

As used herein, the term "double-stranded RNA" or "dsRNA" refers to an iRNA comprising an RNA molecule or molecular complex having a hybrid duplex region comprising two anti-parallel and substantially complementary nucleic acid strands that will be referred to as having "sense" and "antisense" orientations relative to a target RNA. The duplex region may be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, but will generally range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. With respect to duplexes between 9 and 36 base pairs, the duplex can have any length within this range, e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any subrange therebetween, including but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNA produced in cells by processing with Dicer and similar enzymes typically has a base pair length in the range of 19-22. One strand of the duplex region of the dsDNA comprises a sequence that is substantially complementary to a region of the target RNA. The two strands forming the duplex structure may be from a single RNA molecule having at least one region of self-complementarity, or may be formed from two or more separate RNA molecules. Where the duplex region consists of two strands of a single molecule, the molecule may have the duplex region separated by a single nucleotide strand (referred to herein as a "hairpin loop") between the 3 '-end of one strand and the 5' -end of the respective other strand forming the duplex structure. The hairpin loop may comprise at least one unpaired nucleotide; in some embodiments, a hairpin loop may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 31, or more unpaired nucleotides. In the case where the two substantially complementary strands of the dsRNA are composed of separate RNA molecules, these molecules need not be covalently linked, but may be covalently linked. In the case where two strands are covalently linked by means other than a hairpin loop, the linking structure is referred to as a "linker". The term "siRNA" is also used herein to refer to dsRNA as described above.

The skilled artisan will recognize that the terms "RNA molecule" or "ribonucleic acid molecule" include not only RNA molecules as expressed or present in nature, but also RNA analogs and derivatives comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, "ribonucleosides" include nucleobases and ribose, and "ribonucleotides" are ribonucleosides having 1, 2 or 3 phosphate moieties. However, as used herein, the terms "ribonucleoside" and "ribonucleotide" are considered equivalent. For example, as described below, RNA can be modified in nucleobase structure or in ribose-phosphate backbone structure. However, molecules containing ribonucleoside analogues or derivatives must retain the ability to form duplexes. As non-limiting examples, the RNA molecule can also comprise at least one modified ribonucleoside including, but not limited to, a 2 '-O-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, a non-base nucleoside, a 2 '-deoxy-2' -fluoro modified nucleoside, a 2 '-amino modified nucleoside, a 2' -alkyl modified nucleoside, a morpholino nucleoside, a phosphoramidate, or a nucleoside comprising a non-natural base, or any combination thereof. Alternatively, the RNA molecule may comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more modified ribonucleosides up to the entire length of the dsRNA molecule. For each of these multiple modified ribonucleosides in an RNA molecule, the modifications need not be the same. In one embodiment, the modified RNA contemplated for use in the methods and compositions described herein is a Peptide Nucleic Acid (PNA) that has the ability to form a desired duplex structure and allows or mediates specific degradation of the target RNA via the RISC pathway.

In one aspect, the modified ribonucleosides comprise deoxyribonucleosides. In this case, the iRNA agent may comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang or one or more deoxynucleosides within the double stranded portion of the dsRNA. However, it is self-evident that in any case the term "iRNA" does not cover double stranded DNA molecules.

In one aspect, the RNA interfering agent comprises a single-stranded RNA that interacts with a target RNA sequence to direct cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into plant and invertebrate cells is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al, Genes Dev. (Gene and development) 2001,15: 485). The ribonuclease enzyme Dicer processes dsRNA into 19-23 base pair short interfering RNA with characteristic double base 3' overhangs (Bernstein et al, (2001) Nature (Nature) 409: 363). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases open the siRNA duplex, making it possible for the complementary antisense strand to direct target recognition (Nykanen et al, (2001) Cell 107: 309). Upon binding to the appropriate target mRNA, one or more endonucleases internal to the RISC cleave the target to induce silencing (Elbashir et al, (2001) Genes Dev. (Gene and development) 15: 188). Thus, in one aspect, the invention relates to single stranded RNA that facilitates the formation of a RISC complex that effects target gene silencing.

As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from a duplex structure of an iRNA (e.g., dsRNA). For example, nucleotide overhangs are present when the 3 '-end of one strand of a dsRNA extends beyond the 5' -end of the other strand or vice versa. The dsRNA may comprise an overhang of at least one nucleotide; alternatively, the overhang may comprise at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 or more nucleotides. The nucleotide overhang may comprise or consist of nucleotide/nucleoside analogues (including deoxynucleotides/nucleosides). The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may be present at the 5 'end, the 3' end, or both ends of the antisense or sense strand of the dsRNA.

In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3 'end and/or the 5' end. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3 'end and/or the 5' end. In another embodiment, one or more nucleotides in the overhang are replaced with a nucleoside phosphorothioate.

As used herein in reference to dsRNA, the term "flush" or "blunting" means that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., there is no nucleotide overhang. One or both ends of the dsRNA may be flush. Where the two ends of the dsRNA are flush, the dsRNA is said to be blunt-ended. For clarity, "blunt ended" dsRNA is dsRNA that is flush at both ends, i.e., no nucleotide overhang at any end of the molecule. In most cases, such a molecule will be double stranded over its entire length.

The term "antisense strand" or "guide strand" refers to the strand of an iRNA (e.g., dsRNA) that includes a region of substantial complementarity to a target sequence. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence as defined herein). Where the complementary region is not fully complementary to the target sequence, the mismatch may be present within an internal or terminal region of the molecule. Typically, the most tolerated mismatches are present in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5 'and/or 3' end.

The term "sense strand" or "passenger strand" as used herein refers to a strand of an iRNA (e.g., dsRNA) that includes a region that is substantially complementary to a region of an antisense strand as that term is defined herein.

As used herein, in one embodiment, the term "SNALP" refers to a stable nucleic acid-lipid particle. SNALP represent lipid vesicles that coat a reduced aqueous interior, wherein the aqueous interior comprises a nucleic acid (e.g., an iRNA or a plasmid from which an iRNA is transcribed). SNALP are described, for example, in U.S. patent application publication nos. 20060240093, 20070135372 and in international application No. WO 2009082817. Examples of "SNALP" formulations are described elsewhere herein.

When referring to iRNA, "introduced into a cell" means promoting or effecting uptake or absorption into the cell, as understood by one of ordinary skill in the art. Uptake or uptake of iRNA can occur by unassisted diffusion processes or active cellular processes or by aids or devices. The meaning of this term is not limited to cells in vitro; an iRNA can also be "introduced into a cell," where the cell is part of a living organism. In this case, introduction into the cell will include delivery to the organism. For example, for in vivo delivery, the iRNA may be injected into a tissue site or administered systemically. In vivo delivery may also be by means of β -glucan delivery systems such as those described in U.S. patent nos. 5,032,401 and 5,607,677 and U.S. publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. Included in vitro introduction into cells are methods known in the art, such as electroporation and lipofection. Additional methods are described below or are known in the art.

As used herein, the term "modulating expression" refers to at least partially "inhibiting" or partially "activating" expression of TMPRSS6 gene in cells treated with an iRNA composition as described herein, as compared to the expression of TMPRSS6 in untreated cells.

The terms "activate", "enhance", "upregulate … … expression", "increase … … expression", and the like, as long as they relate to the TMPRSS6 gene, all herein refer to at least partially activating expression of the TMPRSS6 gene, as demonstrated by an increase in the amount of TMPRSS6 mRNA that can be isolated from or detected in a first cell or population of cells in which TMPRSS6 gene is transcribed and which has been treated such that TMPRSS6 gene expression is increased as compared to a second cell or population of cells (control cells) that is substantially identical to the first cell or population of cells but has not been so treated.

In one embodiment, TMPRSS6 gene expression is activated by administration of iRNA as described herein by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%. In some embodiments, the TMPRSS6 gene is activated by administration of iRNA characterized in the present invention by at least about 60%, 70%, or 80%. In some embodiments, TMPRSS6 gene expression is activated by administration of iRNA as described herein by at least about 85%, 90%, or 95% or more. In some embodiments, TMPRSS6 gene expression is increased at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more in a cell treated with an iRNA as described herein compared to expression in an untreated cell. Activation of expression by small dsrnas is described, for example, in Li (plum) et al, 2006proc.natl.acad.sci.u.s.a. (journal of the american national academy of sciences) 103:17337-42 and in US 20070111963 and US 2005226848, each of which is incorporated herein by reference.

The terms "silence", "inhibit expression of … …", "down-regulate expression of … …", "inhibit expression of … …", and the like, as they relate to TMPRSS6 gene, all of which refer herein to at least partial inhibition of expression of TMPRSS6 gene, as demonstrated by a reduction in the amount of TMPRSS6 mRNA that can be isolated from or detected in a first cell or population of cells in which the TMPRSS6 gene is transcribed and which has been treated such that expression of TMPRSS6 gene is inhibited as compared to a second cell or population of cells (control cells) that is substantially identical to the first cell or population of cells but has not been so treated. The degree of inhibition is generally expressed by the following formula

Alternatively, the degree of inhibition may be given in terms of a decrease in a parameter functionally linked to the expression of the TMPRSS6 gene (e.g., the amount of protein encoded by the TMPRSS6 gene or the number of cells showing a certain phenotype (e.g., decreased iron levels or iron absorption)). In principle, TMPRSS6 gene silencing can be determined in any cell expressing TMPRSS6 either constitutively or by genome engineering and by any suitable assay.

For example, in certain instances, TMPRSS6 gene expression is inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of iRNA characterized in the present invention. In some embodiments, the TMPRSS6 gene is inhibited by administering an iRNA as described herein by at least about 60%, 70%, or 80%. In some embodiments, the TMPRSS6 gene is inhibited by administering iRNA as described herein by at least about 85%, 90%, 95%, 98%, 99%, or more.

As used herein in the context of expression of TMPRSS6, the terms "treatment or treatment" (treat and treatment), and the like, refer to attenuation or alleviation from pathological processes mediated by expression of TMPRSS 6. In the context of the present invention, the terms "treat or treat", and the like, in reference to any other condition described below (in addition to the pathological process mediated by TMPRSS6 expression), mean to attenuate or alleviate at least one symptom associated with this condition (hemochromatosis (e.g. β -thalassemia)), or to slow or reverse the progression or expected progression of this condition.

By "decrease" in the context of disease markers or symptoms is meant a statistically significant decrease in such levels. The reduction may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably to a level that is acceptable within the normal range for individuals who do not suffer from such disorders.

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

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

As used herein, the term "thalassemia" refers to an inherited recessive hematologic disorder. Loss of function mutations result in a reduced or non-synthetic rate of one of the globin chains that make up hemoglobin and result in a deficiency of normal globin. Thalassemia patients result in deficiencies in alpha globin (referred to as alpha-thalassemia), beta globin (referred to as beta-thalassemia), or in rare cases delta globin. In α -thalassemia, excess β chains form unstable tetramers with abnormal oxygen dissociation curves. Beta-thalassemia may be mild, severe or intermediate.

The beta globin chain is encoded by a single gene called the HBB (hemoglobin, beta) gene. Beta-thalassemia minor occurs in patients carrying one mutant beta-thalassemia allele and one wild-type allele. This disease does not affect blood iron levels and the patient does not require treatment. Beta-thalassemia major occurs when a patient carries two knockout mutant beta-thalassemia alleles. Excess iron accumulates in these patients and is stored primarily in the hypertrophic kupffer cells. Patients with severe beta-thalassemia are typically treated with long-term blood transfusion therapy, iron chelation, splenectomy, and allogeneic hematopoietic transplantation. Beta-thalassemia intermedia occurs when a patient carries one knockout allele and one partially dysfunctional allele of the beta-thalassemia gene. Excess iron accumulates in these patients and is stored primarily in hepatocytes. Patients with thalassemia major and thalassemia intermediate develop anemia (hypoxia), which results in increased EPO (erythropoietin) and thus largely compensatory and ineffective erythropoiesis (production of red blood cells in the bone marrow by stem cells). Patients with intermediate thalassemia sometimes develop hepatosplenomegaly, jaundice, osteopenia, thrombotic episodes, leg ulcers, low blood pressure in the pulmonary circulation, congestive heart failure, diabetes, growth hormone deficiency, hypothyroidism, hypoparathyroidism, hypogonadism, and facial abnormalities.

As used herein, the term "hemochromatosis" refers to a disorder that results in excessive iron absorption from the gastrointestinal tract. Hemochromatosis occurs in two forms: primary and secondary. Primary hemochromatosis, the most common genetic disorder in the united states (estimated to be suffered from 1 out of every 200 to 300 us), is often caused by specific genetic problems that cause excessive iron absorption. Secondary or acquired hemochromatosis can be caused by a variety of diseases (e.g., thalassemia or iron granulocytic anemia). Secondary hemochromatosis sometimes develops in patients with hemolytic anemia and chronic alcoholism. Symptoms of hemochromatosis include abdominal pain, joint pain, weakness, lack of energy, weakness, dull skin (often referred to as "bronzing"), and loss of body hair.

The term "pharmaceutically acceptable carrier" refers to a carrier used 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 media. For orally administered drugs, 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 preserving agents. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binders may include starch and gelatin, while if present, the lubricant will typically be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with various materials (e.g., glyceryl monostearate or glyceryl distearate) to delay absorption in the gastrointestinal tract. The pharmaceutical agents included in the pharmaceutical formulations are described further below.

As used herein, a "subject" is a mammal, such as dogs, horses, cats, and other non-human primates. In a preferred embodiment, the subject is a human.

As used herein, the term "LNPXX," where "XX" is a number, also referred to herein as "AFXX. For example, LNP09 is also known as AF09 and LNP12 is also known or referred to as AF 12.

As used herein, the terms "comprises" or "comprising" (including and comprises) are intended to refer to compositions, methods, and one or more corresponding components thereof, which are essential to the invention, but which are open to the inclusion of unspecified elements (whether or not necessary).

As used herein, the term "consisting of … …" refers to those elements required for a given embodiment. The terms allow for the presence of elements that do not materially affect one or more of the basic and novel or functional features of this embodiment characterized in this invention.

The term "consisting of … …" refers to the compositions, methods, and their respective components as described herein, excluding any elements not mentioned in this description of the example.

II.Double-stranded ribonucleic acid (dsRNA)

iRNA agents that modulate expression of TMPRSS6 gene are described herein. In one embodiment, the iRNA agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of the TMPRSS6 gene in a cell or a mammal (e.g., in a human with elevated iron levels, such as in a patient with β -thalassemia or hemochromatosis). The dsRNA includes an antisense strand having a region of complementarity that is complementary to at least a portion of the mRNA formed in the expression of the TMPRSS6 gene. This region of complementarity is 30 nucleotides in length or less, generally 19-24 nucleotides in length, and wherein when contacted with a cell expressing the TMPRSS6 gene, then the dsRNA inhibits expression of the TMPRSS6 gene by at least 10%, as determined by, for example, PCR or branched dna (bdna) -based methods, or by protein-based methods, as determined by western blotting. In one embodiment, the iRNA agent activates TMPRSS6 gene expression in a cell or mammal. Can be measured by measuring TMPRSS6 mRNA levels, e.g., by bDNA or An assay, or the expression of the TMPRSS6 gene in cell cultures, such as COS cells, HeLa cells, primary hepatocytes, HepG2 cells, primary culture cells or in a biological sample from the subject, is determined by measuring protein levels, e.g., by immunofluorescence analysis, using, e.g., western blotting or flow cytometry.

dsRNA includes two RNA strands that are complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of the dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary to, and typically fully complementary to, the target sequence. The target sequence may be derived from the sequence of mRNA formed during expression of the TMPRSS6 gene. The other strand (the sense strand) includes a region of complementarity to the antisense strand, such that when combined under suitable conditions, the two strands hybridize and form a duplex structure. Typically, the duplex structure is between 15 and 30 and includes 15 and 30 base pairs in length, more typically between 18 and 25 and includes 18 and 25 base pairs in length, yet more typically between 19 and 24 and includes 19 and 24 base pairs in length, and more typically between 19 and 21 and includes 19 and 21 base pairs in length. Typically, the region complementary to the target sequence is between 15 and 30 and includes 15 and 30 nucleotides in length, more typically between 18 and 25 and includes 18 and 25 nucleotides in length, yet more typically between 19 and 24 and includes 19 and 24 nucleotides in length, and more typically between 19 and 21 and includes 19 and 21 nucleotides in length. In some embodiments, the iRNA is between 15 and 20 and includes 15 and 20 nucleotides in length, and in other embodiments, the iRNA is between 25 and 30 and includes 25 and 30 nucleotides in length. As the skilled person will recognise, the targeted region of RNA targeted for cleavage will most often be part of a larger RNA molecule (often an mRNA molecule). In related cases, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). In some cases, dsRNA with duplexes as short as 9 bases can mediate RNAi-directed RNA cleavage. Most often, the target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.

One of ordinary skill in the art will also recognize that the duplex region is the main functional portion of the dsRNA, for example, a duplex region of 9 to 36 base pairs (e.g., 15-30 base pairs). Thus, in one embodiment, to achieve a functional duplex (e.g., 15-30 base pairs) that is processed to direct the desired RNA cleavage, an RNA molecule or RNA molecule complex having a duplex region greater than 30 base pairs is dsRNA. Thus, the skilled artisan will recognize that in one embodiment the miRNA is dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, iRNA agents useful for targeting TMPRSS6 expression are not produced in the target cell by cleavage of larger dsRNA.

The dsRNA as described herein may further comprise one or more single stranded nucleotide overhangs. Such dsRNA can be synthesized by standard methods known in the art as discussed further below, for example, by using an automated DNA synthesizer, such as is commercially available from the company Biosearch, Applied Biosystems, inc. In one embodiment, the TMPRSS6 gene is the human TMPRSS6 gene. In another embodiment, the TMPRSS6 gene is a mouse or rat TMPRSS6 gene. The sequence of mouse TMPRSS6 mRNA can be found under GenBank accession No. NM-027902 (GI:125656151, record date 12 months and 28 days 2010). The sequence of rat TMPRSS6 mRNA can be found under GenBank accession No. NM-001130556.1 (GI:194474097, record date 2011, 1/17). In particular embodiments, the first sequence is a dsRNA sense strand comprising a sense sequence of one of tables 2, 3 or 4 and the second sequence is a dsRNA antisense strand comprising an antisense sequence of one of tables 2, 3 or 4. Alternative dsRNA species targeting elsewhere in the target sequence provided in tables 2, 3 or 4 can be readily determined using the target sequence and flanking TMPRSS6 sequences.

In one aspect, the dsRNA will comprise at least two nucleotide sequences, a sense sequence and an antisense sequence, whereby the sense strand is selected from the group of sequences provided in table 2, 3 or 4. In this aspect, one of the two sequences is complementary to the other of the two sequences, wherein one of the sequences is substantially complementary to the sequence of the mRNA produced in expression of the TMPRSS6 gene. In this regard, a dsRNA will include two oligonucleotides, wherein one oligonucleotide is described as the sense strand in table 2, 3 or 4 and the second oligonucleotide is described as the corresponding antisense strand from the sense strand in table 2, 3 or 4. As described elsewhere herein and as known in the art, the complementary sequence of a dsRNA may also be comprised as a self-complementary region of a single nucleic acid molecule, as opposed to being on a separate oligonucleotide.

It is well known to the skilled person that dsRNAs with duplex structures of between 20 and 23 base pairs, but especially 21 base pairs, have been approved due to the particularly efficient induction of gRNA interference (Elbashir et al, EMBO 2001,20: 6877-. However, others have found that shorter or longer RNA duplex structures may also be effective. In the examples described above, due to the nature of the oligonucleotide sequences provided in tables 2, 3 or 4, the dsRNA described herein may comprise at least one strand of minimum 21nt in length. It is reasonable to expect that shorter duplexes of one of the sequences in tables 2, 3 or 4 with only a few nucleotides short at one or both ends may be similarly effective compared to the dsrnas described above. Thus, dsrnas are contemplated according to the invention which have a partial sequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides from one of the sequences in tables 2, 3 or 4 and which differ from a dsRNA comprising the full sequence in their ability to inhibit expression of the TMPRSS6 gene by no more than 5%, 10%, 15%, 20%, 25% or 30%.

In addition, the RNAs provided in tables 2, 3 or 4 identify sites in the TMPRSS6 transcript that are susceptible to RISC-mediated cleavage. In this regard, the invention further features irnas that target within one of such sequences. As used herein, an iRNA is said to target a particular site within a transcript if it promotes cleavage of the RNA transcript anywhere within that site. Such iRNA will typically comprise at least 15 contiguous nucleotides from one of the sequences provided in tables 2, 3 or 4 linked to an additional nucleotide sequence taken from a region where the selected sequence in the TMPRSS6 gene remains contiguous.

Although the target sequences are generally 15-30 nucleotides in length, there is wide variation in the applicability of a particular sequence within this range to direct cleavage of any given target RNA. The various software packages and guidelines described herein provide guidance for identifying the optimal target sequence for any given gene target, but empirical methods may also be employed in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed on the target RNA sequence in a word-by-word or graphical manner (including, for example, in a computer manner) to identify sequences within a range of sizes that are likely to serve as target sequences. By moving the sequence "window" stepwise by one nucleotide upstream or downstream of the initial target sequence position, the next potential target sequence can be identified until a full set of possible sequences is identified for any given target size selected. This method, in conjunction with systematically synthesizing and testing the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform best, can identify those RNA sequences that mediate the best inhibition of target gene expression when targeted with an iRNA agent. Thus, while the sequences identified, for example, in tables 2, 3 or 4 represent valid target sequences, it is contemplated that further optimization of inhibition efficiency may be achieved by "stepping the window" one nucleotide upstream or downstream of a given sequence to identify sequences with equal or better inhibition characteristics.

In addition, it is contemplated that for any sequence identified, such as in tables 2, 3 or 4, further optimization can be achieved by: the system adds or removes nucleotides to generate longer or shorter sequences and tests those generated by advancing a window of longer or shorter size from that point forward or backward along the target RNA. Again, linking this approach to the generation of new candidate targets, while testing the effectiveness of irnas based on these target sequences in an inhibition assay as known in the art or as described herein, may lead to further improvements in efficiency inhibition. Again, such optimized sequences can be adjusted to further optimize the molecule (e.g., increase serum stability or circulating half-life, increase thermostability, enhance transmembrane delivery, target a particular location or cell type, increase interaction with silencing pathway enzymes, increase endosomal discharge, etc.) as an expression inhibitor, e.g., by introducing modified nucleotides as described herein or as known in the art, adding or altering overhangs, or other modifications as known in the art and/or discussed herein.

An iRNA as described herein can contain one or more mismatches with respect to a target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains a mismatch relative to the target sequence, it is preferred that the mismatched region should not be located within the center of the complementary region. If the antisense strand of the iRNA contains a mismatch relative to the target sequence, it is preferred that the region of mismatch should be limited to within the last 5 nucleotides from the 5 'or 3' end of the complementary region. For example, for an RNA strand of a 23 nucleotide iRNA drug that is complementary to a region of the TMPRSS6 gene, the RNA strand does not typically contain any mismatches within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether iRNA containing a mismatch relative to the target sequence is effective in inhibiting TMPRSS6 gene expression. It is important to consider the efficacy of mismatched irnas in inhibiting expression of TMPRSS6 gene, especially when the specific complementary region in TMPRSS6 gene is known to have polymorphic sequence variation within the population.

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. Such dsrnas having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. In yet another embodiment, the RNA of the iRNA (e.g., dsRNA) is chemically modified to enhance stability or other beneficial characteristics. Nucleic acids characterized in the present invention can 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 (ed.), John Wiley & Sons, Inc. (Wihn Willi-parent-son publishing Co., Ltd.), New York, N.Y., U.S., which are hereby incorporated by reference. Modifications include, for example, (a) terminal modifications, e.g., 5 'terminal modifications (phosphorylation, conjugation, inverted linkage, etc.), 3' terminal modifications (conjugation, DNA nucleotides, inverted linkage, etc.), (b) base modifications, e.g., base substitutions to a stabilizing base, destabilizing base, or base pairing with an extended partner library, base removal (non-base nucleotides), or conjugated base, (c) sugar modifications (e.g., at 2 'or 4' positions) or sugar substitutions, and (d) backbone modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to, RNA containing a modified backbone or no natural internucleoside linkages. RNAs with modified backbones include those that do not have a phosphorus atom in the backbone, among others. For the purposes of this specification and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone may also be considered oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates, including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3' -phosphoramidate and aminoalkyl phosphoramidate esters, thiocarbonylphosphamide esters, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these esters, and those esters having reversed polarity where adjacent pairs of nucleoside units are 3'-5' to 5'-3' or 2'-5' to 5'-2' linked. Different 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. patent nos. 3,687,808; 4,469,863; 4,476,301, respectively; 5,023,243; 5,177,195, respectively; 5,188,897, respectively; 5,264,423; 5,276,019; 5,278,302; 5,286,717, respectively; 5,321,131, respectively; 5,399,676, respectively; 5,405,939, respectively; 5,453,496, respectively; 5,455,233, respectively; 5,466,677, respectively; 5,476,925, respectively; 5,519,126, respectively; 5,536,821, respectively; 5,541,316, respectively; 5,550,111, respectively; 5,563,253, respectively; 5,571,799, respectively; 5,587,361, respectively; 5,625,050, respectively; 6,028,188, respectively; 6,124,445, respectively; 6,160,109, respectively; 6,169,170, respectively; 6,172,209, respectively; 6,239,265, respectively; 6,277,603, respectively; 6,326,199, respectively; 6,346,614, respectively; 6,444,423, respectively; 6,531,590, respectively; 6,534,639, respectively; 6,608,035, respectively; 6,683,167, respectively; 6,858,715, respectively; 6,867,294, respectively; 6,878,805, respectively; 7,015,315, respectively; 7,041,816, respectively; 7,273,933, respectively; 7,321,029, respectively; and U.S. patent RE39464, each of which is incorporated herein by reference.

Wherein the modified RNA backbone, excluding the phosphorus atom, has a backbone formed of short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom internucleoside linkages or heterocyclic internucleoside linkages. These include those having the following structures: morpholino linkages (formed in part from the sugar portion of a nucleoside); a siloxane backbone; sulfide, sulfoxide and sulfone backbones; a formylacetyl and thiocarbonylacetyl backbone; methylene formyl acetyl and thio formyl acetyl backbones; an alkene-containing backbone; amino groupA sulfonate backbone; methylene imino and methylene hydrazino backbones; sulfonate and sulfonamide backbones; an amide backbone; and has a blend of N, O, S and CH₂Other backbones of the component parts.

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, respectively; 5,185,444, respectively; 5,214,134, respectively; 5,216,141, respectively; 5,235,033, respectively; 5,64, 562; 5,264,564, respectively; 5,405,938, respectively; 5,434,257, respectively; 5,466,677, respectively; 5,470,967, respectively; 5,489,677; 5,541,307, respectively; 5,561,225, respectively; 5,596,086, respectively; 5,602,240; 5,608,046, respectively; 5,610,289, respectively; 5,618,704, respectively; 5,623,070, respectively; 5,663,312, respectively; 5,633,360, respectively; 5,677,437, respectively; and 5,677,439, each of which is incorporated herein by reference.

In other RNA mimetics suitable or contemplated for iRNA, the sugar and internucleoside linkages, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with the appropriate nucleic acid target compounds. One such oligomeric compound, known as a Peptide Nucleic Acid (PNA), has been shown to have RNA mimics with superior hybridization properties. In PNA compounds, the sugar backbone of RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases remain and are bound, directly or indirectly, to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teachings on PNA compounds can be found, for example, in Nielsen et al, Science, 1991,254, 1497-1500.

Some examples of characterizations in the present invention include RNA with phosphorothioate backbone and oligonucleosides with heteroatom backbone, and in particular- -CH of U.S. Pat. No. 5,489,677, referenced above₂--NH--CH₂--、--CH₂--N(CH₃)--O--CH₂- - - [ named methylene (methylimino) or MMI backbone]、--CH₂--O--N(CH₃)--CH₂--、--CH₂--N(CH₃)--N(CH₃)--CH₂- - -and- -N (CH) ₃)--CH₂--CH₂- - - - - [ wherein the natural phosphoric acid diester is predominantThe chain is represented by- -O- -P- -O- -CH₂--]And the amide backbone of U.S. Pat. No. 5,602,240 referenced above. In some embodiments, the RNA characterized herein has the morpholino backbone structure of U.S. patent No. 5,034, 506 referenced above.

Modified RNAs may also contain one or more substituted sugar moieties. The irnas (e.g., dsRNA) characterized herein can include one of the following at the 2' position: OH; f; o-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁To C₁₀Alkyl or C₂To C₁₀Alkenyl and alkynyl groups. Exemplary suitable modifications include O [ (CH)₂)_nO]_mCH₃、O(CH₂)._nOCH₃、O(CH₂)_nNH₂、O(CH₂)_nCH₃、O(CH₂)_nONH₂And O (CH)₂)_nON[(CH₂)_nCH₃)]₂Wherein n and m are from 1 to about 10. In other embodiments, the dsRNA includes at the 2' position one of: c₁To C₁₀Lower alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH₃、OCN、Cl、Br、CN、CF₃、OCF₃、SOCH₃、SO₂CH₃、ONO₂、NO₂、N₃、NH₂Heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of an iRNA or groups for improving the pharmacodynamic characteristics of an iRNA, and other substituents with similar properties. In some embodiments, the modification comprises 2 '-methoxyethoxy (2' -O- -CH) ₂CH₂OCH₃Also known as 2'-O- (2-methoxyethyl) or 2' -MOE) (Martin et al, Helv. Chim. acta,1995,78486-504), i.e., alkoxy-alkoxy. Another exemplary modification is 2' -dimethylaminoxyethoxy, i.e., O (CH)₂)₂ON(CH₃)₂The group, also known as 2' -DMAOE, as described in the examples 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)₂Also described in the examples below.

Other modifications include 2 '-methoxy (2' -OCH)₃) 2 '-Aminopropoxy (2' -OCH)₂CH₂CH₂NH₂) And 2 '-fluoro (2' -F). Similar modifications can also be made at other positions on the RNA of the iRNA, particularly at the 3 'terminal nucleotide or at the 3' position of the sugar and the 5 'position of the 5' terminal nucleotide in 2'-5' linked dsRNA. The iRNA may also have a sugar mimetic, e.g., a cyclobutyl moiety 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. patent nos. 4,981,957; 5,118,800, respectively; 5,319,080, respectively; 5,359,044, respectively; 5,393,878, respectively; 5,446,137, respectively; 5,466,786, respectively; 5,514,785, respectively; 5,519,134, respectively; 5,567,811, respectively; 5,576,427, respectively; 5,591,722, respectively; 5,597,909, respectively; 5,610,300, respectively; 5,627,053, respectively; 5,639,873, respectively; 5,646,265, respectively; 5,658,873, respectively; 5,670,633, respectively; and 5,700,920, some of which are commonly owned by the present application and each of which is incorporated herein by reference.

irnas 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-propargyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-mercapto, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, and guanine, 5-halogen, especially 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-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified nucleotides in Biochemistry, Biotechnology and Medicine (Nucleosides Modified in Biochemistry, Biotechnology and Medicine), Herdewijn (Hedwight), P. ed, Wiley-VCH publishing company, 2008; in the sense Encyclopedia Of Polymer Science And Engineering, p.858-859, Kroschwitz, J.L, editors John Wiley & Sons (John Wiley, Willi-son publishing Co., 1990), those disclosed by Englisch (Engelitz) et al, Angewandte Chemie (applied chemistry), International edition, 1991,30,613 And those disclosed by Sanghvi, Y S.ke, Chapter 15, Research And Applications (dsRNA Research And application), p.289-302, Croooker, S.T. And Lebleu (Lebleb Liu), B.editors, CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of oligomeric compounds characterized in the invention. These nucleobases include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propargyluracil and 5-propargylcytosine. 5-methyl cytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 ℃ -1.2 ℃ (Sanghwei, Y.S., crook, S.T. and Lebleu (Leber Liu), B. editions, dsRNA Research and Applications (dsRNA Research and application), CRC Press, Boca Raton, 1993, p. 276 and 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-described modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-described U.S. Pat. nos. 3,687,808, and U.S. Pat. nos. 4,845,205; 5,130, 30; 5,134,066, respectively; 5,175,273, respectively; 5,367,066, respectively; 5,432,272; 5,457,187, respectively; 5,459,255; 5,484,908, respectively; 5,502,177, respectively; 5,525,711, respectively; 5,552,540, respectively; 5,587,469, respectively; 5,594,121,5,596,091; 5,614,617, respectively; 5,681,941, respectively; 6,015,886, respectively; 6,147,200, respectively; 6,166,197, respectively; 6,222,025, respectively; 6,235,887, respectively; 6,380,368, respectively; 6,528,640, respectively; 6,639,062, respectively; 6,617,438, respectively; 7,045,610, respectively; 7,427,672, respectively; and 7,495,088, each of which is incorporated herein by reference, and U.S. patent No. 5,750,692, which is also incorporated herein by reference.

The RNA of the iRNA may also be modified to include one or more Locked Nucleic Acids (LNAs). Locked nucleic acids are nucleotides with a modified ribose moiety, wherein the ribose moiety comprises an additional bridge connecting the 2 'carbon and the 4' carbon. This structure effectively "locks" the ribose in the 3' -internal structural conformation. Addition of locked Nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al, (2005) Nucleic Acids Research 33(1): 439-.

Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. nos. 6,268,490; 6,670, 461; 6,794,499, respectively; 6,998,484; 7,053,207, respectively; 7,084,125, respectively; and 7,399,845, each of which is incorporated herein by reference in its entirety.

Another modification of the RNA of an iRNA characterized in the present invention involves chemically linking the RNA to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties or cellular uptake of the iRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al, Proc. Natl. Acid. Sci. USA (Proc. Natl. Acad. Sci. USA), 1989,86:6553-6556), cholic acid (Manohara. et al, Bio.Med. chem. Let. (Bioorganic and medicinal chemical communication), 1994,4:1053-1060), thioethers such as hexyl-S-tritylmercaptan (beryl-S-tritylthiol) (Manohara. et al, Ann. N.Y. Acad. Sci. (New York. Acad. Sci., 1992, 660: 306-309; Macroha. orchid et al, Bio.Chem. chem. Let. (Biochemical and medicinal chemical communication), cysteine residues (Beishhaas. Achson. TM.) (Beishas. Acad. Sci., 1992., 660: 306-27; Mannohara. Lang. et al, Biorg. chem. Lam. Let. (Biochemical and chemical communication), cysteine residues (Beisha), 2765, Beishhaas. (Beisha), aliphatic probe, Beishhaas. 19820, Beisha, et al, Beisha, such as, Beisha, and so, such as, Beisha, and so on et al, Beisha, such as, Beisha, or a, Beisha, such as, Beisha, such as, a, Beisha, or a, such as a, or a, such as a, EMBO J, 1991, 10: 1111-; kabanov et al, FEBS Lett. (Federation of the European Biochemical society), 1990,259: 327-; svinarchuk et al, Biochimie (Biochemi), 1993,75:49-54), phospholipids, such as dicetyl-rac-glycerol or triethylammonium 1, 2-di-O-cetyl-rac-glycerol-3-phosphonate (Manohara et al, Tetrahedron Lett. (Tetrahedron Communication), 1995,36: 3651-one 3654; shea et al, Nucl. acids Res. (nucleic acids research), 1990,18:3777-3783), polyamine or polyethylene glycol chain (Manohara (Mannohara) et al, Nucleosides & Nucleotides, 1995,14: 969-containing 973), or adamantane acetic acid (Manohara (Mannohara) et al, Tetrahedron Lett. (Tetrahedron communication), 1995,36: 3651-containing 3654), a palmityl moiety (Mishra et al, biom. Biophys. acta (Proc. Biochem. Biophys., Pharma., 1995,1264: 229-containing 237), or an octadecylamine or hexylamino-carbonyloxy cholesterol moiety (Crooke (Kluk et al, J.rmacol. exp. Ther. (Pharmacol. Pharma. Ther. (Pharmacology. Pharmacol., 1996,277-containing 937).

In one embodiment, the ligand alters the distribution, targeting, or longevity of the iRNA drug into which the ligand is incorporated. In preferred embodiments, such a ligand provides enhanced affinity for a selected target, e.g., a molecule, cell, or cell type (e.g., a liver cell, such as a hepatocyte), compartment (e.g., a cell or organ compartment, a body tissue, organ, or region), as compared to, e.g., a species in which such a ligand is not present. Preferred ligands will not participate in duplex pairing in duplex nucleic acids.

Ligands may include naturally occurring substances, such as proteins (e.g., Human Serum Albumin (HSA), Low Density Lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include the following polyamino acids: polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene acid-maleic anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include: polyethyleneimine, Polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, polyamine quaternary salt, or alpha helical peptide.

Ligands may also include targeting groups, e.g., cell or tissue targeting agents that bind to a specified cell type, e.g., kidney cells, e.g., lectins, glycoproteins, lipids, or proteins, e.g., antibodies. The targeting group can be a thyroid stimulating hormone, a melanotropin, a lectin, a glycoprotein, a surfactant protein a, a mucin carbohydrate, a multivalent lactose, a multivalent galactose, an N-acetyl-galactosamine, an N-acetyl-glucosamine multivalent mannose, a multivalent fucose, a glycosylated polyamino acid, a multivalent galactose, a transferrin, a bisphosphonate, polyglutamic acid, polyaspartic acid, a lipid, cholesterol, a steroid, cholic acid, folic acid, vitamin B12, vitamin a, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalators (e.g., acridine), cross-linkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin), thialines (Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxyhexyl, cetyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholic acid, dimethoxytrityl, or phenoxathiin Oxazine peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate esters, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [ MPEG ] peptides]₂Polyamino groups, alkyl groups, substituted alkyl groups, radiolabelled labels, enzymes, haptens (e.g. biotin), transport/absorption enhancers (e.g. aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g. imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of the tetraazamacrocycle), dinitrophenyl, HRP or AP.

The ligand may be a protein, e.g., a glycoprotein, or a peptide, e.g., a molecule having specific affinity for an ancillary ligand, or an antibody, e.g., an antibody that binds to a specified cell type, e.g., a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They may also include non-peptide species such as lipids, lectins, sugars, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose or multivalent fucose. The ligand may be, for example, lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-. kappa.B.

The ligand may be a substance, e.g., a drug, that can increase uptake of the iRNA drug into the cell, e.g., by disrupting the cytoskeleton of the cell (e.g., by disrupting cellular microtubules, microwires, and/or intermediate filaments). The drug may for example be taxol, vincristine, vinblastine, cytochalasin, nocodazole, profilaggrin (japlakinolide), latrunculin a, phalloidin, bryoid (swinhole) a, indanoxine (indocine) or myostatin.

In some embodiments, a ligand linked to an iRNA as described herein acts as a PK modulator. As used herein, "PK modulator" refers to a pharmacokinetic modulator. PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacyl glycerides, phospholipids, sphingolipids, naproxen, ibuprofen (ibuprofen), vitamin E, biotin, and the like. Oligonucleotides comprising a number of phosphorothioate linkages are also known to bind to serum proteins, and therefore short oligonucleotides comprising multiple phosphorothioate linkages in the backbone, for example, oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, are also suitable for use in the present invention as ligands (e.g., as PK modulating ligands). In addition, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

For macromolecular drugs and hydrophilic drug molecules that cannot readily cross bilayer membranes, entrapment within the endosomal lysosomal compartment of the cell is considered to be the biggest obstacle to efficient delivery to their site of action. In recent years, many methods and strategies have been created to solve this problem. For liposomal formulations, the use of fusogenic Lipids in the formulation has been the most common protocol (Singh, R.S., Goncalves, (Goncalves) C. et al, (2004) On the Gene Delivery efficacy of pH-Sensitive Cationic Lipids via Endosomal Protonation, A Chemical Biology Investigation. chem.biol. 11, 713-723.). Other components that exhibit pH-sensitive endosome cleavage activity due to protonation and/or pH-induced conformational changes include charged polymers and peptides. Examples can be found in Hoffman (Hoffman), A.S., Stayton, P.S. et al, (2002) Design of "smart" Polymers that can be used to direct intracellular drug delivery, "Polymers adv.Technol (advanced technology Polymers) 13, 992-999; kakudo (horn), Chaki (Qiagen), T.T., S.et al, (2004). Transferrin-Modified Liposomes equivalent with a pH-Sensitive Fusogenic Peptide: An Artificial Virus-like Delivery System, Biochemistry 436, 5618-; yessing (Asian octanes), M.A. and Leroux (Lu), J.C (2004). Membrane-destabilizing polysaccharides: interactions with lipid bilayers and endosomal escape of biological macromolecules, Adv.drug Deliv. Rev. (advanced drug delivery) reviews 56, 999-; oliveira (orlivira), s., van Rooy (fanreu), i. et al, (2007). The fusion peptide enhances endosomal escape that improves iRNA-induced oncogene silencing. Int.j.pharm. (journal of international pharmaceutical sciences) 331,211-4. They have been used in drug delivery systems in general, such as liposomes or lipid complexes. For folate receptor mediated delivery using liposomal formulations, for example, pH-sensitive fusion peptides have been incorporated into liposomes and shown to enhance activity by improving drug unloading during the uptake process (turn, m.j., Reddy, j.a. et al, (2002) characteristics of novel pH-sensitive peptides that enhance drug release from folate-targeted liposomes at endosomal pH, 1559, 56-68).

In certain embodiments, the endosomal cleavage component of the invention can be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane activity and/or fusibility. A peptidomimetic can be a small protein-like chain designed to mimic a peptide. Peptidomimetics may be derived from modification of existing peptides that aim to alter the properties of the molecule, or peptide-like molecular synthesis using unnatural amino acids or their analogs. In certain embodiments, they have improved stability and/or biological activity when compared to peptides. In certain embodiments, the endosomal lysis component adopts its active conformation at an endosomal pH (e.g., pH 5-6). An "active" conformation is one in which the endosomal lytic component facilitates endosomal lysis and/or the modular composition characterized in the present invention, or any component thereof (e.g., nucleic acid), is transported from the endosome to the cell cytoplasm.

Libraries of compounds can be screened for their differential membrane activity at endosomal pH versus neutral pH using a hemolytic assay. Promising candidates isolated by this method can be used as components of the modular compositions characterized in the present invention. A method of identifying an endosomal lysis component for use in the compositions and methods of the invention can comprise: providing a library of compounds; contacting blood cells with members of the library, wherein the pH of the medium in which the contacting occurs is controlled; determining whether the compound induces differential lysis of blood cells at a low pH (e.g., about pH 5-6) versus a neutral pH (e.g., about pH 7-8).

Exemplary endosomal lytic components include GALA peptides (Subbarao et al, Biochemistry, 1987,26: 2964-. In certain embodiments, the endosomal cleavage component can contain a chemical group (e.g., an amino acid) that will undergo a charge or protonation change in response to a pH change. The endosomal lysis component can be linear or branched. Exemplary primary sequences of endosomal cleavage components include H₂N-(AALEALAEALEALAEALEALAEAAAAGGC)-CO2H(SEQ ID NO:2)；H₂N- (AALAEALAEALAEALAEALAEALAAAAGGC) -CO2H (SEQ ID NO: 3); and H₂N-(ALEALAEALEALAEA)-CONH2(SEQ ID NO:4)。

In certain embodiments, more than one endosomal lysis component can be incorporated into an iRNA agent characterized in the present invention. In some embodiments, this will result in the incorporation of more than one identical endosomal lysis component into the iRNA agent. In some embodiments, this will result in the incorporation of two or more different endosomal cleavage components into the iRNA agent.

These endosomal lytic components can mediate endosomal escape by, for example, altering conformation at endosomal pH. In certain embodiments, the endosomal lytic component can exist in a random coil conformation at neutral pH and rearrange into an amphipathic helix at endosomal pH. As a result of this conformational transition, these peptides can insert into the lipid membrane of the endosome, causing leakage of endosomal contents into the cytoplasm. Because the conformational transition is pH dependent, the endosomal lytic component can exhibit little or no fusogenic activity while circulating in the blood (pH about 7.4). As used herein, "fusogenic activity" is defined as the activity that results in the disruption of a lipid membrane by endosomal lytic components. One example of a fusion activity is disruption of the endosomal membrane by endosome lytic components, resulting in endosome lysis or leakage and translocation of one or more components of the modular composition (e.g., nucleic acid) characterized in the present invention from the endosome into the cytoplasm.

In addition to hemolysis assays, suitable endosomal lysis components can be tested and identified by the skilled artisan using other methods, as described herein. For example, a compound can be tested for its ability to respond to a change in charge, for example, according to a pH environment, by routine methods (e.g., in a cellular assay). In certain embodiments, a test compound is combined or contacted with a cell and the cell is allowed to internalize the test compound, e.g., by endocytosis. Endosomal preparations from control cells can then be compared by contacting the cells with the endosomal preparation to produce an endosomal preparation. A change, e.g., a decrease, in the endosomal fraction from the contacted and control cells indicates that the test compound can function as a fusogenic agent. Alternatively, the contacted cells and control cells can be evaluated, e.g., by microscopy, e.g., by optical or electron microscopy, to determine differences in the endosomal population in the cells. The test compound and/or endosome may be labeled, for example, to quantify endosome leakage.

In another type of assay, an iRNA agent as described herein is constructed using one or more test or putative fusion agents. iRNA drugs can be labeled for easy visualization. Once the iRNA drug is taken up by the cell, the ability of the endosomal lysis component to facilitate endosomal escape can be assessed, for example, by preparing an endosomal preparation, or by microscopic techniques, which enable visualization of labeled iRNA drug in the cell cytoplasm. In certain other embodiments, inhibition of gene expression or any other physiological parameter may be used as a surrogate marker for endosomal escape.

In other embodiments, circular dichroism spectroscopy may be used to identify compounds that exhibit pH-dependent structural transformations.

A two-step assay may also be performed in which a first assay evaluates the ability of an individual test compound to respond to a pH change and a second assay evaluates the ability of a modular composition comprising the test compound to respond to a pH change.

Lipid conjugates

In one ligand, the ligand or conjugate is a lipid or lipid-based molecule. Such lipids or lipid-based molecules preferably bind to serum proteins, e.g., Human Serum Albumin (HSA). The HSA-binding ligand allows the conjugate to distribute to a target tissue, e.g., a non-renal target tissue of the body. For example, the target tissue may be liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to modulate binding to a serum protein (e.g., HSA).

Lipid-based ligands can be used to modulate (e.g., control) the binding of the conjugate to the target tissue. For example, lipids or lipid-based ligands that bind more strongly to HSA will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to HAS can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds HSA with sufficient affinity that the conjugate will preferentially distribute to non-renal tissue. However, it is preferred that this affinity is not so strong that HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid-based ligand binds HSA weakly or not at all, so that the conjugate will preferentially distribute to the kidney. Other moieties that target kidney cells may also be used instead of or in addition to the lipid-based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, that is taken up by a target cell (e.g., a proliferating cell). These are particularly useful for treating disorders characterized by undesired cellular (e.g., malignant or non-malignant types, e.g., cancer cells) proliferation. Exemplary vitamins include vitamins A, E and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HSA and Low Density Lipoprotein (LDL).

In another aspect, the ligand is a cell penetrating agent, preferably a helical cell penetrating agent. Preferably, the osmotic agent is amphiphilic. One exemplary agent penetrant is a peptide such as tat or antennapedia protein. If the osmotic agent is a peptide, it may be modified, including the use of peptidyl mimetics, inverso, non-peptide or pseudopeptide bonds, and D-amino acids. The spiral osmotic agent is preferably an alpha-spiral osmotic agent, which preferably has lipophilic and lipophobic phases.

Cell penetrating peptides

Peptides suitable for use with the present invention may be natural peptides, for example, tat or antennapedia peptides, synthetic peptides or peptidomimetics. Furthermore, such peptides may be modified peptides, for example peptides may comprise non-peptide or pseudopeptide bonds and D-amino acids. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that are capable of folding into a defined three-dimensional structure similar to a native peptide. Conjugation of peptides and peptidomimetics to iRNA drugs can affect the pharmacokinetic profile of iRNA, for example, by enhancing cell recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

The peptide or peptidomimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., consisting essentially of Tyr, Trp, or Phe). The peptide moiety may be a dendrimer peptide, constrained peptide or cross-linked peptide. In another alternative, the peptide portion may include a hydrophobic Membrane Transport Sequence (MTS). An exemplary peptide containing a hydrophobic MTS is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 5). RFGF analogs containing a hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:6)) can also be targeting moieties. The peptide moiety may be a "delivery" peptide, which may carry bulky polar molecules (including peptides, oligonucleotides and proteins) across the cell membrane. For example, sequences from the HIV Tat protein (GRKKRRQRRPPQ (SEQ ID NO:7)) and the Drosophila antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:8)) have been found to be able to function as delivery peptides. Peptides or peptidomimetics can be encoded by random DNA sequences, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al, Nature, 354:82-84,1991). Preferably, the peptide or peptidomimetic tethered to a lipid is a cell targeting peptide, such as an arginine-glycine-aspartic acid (RGD) peptide or RGD mimetic. The peptide moiety may range from about 5 amino acids to about 40 amino acids in length. The peptide moiety may have structural modifications, for example to increase stability or to direct conformational properties. Any of the structural modifications described below may be utilized.

The RGD peptide moiety can be used to target tumor cells, such as endothelial tumor cells or breast Cancer tumor cells (Zitzmann et al, Cancer Res., 62: 5139-. RGD peptides can facilitate targeting of dsRNA agents to tumors in a variety of other tissues, including lung, spleen or liver (Aoki et al, Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of the iRNA agent to the kidney. The RGD peptide may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a particular tissue. For example, glycosylated RGD peptides can deliver iRNA drugs to express alpha_Vβ₃The tumor cells of (Haubner et al, Jour. Nucl. Med. (J. Nuclear medicine), 42: 326-.

Labeled peptides that target enrichment in proliferating cells may be used. For example, RGD-containing peptides and peptidomimetics can target cancer cells, particularly cells displaying α ν β 3 integrin. Therefore, RGD peptides, RGD-containing cyclic peptides, RGD peptides containing D-amino acids, and synthetic RGD mimetics may be used. In addition to RGD, other moieties targeting the α v β 3 integrin ligand may be used. In general, such ligands can be used to control proliferating cells and angiogenesis.

A "cell penetrating peptide" is capable of penetrating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, e.g., a human cell. The microbial cell penetrating peptide may be, for example, an α -helical linear peptide (e.g., LL-37 or cecropin P1), a disulfide bond-containing peptide (e.g., α -defensin, β -defensin or transantibacterial peptide), or a peptide containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). The cell penetrating peptide may also comprise a Nuclear Localization Signal (NLS). For example, the cell penetrating peptide may be a amphiphilic peptide such as MPG, which is derived from the fusion peptide domain of HIV-1gp 41 and the NLS of the SV 40 large T antigen (Simeoni et al, nucleic acids Res. (nucleic acids research) 31:2717-2724, 2003).

Glycoconjugates

In some embodiments, the iRNA oligonucleotides described herein further comprise a glycoconjugate. As described herein, glycoconjugates facilitate in vivo delivery of nucleic acids as well as compositions suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to a compound that is itself a carbohydrate composed of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least 6 carbon atoms with oxygen, nitrogen, or sulfur atoms bonded to each carbon atom; or a compound having as a part thereof a carbohydrate consisting of one or more monosaccharide units (which may be linear, branched or cyclic) having at least six carbon atoms with an oxygen, nitrogen or sulfur atom bound to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides and oligosaccharides containing about 4-9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose and polysaccharide gums. The specific monosaccharide includes C ₅Above (preferably C)₅-C₈) A sugar; disaccharides and trisaccharides include sugars with two or three monosaccharide units (preferably C)₅-C₈)。

In one embodiment, the glycoconjugate is selected from the group consisting of:

i.e., formula II-formula XXII.

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

when one of X or Y is an oligonucleotide, the other is hydrogen.

In some embodiments, the glycoconjugates further comprise other ligands such as, but not limited to, PK modulators, endosomolytic ligands, and cell penetrating peptides.

Joint

In some embodiments, the conjugates described herein can be linked to an iRNA oligonucleotide via various linkers, which can be cleavable or non-cleavable.

The term "linker" or "linking group" means an organic moiety that links two moieties of a compound. The linker typically comprises a direct bond or atom, e.g. oxygen or sulphur, unit, e.g. 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, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkyl Heteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylene groups may be interrupted or terminated O, S, S (O), SO, and₂、N(R⁸) C (o), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl; wherein R is⁸Is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker has 1-24 atoms, preferably 4-24 atoms, preferably 6-18 atoms, more preferably 8-18 atoms, and most preferably 8-16 atoms.

A cleavable linker is a group that is sufficiently stable outside the cell, but is cleaved upon entry into the target cell to release the two moieties held together by the linker. In a preferred embodiment, the cleavable linker cleaves at least 10-fold or more preferably at least 100-fold faster in the target cell or under a first reference condition (which may, e.g., be selected to mimic or represent an intracellular condition) than in the blood of the subject or under a second reference condition (which may, e.g., be selected to mimic or represent a condition present in blood or serum).

The cleavable linker is labile to a cleaving agent (e.g., pH, redox potential, or presence of a degradable molecule). Typically, the cleavage agent is more prevalent or found to be present at a higher level or activity inside the cell than in serum or blood. Examples of such degradation agents include: a redox agent selected for a particular substrate or not having substrate specificity, e.g., including an oxidase or reductase present in the cell or a reducing agent that can degrade a redox cleavable linker by reduction, e.g., a thiol; an esterase; endosomes or substances that can create an acidic environment, e.g., those that create a pH of five or less; enzymes that hydrolyze or degrade acid-cleavable linkers can function as generalized acids, peptidases (which may have substrate specificity), and phosphatases.

The cleavable bond group, such as a disulfide bond, may be pH labile. The pH of human serum was 7.4, while the intracellular average pH was 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 about 5.0. Some linkers will have a cleavable linker that is cleaved at a preferred pH, thus releasing the cationic lipid from the ligand inside the cell or to a desired cellular compartment.

The linker may include a cleavable linker cleavable by a particular enzyme. The type of cleavable linker incorporated into the linker may depend on the cell to be targeted. For example, a liver-targeting ligand may be linked to a cationic lipid via a linker comprising an ester group. Hepatocytes are rich in esterases and therefore this linker will cleave more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterase include cells of the lung, renal cortex and testis.

In targeting esterase-rich cell types (e.g., liver cells and synovial cells), linkers containing peptide bonds can be used.

In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of the degrading agent (or condition) to cleave the candidate linker. It would also be desirable to also test candidate cleavable linkers for their ability to resist cleavage when in blood or in contact with other non-target tissues. Thus, the relative sensitivity to cleavage between a first and a second condition may be determined, wherein the first condition is selected to indicate cleavage in the target cell and the second condition is selected to indicate cleavage in other tissues or biological fluids such as blood or serum. The evaluation can be carried out in a cell-free system, in cells, in cell culture, in organ or tissue culture or in whole animals. It may be useful to make a preliminary evaluation under cell-free or culture conditions and to confirm it by further evaluation in whole animals. In preferred embodiments, a useful candidate compound is cleaved at least 2, 4, 10, or 100 fold in a cell (or in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or in vitro conditions selected to mimic extracellular conditions).

Redox cleavable linkers

One class of cleavable linkers are redox cleavable linkers that cleave upon reduction or oxidation. An example of a reductively cleavable linker is a disulfide linker (-S-S-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker" or, for example, is suitable for use with a particular iRNA moiety and a particular targeting agent, the methods described herein can be interrogated. For example, a candidate can be evaluated by incubating with Dithiothreitol (DTT) or other reducing agent an agent known in the art that mimics the cleavage rate observed in a cell (e.g., a target cell). The candidate may also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved in blood by up to 10%. In preferred embodiments, a useful candidate compound degrades in cells (or in vitro conditions selected to mimic intracellular conditions) by at least a factor of 2, 4, 10, or 100 compared to blood (or in vitro conditions selected to mimic extracellular conditions). The cleavage rate of a candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular mediators and compared to conditions selected to mimic extracellular mediators.

Phosphate-based cleavable linkers

The phosphate-based cleavable linker is cleaved by a substance that degrades or hydrolyzes the phosphate group. Examples of substances which cleave phosphate groups in cells are enzymes, such as phosphatases in cells. Examples of phosphate-based linkers are-O-P (O) (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 (ORk) -O-, -O-P (O) (Rk) -O-, -O-P (S) (Rk) -O-, -S-P (S) (Rk) -O-), (Rk) S-, -O-P (S) (Rk) S-. Preferred embodiments are-O-P (O) (OH) -, -O-P (S) (SH) -, -O-, -S-P (O) (OH) -, -O-P (O) (OH) -, -S-P (O) (OH) -, -S-, -O-P (S) (OH) -, -S-P (S) (OH) -, -O-P (O) (H) -, -O-P (S) (H) -, -O-, -S-P (O) (H) -, -O-, -S-P (S) (H) -, -O- & S (H) -, (H) -S-, -O-P (S) and (H) -S-. A preferred embodiment is-O-P (O) (OH) -O-. These candidates can be evaluated using methods similar to those described above.

Acid cleavable linkers

An acid-cleavable linker is a linker that is cleaved under acidic conditions. In preferred embodiments, the acid-cleavable linker is cleaved in an acidic environment at a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less) or by a substance (e.g., an enzyme) that can act as a generalized acid. In cells, certain low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and amino acid esters. The acid cleavable group may have the general formula-C ═ NN-, C (O) O, or-oc (O). Preferred examples are when the carbon (alkoxy) attached to the oxygen of the ester is aryl, substituted alkyl or tertiary alkyl, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

Ester-based linking groups

The ester-based cleavable linker is cleaved by an enzyme (e.g., esterase and amidase in a cell). Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester cleavable linker has the general formula-C (O) O-or-OC (O) -. These candidates can be evaluated using methods similar to those described above.

Peptide-based cleavage groups

The cleavable linker based on the peptide is cleaved by enzymes (e.g., peptidases and proteases in the cell). A cleavable linker based on a peptide is a peptide bond formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not include an amide group (-C (O) NH-). Amide groups may be formed between any alkylene, alkenylene or alkynylene groups. Peptide bonds are a particular type of amide bond formed between amino acids to produce peptides and proteins. Base ofCleavage groups on peptides are generally limited to the formation of peptide bonds (i.e., amide bonds) between amino acids that produce peptides and proteins and do not include intact amide functionality. The peptide-based cleavable linker has the general formula NHCHR^AC(O)NHCHR^BC (O) -, wherein R^AAnd R^BAre the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

Representative glycoconjugates wherein the linker includes but is not limited to,

when one of X or Y is an oligonucleotide, the other is hydrogen.

Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. patent nos. 4,828,979; 4,948,882, respectively; 5,218,105; 5,525,465, respectively; 5,541,313, respectively; 5,545,730, respectively; 5,552,538, respectively; 5,578,717, 5,580,731; 5,591,584, respectively; 5,109,124, respectively; 5,118,802, respectively; 5,138,045; 5,414,077, respectively; 5,486,603, respectively; 5,512,439, respectively; 5,578,718, respectively; 5,608,046, respectively; 4,587,044, respectively; 4,605,735, respectively; 4,667,025, respectively; 4,762,779, respectively; 4,789,737, respectively; 4,824,941, respectively; 4,835,263, respectively; 4,876,335, respectively; 4,904,582, respectively; 4,958,013, respectively; 5,082,830; 5,112,963, respectively; 5,214,136, respectively; 5,082,830; 5,112,963, respectively; 5,214,136, respectively; 5,245,022, respectively; 5,254,469, respectively; 5,258,506, respectively; 5,262,536, respectively; 5,272,250, respectively; 5,292,873, respectively; 5,317,098, respectively; 5,371,241, respectively; 5,391,723, respectively; 5,416,203, 5,451, 463; 5,510,475, respectively; 5,512,667, respectively; 5,514,785, respectively; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726, respectively; 5,597,696; 5,599,923, respectively; 5,599,928 and 5,688,941; 6,294,664, respectively; 6,320,017; 6,576,752, respectively; 6,783,931, respectively; 6,900,297, respectively; 7,037,646, respectively; each of which is incorporated herein by reference.

All positions in a given compound need not be uniformly modified, and in fact more than one of the foregoing modifications can be incorporated in a single compound or even at a single nucleoside within the iRNA. The present invention also includes iRNA compounds as chimeric compounds. In the context of the present invention, a "chimeric" iRNA compound or "chimera" is an iRNA compound containing two or more chemically distinct regions, preferably dsRNA, each of which is composed of at least one monomeric unit (i.e. nucleotides in the case of dsRNA compounds). These irnas typically contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased target nucleic acid binding affinity to the iRNA. The additional region of the iRNA may serve as a substrate for an enzyme capable of cleaving RNA: DNA or RNA: RNA hybrid molecules. By way of example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA-DNA duplex. Thus, activation of rnase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency with which iRNA inhibits gene expression. Thus, comparable results can be obtained with often shorter irnas when using chimeric dsrnas, compared to phosphorothioate deoxydsrnas that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if desired, related nucleic acid hybridization techniques known in the art.

In some cases, the RNA of the iRNA may be modified with non-ligand groups. Many non-ligand molecules have been conjugated to irnas in order to enhance iRNA activity, cellular distribution or cellular uptake, and procedures for performing such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Kubo (Long-lived), T.et al, biochem. Biophys. Res. Comm. (Biochemical and biophysical research communication), 2007, 365(1): 54-61; Letsinger (Letsinger) et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci. USA), 1989,86:6553), cholic acid (Manohara (Mannhei) et al, Bioorg. Med. chem. Lett. (Bioorganic and medicinal chemical communication), 1994,4:1053), thioethers such as hexyl-S-tritylmercaptan (Manohara. Lane., Ann. Y. Acad. Sci. (N.Y., Neuro. Col. Acad. C., 1992,660: 306; Manohara. Mannohara. Lane.g., Saohara. Maryla., Merck. March. Marohol, Bioohol et al. Acad. Col. Marohol (Bioohol, Biochemical and chemical research communication), Bioohol et al., Biochemical research communication, Lesson. Nu. Nakayas. Nakaur. Nakayas. Nakaki et al., Ben. Nakayak. 20, Bioohol et al., Bioohol (Bioohol, C., USA. Nakayak., USA., 1992,660, Bioohol, C., USA, Bioohol, C., USA, Bioohol, C., USA, C, EMBO J.,1991,10: 111; kabanov et al, FEBS Lett (Federation of the European Biochemical Association), 1990,259: 327; svinarchuk et al, Biochimie (Biochemi), 1993,75:49), phospholipids, such as dicetyl-rac-glycerol or triethylammonium 1, 2-di-O-cetyl-rac-glycerol-3-phosphonate (Manohara et al, Tetrahedron Lett. (Tetrahedron Communication), 1995,36: 3651; see et al, Nucl. acids Res. (nucleic acids research), 1990, 18:3777), polyamine or polyethylene glycol chains (Manohara (Mannohara, et al, Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manohara (Mannohara), et al, Tetrahedron Lett. (Tetrahedron communication), 1995,36:3651), a palm-based moiety (Mishra, et al, Biochim. Biophys. acta (Proc. biochem., 1995,1264:229), or an octadecyl amine or hexyl amino-carbonyloxy cholesterol moiety (Crooke, et al, J.Pharmacol. exp.r. (Pharmacol. J.Physiol., J.Physics., 1996,277: 923). Representative U.S. patents that teach the preparation of such RNA conjugates have been listed above. Common conjugation schemes involve the synthesis of RNA bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule being conjugated using a suitable coupling or activation reagent. In the solution phase, the conjugation reaction can be carried out with the RNA still bound to the solid support or after cleavage of the RNA. Purification of the RNA conjugate by HPLC (high performance liquid chromatography) generally provides a pure conjugate.

Delivery of iRNA

Delivery of iRNA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be made directly by administering to the subject a composition comprising iRNA (e.g., dsRNA). Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct expression of the iRNA.

Direct delivery of iRNA compositions

Generally, any method of delivering a nucleic acid molecule can be adapted for use with iRNA (see, e.g., Akhtar (ahertal) S. and Julian (Julian) RL. (1992) Trends cell. biol. (Trends in cell biology) 2(5):139-144 and WO 94/02595, which are incorporated herein by reference in their entirety). However, there are three factors that are thought to be important for successful delivery of iRNA molecules in vivo: (a) biological stability of the delivered molecule, (2) prevention of non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. Nonspecific effects of siRNA can be minimized by local administration, for example by direct injection or implantation into tissue (as a non-limiting example, a tumor) or by local administration of the preparation. Local administration to the treatment site maximizes the local concentration of the agent, limits exposure of the drug to systemic tissues that might otherwise be damaged by or degrade the agent, and allows for lower total doses of iRNA molecules to be administered. Several studies have shown successful knock-down of gene products when irnas are administered locally. For example, intravitreal delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ. et al, (2004) Retina (Retina) (24:132- G, et al, (2004) Nucleic Acids 32: e 49; tan (Tan), PH. et al, (2005) Gene Ther (Gene therapy) 12: 59-66; makimura, h. et al, (2002) BMC Neurosci (BMC neuroscience) 3: 18; shishkina (Hishikina), GT. et al, (2004) Neuroscience 129: 521-; thaker (Taker), ER. et al, (2004) Proc. Natl. Acad. Sci. U.S.A. (Proc. Natl. Acad. Sci. USA) 101: 17270-17275; akaneya (Acania), Y, et al, (2005) J.Neurophylliol. (journal of neurophysiology) 93: 594. (journal of neurophysiology) 602 and was successfully delivered to the lung by intranasal administration (Howard, KA. et al, (2006) mol.Ther. (molecular therapy) 14: 476-. For systemic administration of iRNA for treatment of disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods play a role in preventing dsRNA from being rapidly degraded by endonucleases and exonucleases in vivo. Modification of the RNA or pharmaceutically acceptable carrier can also allow the iRNA composition to be targeted to a target tissue and avoid unwanted off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups (e.g., cholesterol) to enhance cellular uptake and prevent degradation. For example, iRNA conjugated to a lipophilic cholesterol moiety against ApoB was systemically injected into mice and resulted in the knock-down of ApoB mRNA in the liver and jejunum (Soutschek, j. et al, (2004) Nature 432: 173-. Conjugation of iRNAs to aptamers has been shown to inhibit tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, JO. et al, (2006) nat. Biotechnol. (Nature Biotechnology) 24: 1005-. In alternative embodiments, iRNA can be delivered using a drug delivery system (e.g., a nanoparticle, dendrimer, polymer, liposome, or cationic delivery system). The positively charged cation delivery system facilitates the binding of the (negatively charged) iRNA molecules and also enhances the interaction of the negatively charged cell membrane to allow efficient uptake of the iRNA by the cell. Cationic lipids, dendrimers, or polymers can bind to iRNA or be induced to form vesicles or micelles that package siRNA (see, e.g., Kim SH. et al, (2008) Journal of Controlled Release 129(2): 107-. The formation of vesicles or micelles further prevents degradation of the iRNA upon systemic administration. Methods for making and administering cation-iRNA complexes are well within the capabilities of those of ordinary skill in the art (see, e.g., Sorensen, DR., et al, (2003) J.Mol.biol (J. Mol. biol.) 327: 761-766; Verma (Verma), UN., et al, (2003) Clin.cancer Res. (clinical cancer research) 9: 1291-1300; Arnold, AS, et al, (2007) J.Hypertens. (J. hypertension) 25:197-205, which is incorporated herein by reference in its entirety). Some non-limiting examples of drug delivery systems that may be used to deliver iRNA systemically include DOTAP (Sorensen, sonsen, DR. et al, (2003), supra; Verma (Verma), UN. et al, (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al, (2006) Nature (natural N)441:111-, s. (2006) mol. pharm. (molecular pharmaceuticals) 3: 472-; yoo (Liu), H. et al, (1999) pharm. Res. (pharmaceutical research) 16: 1799-1804). In some embodiments, the iRNA forms a complex with a cyclodextrin for systemic administration. Methods for administering pharmaceutical compositions of iRNA and cyclodextrin can be found in U.S. patent No. 7,427,605, which is incorporated herein by reference in its entirety.

Vector-encoded iRNA

In another aspect, an iRNA targeting the TMPRSS6 gene may be expressed from a transcriptional unit inserted into a DNA or RNA vector (see, e.g., Couture (kutalr), a et al, TIG. (1996),12: 5-10; skilern (schulkelen), a. et al, international PCT publication No. WO 00/22113, Conrad (Conrad), PCT publication No. WO 00/22114, and Conrad (Conrad), U.S. patent No. 6,054,299). Depending on the particular construct and target tissue or cell type used, expression may be transient (on the order of hours to weeks) or persistent (weeks to months or longer). These transgenes may be introduced as linear constructs, circular plasmids, or viral vectors which may be either integrated or non-integrated vectors. The transgene may also be constructed to allow it to be inherited as an extrachromosomal plasmid (Gassmann et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci.) (1995)92: 1292).

The single strand or multiple strands of the iRNA can be transcribed from a promoter on the expression vector. Where two separate strands are to be expressed to produce, for example, dsRNA, the two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively, each individual strand of the dsRNA may be transcribed from a promoter that are both located on the same expression plasmid. In one embodiment, the strand of the dsRNA is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence such that the dsRNA has a stem-loop structure.

iRNA expression vectors are typically DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to generate recombinant constructs that express irnas as described herein. Eukaryotic expression vectors are well known in the art and are available from a variety of commercial sources. Typically, such vectors are provided which contain convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of the iRNA-expressing vector can be systemic, e.g., by intravenous or intramuscular administration, by administration to target cells that are transplanted from the patient and subsequently reintroduced into the patient, or by any other means that allows introduction to the desired target cells.

The iRNA expression plasmid can be used as a vector with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO)^TM) The complex of (a) is transfected into a target cell. Multiple lipofections for iRNA-mediated knockdown targeting different regions of a target RNA over a one week or longer time frame are also contemplated by the present invention. Successful introduction of the vector into the host cell can be monitored using different known methods. For example, transient transfection may be signaled by a reporter, such as a fluorescent label, e.g., Green Fluorescent Protein (GFP). Markers can be used to ensure stable turnover of cells ex vivo Staining, wherein the marker provides the transfected cells with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, etc.; (c) an adeno-associated virus vector; (d) a herpes simplex virus vector; (e) SV40 vector; (f) a polyoma viral vector; (g) a papillomavirus vector; (h) a small RNA viral vector; (i) poxvirus vectors such as orthopoxvirus, e.g., vaccinia virus vector or avipoxvirus, e.g., canarypox virus or fowlpox virus; and (j) helper-dependent or entero-free adenovirus. Replication-defective viruses may also be advantageous. Different vectors will or will not be incorporated into the genome of the cell. If desired, the construct may include viral sequences for transfection. Alternatively, the constructs may be incorporated into vectors capable of episomal replication (e.g., EPV and EBV vectors). Constructs for recombinant expression of irnas will typically require regulatory elements, e.g., promoters, enhancers, etc., to ensure expression of the RNA in the target cell. Other aspects contemplated for vectors and constructs are described further below.

Vectors useful for delivering iRNA will include regulatory elements (promoters, enhancers, etc.) sufficient to express the iRNA in the desired target cell or tissue. The regulatory elements may be selected to provide either constitutive or regulated/inducible expression.

The expression of iRNA can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to certain physiological regulators (e.g., circulating glucose levels or hormones) (Doherty et al, 1994, FASEB J.8: 20-24). Such inducible expression systems suitable for controlling the expression of dsRNA in a cell or in a mammal include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). One of ordinary skill in the art will be able to select the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.

In a particular embodiment, a viral vector containing a nucleic acid sequence encoding an iRNA can be used. For example, retroviral vectors can be used (see Miller et al, meth. enzymol. (methods in enzymology) 217:581-599 (1993)). These retroviral vectors contain the components necessary for the proper packaging and integration of the viral genome into the host cell DNA. The nucleic acid sequence encoding the ingiRNA is cloned into one or more vectors that facilitate delivery of the nucleic acid into the patient. More details on retroviral vectors can be found, for example, in Boesen et al, Biotherapy 6:291-302(1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells in order to render the stem cells more resistant to chemotherapy. Other references that illustrate the use of retroviral vectors in gene therapy are: clowes et al, J.Clin.Invest. (J.Clin.) (J.Clin. Clin.). 93: 644-; kiem et al, Blood 83: 1467-; salmonos (Salamons) and Gunzberg (Gonzburg), Human Gene Therapy (Human Gene Therapy) 4:129-141 (1993); and Grossman and Wilson, curr. Opin Genetics and Devel 3: 110-. Lentiviral vectors contemplated for use include, for example, HIV-based vectors, such as those described in U.S. patent nos. 6,143,520; 5,665,557, respectively; and 5,981,276, which are incorporated herein by reference.

Adenoviruses are also contemplated for delivery of iRNA. Adenoviruses are a particularly attractive vehicle, for example, for delivering genes to the respiratory epithelium. Adenoviruses naturally infect the respiratory epithelium, where they cause mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenovirus has the advantage of being able to infect non-dividing cells. An overview of adenovirus gene therapy is presented by Kozarsky and Wilson, Current Opinion in Genetics and Development (New Genetics and Development) 3:499-503 (1993). Bout (Butt et al, Human Gene Therapy 5:3-10 (1994)) demonstrated the use of adenoviral vectors to transfer genes to the respiratory epithelium of rhesus monkeys. Using adenoviruses in gene therapyOther examples can be found in Rosenfeld et al, Science 252: 431-; rosenfeld et al, Cell 68:143-155 (1992); mastrangeli (Masterland Jelliy) et al, J.Clin.Invest. (J.Clin. Clin.) 91: 225. 234 (1993); PCT publications WO 94/12649; and Wang et al, Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing irnas characterized in this invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia (Xia) H et al, (2002), nat. biotech (natural biotechnology) 201006 and 1010.

The use of adeno-associated virus (AAV) vectors (Walsh) et al, Proc. Soc. exp. biol. Med. (Proc. Biol. Proc. and Proc. Congress. Proc. 300 (1993); U.S. Pat. No. 5,436,146) is also contemplated. In one embodiment, the iRNA may be expressed as two separate complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoter or the Cytomegalovirus (CMV) promoter. Suitable AV vectors for expressing the dsRNA characterized in the present invention, methods for constructing recombinant AV vectors and methods for delivering the vectors into target cells are described in Samulski (Samoolski) R et al, (1987), J.Virol (J.Virol) 61: 3096-3101; fisher (Fisher) K J et al, (1996), J.Virol (J.Virol), 70: 520-; samulski (Samoolski) R et al, (1989), J.Virol (J.Virol.) 63: 3822-3826; U.S. patent nos. 5,252,479; U.S. Pat. nos. 5,139,941; international patent application No. WO 94/13788; and international patent application No. WO 93/24641, the complete disclosure of which is incorporated herein by reference

Another preferred viral vector is a poxvirus, e.g.a vaccinia virus, e.g.an attenuated vaccinia virus such as modified Ankara virus (MVA) or NYVAC, an avipox virus, e.g.a fowlpox virus or canarypox virus.

The tropism of a viral vector can be adjusted by pseudoviral infection of the vector with envelope proteins or other surface antigens from other viruses or by replacement of different viral capsid proteins as appropriate. For example, lentiviral vectors can be pseudoviralized with surface proteins from Vesicular Stomatitis Virus (VSV), rabies virus, ebola virus, Mokola, and the like. AAV vectors can be targeted to different cells by engineering the vectors to express capsid proteins of different serotypes; see, for example, Rabinowitz J E et al, (2002), J Virol (J. Virol) 76:791-801, the entire disclosure of which is incorporated herein by reference.

The pharmaceutical preparation of the carrier may comprise the carrier in an acceptable diluent, or may comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical formulation may comprise one or more cells producing such a gene delivery system.

III.Pharmaceutical compositions containing iRNA

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

The pharmaceutical compositions characterized herein are administered at a dose sufficient to inhibit expression of the TMPRSS6 gene. Typically, a suitable dose of iRNA will be in the range of 0.01 to 200.0 mg per kg body weight of the recipient per day, typically in the range of 1 to 50mg per kg body weight per day. For example, dsRNA can be administered at 0.05mg/kg, 0.5mg/kg, 1mg/kg, 1.5mg/kg, 2mg/kg, 3mg/kg, 10mg/kg, 20mg/kg, 30mg/kg, 40mg/kg or 50mg/kg per single dose. The pharmaceutical composition may be administered once daily, or once weekly, or once monthly, or once every other month. The composition may alternatively be administered 2 times weekly or 2 times monthly or once every 2, 3 or 4 weeks. In some embodiments, the iRNA is administered as two, three or more sub-doses at appropriate intervals throughout the day or by controlled release formulation, even using continuous infusion or delivery methods. In this case, the iRNA contained in each sub-dose must be correspondingly less in order to achieve a total daily dose. Dosage units may also be formulated for delivery over several days, for example, using conventional sustained release formulations that provide sustained iRNA release over a time range of several days. Sustained release formulations are well known in the art and are particularly useful for delivering drugs at a specific site, such as may be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding plurality of daily doses.

The effect of a single dose on the level of TMPRSS6 may be long lasting such that subsequent doses are administered at intervals of no more than 3, 4 or 5 days or at intervals of no more than 1, 2, 3, or 4 weeks.

The skilled artisan will appreciate that certain factors may affect the dosage and timing schedule required to effectively treat a subject, including but not limited to the severity of the disease or disorder, prior treatments, the overall health and/or age of the subject, and other diseases present. In addition, treating a subject with a therapeutically effective amount of the composition can include a single treatment or a series of treatments. Effective dosages and in vivo half-lives for each iRNA encompassed by the present invention can be estimated using conventional methods or based on in vivo testing using appropriate animal models, as described elsewhere herein.

Advances in mouse genetics have resulted in multiple mouse models for studying various human diseases (e.g., pathological processes mediated by TMPRSS6 expression). Such models can be used for in vivo testing of iRNA, as well as for determining therapeutically effective doses. A suitable mouse model is, for example, a mouse containing a transgene expressing human TMPRSS 6.

The invention also includes pharmaceutical compositions and formulations comprising the iRNA compounds characterized in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending on whether local or systemic treatment is required and on the area to be treated. Administration may be topical (e.g., via transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including with a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion administration; subcutaneous administration, e.g., with an implanted device; or intracranial administration, e.g., by intraparenchymal, intrathecal, or intraventricular administration.

irnas can be delivered in such a manner to target a particular tissue, such as the liver (e.g., hepatocytes of the liver).

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, powdered or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the iRNA characterized in this invention is mixed 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., dioleoylphosphatidydope ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic lipids and liposomes (e.g., dioleoyltrimethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The irnas characterized in the present invention can be encapsulated within liposomes or can form complexes therewith, particularly with cationic liposomes. Alternatively, the iRNA may be complexed with a lipid, particularly with a cationic lipid. 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-monodecate, 1-dodecylazepan-2-one, acylcarnitine, acylcholine, or C _1-20Alkyl esters (e.g., isopropyl myristate IPM), monoacylglycerols, diacylglycerols, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. patent No. 6,747,014, which is incorporated herein by reference.

Liposome formulations

In addition to microemulsions, there are many organized surface active structures that have been studied and used in pharmaceutical formulations. These include monolayers, micelles, bilayers, and vesicles. Vesicles, such as liposomes, have attracted great interest due to their specificity and the duration of action they provide from a drug delivery standpoint. As used herein, the term "liposome" means a vesicle composed of amphipathic lipids arranged in a globular bilayer or bilayer.

Liposomes are unilamellar or multilamellar vesicles having a membrane formed by 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 with the cell wall. Non-cationic liposomes, while unable to fuse efficiently with the cell wall, are taken up by macrophages in vivo.

In order to penetrate intact mammalian skin, lipid vesicles must pass through a series of pores, each having a diameter of less than 50nm, under the influence of a suitable transdermal gradient. It is therefore desirable to use liposomes that are highly deformable and capable of passing through such pores.

Other advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide variety of water-soluble and lipid-soluble drugs; liposomes can protect encapsulated drugs from metabolism and degradation in their internal compartments (Rosoff, incorporated by reference from Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (editors), 1988, Marcel Dekker, inc. company, new york, state of new york, volume 1, page 245). Important considerations in the preparation of liposomal formulations are the lipid surface charge, vesicle size and aqueous volume of the liposomes.

Liposomes can be used to transfer and deliver active ingredients to the site of action. Because the liposome membrane is similar in structure to a biological membrane, when liposomes are applied to tissue, the liposomes begin to coalesce with the cell membrane and as the coalescence of liposomes and cells progresses, the liposome contents pour into the cells where the effective drug can act.

As a mode of delivery for many drugs, liposome formulations have been the focus of intensive research. There is growing evidence that: for topical administration, liposomes offer several advantages over other formulations. Such advantages include a reduction in side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer multiple (hydrophilic and hydrophobic) drugs 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 DNA) have been administered to the skin. Most applications result in targeting the epithelium

Liposomes fall into two broad categories. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form stable complexes. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in the endosome. Due to the acidic pH inside the endosome, liposomes burst, releasing their contents into the cell cytoplasm (Wang et al, biochem. Biophys. Res. Commun., 1987,147, 980-.

The pH-sensitive or negatively charged liposomes entrap the nucleic acid without complexing it. Since both DNA and lipids carry similar charges, repulsion occurs rather than complex formation. However, some of the DNA is embedded within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding thymidine kinase genes to cultured cell monolayers. Expression of the foreign gene was detected in the target cells (Zhou et al, Journal of Controlled Release, 1992,19, 269-274).

One major type of liposome composition includes phospholipids in addition to naturally derived phosphatidylcholines. Neutral liposome compositions can be formed, for example, from Dimyristoylphosphatidylcholine (DMPC) or Dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are typically formed from dimyristoyl phosphatidylglycerol, whereas anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from Phosphatidylcholine (PC) (e.g., like soy PC and egg PC). The other type is formed by a mixture of phospholipids and/or phosphatidylcholine and/or cholesterol.

Several studies have evaluated the topical delivery of liposomal pharmaceutical formulations to the skin. The application of interferon-containing liposomes to guinea pig skin results in a reduction in cutaneous herpes pain, while delivery of interferon via other means (e.g., as a solution or as an emulsion) is ineffective (Weiner et al, Journal of Drug Targeting, 1992,2, 405-410). In addition, an additional study tested the efficacy of interferon relative to that administered as part of a liposomal formulation when the interferon was administered using an aqueous system, and concluded that such liposomal formulations were 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 use in delivering drugs to the skin, particularly systems that include non-ionic surfactants and cholesterol. Containing Novasome^TMI (glyceryl dilaurate/Cholesterol/polyoxyethylene-10-octadecyl Ether) and Novasome^TMA non-ionic liposome formulation of II (glyceryl distearate/cholesterol/polyoxyethylene-10-octadecyl ether) was used to deliver cyclosporin into the dermis of the mouse skin. The results show that such nonionic liposome systems are effective in promoting cyclosporin A deposition into different layers of the skin (Hu et al, S.T.P.Pharma.Sci. (pharmaceutical science, technology and practice; pharmacy), 1994,4,6, 466).

Liposomes also include "sterically stabilized" liposomes, as the term is used herein, which refers to liposomes encapsulating one or more specialized lipids that, when incorporated into the liposome, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilised liposomes are those in which part (A) of the vesicle-forming lipid fraction of the liposome comprises one or more glycolipids, such as the monosialoganglioside GM₁Or (B) derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. While not wishing to be bound by any particular theory, it is believed in the art that In the case of less sterically stabilized liposomes containing gangliosides, sphingomyelin or PEG-derivatized lipids, the enhanced circulatory half-life of these sterically stabilized liposomes results from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al, FEBS Letters (FeBS Federation of the European society of biochemistry Commission), 1987,223, 42; Wu et al, Cancer Research (Cancer Research), 1993,53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjoulos et al (ann.n.y.acad.sci. (proceedings of the new york academy of sciences), 1987, 507,64) reported monosialoganglioside GM₁Galactocerebroside sulfate and phosphatidylinositol improve the ability of the liposomes to have a blood half-life. The results of these studies are illustrated by Gabizon et al (Proc. Natl. Acad. Sci. U.S.A. (Proc. Natl. Acad. Sci., USA Proc., 1988, 85, 6949). U.S. Pat. Nos. 4,837,028 and WO 88/04924, both to Allen et al, disclose compositions comprising (1) sphingomyelin and (2) the ganglioside GM₁Or liposomes of galactocerebroside sulfate. U.S. Pat. No. 5,543,152(Webb et al) discloses liposomes containing 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 for their preparation are known in the art. Sunamoto et al (Bull. chem. Soc. Jpn. (published by the Japanese society of chemistry), 1980, 53,2778) describe compositions comprising a nonionic detergent 2C_1215GThe liposome of (1), said liposome comprising a PEG moiety. Illum et al (FEBS Lett. (Federation of European Biochemical society), 1984,167,79) reported that hydrophilic coating of polystyrene particles with polymeric glycols resulted in significantly increased blood half-life. Synthetic phospholipids modified by conjugation to carboxyl groups of polyglycols (e.g., PEG) are described by Sears (siers) (U.S. patent nos. 4,426,330 and 4,534,899). Klibanov et al (FEBS Lett. (Federation of European Biochemical society), 1990,268,235) describe experiments that demonstrate the following: liposomes comprising Phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increasesBlood circulation half-life. Blume et al (Biochimica et Biophysica Acta (report on biochemistry and biophysics), 1990,1029,91) extend this observation to other PEG-derivatized phospholipids, for example, DSPE-PEG consisting of a combination of Distearoylphosphatidylethanolamine (DSPE) and PEG. European patent nos. EP 0445131B 1 and WO 90/04384 to Fisher (Fisher) describe liposomes having covalently bound PEG moieties on their outer surface. Liposome compositions containing 1-20 mole% of PE derivatized with PEG and methods of use thereof are described by Woodle et al (U.S. patent nos. 5,013,556 and 5,356,633) and Martin et al (U.S. patent No. 5,213,804 and european patent No. EP 0496813B 1). Liposomes containing many other lipid 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). WO 96/10391(Choi et al) describes liposomes comprising PEG-modified ceramide lipids. U.S. patent No. 5,540,935(Miyazaki et al) and U.S. patent No. 5,556,948(Tagawa et al) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surface.

A variety of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al discloses a method for encapsulating high molecular weight nucleic acids in liposomes. U.S. patent No. 5,264,221 to Tagawa et al discloses liposomes that bind proteins and states that the contents of such liposomes may include dsRNA. U.S. patent 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 dsRNA targeted to raf genes.

Transfersomes are yet another type of liposome and are highly deformable aggregates of lipids that are attractive candidates for drug delivery vehicles. The transfersomes can be described as lipid droplets that are so highly deformable that they can easily penetrate pores smaller than the droplets. Transfersomes are suitable for use in environments where they are used, for example, they are self-optimizing (adapting to the shape of pores in the skin), have self-replenishing properties, often reach their target without fragmentation and are often self-loading. To make transfersomes, it is possible to add surface-edge activators, typically surfactants, to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Carrier-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of many different types of surfactants, both natural and synthetic, is to use the hydrophilic/lipophilic balance (HLB). The nature of the hydrophilic group (also called the "head") provides the most useful means of classifying the different surfactants used in the formulation (Rieger, incorporated by Pharmaceutical Dosage Forms, Marcel Dekker, inc. new york, state of new york, 1988, page 285).

If the surface active molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceutical and cosmetic products and are available over a wide range of pH values. Generally, 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. Also included in this category are nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols and ethoxylated block polymers. Polyoxyethylene surfactants are the most popular members of the class of nonionic surfactants.

Surfactants of this type are classified as anionic if they carry a negative charge when dissolved or dispersed in water. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, sulfates (e.g., alkyl sulfates and ethoxylated alkyl sulfates), sulfonates (e.g., alkyl benzene sulfonates), acyl isethionates, acyl taurates and sulfosuccinates, and phosphate esters. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

Surfactants of this type are classified as cationic if they carry a positive charge when dissolved or dispersed in water. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most common members of this class.

Surfactants are classified as amphoteric if the surface active molecule has the ability to carry either a positive or negative charge. Amphoteric surfactants include acrylic acid derivatives, substituted alkyl groups, N-alkyl betaines and phosphorus esters.

The use of surfactants in pharmaceuticals, formulations and in emulsions has been reviewed (Rieger, incorporated by reference into Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, page 285).

Nucleic acid lipid particles

In one embodiment, the TMPRSS6 dsRNA characterized in the invention are fully encapsulated in a lipid formulation, e.g., to form SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles. 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. SNALP and SPLP typically contain cationic lipids, non-cationic lipids, and lipids that prevent aggregation of the particles (e.g., PEG-lipid conjugates). SNALP and SPLP are extremely useful for systemic applications because they exhibit extended circulatory life and accumulate at a distal site (e.g., a site physically separate from the site of administration) following intravenous (i.v.) injection. SPLP comprises "pSPLP" which comprises an encapsulated condensing agent-nucleic acid complex, as described in PCT publication No. WO 00/70090. The particles of the present invention typically have an average diameter of from about 50nm to about 150nm, more typically from about 60nm to about 130nm, more typically from about 70nm to about 110nm, most typically from about 70nm to about 90nm, and are substantially non-toxic. Furthermore, when present in the nucleic acid-lipid particles of the invention, the nucleic acid is resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are described in, for example, U.S. patent nos. 5,976,567; 5,981,501, respectively; 6,534,484, respectively; 6,586,410, respectively; 6,815,432, respectively; 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 a range 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 from about 6:1 to about 9: 1.

The cationic lipid may be, for example, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleyloxy) propylamine (DODMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLInDMA), 1, 2-dilinolylenyloxy-N, N-dimethylaminopropane (DLenDMA), 1, 2-dioleylcarbamoyloxy-3-dimethylaminopropane (DLin-C) -DAP), 1, 2-dioleyloxy-3- (dimethylamino) acetoxypropane (DLin-DAC), 1, 2-dioleyloxy-3-morpholinopropane (DLin-MA), 1, 2-dioleoyl-3-dimethylaminopropane (DLInDAP), 1, 2-dioleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-oleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleyloxy-3-trimethylaminopropane hydrochloride (DLin-TMA. Cl), 1, 2-dioleyl-3-trimethylaminopropane hydrochloride (DLin-TAP. Cl), 1, 2-dioleyloxy-3- (N-methylpiperazine) propane (DLin-MPZ), or 3- (N, N-dioleylamino) -1, 2-propanediol (DLINAP), 3- (N, N-dioleylamino) -1, 2-propanediol (DOAP), 1, 2-dioleyloxy-3- (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLINDMA), 2-dioleyl-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS) -N, n-dimethyl-2, 2-bis ((9Z,12Z) -octadec-9, 12-dienyl) tetrahydro-3 aH-cyclopenta [ d ] [1,3] dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z) -thirty-seven-carbon-6, 9,28, 31-tetraen-19-yl 4- (dimethylamino) butanoate (MC3), 1' - (2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethylazaldi) didodecan-2-ol (Tech G1), or mixtures thereof. The cationic lipid may comprise from about 20 mol% to about 50 mol% or about 40 mol% of the total lipid present in the particle.

In another example, the compound 2, 2-dioleyl-4-dimethylaminoethyl- [1,3] -dioxolane can be used to prepare lipid-siRNA nanoparticles. The synthesis of 2, 2-dioleyl-4-dimethylaminoethyl- [1,3] -dioxolane is described in U.S. provisional patent application No. 61/107,998, filed on 23.10.2008, which is incorporated herein by reference.

In one embodiment, the lipid particle comprises 40% 2, 2-dioleyl-4-dimethylaminoethyl- [1,3] -dioxolane: 10% DSPC: 40% cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0 + -20 nm and a siRNA/lipid ratio of 0.027.

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), Dipalmitoylphosphatidylethanolamine (DOPE), palmitoleoylphosphatidylcholine (POPC), palmitoleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl-cyclohexane-1-carboxylate (DOPE-mal), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE), Distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid may comprise 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 can be, for example, a polyethylene glycol (PEG) -lipid, including, but not limited to, PEG-Diacylglycerol (DAG), PEG-Dialkyloxypropyl (DAA), PEG-phospholipid, PEG-cerebroside (Cer), or mixtures thereof. The PEG-DAA conjugate can be, for example, PEG-bisLauryloxypropyl (Ci)₂) PEG-dimyristyloxypropyl (Ci)₄) PEG-dipalmityloxypropyl (Ci)₆) Or PEG-distearyloxypropyl (C)]₈). The conjugated lipid that prevents aggregation of the particles may comprise from 0 mol% to about 20 mol% or about 2 mol% of the total lipid present in the particles.

In some embodiments, the nucleic acid-lipid particle further comprises cholesterol at, e.g., about 10 mol% to about 60 mol% or about 48 mol% of the total lipid present in the particle.

LNP01

In one example, lipid nd98.4hcl (MW 1487) (see U.S. patent application No. 12/056,230 filed on 26/3/2008, which is incorporated herein by reference in its entirety), cholesterol (Sigma-Aldrich), and PEG-cerebroside C16(Avanti polar lipid) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). A mother liquor of each component in ethanol can be prepared as follows: ND98, 133 mg/ml; cholesterol, 25mg/ml, PEG-cerebroside C16, 100 mg/ml. ND98, cholesterol, and PEG-cerebroside C16 mother liquor may then be combined at, for example, a 42:48:10 molar ratio. The combined lipid solution may be mixed with aqueous dsRNA (e.g., in sodium acetate at pH 5) such that the final concentration of ethanol is about 35% -45% and the final concentration of sodium acetate is about 100-300 mM. Upon mixing, lipid-dsRNA nanoparticles typically form spontaneously. Depending on the desired particle size distribution, the resulting nanoparticle mixture may be extruded through a polycarbonate membrane (e.g., 100nm cutoff) using, for example, a hot barrel extruder, such as a Lipex extruder (Northern Lipids, Inc.). In some cases, the extrusion step may be omitted. Ethanol removal and simultaneous buffer exchange can be achieved by, for example, dialysis or tangential flow filtration. The buffer may be exchanged with, for example, Phosphate Buffered Saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

Formulations of LNP01 are described, for example, in international application publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are as follows:

DSPC: distearoyl phosphatidylcholine

DPPC: dipalmitoyl phosphatidylcholine

PEG-DMG: PEG-dimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with average molecular weight of 2000)

PEG-DSG: PEG-Distyryl Glycerol (C18-PEG, or PEG-C18) (PEG with an average molecular weight of 2000)

PEG-cDMA: PEG-carbamoyl-1, 2-dimyristyloxypropylamine (PEG with an average molecular weight of 2000)

Formulations comprising SNALP (1, 2-di-linolenyloxy-N, N-dimethylaminopropane (DLinDMA)) are described in international publication No. WO2009/127060 filed 4, 15, 2009, which is hereby incorporated by reference.

XTC-containing formulations such as U.S. provisional serial No. 61/148,366 filed on 1/29 of 2009; united states provisional serial No. 61/156,851 filed on 3/2 in 2009; U.S. provisional serial No. filed on day 10, 6 months 2009; us provisional serial No. 61/228,373 filed on 24/7/2009; U.S. provisional serial No. 61/239,686 filed on 9/3/2009 and international application No. PCT/US2010/022614 filed on 1/29/2010, which are hereby incorporated by reference.

Formulations comprising MC3 are described, for example, in U.S. provisional serial No. 61/244,834 filed on day 9/22 2009, U.S. provisional serial No. 61/185,800 filed on day 6/10 2009, and international application No. PCT/US10/28224 filed on day 6/10 2009, which are hereby incorporated by reference.

Formulations comprising ALNY-100 are described, for example, in international patent application No. PCT/US09/63933 filed 11/10/2009, which is hereby incorporated by reference.

Formulations containing C12-200 are described, for example, in U.S. provisional serial No. 61/175,770 filed 5.5.2009, international application No. PCT/US10/33777 filed 5.5.2010, which are hereby incorporated by reference.

As used herein, the term "LNPXX", where "XX" is a number, is also referred to herein as "AFXX". For example, LNP09 is also known as AF09 and LNP12 is also known or referred to as AF 12.

Synthesis of cationic lipids

Any compound used in the nucleic acid particles characterized in the present invention, e.g., cationic lipids, etc., can be prepared by known organic synthesis techniques, including the methods described in more detail in the examples. Unless otherwise indicated, all substituents are defined below.

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

"alkenyl" means an alkyl group as defined above containing at least one double bond between adjacent carbon atoms. Alkenyl includes cis and trans isomers. Representative straight and branched alkenyls include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2, 3-dimethyl-2-butenyl, and the like.

"alkynyl" means any alkyl or alkenyl group as defined above additionally containing at least one triple bond between adjacent carbons. Representative straight and branched alkynyl groups include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

"acyl" means any alkyl, alkenyl or alkynyl group in which the carbon at the point of attachment is replaced with an oxo group as defined below. For example, -C (═ O) alkyl, -C (═ O) alkenyl, and-C (═ O) alkynyl are acyl groups.

"heterocycle" means a 5-to 7-membered monocyclic or 7-to 10-membered bicyclic heterocycle which is either saturated, unsaturated or aromatic and contains 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the foregoing heterocycles are fused to a benzene ring. The heterocyclic ring may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl (piperizinyl), hydantoinyl (hydantoinyl), valerolactam (valrolactoamyl), oxirane, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothienyl, tetrahydrothiopyranyl, etc.

The terms "optionally substituted alkyl", "optionally substituted alkenyl", "optionally substituted alkynyl", "optionally substituted acyl", and "optionally substituted heterocycle" mean that, upon substitution, at least one hydrogen atom is replaced by a substituent. In the case of an oxo substituent (═ O), two hydrogen atoms are replaced. In this aspect, substituents include oxygen, halogen, heterocycle, -CN, -OR^x、-NR^xR^y、-NR^xC(＝O)R^y、-NR^xSO₂R^y、-C(＝O)R^x、-C(＝O)OR^x、-C(＝O)NR^xR^y、–SO_nR^xand-SO_nNR^xR^yWherein n is 0, 1 or 2, R^xAnd R^yAre the same or different and are independently hydrogen, alkyl or heterocyclic, and each of the alkyl and heterocyclic substituents may be further substituted with one or more of the following substituents: oxo, halogen, -OH, -CN, alkyl, -OR^xHeterocyclic ring, -NR^xR^y、-NR^xC(＝O)R^y、-NR^xSO₂R^y、-C(＝O)R^x、-C(＝O)OR^x、-C(＝O)NR^xR^y、-SO_nR^xand-SO_nNR^xR^y。

"halogen" means fluorine, chlorine, bromine and iodine.

In some embodiments, the methods characterized in the present invention may require the use of protecting groups. Protecting group methodologies are well known to those of ordinary skill in the art (see, e.g., Protective Groups in Organic Synthesis, Green (Green), t.w. et al, Wiley-Interscience, new york city, 1999). In short, within the context of the present invention, a protecting base is any group that reduces or eliminates the unwanted reactivity of the functional group. Protecting groups may be added to functional groups to mask their reactivity during certain reactions and subsequently removed to reveal the original functional groups. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates the unwanted reactivity of an alcohol functional group. Protecting groups may be added and removed using techniques well known in the art.

Synthesis of formula A

In some embodiments, the nucleic acid-lipid particles characterized in the present invention are formulated using a cationic lipid of formula a:

wherein R is₁And R₂Independently is alkyl, alkenyl or alkynyl, each of which may be optionally substituted, and R₃And R₄Independently is lower alkyl or R₃And R₄May be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2, 2-dioleyl-4-dimethylaminoethyl- [1, 3)]-dioxolane). Generally, lipids of formula a above can be made by the following reaction schemes 1 or 2, wherein all substituents are as defined above unless otherwise indicated.

Scheme 1

Lipid A can be prepared according to scheme 1, wherein R₁And R₂Independently is alkyl, alkenyl or alkynyl, each of which may be optionally substituted, and R₃And R₄Independently is lower alkyl or R₃And R₄May be taken together to form an optionally substituted heterocyclic ring. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 produced ketal 3. Treatment of ketal 3 with amine 4 produces lipids of formula a. Lipids of formula a can be converted to the corresponding ammonium salts with organic salts of formula 5, wherein X is an anionic counter ion selected from the group consisting of halogen, hydroxide, phosphate, sulfate, and the like.

Scheme 2

Alternatively, the ketone 1 starting material may be prepared according to scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. The reaction of 6 and 7 produces ketone 1. Ketone 1 is converted to the corresponding lipid of formula a as described in scheme 1.

Synthesis of MC3

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

Synthesis of ALNY-100

The synthesis of ketal 519[ ALNY-100] was performed using scheme 3 below:

515 Synthesis

To a stirred suspension of LiAlH4(3.74g, 0.09852mol) in 200mL of anhydrous THF in a two-necked RBF (1L) under nitrogen atmosphere at 00C was slowly added a solution of 514(10g, 0.04926mol) in 70mL of THF. After the addition was complete, the reaction mixture was warmed to room temperature and then heated to reflux for 4 hours. The progress of the reaction was monitored by TLC. After completion of the reaction (checked by TLC), the mixture was cooled to 00C and quenched by careful addition of saturated Na2SO4 solution. The reaction mixture was stirred at room temperature for 4h and filtered off. The residue was washed thoroughly with THF. The filtrate and washings were mixed and diluted with 400mL dioxane and 26mL concentrated HCl and stirred at room temperature for 20 minutes. The volatiles were stripped under vacuum to afford 515 hydrochloride as a white solid. Yield: 7.12g¹H-NMR (DMSO,400 MHz): δ — 9.34 (width, 2H),5.68(s,2H),3.74(m,1H),2.66-2.60(m,2H),2.50-2.45(m, 5H).

516 Synthesis

To a stirred suspension of compound 515 in 250mL of two-necked RBF in 100mL of anhydrous DCM was added NEt3(37.2mL, 0.2669mol) and cooled to 00C under nitrogen atmosphere. After the slow addition of N- (benzyloxycarbonyloxy) -succinimide in 50mL dry DCM (20g, 0.08007mol), the reaction mixture was allowed to warm to room temperature. After the reaction is complete (detection by TLC for 2-3 hours), the mixture is taken up in 1N HCl solution (1X 100) mL) and a saturated NaHCO3 solution (1X 50 mL). The organic layer was then dried with anhydrous Na2SO4 and the solvent was evaporated to give a crude material which was purified by silica gel column chromatography to obtain 516 as a viscous substance. Yield: 11g (89%).¹H-NMR(CDCl3,400MHz):δ＝7.36-7.27(m,5H),5.69(s,2H),5.12(s,2H),4.96(br.,1H)2.74(s,3H),2.60(m,2H),2.30-2.25(m,2H)。LC-MS[M+H]-232.3(96.94％)。

517A and 517B Synthesis

Cyclopentene 516(5g, 0.02164mol) was dissolved in a solution of 220mL acetone and water (10:1) in a single neck 500mL RBF and N-methylmorpholine-N-oxide (7.6g, 0.06492mol) was added thereto at room temperature followed by 4.2mL of a 7.6% OsO4 solution in t-butanol (0.275g, 0.00108 mol). After the reaction was complete (about 3 hours), the mixture was quenched by addition of solid Na2SO3 and the resulting mixture was stirred at room temperature for 1.5 hours. The reaction mixture was diluted with DCM (300mL) and washed with water (2 × 100mL), followed by saturated NaHCO3(1 × 50mL) solution, water (1 × 30mL) and finally brine (1 × 50 mL). The organic phase was dried over anhydrous Na2SO4 and the solvent was removed in vacuo. Silica gel column chromatographic purification of the crude material provided a mixture of diastereomers, which was separated by preparative HPLC. Yield: about 6g of crude

517A-Peak-1 (white solid), 5.13g (96%). ¹H-NMR(DMSO,400MHz)：δ＝7.39-7.31(m,5H),5.04(s,2H),4.78-4.73(m,1H),4.48-4.47(d,2H),3.94-3.93(m,2H),2.71(s,3H),1.72-1.67(m,4H)。LC-MS-[M+H]-266.3，[M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry was confirmed by X-ray.

518 Synthesis

Using a procedure analogous to that described for the synthesis of compound 505, compound 518(1.2g, 41%) was obtained as colorless oil.¹H-NMR(CDCl3,400MHz):δ＝7.35-7.33(m,4H),7.30-7.27(m,1H),5.37-5.27(m,8H),5.12(s,2H),4.75(m,1H),4.58-4.57(m,2H),2.78-2.74(m,7H),2.06-2.00(m,8H),1.96-1.91(m,2H),1.62(m,4H),1.48(m,2H),1.37-1.25(br m,36H),0.87(m,6H)。HPLC-98.65％。

General procedure for the Synthesis of Compound 519

A solution of compound 518(1eq) in hexane (15mL) was added dropwise to an ice-cold solution of LAH in THF (1M, 2 equivalents). After complete addition, the mixture was heated at 40 ℃ for 0.5h and then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 solution, then filtered through a filter and reduced to oil. Column chromatography afforded pure 519(1.3g, 68%) as a colorless oil.¹³C NMR 130.2,130.1(x2),127.9(x3),112.3,79.3,64.4,44.7,38.3,35.4,31.5,29.9(x2),29.7,29.6(x2),29.5(x3),29.3(x2),27.2(x3),25.6,24.5,23.3,226, 14.1; electrospray MS (+ ve): the calculated molecular weight of (M + H) + C44H80NO2 was 654.6, with a molecular weight of 654.6 observed.

Formulations prepared by either standard methods or extrusion-free methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free of aggregates or deposits. The particle size and particle size distribution of the lipid-nanoparticles can be measured by light scattering using, for example, Malvern Zetasizer Nano ZS (Malvern corporation, usa). The particles should be about 20-300nm in size, for example 40-100 nm. The particle size distribution should be monomodal. Total dsRNA concentration in the formulation together with the encapsulated fraction was estimated using a dye exclusion assay. Samples of formulated dsRNA can be incubated with RNA binding dyes, such as Ribogreen (molecular probe), in the presence or absence of formulation-damaging surfactants (e.g., 0.5% Triton-X100). The total dsRNA in the formulation can be determined by the signal from the surfactant-containing sample relative to a standard curve. The encapsulated fraction was determined by subtracting the "free" dsRNA content (as measured by signal in the absence of surfactant) from the total dsRNA content. The percentage of encapsulated dsRNA is typically greater than 85%. For the SNALP formulation, the particle size is at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, at least 90nm, at least 100nm, at least 110nm, and at least 120 nm. Suitable ranges are typically from about at least 50nm to about at least 110nm, from about at least 60nm to about at least 100nm, or from about at least 80nm to about at least 90 nm.

Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in aqueous or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which the dsRNA characterized in the invention is administered together with one or more penetration enhancing surfactants and a chelating agent. Suitable surfactants include fatty acids and/or esters or salts thereof, cholic acids and/or salts thereof. Suitable cholic acids include chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, gluccholic acid, glycocholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24, 25-dihydrofusidate and sodium dihydrofusidate. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, undecanoic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, didecanoate, tricaprate, monoolein, dilaurin, glyceryl 1-monodecanoate, 1-dodecylazepan-2-one, acylcarnitine, acylcholine, or monoacylglycerol, diacylglycerol, or pharmaceutically acceptable salts (e.g., sodium salt) thereof. In some embodiments, for example, a fatty acid/salt is used in combination with a bile acid/salt using a penetration enhancer combination. An exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Other penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Dsrnas characterized in the present invention can be delivered or complexed orally in particulate form, including spray-dried particles, to form microparticles or nanoparticles. The DsRNA complexing agent comprises polyamino acid; a polyimine; a polyacrylate; polyalkyl acrylates, polyoxyethanes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG), and starch; polyalkylcyanoacrylate; DEAE derivatized polyimine, pullulan, cellulose, and starch. Suitable complexing agents include chitosan, N-trimethyl chitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethyl ethylene P (TDAE), polyaminostyrenes (e.g., p-amino), poly (methyl cyanoacrylate), poly (ethyl cyanoacrylate), poly (butyl cyanoacrylate), poly (isobutyl cyanoacrylate), poly (isohexyl cyanoacrylate), DEAE-methacrylate, DEAE-hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexamethylene acrylate, poly (d, l-lactic acid)), poly (DL-lactic acid-co-glycolic acid (PLGA), alginate and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are detailed in U.S. patent 6,887,906, U.S. publn.no.20030027780, and U.S. patent 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 mixtures, and other pharmaceutically acceptable carriers or excipients.

The pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Particularly preferred are formulations that target the liver in the treatment of liver disorders, such as liver cancer.

The pharmaceutical formulations of the present invention, which may be conveniently 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 the active ingredient into association with one or more pharmaceutically acceptable carriers or excipients. Typically, the formulation is prepared by: the active ingredient is uniformly and intimately admixed with a liquid carrier or a finely divided solid carrier or both, and the product is then shaped as desired.

The compositions of the present invention may be formulated into any of a number of 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 sodium carboxymethyl cellulose, sorbitol, and/or dextran. The suspension may also contain stabilizers.

Additional formulations

Emulsion formulation

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous Systems in which a liquid is dispersed in another in the form of droplets generally exceeding 0.1 μm in diameter (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems), Allen (Allen), LV., Popovich (boboverqi) ng, and Ansel (Ansel) HC.,2004, Lippincott Williams & Wilkins press (8 th edition), new york state; Idson (edson), incorporated from Pharmaceutical Dosage Forms, Lieberman (liberman), Rieger (Rieger) and Banker (bond editors), 1988, cell Dekker, mark. company, new york state, page 1, page 199; solfa (r), incorporated from roughson, inc (r) and roll of drugs (r) editors, incorporated from rockwell, inc (r et al, inc. r et al, inc (r, r et al, inc (r, r et al), a first page 245; block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (edited), 1988, Marcel Dekker, inc, new york, volume 2, page 335; higuchi et al, Remington's Pharmaceutical Sciences, Mack Publishing Co., Inc., Easton, Pa., 1985, p 301. Emulsions are often biphasic systems comprising two immiscible liquids intimately mixed and dispersed in each other. In general, emulsions may be of the water-in-oil (w/o) or oil-in-water (o/w) variety. When the aqueous phase is finely dispersed into the bulk oil phase and dispersed as a micro-liquid into the bulk oil phase, the resulting composition is referred to as a water-in-oil (w/o) emulsion. Alternatively, when the oil phase is finely dispersed into the bulk aqueous phase and dispersed as a micro-liquid into the bulk aqueous phase, the resulting composition is referred to as an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phase and the active drug may be present as a solution in the aqueous phase, the oil phase, or as a separate phase itself. Pharmaceutical excipients, such as emulsifiers, stabilizers, dyes and antioxidants, may also be present in the emulsion, if desired. The pharmaceutical emulsion may also be a multiple emulsion consisting of more than two phases, for example, in the case of oil-in-water (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages not available with simple binary emulsions. Multiple emulsions in which a single oil droplet of the o/w emulsion surrounds a small water droplet constitute the w/o/w emulsion. Likewise, a system of enclosed oil droplets in stable water globules in an oleaginous continuous phase provides an o/w/o emulsion.

Emulsions are characterized by low or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed in the external or continuous phase and is maintained in this form by the emulsifier or formulation viscosity. Either phase of the emulsion may be semi-solid or solid, as is the case with emulsion-type ointment bases and creams. Other means of stabilizing emulsions require the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can be roughly classified into 4 types: synthetic surfactants, naturally occurring emulsifiers, absorption matrices and finely divided solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich (Pobovich) NG. and Ansel HC.,2004, Lippincott Williams & Wilkins Press (8 th edition), New York, Idson (Edson), incorporated by reference from Pharmaceutical Dosage Forms, Lieberman (Liberman), Rieger (Rienger) and Bank (eds.), 1988, Marcel Dekker, Inc., New York, state, Vol.1, p.199).

Synthetic surfactants, also known as surface active agents, have found widespread use in the formulation of emulsions and have been reviewed in the literature (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems), Allen (Allen), LV., Popovich (boboverqi) ng, and Ansel (Ansel) HC.,2004, lippinco Williams & Wilkins press (8 th edition), new york state; Rieger (Rieger), incorporated from Pharmaceutical Dosage Forms, Lieberman (leberman), Rieger (Rieger) and Banker (bank) (editors), 1988, Marcel Dekker, inc. company, new york, volume 1 son, page # p.; ideben from ideben (ideben), incorporated from redware (r), incorporated from company (forberg), incorporated (r) and incorporated (forberg), new york, 1988, volume 1, page 199). Surfactants are generally amphoteric and comprise a hydrophilic portion and a hydrophobic portion. The ratio of hydrophilic to hydrophobic properties of a surfactant has been referred to as the hydrophilic/lipophilic balance (HLB) and is a valuable tool for surfactant classification and selection in the preparation of formulations. Surfactants can be based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphiphilic are divided into different classes (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich (Pobovich) NG. and Ansel (Anser) HC.,2004, Lippincott Williams & Wilkins Press (8 th edition), New York, Rieger (Rieger), incorporated by Pharmaceutical Dosage Forms, Lieberman (Lieberman), Rieger (Rieger), and Bank (Ricker) (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, p.285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. The absorbent matrices possess hydrophilic properties such that they can absorb water to form w/o emulsions while still retaining their semi-solid consistency, e.g., anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers, especially in combination with surfactants and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminium silicate and colloidal magnesium aluminium silicate, pigments and non-polar solids such as carbon or glyceryl tristearate.

A variety of non-emulsifying materials are also included in the emulsion and contribute to the characteristics of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrocolloids, preservatives and antioxidants (Block (Brooks), from Pharmaceutical Dosage Forms, Lieberman, Rieger and Bank (Ponker) (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, p.335; Idson (Edson), from Pharmaceutical Dosage Forms, Lieberman, Rieger and Bank (Ponker) (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol.1, p.199).

Hydrocolloids or hydrocolloids include naturally occurring gums and synthetic polymers, such as polysaccharides (e.g., gum arabic, agar, alginic acid, carrageenan, guar gum, carrageenan, and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomers, cellulose ethers, and carboxyvinyl polymers). These colloids are dispersed or swollen in water to form a colloidal solution that stabilizes the emulsion by forming a firm interfacial film around the dispersed droplets and by increasing the viscosity of the outer phase.

Since emulsions often contain many ingredients that can readily support microbial growth, such as sugars, proteins, sterols, and phospholipids, these formulations often incorporate preservatives. Common preservatives included in the emulsions include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid and boric acid. Antioxidants are also often added to emulsion formulations to prevent deterioration of the formulation. The antioxidants used may be radical scavengers, such as tocopherol, alkyl gallate, 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 use of emulsion formulations via the cutaneous, oral and gastrointestinal routes and methods for their manufacture have been reviewed in the literature (see, e.g., Ansel's Pharmaceutical Delivery Forms and Drug Delivery Systems, Allen, LV., Popovich (Pobovich) NG. and Ansel HC.,2004, Lippincott Williams & Wilkins publications and (8 th edition), New York; Idson (Edison), from Pharmaceutical Delivery Forms, Lieberman (Liberman), Rieger (Riegger) and Bank (Bangkang) (eds.), 1988, cel Dekker, Inc., New York, Vol.1, page 199). Emulsion formulations for oral Delivery have been used very widely due to ease of formulation as well as being effective from an absorption and bioavailability standpoint (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems), Allen (Allen), LV., popivich (boboverqi) ng, and Ansel (Ansel) HC.,2004, Lippincott Williams & Wilkins press (8 th edition), new york, Rosoff (rosso), from Pharmaceutical Dosage Forms, Lieberman (liberman), Rieger (Rieger) and Banker (bond editors), 1988, cell Dekker, mark. company, new york, volume 1, page number; pharamson (r), idegren 245, from idegrex (iderman), inc (dieberg), inc (1988, inc), volume 1, page 199, new york). Mineral oil-based laxatives, oil-soluble vitamins and high fat nutritional formulations are among the materials commonly administered orally as o/w emulsions.

In one embodiment of the invention, the composition of iRNA and nucleic acid is formulated as a microemulsion. Microemulsions may be defined as a system of water, oil and amphiphilic molecules that is an optically isotropic and thermodynamically stable single liquid solution (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich (Bobovich) NG. and Ansel (Anser) HC.,2004, Lippinco Williams & Wilkins Press (8 th edition), New York, Rosoff (Rosofv), incorporated from Pharmaceutical Dosage Forms, Lieberman, Rieger (Rieger) and Bank (Ponkg) (ed., 1988, Marcel Dekker, Inc., New York, Vol.1, p.245). Typically, microemulsions are systems prepared by: the oil is first dispersed in an aqueous surfactant solution and then a sufficient amount of a fourth component (typically a medium chain length alcohol) is added to form a clear system. Microemulsions have therefore also been described as thermodynamically stable isotropic clear dispersions of two immiscible liquids, stabilized by an interfacial film of surfactant molecules (Leung and Shah, from Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M. ed., 1989, VCH Publishers, New York, p.185-215). Microemulsions are typically prepared by combining three to five components, including oil, water, surfactant, co-surfactant, and electrolyte. Whether microemulsions are of the water-in-oil (w/o) or oil-in-water (o/w) type depends on the nature of the oil and surfactant used and on the structure and geometric packing of the polar head and hydrocarbon tail of the surfactant molecule (Schott, available from Remington's Pharmaceutical Sciences, Mack Publishing co. inc., Easton, pa, p.s.271).

The phenomenological approach to the use of phase diagrams has been extensively studied and has resulted in extensive knowledge of how to formulate microemulsions to those of ordinary skill in the art (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen (Allen), LV., Popovich (Bobovich) NG. and Ansel (Anser) HC.,2004, Lippinco Williams & Wilkins Press (8 th edition), New York, Rosoff (Rosofv), incorporated from Pharmaceutical Dosage Forms, Lieberman (Liberman), Rieger (Rieger) and Bank (Bangke) (eds.), 1988, Marcel Dekker, Inc., New York, Vol.1, pp.245; Blocker (Blocker), incorporated from Blocket al, Inc. (Forkliner, Inc.), incorporated, Inc. (Forkliner, Inc. (1988, new york, new york state, volume 1, page 335). Microemulsions offer the following advantages over conventional emulsions: dissolving a water-insoluble drug in a formulation of thermodynamically stable droplets that form spontaneously.

Surfactants used in preparing the microemulsion include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sesquioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with co-surfactants. The co-surfactant (typically a short chain alcohol such as ethanol, 1-propanol and 1-butanol) acts to increase interfacial fluidity by penetrating into the surfactant film and thus creating a disordered film due to the void space created between the surfactant molecules. However, microemulsions may be prepared without the use of co-surfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. Typically, the aqueous phase may be, but is not limited to, water, aqueous solutions of drugs, glycerol, PEG300, PEG400, polyglycerol, propylene glycol and derivatives of ethylene glycol. The oil phase may include, but is not limited to, a variety of materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolyzed glycerides, saturated polyglycolyzed C8-C10 glycerides, vegetable oils, and silicone oils.

Microemulsions are particularly interesting from the standpoint of drug solubilization and enhanced drug absorption. Lipid-based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see, e.g., U.S. Pat. No. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantidindes et al, Pharmaceutical Research, 1994,11, 1385-. Microemulsions offer the following advantages: improved drug dissolution, protection of the drug from enzymatic hydrolysis, enhanced drug absorption, possibly due to surfactant-induced membrane fluidity and permeability changes, ease of manufacture, ease of oral administration over solid dosage forms, improved clinical efficacy and reduced toxicity (see, e.g., U.S. Pat. No. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantides et al, Pharmaceutical Research, 1994,11,1385; Ho et al, J.Pharm.Sci. (J.Med. 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 heat labile drugs, peptides or irnas. Microemulsions have also been effective in transdermal delivery of active ingredients in cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will promote increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract and improve local cellular uptake of iRNA and nucleic acids.

Microemulsions of the invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve formulation properties and enhance the uptake of iRNA and nucleic acids of the invention. The penetration enhancers used in the microemulsions of the present invention may be divided into one of five broad categories- -surfactants, fatty acids, bile salts, chelators and non-chelating non-surfactants (Lee (Li) et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92). Each of these categories has been discussed above.

Penetration enhancer

In one embodiment, the present invention employs different penetration enhancers to achieve efficient delivery of nucleic acids, particularly iRNA, to the skin of an animal. Most drugs exist in solution in both ionized and non-ionized forms. However, usually only lipid soluble or lipophilic drugs pass easily through the cell membrane. It has been found that even non-lipophilic drugs can cross the cell membrane if the membrane to be crossed is treated with a permeation enhancer. In addition to assisting the diffusion of non-lipophilic drugs across cell membranes, permeation enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad classes, namely, surfactants, fatty acids, bile salts, chelators, and non-chelating non-surfactants (see, e.g., Malmsten, M.surfactants and polymers in Drug delivery, Informata Health Care, New York, N.Y., 2002; Lee (Li) et al, clinical Reviews in Therapeutic Drug delivery Systems, 1991, page 92). Each of the categories of permeability enhancers mentioned above are described in more detail below.

Surfactant (b): in connection with the present invention, a surfactant (or "surface active agent") is a chemical entity that, when dissolved in an aqueous solution, reduces the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in enhanced uptake of iRNA through the mucosa. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether) (see, for example, Malmsten, m.surfactants and polymers in drug delivery, information Health Care, n.y., 2002; lee (Li) et al, clinical Reviews in Therapeutic Drug carriers Systems (review for medical Carrier Systems), 1991, page 92); and perfluorinated chemical emulsions, such as FC-43. Takahashi et al, j.pharm.pharmacol (journal of pharmacology), 1988,40, 252).

Fatty acid: various fatty acids and their derivatives acting as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-capric 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-monodecanoate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C thereof _1-20Alkyl esters (e.g., methyl, isopropyl, and t-butyl esters) and mono-and diglycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see, e.g., touutou (du, fig.), e. et al, Enhancement in Drug Delivery), CRC Press, Danvers, ma, 2006; Lee, et al, Critical Reviews in Therapeutic Drug carriers Systems (review of pharmaceutical Carrier Systems), 1991, page 92; muranhari, pharmaceutical Reviews in Therapeutic Drug carriers Systems (review of pharmaceutical Carrier Systems), 1990,7, 1-33; haririi et al, jJournal of science and pharmacology), 1992,44, 651-654).

Bile salt: physiological roles of bile include promoting dispersion and absorption of lipid and fat vitamins (see, e.g., Malmsten, M.surfactants and polymers in drug delivery, information Health Care, N.Y., N.2002; Brunton, Chapter 38, from Goodman & Gilman's The Pharmacological Basis of Therapeutics, Goodman, 9 th edition, Hardman et al, McGraw-Hill, N.Y., 1991, p. 934 and 935). Different natural bile salts and their synthetic derivatives act as penetration enhancers. Thus, the term "bile salts" includes any of the naturally occurring bile components as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or a pharmaceutically acceptable sodium salt thereof, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycocholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24, 25-dihydrofusidate (STDHF), sodium dihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (see, for example, Malmsten, M.Surfactants and polymers in drug delivery, Info Health Care, N.Y., N.2002; Lee (Li), et al, critical Reviews in Therapeutic Drug carriers Systems (review for medical Carrier Systems), 1991, page 92; swinyard (Schnayard), chapter 39, from: remington's Pharmaceutical Sciences, 18 th edition, Gennaro (Gennaro) ed., Mack Publishing co., Easton, pa, 1990, page 782-783; muranishi (mura), clinical Reviews in Therapeutic Drug carriers Systems (review on Drug Carrier Systems), 1990,7, 1-33; yamamoto et al, j.pharm.exp.ther, (journal of pharmacology and experimental therapeutics), 1992,263, 25; yamashita et al, J.Pharm.Sci. (J.Pharma. Sci.), 1990,79, 579-.

Chelating agent: chelators, as used in connection with the present invention, may be defined as compounds that remove metal ions from solution by forming complexes with the metal ions, resulting in enhanced absorption of iRNA through the mucosa. Chelating agents have additional advantages with respect to their use as penetration enhancers in the present invention: also act as dnase inhibitors, since most characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelators (Jarrett, j. chromatogr. (journal of chromatography), 1993,618, 315-339). Suitable chelating agents include, but are not limited to, disodium Ethylenediamine (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and Hierarchia salicylate), N-acyl derivatives of collagen, N-aminoacyl derivatives of laureth-9 and beta-diketone (enamines) (see, e.g., Katdare (Katedder), A. et al, Excipient development for pharmaceutical, biotechnology, and Drug delivery system excipients, CRC Press, Danvers, Massachusetts, 2006; Lee (Li) et al, clinical Reviews in Therapeutic Drug delivery Systems (review of medical Carrier Systems), 1991, page 92; Muranishi, Critical in Therapeutic Drug delivery Systems (review of medical Carrier Systems), review of medical Carrier Systems (1990), 1 to 33; buur et al, j.control Rel (journal of controlled release), 1990,14, 43-51).

Non-chelating non-surfactant: as used herein, a non-chelating non-surfactant penetration enhancing compound may be defined as a compound that exhibits no significant activity as a chelator or as a surfactant but rather enhances the uptake of iRNA through the mucosal membrane of the digestive tract (see, e.g., Muranishi (Muranishi), clinical Reviews in Therapeutic Drug Carrier Systems (review of medical Carrier Systems), 1990,7, 1-33). Penetration enhancers of this class include, for example, unsaturated cyclic ureas, 1-alkyl-and 1-alkenyl azacyclo-alkanone derivatives (Lee (Li) et al, clinical Reviews in Therapeutic Drug carriers Systems (review of medical Carrier Systems, 1991, page 92), and non-steroidal anti-inflammatory agents, such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al, J.Pharm.Pharmacol. (J.Pharmacol., 1987,39, 621-626).

Substances that enhance uptake of iRNA at the cellular level may also be added to the pharmaceutical and other compositions of the invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. patent No. 5,705,188), cationic glycerol derivatives, and polycationic molecules such as polylysine (Lollo et al, PCT application WO 97/30731) are also known to enhance cellular uptake of dsRNA. Examples of commercially available transfection reagents include, for example, Lipofectamine ^TM(Invitrogen; Carlsbad, Calif.), Lipofectamine 2000^TM(Invitrogen; Carlsbad, Calif.), 293fectin^TM(Invitrogen; Carlsbad, Calif.), Cellffectin^TM(Invitrogen; Carlsbad, Calif.), DMRIE-C^TM(Invitrogen; Carlsbad, Calif.), FreeStyle^TMMAX (Invitrogen; Carlsbad, Calif.), Lipofectamine^TM2000CD (Invitrogen; Carlsbad, Calif.), Lipofectamine^TM(Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine^TM(Invitrogen; Carlsbad, Calif.), Optifect^TM(Invitrogen; Carlsbad, Calif.), X-tremagene Q2 transfection reagent (Roche; Grenzachstrysee, Switzerland), DOTAP lipofection reagent (Grenzachstrysee, Switzerland), DOSPER lipofection reagent (Grenzachstrysee, Greenzarkel), Switzerland) or Fugene (Grenzachstrysene, Switzerland), Reagents (Promega Corp.; Madison, Wis.), TransFast^TMTransfection reagent (Promega Corp.; Madison, Wis.), Tfx^TM20 reagents (Promega Corp.; Madison, Wis.), Tfx^TM50 reagents (Promega Corp.; Madison, Wis.), DreamFect^TM(OZ Biosciences; Marseilles, France), EcoTransfect (OZ Biosciences; Marseilles, France), TransPassa D1 transfection reagent (New England Biolabs; Ipswich, Ipswech, Mass., USA), LyoVec^TM/LipoGen^TM(Invivogen, Inc.; san Diego, Calif., USA), PerFectin transfection reagent (Genlantis, san Diego, Calif., USA), NeuroPORTER transfection reagent (Genlantis, san Diego, Calif., USA), Geneporter 2 transfection reagent (Genlantis, san Diego, Calif., USA), Cytofectin transfection reagent (Genlantis, san Diego, Calif., USA), BaculoPORTER transfection reagent (Genlantis, san Diego, Calif., USA), Trojanoparter ^TMTransfection reagents (Genlantis, Inc.; san Diego, Calif., USA), RiboFect (Bioline, Taunton, Massachusetts, USA), PlasFect (Bioline, Taunton, Massachusetts, USA), UniFECTOR (B-Bridge International, mountain View, Calif., USA), Surefactor (B-Bridge International, mountain View, Calif., USA), or HiFect^TM(B-Bridge International, mountain View, Calif., USA), among others

Other substances may be used to enhance permeation of the administered nucleic acid, including glycols, such as ethylene glycol and propylene glycol, pyrroles, such as 2-pyrrole, azone, and terpenes, such as limonene and menthone.

Carrier

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, a "carrier compound" or "carrier" can refer to a nucleic acid or analog thereof that is inert (i.e., does not possess biological activity itself), but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a biologically active nucleic acid by, for example, degrading the biologically active nucleic acid or facilitating its removal from circulation. Coadministration of nucleic acid and carrier compound (typically with an excess of the latter substance) can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidney or other external circulating reservoirs, presumably due to competition between the carrier compound and the nucleic acid for the co-receptor. For example, recovery of partially phosphorothioated dsRNA in liver tissue can be reduced when co-administered with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4 'isothiocyanatostilbene-2, 2' -disulfonic acid (Miyao et al, DsRNA Res. Dev. (DsRNA research and development), 1995,5, 115-.

Excipient

In contrast to a carrier compound, a "pharmaceutically acceptable 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 excipients may be liquid or solid and are selected according to the intended mode of administration in mind, to provide the desired volume, consistency, etc. when combined with the nucleic acid and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, dibasic calcium phosphate, or the like); lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not deleteriously react with the nucleic acid may also be used to formulate the compositions of the 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, hydroxymethyl cellulose, 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 nucleic acids in liquid or solid oil bases. The solution may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not deleteriously react with the nucleic acid may be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, and the like.

Other Components

The compositions of the present invention may additionally contain other auxiliary components conventionally present in pharmaceutical compositions at usage levels established in the art. Thus, for example, the compositions may contain additional compatible pharmaceutically active materials, such as antipruritics, hemostatics, local anesthetics, or anti-inflammatory agents, or may contain additional materials useful in the actual formulation of different dosage forms of the compositions of the present invention, such as dyes, flavors, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials should not unduly interfere with the biological activity of the components of the compositions of the present invention when added. The formulation can be sterilized and, if desired, mixed with auxiliaries which do not adversely interact with the nucleotide or nucleotides of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors and/or aromatic substances and the like.

Aqueous suspensions may contain substances which increase the viscosity of the suspension, including sodium carboxymethyl cellulose, sorbitol, and/or dextran. The suspension may also contain stabilizers.

In some embodiments, the pharmaceutical compositions characterized in this invention comprise (a) one or more iRNA compounds and (b) one or more anti-cytokine biologic agents that act through non-RNAi mechanisms. Examples of such biologicals include biologicals targeted to IL1 β (e.g., anakinra), IL6 (e.g., tocilizumab), or TNF (e.g., etanercept, infliximab, adalimumab, or certolizumab (certolizumab)).

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining LD50 (the dose lethal to 50% of the population) and 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 LD50/ED50 ratio. Compounds that exhibit high therapeutic indices are preferred.

Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. Dosages of the compositions characterized herein are generally within the range of circulating concentrations that include ED50 with low or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration employed. For any compound used in the methods characterized in this invention, a therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range (e.g., to achieve a reduction in the polypeptide concentration) of a compound or, where appropriate, a polypeptide product of a target sequence, wherein the plasma concentration includes IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine the dosage for use in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the irnas described herein may be administered in combination with other known agents effective in treating pathological processes mediated by TMPRSS6 expression. In any case, the amount and timing of iRNA administration can be adjusted by the administering physician based on results observed using standard efficacy values known in the art or described herein.

Methods for treating diseases caused by expression of TMPRSS6 gene

The invention specifically relates to irnas targeting TMPRSS6 and compositions containing at least one such iRNA for use in treating TMPRSS6 mediated disorders or diseases. For example, compositions containing irnas targeting the TMPRSS6 gene are useful for treating disorders associated with elevated iron levels, such as thalassemia (e.g., beta-thalassemia intermedia or alpha-thalassemia), primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, sideroblasts anemia, hemolytic anemia, erythropoietic alloanemia, or sickle cell anemia. In one embodiment, TMPRSS6 iRNA is used to treat hemoglobinopathies. TMPRSS6 iRNA characterized in the present invention may also be used to treat elevated iron levels due to other conditions (e.g., chronic alcoholism).

In thalassemia, hemoglobin chains with inadequate bone marrow synthesis; this in turn reduces the production of red blood cells and causes anemia. Either the alpha or beta chain can be affected, but beta thalassemia is more common; newborn infants are healthy because their body still produces HbF without beta chains; during the first months of life, the bone marrow turns to produce HbA and symptoms begin to appear.

β^-Thalassemia is not expressed (beta) by the allele associated with the HBB gene⁰) Or low expression (. beta.)⁺) Is caused by the mutation(s). Beta is a^-Thalassemia varies in severity, depending on genotype, and includes mild/trait beta _ thalassemia (beta/beta)⁰Or beta/beta +, intermediate form beta^-Thalassemia (. beta.)⁰Beta +) and heavy beta^-Thalassemia (. beta.)⁰/β⁰Or beta⁺/β⁺)。

Thalassemia Intermedia (TI) typically presents mild hemolysis, while beta-Thalassemia Major (TM) is typically accompanied by massive hemolysis that causes, for example, anemia and splenomegaly; and highly ineffective erythropoiesis, which results in bone marrow drive (skeletal muscle changes,Bone mass reduction), increased erythropoietin synthesis, hepato-splenomegaly, consumption of blood tonics (megaloblastic anemia), and high uric acid in the blood. Irnas characterized in the present invention, e.g., TMPRSS6 iRNA, are more suitable for treating iron overload typically associated with more TI-like thalassemia (e.g., for treating beta-bearing thalassemia ⁰/β+、β/β⁰Or individuals of the beta/beta + genotype).

Symptoms of beta-thalassemia also include complications due to therapy, for example, iron excess, which causes endocrinopathy, liver fibrosis, and myocardial fibrosis. Administration of iRNA agents targeting TMPRSS6 may be effective to treat one or more of these symptoms.

Alpha-thalassemia is not expressed due to the presence of the allele of HBA1 or HBA2 gene (alpha)⁰) Or low expression (alpha)⁺) Is caused by the mutation(s). Alpha-thalassemia varies in severity depending on genotype, and includes mild (-alpha/alpha), hemoglobin Bart fetal edema (alpha)⁰/α⁰) Alpha-thalassemia minor (-/alpha), (-alpha/-alpha) and HbH disease (-/- -alpha). Lower alpha-globin chain production, resulting in an excess of beta chain in adults and an excess of gamma chain in newborns. The excess beta chain forms an unstable tetramer with an abnormal oxygen dissociation curve (called 4 beta chain hemoglobin H or HbH). Administration of iRNA agents targeting TMPRSS6 may be effective to treat iron overdose in subjects with alpha-thalassemia.

Symptoms of hemochromatosis include, for example, abdominal pain, joint pain, weakness, lack of energy, weakness, dull skin (often referred to as "bronzing"), and loss of body hair. Administration of iRNA agents targeting TMPRSS6 may be effective to treat one or more of these symptoms.

Other symptoms associated with iron overload include an increased risk of liver disease (cirrhosis, cancer), heart attack or failure, diabetes, osteoarthritis, osteoporosis, metabolic syndrome, hypothyroidism, hypogonadism, and in some cases premature death. Inappropriate management leading to excess iron may also accelerate such neurodegenerative diseases, such as alzheimer's disease, early onset parkinson's disease, huntington's disease, epilepsy and multiple sclerosis. Administration of irnas targeting TMPRSS6, e.g., irnas described in tables 2, 3, or 4, may treat one or more of these symptoms, or prevent the development or progression of a disease or disorder that is exacerbated by increased iron levels.

The invention further relates to the use of iRNA or pharmaceutical compositions thereof, for example, in combination with other drugs and/or other therapeutic methods, for example, with known drugs and/or known therapeutic methods (e.g., those currently used to treat such disorders), for treating disorders associated with elevated iron levels. For example, in certain embodiments, iRNA targeting TMPRSS6 is administered in combination with, for example, an iron chelator (e.g., deferoxamine), folic acid, a blood transfusion, phlebotomy, an agent that controls ulcers, an agent that increases fetal hemoglobin levels (e.g., hydroxyurea), an agent that controls infection (e.g., antibiotics and antiviral agents), an agent that treats thrombogenic conditions, or stem cell or bone marrow transplantation. Stem cell transplantation may utilize stem cells from the umbilical cord (e.g., from an autophily, e.g., siblings). Exemplary iron chelators include deferoxamine, deferasirox (Exjade), deferiprone, vitamin E, wheat germ oil, tocofersolan (tocophersolan), and opuntia ficus indica xanthins (indica).

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

The invention features methods of administering iRNA agents targeting TMPRSS6 to patients having diseases or disorders mediated by expression of TMPRSS6 (e.g., disorders associated with elevated iron levels). Administration of the dsRNA can reduce iron levels, reduce ferritin levels, and/or reduce transferrin saturation levels. For example, administration of dsRNA can reduce serum iron levels and/or reduce serum ferritin levels. Transferrin saturation levels can be reduced by 5%, 10%, 15%, 20%, 25% or more. Transferrin saturation levels can be reduced to below 50%, below 45%, below 40%, below 35% or lower. Transferrin saturation is a measure of the amount of iron bound to serum transferrin and corresponds to the ratio of serum iron to total iron binding capacity.

By "decrease" in this context is meant a statistically significant decrease in such levels. The reduction may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably to a level that is accepted as within the normal range for individuals not suffering from this disorder.

The efficacy of treating or preventing a disease can be assessed, for example, by measuring the level of disease progression, disease remission, symptom severity, pain reduction, quality of life, drug dosage required to maintain therapeutic efficacy, disease markers appropriate for a given disease being treated or targeted for prevention, or any other measurable parameter. It is well within the ability of one of ordinary skill in the art to monitor the efficacy of treatment or prevention by measuring any one of such parameters or any combination of parameters. For example, transferrin saturation levels or serum ferritin levels may be monitored for the efficacy of a given treatment regimen.

Iron level tests are typically performed on samples of the patient's blood. The iron level test measures the amount of iron carried by transferrin in serum. The TIBC (total iron binding capacity) assay measures the amount of iron that blood will carry when transferrin is fully saturated. Since transferrin is produced by the liver, TIBCs can be used to monitor liver function and nutrition in a subject. The transferrin test is a direct measure of the level of transferrin (also known as transferrin) in the blood. The saturation level of transferrin can be calculated by dividing the serum iron level by TIBC. The ferritin test measures the level of proteins in the blood that store iron for later use by the body.

iRNA treatment described herein can be used to treat individuals having elevated levels of iron, as may be indicated by iron levels in serum, e.g., iron levels measured at greater than 350 μ g/dL, greater than 500 μ g/dL, greater than 1000 μ g/dL, or higher. In one embodiment, the elevated level of iron in serum is, for example, greater than 15, 20, 25 or 30mg/g dry weight.

iRNA treatment described herein may be used to treat subjects having elevated ferritin levels, as may be indicated by elevated ferritin levels in serum, for example, ferritin levels measured at greater than 300 μ g/L, greater than 500 μ g/L, greater than 1000 μ g/L, greater than 1500 μ g/L, greater than 2000 μ g/L, greater than 2500 μ g/L, or 3000 μ g/L or higher.

iRNA treatment described herein can be used to treat individuals having elevated levels of iron, as may be indicated by elevated transferrin levels in serum, e.g., transferrin levels measured at greater than 400mg/dL, greater than 500mg/L, greater than 1000mg/dL, or higher.

iRNA treatment described herein can be used to treat individuals with moderately elevated iron levels, as may be indicated by moderately elevated transferrin saturation levels, e.g., 40%, 45%, or 50% or higher saturation levels. In addition, the treatments described herein can also be used to prevent elevated iron levels in individuals with only a small increase in transferrin saturation. Those of ordinary skill in the art can readily monitor transferrin saturation levels in subjects receiving treatment with iRNA as described herein and determine that transferrin saturation levels are reduced by at least 5% or 10%.

iRNA treatment described herein can be used to treat individuals with elevated iron levels, as may be indicated by TIBC values greater than 400 μ g/dL, greater than 500 μ g/dL, or greater than 1000 μ g/dL or higher.

In some embodiments, a subject in need of treatment with TMPRSS6 siRNA has a reduced hematocrit level, a reduced hemoglobin level, an increased width of red blood cell distribution, an increased number of reticulocytes, a reduced number of mature red blood cells, an increased unsaturated iron binding capacity, a reduced ineffective erythropoiesis, a reduced extramedullary hematopoiesis, and/or a reduced level of HAMP1 expression.

The patient may be further monitored by determining blood glucose (glucose) levels or alpha fetoprotein levels, by echocardiography (e.g., to examine heart function), Electrocardiogram (ECG) (e.g., to examine electrical activity of the heart), imaging tests (e.g., CT scans, MRI and ultrasound), and liver function tests. Excess iron staining or iron concentration may be measured on liver biopsies to confirm the extent of liver injury, e.g., stage of liver disease.

A therapeutic or prophylactic effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or by the absence of worsening or the development of multiple symptoms, where they would otherwise be expected. As an example, a favorable change in a measurable parameter of a disease of at least 10% and preferably at least 20%, 30%, 40%, 50% or more may indicate an effective treatment. The efficacy of a given iRNA drug or formulation of such a drug can also be determined using experimental animal models of a given disease as known in the art. When using experimental animal models, the efficacy of the treatment was confirmed when a statistically significant reduction in the markers or symptoms was observed.

Alternatively, the efficacy may be measured by a severity-reducing measure of the disease, as determined by one skilled in the diagnostic art based on clinically accepted disease severity grading scales.

Therapeutic amounts of iRNA, such as 0.01mg/kg, 0.05mg/kg, 0.1mg/kg, 0.5mg/kg, 1.0mg/kg, 1.5mg/kg, 2.0mg/kg, or 2.5mg/kg dsRNA, can be administered to a patient. The iRNA may be administered by intravenous infusion for a sustained period of time, e.g., for a period of 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes. Administration will be repeated, for example, on a regular basis, e.g., every two weeks (i.e., every two weeks) for a month, two months, three months, four months, or longer. After the initial treatment regimen, treatment may be given on a less frequent basis. For example, administration may be repeated once a month for six months or a year or more after administration for three months every two weeks. Administration of iRNA can reduce TMPRSS6 levels, e.g., 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 in a cell, tissue, blood, urine, or other compartment of a patient.

A smaller dose, e.g., 5% infusion response, may be administered to the patient before the full dose of iRNA is administered, and monitored for side effects, e.g., allergy or worsening of symptoms. In another example, the patient may be monitored for an unwanted immunostimulatory effect, such as increased levels of cytokines (e.g., TNF- α or INF- α).

Many of the disorders associated with elevated iron levels are genetic. Thus, patients in need of TMPRSS6 iRNA can be identified by taking a family history. A healthcare provider, such as a physician, nurse, or family member, may obtain a family history prior to prescribing or administering TMPRSS6 dsRNA. DNA assays can also be performed on patients to identify mutations in the TMPRSS6 gene prior to administration of the TMPRSS6 dsRNA to the patient. For example, the diagnosis of hereditary hemochromatosis can be confirmed by identifying the two HFE (hemochromatosis) gene mutations C282Y and H63D according to GenBank accession number CAB07442.1(GI:1890180, recorded in 23.10.2008).

Due to the inhibitory effect on the expression of TMPRSS6, the composition according to the present invention or the pharmaceutical composition prepared therefrom may improve the quality of life.

Methods for modulating expression of TMPRSS6 gene

In yet another aspect, the invention provides a method of modulating (e.g., inhibiting or activating) TMPRSS6 gene expression in a mammal.

In one embodiment, the method comprises administering a composition characterized in the invention to the mammal such that expression of the target TMPRSS6 gene is reduced, e.g., for an extended duration, e.g., at least two days, three days, four days, or longer, e.g., one week, two weeks, three weeks, or four weeks or longer. The effect of reducing the expression of the target TMPRSS6 gene preferably results in a reduction in iron uptake and/or mobilization in vivo. Reduced iron absorption or mobilization may be manifested by the observation of reduced serum ferritin levels, serum or liver iron levels, and/or serum transferrin saturation levels. In some embodiments, one or more of serum ferritin levels, serum or liver iron levels, or serum transferrin saturation levels is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 60% or more compared to pre-treatment levels. In some embodiments, serum ferritin levels are reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 60% or more compared to pre-treatment levels.

In another embodiment, the method comprises administering a composition as described herein to the mammal such that expression of the target TMPRSS6 gene is increased, e.g., by at least 10%, as compared to an untreated animal. In some embodiments, activation of TMPRSS6 occurs over an extended duration, e.g., at least two, three, four or more days, e.g., one, two, three, four or more weeks. Without wishing to be bound by theory, iRNA may activate TMPRSS6 expression by stabilizing TMPRSS6 mRNA transcripts, interacting with promoters in the genome, and/or inhibitors that inhibit TMPRSS6 expression.

iRNA useful for the methods and compositions characterized in this invention specifically targets the (primary or processed) RNA of the TMPRSS6 gene of interest. Compositions and methods for inhibiting the expression of these TMPRSS6 genes using irnas may be prepared and implemented as described elsewhere herein.

In one embodiment, the method comprises administering a composition comprising an iRNA, wherein the iRNA comprises a nucleotide sequence that is complementary to at least a portion of an RNA transcript of TMPRSS6 gene of the mammal to be treated. When the organism to be treated is a mammal (e.g., a human), the composition can be administered by any means known in the art, including, but not limited to, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, these compositions are administered by intravenous infusion or injection.

Unless defined otherwise, 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 iRNA's and methods characterized in the present 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 of the invention

Example 1 Interfering RNA (iRNA) Synthesis

Sources of reagents

Where the source of the reagents is not specified herein, such reagents may be obtained from any molecular biology reagent supplier, with quality/purity standards for molecular biology applications.

Oligonucleotide synthesis

Applicants have used several different methods to generate iRNA molecules described herein. This example describes one approach that has been used. The iRNA as described herein can be prepared by one of ordinary skill using any method known in the art.

Oligonucleotides were synthesized on an AKTAoligopilot synthesizer. A commercially available controlled pore glass solid support (dT-CPG,prime Synthesis) and RNA phosphoryl imine with standard protecting groups, 5' -O-dimethoxytrityl N6-benzoyl-2 ' -tert-butyldimethylsilyl-adenosine-3 ' -O-N, N ' -diisopropyl-2-cyanoethyl phosphoryl imine, 5' -O-dimethoxytrityl-N4-acetyl-2 ' -tert-butyldimethylsilyl-cytidine-3 ' -O-N, N ' -diisopropyl-2-cyanoethyl phosphoryl imine, 5' -O-dimethoxytrityl-N2-isobutyryl-2 ' -tert-butyldimethylsilyl-guanosine-3 ' -O-N, n ' -diisopropyl-2-cyanoethyl phosphoryl imine and 5' -O-dimethoxytrityl-2 ' -tert-butyldimethylsilyl-uridine-3 ' -O-N, N ' -diisopropyl-2-cyanoethyl phosphoryl imine (Pierce Nucleic Acids Technologies) were used for oligonucleotide synthesis. 2' -Fphosphorylimine, 5' -O-dimethoxytrityl-N4-acetyl-2 ' -fluoro-cytidine-3 ' -O-N, N ' -diisopropyl-2-cyanoEthyl-phosphoryl imine and 5 '-O-dimethoxytrityl-2' -fluoro-uridine-3 '-O-N, N' -diisopropyl-2-cyanoethyl-phosphoryl imine were purchased from Promega corporation. All phosphorus imides in acetonitrile (CH) ₃CN) was used, except for guanosine used at a concentration of 0.2M in 10% THF/ANC (v/v). A 16 minute coupling/recycle time was used. The activator was 5-ethylthiotetrazole (0.75M, American International Chemicals); for PO-oxidation iodine/water/pyridine was used and for PS-oxidation PADS (2%) in 2, 6-lutidine (1:1v/v) was used.

3' -ligand conjugated strands are synthesized using a solid support containing the corresponding ligand. For example, the introduction of cholesterol units in the sequence is performed from hydroxyprolinol-cholesterol phosphinimine. Cholesterol is tethered via a 6-aminocaproic acid ester linkage to trans-4-hydroxyprolinol to obtain a hydroxyprolinol-cholesterol moiety. 5' -terminal Cy-3 and Cy-5.5 (fluorophore) -labeled iRNA were synthesized from the corresponding Quasar-570(Cy-3) purchased from Biosearch Technologies, Inc. Conjugation of ligands to the 5' -terminal and or internal positions is achieved by using appropriately protected ligand-phosphoimide building blocks. In the presence of 5- (ethylthio) -1H-tetrazole activator, for an extended period of 15 minutes, to remove anhydrous CH₃A0.1M solution of phosphoramidite in CN was coupled to the solid support bound oligonucleotide. Internucleotide phosphite oxidation to phosphate was performed as reported (1) using standard iodine-water or by conjugating oligonucleotides with a 10 minute oxidation wait time by treatment with t-butyl hydroperoxide/acetonitrile/water (10:87: 3). The phosphorothioates are incorporated by oxidizing the phosphite to the phosphorothioate using a sulfur transfer reagent such as DDTT (available from AM Chemicals), PADS, and or Beaucage reagents. Cholesterol phosphoimines were synthesized by itself and used at a concentration of 0.1M in dichloromethane. The coupling time for cholesterol phosphoimide was 16 minutes.

Deprotection of I (nucleobase deprotection))

After completion of the synthesis, the support was transferred to a 100mL glass Vial (VWR). The oligonucleotide was excised from the support while deprotecting the base and phosphate groups with 80mL of an ethanol-ammonia mixture [ ammonia: ethanol (3:1) ] at 55 ℃ for 6.5 hours. The bottle was cooled briefly on ice and then the ethanol-ammonia mixture was filtered to a new 250-mL bottle. CPG was washed with 2X 40mL portions of ethanol/water (1:1 v/v). The volume of the mixture was then reduced to about 30mL by a rotary evaporator. The mixture was then frozen on dry ice and dried under vacuum on a centrifugal vacuum concentrator.

Deprotection of II (removal of 2' -TBDMS group))

The dried residue was resuspended in 26mL of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60 ℃ for 90 minutes to remove the tert-butyldimethylsilyl group (TBDMS) at the 2' position. The reaction was then quenched with 50mL of 20mM sodium acetate and the pH adjusted to 6.5. The oligonucleotides were stored in a freezer until purification.

Analysis of

The oligonucleotides were analyzed by High Performance Liquid Chromatography (HPLC) prior to purification, and the choice of buffer and column depends on the sequence and or the nature of the conjugated ligand.

HPLC purification

The ligand conjugated oligonucleotides were purified by reverse phase preparative HPLC. Unconjugated oligonucleotides were purified by anion-exchange HPLC on self-packed TSK gel columns. The buffer is 10% CH₃20mM sodium phosphate (pH 8.5) (buffer A) and 10% CH in CN₃CN, 20mM sodium phosphate (pH 8.5) in 1M NaBr (buffer B). Fractions containing full-length oligonucleotides were pooled, desalted and lyophilized. Approximately 0.15OD of the desalted oligonucleotide was diluted in water to 150. mu.L and then pipetted into dedicated vials for CGE and LC/MS analysis. The compounds were then analyzed by LC-ESMS and CGE.

iRNA preparation

For the general preparation of iRNA, equimolar amounts of the sense and antisense strands were heated in 1xPBS at 95 ℃ for 5min and slowly cooled to room temperature. The integrity of the duplex was confirmed by HPLC analysis.

Nucleic acid sequences are represented using standard nomenclature and the exact abbreviations of table 1.

Table 1: abbreviations for nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in the oligonucleotide, are interconnected by 5'-3' -phosphodiester linkages.

Example 2 TMPRSS6 SiRNA design

Transcript

Sirnas targeting TMPRSS6 were designed and synthesized. The design used the human transcript from NCBI Refseq Collection (NM-153609.2, SEQ ID NO:1, FIG. 1).

The siRNA duplexes were designed to have 100% identity to the TMPRSS6 gene.

A total of 655 sense siRNA oligomers and 655 antisense siRNA oligomers derived from human TMPRSS6 were designed. The oligomers are presented in table 2. Additional human TMPRSS6 derived sense and antisense siRNA oligomers are presented in table 3. Sense and antisense siRNA oligomers derived from human TMPRSS6 with modifications are presented in table 4.

TABLE 2 human TMPRSS6 Sense and antisense strand sequences of dsRNA

TABLE 3 human TMPRSS6 Unmodified sense and antisense strand sequences of dsRNA

TABLE 4 human TMPRSS6 Modified sense and antisense strand sequences of dsRNA

Synthesis of the TMPRSS6 sequence

The TMPRSS6 iRNA sequence can be synthesized on a MerMade192 synthesizer on a 1. mu. mol scale.

Endo light chemistry can be applied as detailed below.

All pyrimidines in the sense strand (cytosine and uridine) contain a 2' -O-methyl base (2' O-methyl C and 2' -O-methyl U).

In the antisense strand, pyrimidines adjacent to (pointing to the 5' position) the riboa nucleosides can be replaced with their corresponding 2-O-methyl nucleosides.

A two base dTsdT extension may be introduced at the 3' end of both the sense and antisense sequences.

The sequence file may be converted into a text file to make it compatible for loading in the MerMade192 synthesis software.

Synthesis, cleavage and deprotection

TMPRSS6 sequences were synthesized using solid phase supported oligonucleotide synthesis using phosphoimide chemistry.

The synthesis of the above sequences can be performed in 96-well plates on a 1 μm scale. The imide solution may be prepared at a concentration of 0.1M and ethylthiotetrazole (0.6M in acetonitrile) may be used as the activator.

The synthesized sequence can be cleaved and deprotected in a 96-well plate using methylamine in the first step and a fluoride reagent in the second step. The crude sequence can be precipitated using an acetone: ethanol (80:20) mixture and the precipitate resuspended in 0.02M sodium acetate buffer. Samples from each sequence can be analyzed by LC-MS to confirm identity, by UV analysis for quantification. The selected sample set can also be analyzed by IEX chromatography to determine purity.

Purification and desalination

All sequences were purified on an AKTA seeker purification system using Source 15Q columns. Sample injection and collection can be performed in 96-well (1.8mL deep well) plates. A single peak corresponding to the full-length sequence can be collected in the eluent. The purified sequence can be desalted on a SephadexG25 column using an AKTA purificator. The desalted TMPRSS6 sequences can be analyzed for concentration (by UV measurement at a 260) and purity (by ion exchange HPLC). The single strand may then be renatured.

Example 3. Pair TMPRSS6 In vitro screening of siRNA duplexes for TMPRSS6 knockdown

TMPRSS6 siRNA duplexes were screened for the ability to knock down expression of TMPRSS6 in vitro. Single dose screens, dose response screens and host cell viability were evaluated.

In vitro screening：

Cell cultures and transfections for single dose studies and dose response studies：

HeLa or Hep3B cells (ATCC, Manassas, Va.) were incubated at 37 ℃ in 5% CO₂Grown under atmosphere to near confluence in X (ATCC company) supplemented with 10% FBS, streptomycin and glutamine (ATCC), and then released from the plates by trypsinization. Transfection was performed by adding 14.8. mu.l of Opti-MEM plus 0.2. mu.l of Lipofectamine RNAiMax (Invitrogen, Calif. Bard., Calif. Cat. No. 13778-150) per well to 5. mu.l of siRNA duplexes per well into 96-well plates and incubating for 15 minutes at room temperature. Subsequent addition of a catalyst containing about 2X 10⁴80 μ l of antibiotic-free complete growth medium of HeLa or Hep3B cells to the siRNA mixture. Cells were incubated for 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10nM and 0.1nM duplex final concentrations and dose response experiments were performed at 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, 0.00001nM duplex final concentrations.

Use of mRNA isolation kit (Invitrogen corporation, product No. 610-12))Separation of Total RNA：

Cells were harvested and lysed in 150 μ l lysis/binding buffer before useThermomixer (mixing speed was the same throughout the process) was mixed at 850rpm for 5 minutes. Ten microliters of the mixture of magnetic beads and 80. mu.l lysis/binding buffer was added to the round bottom plate and mixed for 1 minute. Magnetic beads are captured using a magnetic support and are not presentThe supernatant was removed with agitation of the beads. After removing the supernatant, the lysed cells were added to the remaining magnetic beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads were washed twice with 150 μ l of wash buffer a and mixed for one minute. The magnetic beads were captured again and the supernatant was removed. The beads were then washed with 150 μ l wash buffer B, captured, and the supernatant removed. Next, the beads were washed with 150 μ Ι elution buffer, captured, and the supernatant was removed. The magnetic beads were then allowed to dry for two minutes. After drying, 50 μ l elution buffer was added and mixed for five minutes at 70 ℃. The magnetic beads were captured on the magnet for five minutes. 40 μ l of supernatant was removed and added to another 96-well plate.

Using ABI high Capacity cDNA reverse transcription kit (Applied) Biosystems, Foster City (Foster City) Temmit), California, catalog No. 4368813) was synthesized：

Add mastermix per reaction: mu.l 10 Xbuffer, 0.8. mu.l 25 XdNTPs, 2. mu.l random primers, 1. mu.l reverse transcriptase, 1. mu.l RNase inhibitor and 3.2. mu. l H2 in 2O to 10. mu.l total RNA. Using a Bio-RadC-1000 or S-1000 thermal cycler (Hercules, Calif.) the following procedure was followed: cDNA was generated at 25 ℃ for 10min, 37 ℃ for 120min, 85 ℃ for 5sec, and 4 ℃ hold.

Real-time PCR：

Mu.l of cDNA was added to 50 384-well plates (catalog No. 04887301001, Roche Co.))Each well contained 0.5. mu.l of GAPDH TaqMan probe (Cat. No. 4326317E of Applied Biosystems Co., Ltd.))0.5. mu.l TMPRSS6 TaqMan probe (Applied Biosystems catalog number Hs00542184_ m 1))And 5. mu.l Lightcycler 480 Probe Master mix (Roche Cat No. 04887301001))The master mixture of (1). Use of Δ Δ Ct (RQ))Assays were performed on ABI7900HT real-time PCR System (Applied Biosystems Co., Ltd.))Real-time PCR was performed. Each duplex was tested in two independent transfections and assayed in duplicate for each transfection, unless otherwise indicated in the summary table.

To calculate relative fold change, real-time data was analyzed using the Δ Δ Ct method and classified for the following assays Normalization, the assay was performed with cells transfected with 10nM AD-1955 or mock transfected cells. IC calculation with 4-parameter fitting model Using XLFit₅₀And normalized to cells transfected with AD-1955 at the same dose range or to its own lowest dose.

And (4) screening the viability. HeLa or Hep3B cells s (ATCC company, Manassas, Va.) were cultured in culture medium)At 37 ℃ in 5% CO₂Supplemented with 10% FBS, streptomycin and glutamine (ATCC) under atmosphere)X (ATCC))After growth to near confluence, release from the plate by trypsinization. Cell viability was measured in HeLa cells and Hep3B cells after transfection with 100, 10, 1, 0.1, 0.01 and 0.0001nM siRNA on days 3 and 5. Cells were plated at 2.5X 10 per well³-5×10³The density of individual cells was plated in 96-well plates. Each siRNA was assayed in triplicate and the data averaged. siRNAs targeting PLK1 and AD-19200 were included as positive controls for loss of viability and AD-1955 as negative controls. PLK1 and AD-19200 caused a dose-dependent loss of viability. To measure viability, 20ul CellTiter blue (Promega Corp.) was added)Added to each well of a 96-well plate after 3 days and 5 days, and incubated at 37 ℃ for 2 hours. Then, the mixture was measured in a spectrophotometer (Molecular Devices Co., Ltd.) )Middle at 560_Ex/590_EmThe plate is read. Viability was expressed as the mean of light units from triplicate transfections +/-standard deviation.

By TMPRSS6 siRNA duplex in vitro knockdown of TMPRSS6 expression

Table 5 presents data showing the knockdown of TMPRSS6 in Hep3B cells transfected with sirnas targeting TMPRSS6, Hep3B cells. Data are expressed as the fraction of TMPRSS6 message remaining in cells transfected with siRNA targeting TMPRSS6 relative to cells transfected with negative control siRNA (AD-1955). Untreated cells ("untreated" cells) served as a second negative control. All sirnas were tested at least 2 times and qPCR reactions were also performed in duplicate. Single dose experiments were performed at 10nM and 0.1nM siRNA duplex final concentrations.

TABLE 5 in vitroExpression of TMPRSS6 in a single dose screen

Selected TMPRSS6 ₅₀IC in vitro dose response screening of siRNA duplexes

Table 6 presents the IC of selected TMPRSS6 siRNA duplexes determined from in vitro dose response screening₅₀The value is obtained. TMPRSS6 siRNA duplexes (table 5) effective in 10nM and 0.1nM single dose screens at day 1 and day 5 post-transfection in Hep3B cells were tested for TMPRSS6 knockdown in a dose-response manner. Dose response experiments were performed at 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, 0.00001nM siRNA duplex final concentrations. For normalization, the knockdown of TMPRSS6 was measured relative to the non-targeting control AD-1955, or the value obtained at the lowest siRNA concentration for each duplex was tested.

TABLE 6 selected TMPRSS6 ₅₀IC in vitro dose response screening of siRNA duplexes

Using TMPRSS6 In vitro viability screening of HeLa and HEP3B cell lines transfected with siRNA duplexes.

Table 7 presents viability data for HeLa and HEP3B cell lines transfected with TMPRSS6 siRNA duplexes. Viability data were expressed as mean raw fluorescence units, with smaller values representing lower viability. Errors are expressed as standard deviation from triplicate transfections.

TABLE 7 use of TMPRSS6 Viability of HeLa and HEP3B cell lines transfected with siRNA duplexes.

Example 4 TMPRSS6 siRNA duplex leader selection.

To select a particular TMPRSS6 siRNA for further in vivo experiments, the chemically modified siRNA was screened for TMPRSS6 gene silencing activity by transfection in HEP3B human hepatoma cells. Two highly potent sirnas were selected for in vivo evaluation with minimal predicted off-target potential and multi-species reactivity, including reactivity to humans, cynomolgus monkeys, rats, and mice. The efficacy of two selected TMPRSS6 siRNAs was also confirmed in primary mouse hepatocytes, where TMPRSS6 siRNA-1(AD-46273) and TMPRSS6 siRNA-2(AD-46286) both displayed potent TMPRSS6 gene silencing activity, while TMPRSS6 siRNA-1(AD-46273) displayed an IC of 70pM ₅₀(FIG. 2A) and TMPRSS6 siRNA-2(AD-46286) showed an IC of 140pM₅₀(FIG. 2B).

Example 5.WT TMPRSS6 in C57BL/6 mice siRNA mediated silencing of TMPRSS 6.

TMPRSS6 siRNA to WT TMPRSS6 and HAMP1 in C57BL/6 mice The effect of mRNA expression.

To evaluate the in vivo effects of LNP-TMPRSS6 siRNA-1(AD-46273) and LNP-TMPRSS6 siRNA-2(AD-46286), 8 week old female WT C57BL/6 mice were dosed with 1mg/kg LNP-TMPRSS6 siRNA-1(AD-46273) or LNP-TMPRSS6 siRNA-2(AD-46286) or LNP-AD-1955l (siRNA targeting non-mammalian gene luciferase) via tail vein IV injection. TMPRSS6 siRNA was formulated with LNP11(MC 3). Mice were sacrificed 24 hours after dosing and livers were removed, snap frozen and ground to powder. Small amounts (about 20mg) of liver powder were broken up in lysis buffer and usedThe mRNA analysis of (1). A total of five mice were used per group. Data are expressed as LNP-Luc control percentage of target TMPRSS6 mRNA versus β -actin mRNA. As shown in FIG. 3A, LNP-TMPRSS6 siRNA-1(AD-46273) and LNP-TMPRSS6 siRNA-2(AD-46286) specifically and potently inhibited hepatic TMPRSS6 mRNA expression in a dose-dependent manner (data represent mean +/-standard deviation), ED-S ₅₀0.035mg/kg and ED₅₀0.18 mg/kg. As shown in FIG. 3B, LNP-TMPRSS6 siRNA-1(AD-46273) and LNP-TMPRSS6 siRNA-2(AD-46286) also inhibited hepatic HAMP1 mRNA expression in a dose-dependent manner.

WT TMPRSS6 in C57BL/6 mice SiRNA-mediated persistence of TMPRSS6 and HAMP1 gene expression silencing And (4) period.

To evaluate the duration of TMPRSS6 siRNA mediated knockdown of TMPRSS6 and HAMP1 gene expression, eight week old WT C57BL/6 mice were administered LNP-TMPRSS6 siRNA-1(AD-46273), or LNP-Luc control (LNP-AD-1955), or PBS via tail vein IV injection at a single 1mg/kg dose; all siRNA agents were delivered as LNP11 formulations. Mice were sacrificed at 6 hours, 24 hours, 48 hours, 3 days, 7 days, and 14 days. Use ofThe assay analyzed mRNA expression levels of TMPRSS6 and HAMP1 in the liver and normalized against B-actin. 5 mice were used per group, andand the data are presented as mean +/-standard deviation in figure 4. As shown in figure 4, a single dose of 1mg/kg LNP-TMPRSS6 siRNA-1(AD-46273) knockdown TMPRSS6 mRNA expression as early as 6 hours post-dose and decreased TMPRSS6 mRNA expression to about 90% of LNP-Luc control or PBS control for a period of 2 weeks. HAMP1 gene expression began to increase at 24 hours post-dose and was maintained for the duration of the 2-week period, with a maximum increase of 200% control occurring at 14 days post-dose (fig. 4). In addition, serum iron levels were determined as percent transferrin (Tf) saturation using Olympus AU 400. Transferrin saturation levels were calculated as the ratio of serum iron to Total Iron Binding Capacity (TIBC) and expressed as percent transferrin saturation. Starting 24 hours after administration, the percent transferrin saturation was reduced by about 50% and maintained over a 2 week period, indicating a reduction in circulating iron levels in serum (figure 4). The level of TMPRSS6 siRNA mediated silencing of TMPRSS6 was necessary to maintain the TMPRSS6 siRNA mediated effect on HAMP1 gene expression and serum iron levels in WT C57BL/6 mice.

To evaluate the level of TMPRSS6 siRNA mediated TMPRSS6 silencing necessary to maintain TMPRSS6 siRNA mediated effects on HAMP1 gene expression and serum iron levels in WT C57BL/6 mice, C57BL/6 mice were administered with 0.3mg/kg LNP-TMPRSS6 siRNA-1(AD-46273), or LNP-Luc control or PBS; all siRNA agents were delivered as LNP11 formulations. Mice were sacrificed at 5 hours, 24 hours, 48 hours, 3 days, 7 days, 14 days, 21 days, and 28 days post-dose. Use ofThe assay analyzed mRNA expression levels of TMPRSS6 and HAMP1 and normalized against B-actin. Five mice were used per group and the data are presented as mean +/-standard deviation in figure 5. As shown in figure 5, the maximal reduction in TMPRSS6 gene expression was achieved by 90% 24 hours post-dose and was maintained until 3 days post-dose. On day seven post-dose, TMPRSS6 gene expression decreased by about 85%; HAMP1 gene expression was induced to about 250% of control; and transferrin saturation (%) decreased by about 50% (fig. 5). On day 21 post-dose, TMPRSS6 gene expression decreased by about 40%; hAMP1 gene expression has been normalized; and serum iron levels, as measured by transferrin saturation (%), began to return to normal levels (fig. 5). In summary, the maximum knockdown of TMPRSS6 mRNA expression was achieved at 24 hours post-dose and returned to about 50% of normal expression levels by 3 weeks post-dose; hepcidin mRNA levels increased as early as 24 hours post-dose and were maintained up to seven days post-dose; the hepcidin levels returned to the control level on the fourteenth day after dosing; and transferrin saturation (as an indication of circulating iron levels) decreased to 50% of control levels as early as 24 hours post-dose and normalized near the fourth week. Thus the data presented in figure 5 illustrates that more than 50% of TMPRSS6 silencing is required to maintain LNP-TMPRSS6 siRNA-1(AD-46273) mediated effects on HAMP1 gene expression and serum iron levels.

TMPRSS6 siRNA-mediated TMPRSS6 silencing of WT Influence of hematological parameters in C57BL/6 mice.

To evaluate the effect of TMPRSS6siRNA mediated TMPRSS6 silencing on hematological parameters including Hemoglobin (HGB) and hematocrit; WT C57BL/6 mice were dosed with a single 1mg/kg dose of TMPRSS6siRNA-1(AD-46273) or LNP-Luc control or PBS; and then sacrificed at various time points up to two weeks after dosing. Hematology parameters including Hemoglobin (HGB), hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC) reticulocyte hemoglobin content (Chr) were determined using an Advia120 analyzer. As shown in fig. 6A and 6B, silencing of TMPRSS6 in Th3/+ mice resulted in a reduction in HGB (fig. 6A), and a reduction in hematocrit in WT C57BL/6 mice (fig. 6B). There were similar effects on Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC) and reticulocyte hemoglobin content (Chr).

Example 6 Mediterranean anemia mice (Th3/+)Effect of TMPRSS6 silencing mediated by TMPRSS6siRNA

Thalassemia mouse (Th3/+ )TMPRSS6 siRNA-mediated TMPREffect of SS6 silencing on serum iron parameters And (6) sounding.

To evaluate the effect of TMPRSS6 siRNA mediated silencing of TMPRSS6 on serum iron parameters including iron levels, Unsaturated Iron Binding Capacity (UIBC) and Tf saturation in thalassemic mice (Th 3/+); six-week-old Th3/+ mice were administered via tail vein injection with a single dose of 1mg/kg LNP-TMPRSS6 siRNA-1(AD-46273) or LNP-Luc control or PBS; and mice were sacrificed two weeks after dosing. Five mice were used per group and data are presented as mean +/-standard deviation in figure 7, where x represents P value < 0.01 and x represents P value < 0.001. As shown in figure 7, silencing of TMPRSS6 in Th3/+ mice resulted in a significant reduction in serum iron, UIBC and Tf saturation compared to the control PBS group.

Thalassemia mouse (Th3/+)Medium TMPRSS6 siRNA mediated TMPRSS6 silencing of reticulocytes and Red Influence of cellular parameters.

To evaluate the effect of TMPRSS6 siRNA mediated silencing of TMPRSS6 on reticulocyte and erythrocyte parameters, including reticulocyte number, reticulocyte hemoglobin content (CHr) and erythrocyte number (RBC), in thalassemia mice (Th3/+) six-week-old Th3/+ mice were administered via tail vein injection with a single dose of 1mg/kg LNP-TMPRSS6 siRNA-1(AD-46273) or LNP-Luc control or PBS; and mice were sacrificed two weeks after dosing. Reticulocyte and red blood cell parameters were determined using an Advia120 analyzer, including reticulocyte number, reticulocyte hemoglobin content (CHr), and red blood cell number (RBC). Five mice were used per group and data are presented as mean +/-standard deviation in figures 8A-8C, where x represents P value < 0.01 and x represents P value < 0.001. As shown in fig. 8A and 8B, silencing of TMPRSS6 in Th3/+ mice resulted in a significant reduction in reticulocyte number along with reticulocyte hemoglobin content (Chr). Furthermore, silencing of TMPRSS6 in Th3/+ mice resulted in a significant increase in mature red blood cell number (RBC) (fig. 8C), indicating a significant improvement in ineffective erythropoiesis, extramedullary hematopoiesis, and red blood cell production.

Thalassemia mouse (Th3/+)Medium TMPRSS6 siRNA-mediated TMPRSS6 silencing in HematologyShadow of parameter And (6) sounding.

To evaluate the effect of TMPRSS6 siRNA mediated silencing of TMPRSS6 on hematological parameters including Hematocrit (HCT), Hemoglobin (HGB), red cell distribution width (RDW) and mean red cell volume value (MCV) in thalassemic mice (Th 3/+); six-week-old Th3/+ mice were administered via tail vein injection with a single dose of 1mg/kg LNP-TMPRSS6 siRNA-1(AD-46273) or LNP-Luc control or PBS; and mice were sacrificed two weeks after dosing. Hematology parameters including Hematocrit (HCT), Hemoglobin (HGB), red blood cell distribution width (RDW), and mean red blood cell volume value (MCV) were determined using an Advia 120 analyzer. Five mice were used per group and data are presented as mean +/-standard deviation in figure 9, where x represents P value < 0.01 and x represents P value < 0.001. Silencing of TMPRSS6 in Th3/+ mice resulted in a significant increase in HCT (fig. 9A), a significant increase in HGB (fig. 9B), a significant decrease in RDW (fig. 9C), and a significant decrease in MCV (fig. 9D). The data presented in FIG. 9 shows the normalization of the β -thalassemia phenotype of these hematological parameters following administration of LNP-TMPRSS6 siRNA-1 (AD-46273).

Thalassemia mouse (Th3/+)Midamascens of TMPRSS6 siRNA-mediated silencing of TMPRSS6 on the morphology of the peripheral blood Influence.

To evaluate the effect of TMPRSS6 siRNA mediated TMPRSS6 silencing in thalassemic mice (Th3/+) on peripheral blood morphology; six-week-old Th3/+ mice were administered via tail vein injection with a single dose of 1mg/kg LNP-TMPRSS6 siRNA-1(AD-46273) or LNP-Luc control; and mice were sacrificed two weeks after dosing. The meyer-ge/ji staining at 10X magnification showed a significant decrease in staining in Th3/+ mice treated with TMPRSS6 siRNA compared to the control, indicating a decrease in reticulocyte number along with a general trend toward normalization of mature erythrocyte morphology. The meyer-graze/ji staining at 10X magnification also showed that animals with wild-type TMPRSS6 siRNA caused slight red cell size heterogeneity when compared to wild-type control animals.

Thalassemia mouse (Th3/+)Effect of moderate TMPRSS6 siRNA-mediated TMPRSS6 silencing on spleen architecture

To evaluate the effect of TMPRSS6 siRNA mediated TMPRSS6 silencing on spleen architecture in thalassemic mice (Th 3/+); six-week-old Th3/+ mice were administered via tail vein injection with a single dose of 1mg/kg LNP-TMPRSS6 siRNA-1(AD-46273) or LNP-Luc control or PBS; and mice were sacrificed two weeks after dosing. Hematoxylin and eosin (H & E) staining at 10X magnification showed that Th3/+ mice treated with TMPRSS6 siRNA had normalization of spleen architecture, including reduction of sinus-like extramedullary erythropoiesis and re-emergence of white marrow nodules, compared to controls.

Thalassemia mouse (Th3/+)TMPRSS6 silencing mediated by TMPRSS6 siRNA on spleen and liver iron The influence of the amount.

To evaluate the effect of TMPRSS6 siRNA mediated silencing of TMPRSS6 on spleen and liver iron content in thalassemia mice (Th 3/+); six-week-old Th3/+ mice were administered via tail vein injection with a single dose of 1mg/kg LNP-TMPRSS6 siRNA-1(AD-46273) or LNP-Luc control or PBS; and mice were sacrificed two weeks after dosing. 5 mice were used per group and data are presented as mean +/-standard deviation in figures 10A-10C, where x represents P value < 0.01 and x represents P value < 0.001. Silencing of TMPRSS6 in Th3/+ mice resulted in significant reductions in spleen iron content and spleen weight (fig. 10A and 10B, respectively), indicating normalization of extramedullary hematopoiesis. A trend was also observed for reduced liver iron content, but it was not statistically significant (fig. 10C).

The above results indicate that silencing of TMPRSS6 by systemic administration of formulated siRNA increases HAMP expression to a level sufficient to improve the phenotype in a mouse model of beta-thalassemia intermedia. Thus, LNP-TMPRSS 6-sirnas are being developed for congenital iron excess disorders characterized by abnormally low hepcidin levels (e.g., beta-thalassemia intermedia and hereditary hemochromatosis).

Equivalents of

Those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Sequence listing

<110> Alnilam Pharmaceuticals, Inc. (Alnylam Pharmaceuticals, Inc.)

<120> compositions and methods for inhibiting expression of TMPRSS6 gene

<130> 2108968US01

<140> PCT/US2012/030786

<141> 2012-03-28

<150> 61/568,942

<151> 2011-12-09

<150> 61/468,830

<151> 2011-03-29

<160> 611

<170> PatentIn version 3.5

<210> 1

<211> 3212

<212> DNA

<213> Intelligent (Homo sapiens)

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ttactcttcc actccaaaag gatgcccgtg gccgaggccc cccaggtggc tggcgggcag 180

ggggacggag gtgatggcga ggaagcggag ccggagggga tgttcaaggc ctgtgaggac 240

tccaagagaa aagcccgggg ctacctccgc ctggtgcccc tgtttgtgct gctggccctg 300

ctcgtgctgg cttcggcggg ggtgctactc tggtatttcc tagggtacaa ggcggaggtg 360

atggtcagcc aggtgtactc aggcagtctg cgtgtactca atcgccactt ctcccaggat 420

cttacccgcc gggaatctag tgccttccgc agtgaaaccg ccaaagccca gaagatgctc 480

aaggagctca tcaccagcac ccgcctggga acttactaca actccagctc cgtctattcc 540

tttggggagg gacccctcac ctgcttcttc tggttcattc tccaaatccc cgagcaccgc 600

cggctgatgc tgagccccga ggtggtgcag gcactgctgg tggaggagct gctgtccaca 660

gtcaacagct cggctgccgt cccctacagg gccgagtacg aagtggaccc cgagggccta 720

gtgatcctgg aagccagtgt gaaagacata gctgcattga attccacgct gggttgttac 780

cgctacagct acgtgggcca gggccaggtc ctccggctga aggggcctga ccacctggcc 840

tccagctgcc tgtggcacct gcagggcccc aaggacctca tgctcaaact ccggctggag 900

tggacgctgg cagagtgccg ggaccgactg gccatgtatg acgtggccgg gcccctggag 960

aagaggctca tcacctcggt gtacggctgc agccgccagg agcccgtggt ggaggttctg 1020

gcgtcggggg ccatcatggc ggtcgtctgg aagaagggcc tgcacagcta ctacgacccc 1080

ttcgtgctct ccgtgcagcc ggtggtcttc caggcctgtg aagtgaacct gacgctggac 1140

aacaggctcg actcccaggg cgtcctcagc accccgtact tccccagcta ctactcgccc 1200

caaacccact gctcctggca cctcacggtg ccctctctgg actacggctt ggccctctgg 1260

tttgatgcct atgcactgag gaggcagaag tatgatttgc cgtgcaccca gggccagtgg 1320

acgatccaga acaggaggct gtgtggcttg cgcatcctgc agccctacgc cgagaggatc 1380

cccgtggtgg ccacggccgg gatcaccatc aacttcacct cccagatctc cctcaccggg 1440

cccggtgtgc gggtgcacta tggcttgtac aaccagtcgg acccctgccc tggagagttc 1500

ctctgttctg tgaatggact ctgtgtccct gcctgtgatg gggtcaagga ctgccccaac 1560

ggcctggatg agagaaactg cgtttgcaga gccacattcc agtgcaaaga ggacagcaca 1620

tgcatctcac tgcccaaggt ctgtgatggg cagcctgatt gtctcaacgg cagcgacgaa 1680

gagcagtgcc aggaaggggt gccatgtggg acattcacct tccagtgtga ggaccggagc 1740

tgcgtgaaga agcccaaccc gcagtgtgat gggcggcccg actgcaggga cggctcggat 1800

gaggagcact gtgactgtgg cctccagggc ccctccagcc gcattgttgg tggagctgtg 1860

tcctccgagg gtgagtggcc atggcaggcc agcctccagg ttcggggtcg acacatctgt 1920

gggggggccc tcatcgctga ccgctgggtg ataacagctg cccactgctt ccaggaggac 1980

agcatggcct ccacggtgct gtggaccgtg ttcctgggca aggtgtggca gaactcgcgc 2040

tggcctggag aggtgtcctt caaggtgagc cgcctgctcc tgcacccgta ccacgaagag 2100

gacagccatg actacgacgt ggcgctgctg cagctcgacc acccggtggt gcgctcggcc 2160

gccgtgcgcc ccgtctgcct gcccgcgcgc tcccacttct tcgagcccgg cctgcactgc 2220

tggattacgg gctggggcgc cttgcgcgag ggcggcccca tcagcaacgc tctgcagaaa 2280

gtggatgtgc agttgatccc acaggacctg tgcagcgagg tctatcgcta ccaggtgacg 2340

ccacgcatgc tgtgtgccgg ctaccgcaag ggcaagaagg atgcctgtca gggtgactca 2400

ggtggtccgc tggtgtgcaa ggcactcagt ggccgctggt tcctggcggg gctggtcagc 2460

tggggcctgg gctgtggccg gcctaactac ttcggcgtct acacccgcat cacaggtgtg 2520

atcagctgga tccagcaagt ggtgacctga ggaactgccc ccctgcaaag cagggcccac 2580

ctcctggact cagagagccc agggcaactg ccaagcaggg ggacaagtat tctggcgggg 2640

ggtgggggag agagcaggcc ctgtggtggc aggaggtggc atcttgtctc gtccctgatg 2700

tctgctccag tgatggcagg aggatggaga agtgccagca gctgggggtc aagacgtccc 2760

ctgaggaccc aggcccacac ccagcccttc tgcctcccaa ttctctctcc tccgtcccct 2820

tcctccactg ctgcctaatg caaggcagtg gctcagcagc aagaatgctg gttctacatc 2880

ccgaggagtg tctgaggtgc gccccactct gtacagaggc tgtttgggca gccttgcctc 2940

cagagagcag attccagctt cggaagcccc tggtctaact tgggatctgg gaatggaagg 3000

tgctcccatc ggaggggacc ctcagagccc tggagactgc caggtgggcc tgctgccact 3060

gtaagccaaa aggtggggaa gtcctgactc cagggtcctt gccccacccc tgcctgccac 3120

ctgggccctc acagcccaga ccctcactgg gaggtgagct cagctgccct ttggaataaa 3180

gctgcctgat caaaaaaaaa aaaaaaaaaa aa 3212

<210> 2

<211> 29

<212> PRT

<213> Unknown (Unknown)

<220>

<223> "unknown" description of exemplary Primary peptides of endosomal cleavage Components

<400> 2

Ala Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Glu Ala

1 5 10 15

Leu Glu Ala Leu Ala Glu Ala Ala Ala Ala Gly Gly Cys

20 25

<210> 3

<211> 30

<212> PRT

<213> Unknown (Unknown)

<220>

<223> "unknown" description of exemplary Primary peptides of endosomal cleavage Components

<400> 3

Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala

1 5 10 15

Glu Ala Leu Ala Glu Ala Leu Ala Ala Ala Ala Gly Gly Cys

20 25 30

<210> 4

<211> 15

<212> PRT

<213> Unknown (Unknown)

<220>

<223> "unknown" description of exemplary Primary peptides of endosomal cleavage Components

<400> 4

Ala Leu Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Glu Ala

1 5 10 15

<210> 5

<211> 16

<212> PRT

<213> Unknown (Unknown)

<220>

<223> "unknown" description of hydrophobic Membrane translocation peptides

<400> 5

Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro

1 5 10 15

<210> 6

<211> 11

<212> PRT

<213> Unknown (Unknown)

<220>

<223> "unknown" description of hydrophobic Membrane translocation peptides

<400> 6

Ala Ala Leu Leu Pro Val Leu Leu Ala Ala Pro

1 5 10

<210> 7

<211> 13

<212> PRT

<213> Human immunodeficiency virus (Human immunodeficiency virus)

<400> 7

Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln

1 5 10

<210> 8

<211> 16

<212> PRT

<213> Drosophila sp (Drosophila sp.)

<400> 8

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210> 9

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 9

cucuggugcg agcugaccu 19

<210> 10

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 10

aggucagcuc gcaccagag 19

<210> 11

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 11

agcugaccug agaugcacu 19

<210> 12

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 12

agugcaucuc aggucagcu 19

<210> 13

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 13

ucugugagcu gucucggca 19

<210> 14

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 14

ugccgagaca gcucacaga 19

<210> 15

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 15

agcugucucg gcacccacu 19

<210> 16

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 16

agugggugcc gagacagcu 19

<210> 17

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 17

gcugucucgg cacccacuu 19

<210> 18

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 18

aagugggugc cgagacagc 19

<210> 19

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 19

agucacugcc gccugaugu 19

<210> 20

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 20

acaucaggcg gcagugacu 19

<210> 21

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 21

acugccgccu gauguuguu 19

<210> 22

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 22

aacaacauca ggcggcagu 19

<210> 23

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 23

cugccgccug auguuguua 19

<210> 24

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 24

uaacaacauc aggcggcag 19

<210> 25

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 25

gccgccugau guuguuacu 19

<210> 26

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 26

aguaacaaca ucaggcggc 19

<210> 27

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 27

gccugauguu guuacucuu 19

<210> 28

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 28

aagaguaaca acaucaggc 19

<210> 29

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 29

cucuuccacu ccaaaagga 19

<210> 30

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 30

uccuuuugga guggaagag 19

<210> 31

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 31

acuccaaaag gaugcccgu 19

<210> 32

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 32

acgggcaucc uuuuggagu 19

<210> 33

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 33

gugaggacuc caagagaaa 19

<210> 34

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 34

uuucucuugg aguccucac 19

<210> 35

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 35

cuucggcggg ggugcuacu 19

<210> 36

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 36

aguagcaccc ccgccgaag 19

<210> 37

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 37

ucggcggggg ugcuacucu 19

<210> 38

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 38

agaguagcac ccccgccga 19

<210> 39

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 39

gcgggggugc uacucuggu 19

<210> 40

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 40

accagaguag cacccccgc 19

<210> 41

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 41

gggggugcua cucugguau 19

<210> 42

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 42

auaccagagu agcaccccc 19

<210> 43

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 43

ggggugcuac ucugguauu 19

<210> 44

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 44

aauaccagag uagcacccc 19

<210> 45

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 45

ucugguauuu ccuagggua 19

<210> 46

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 46

uacccuagga aauaccaga 19

<210> 47

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 47

ugguauuucc uaggguaca 19

<210> 48

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 48

uguacccuag gaaauacca 19

<210> 49

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 49

gguauuuccu aggguacaa 19

<210> 50

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 50

uuguacccua ggaaauacc 19

<210> 51

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 51

ggucagccag guguacuca 19

<210> 52

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 52

ugaguacacc uggcugacc 19

<210> 53

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 53

agccaggugu acucaggca 19

<210> 54

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 54

ugccugagua caccuggcu 19

<210> 55

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 55

guacucaggc agucugcgu 19

<210> 56

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 56

acgcagacug ccugaguac 19

<210> 57

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 57

acucaggcag ucugcgugu 19

<210> 58

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 58

acacgcagac ugccugagu 19

<210> 59

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 59

caggcagucu gcguguacu 19

<210> 60

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 60

aguacacgca gacugccug 19

<210> 61

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 61

ggcagucugc guguacuca 19

<210> 62

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 62

ugaguacacg cagacugcc 19

<210> 63

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 63

gcagucugcg uguacucaa 19

<210> 64

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 64

uugaguacac gcagacugc 19

<210> 65

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 65

cagucugcgu guacucaau 19

<210> 66

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 66

auugaguaca cgcagacug 19

<210> 67

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 67

ugcguguacu caaucgcca 19

<210> 68

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 68

uggcgauuga guacacgca 19

<210> 69

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 69

cguguacuca aucgccacu 19

<210> 70

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 70

aguggcgauu gaguacacg 19

<210> 71

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 71

guguacucaa ucgccacuu 19

<210> 72

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 72

aaguggcgau ugaguacac 19

<210> 73

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 73

guacucaauc gccacuucu 19

<210> 74

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 74

agaaguggcg auugaguac 19

<210> 75

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 75

cacuucuccc aggaucuua 19

<210> 76

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 76

uaagauccug ggagaagug 19

<210> 77

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 77

gaucuuaccc gccgggaau 19

<210> 78

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 78

auucccggcg gguaagauc 19

<210> 79

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 79

ucuuacccgc cgggaaucu 19

<210> 80

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 80

agauucccgg cggguaaga 19

<210> 81

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 81

cuuacccgcc gggaaucua 19

<210> 82

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 82

uagauucccg gcggguaag 19

<210> 83

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 83

uacccgccgg gaaucuagu 19

<210> 84

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 84

acuagauucc cggcgggua 19

<210> 85

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 85

cgccgggaau cuagugccu 19

<210> 86

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 86

aggcacuaga uucccggcg 19

<210> 87

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 87

gccgggaauc uagugccuu 19

<210> 88

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 88

aaggcacuag auucccggc 19

<210> 89

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 89

uccgcaguga aaccgccaa 19

<210> 90

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 90

uuggcgguuu cacugcgga 19

<210> 91

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 91

ccgcagugaa accgccaaa 19

<210> 92

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 92

uuuggcgguu ucacugcgg 19

<210> 93

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 93

cgccugggaa cuuacuaca 19

<210> 94

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 94

uguaguaagu ucccaggcg 19

<210> 95

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 95

gccugggaac uuacuacaa 19

<210> 96

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 96

uuguaguaag uucccaggc 19

<210> 97

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 97

cugggaacuu acuacaacu 19

<210> 98

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 98

aguuguagua aguucccag 19

<210> 99

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 99

uacaacucca gcuccgucu 19

<210> 100

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 100

agacggagcu ggaguugua 19

<210> 101

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 101

acaacuccag cuccgucua 19

<210> 102

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 102

uagacggagc uggaguugu 19

<210> 103

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 103

aacuccagcu ccgucuauu 19

<210> 104

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 104

aauagacgga gcuggaguu 19

<210> 105

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 105

uuuggggagg gaccccuca 19

<210> 106

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 106

ugaggggucc cuccccaaa 19

<210> 107

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 107

ggaccccuca ccugcuucu 19

<210> 108

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 108

agaagcaggu gaggggucc 19

<210> 109

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 109

gcuucuucug guucauucu 19

<210> 110

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 110

agaaugaacc agaagaagc 19

<210> 111

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 111

ucuucugguu cauucucca 19

<210> 112

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 112

uggagaauga accagaaga 19

<210> 113

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 113

agcaccgccg gcugaugcu 19

<210> 114

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 114

agcaucagcc ggcggugcu 19

<210> 115

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 115

uccccuacag ggccgagua 19

<210> 116

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 116

uacucggccc uguagggga 19

<210> 117

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 117

ccuacagggc cgaguacga 19

<210> 118

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 118

ucguacucgg cccuguagg 19

<210> 119

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 119

acagggccga guacgaagu 19

<210> 120

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 120

acuucguacu cggcccugu 19

<210> 121

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 121

gggccgagua cgaagugga 19

<210> 122

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 122

uccacuucgu acucggccc 19

<210> 123

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 123

ccgagggccu agugauccu 19

<210> 124

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 124

aggaucacua ggcccucgg 19

<210> 125

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 125

cagugugaaa gacauagcu 19

<210> 126

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 126

agcuaugucu uucacacug 19

<210> 127

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 127

gaauuccacg cuggguugu 19

<210> 128

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 128

acaacccagc guggaauuc 19

<210> 129

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 129

aauuccacgc uggguuguu 19

<210> 130

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 130

aacaacccag cguggaauu 19

<210> 131

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 131

acgcuggguu guuaccgcu 19

<210> 132

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 132

agcgguaaca acccagcgu 19

<210> 133

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 133

cgcuggguug uuaccgcua 19

<210> 134

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 134

uagcgguaac aacccagcg 19

<210> 135

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 135

cuggguuguu accgcuaca 19

<210> 136

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 136

uguagcggua acaacccag 19

<210> 137

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 137

gguuguuacc gcuacagcu 19

<210> 138

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 138

agcuguagcg guaacaacc 19

<210> 139

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 139

guuaccgcua cagcuacgu 19

<210> 140

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 140

acguagcugu agcgguaac 19

<210> 141

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 141

aggaccucau gcucaaacu 19

<210> 142

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 142

aguuugagca ugagguccu 19

<210> 143

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 143

ucaugcucaa acuccggcu 19

<210> 144

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 144

agccggaguu ugagcauga 19

<210> 145

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 145

aucaccucgg uguacggcu 19

<210> 146

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 146

agccguacac cgaggugau 19

<210> 147

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 147

accucggugu acggcugca 19

<210> 148

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 148

ugcagccgua caccgaggu 19

<210> 149

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 149

aucauggcgg ucgucugga 19

<210> 150

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 150

uccagacgac cgccaugau 19

<210> 151

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 151

ucauggcggu cgucuggaa 19

<210> 152

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 152

uuccagacga ccgccauga 19

<210> 153

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 153

gcuacuacga ccccuucgu 19

<210> 154

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 154

acgaaggggu cguaguagc 19

<210> 155

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 155

ccgugcagcc gguggucuu 19

<210> 156

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 156

aagaccaccg gcugcacgg 19

<210> 157

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 157

ucuuccaggc cugugaagu 19

<210> 158

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 158

acuucacagg ccuggaaga 19

<210> 159

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 159

gccugugaag ugaaccuga 19

<210> 160

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 160

ucagguucac uucacaggc 19

<210> 161

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 161

gugaagugaa ccugacgcu 19

<210> 162

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 162

agcgucaggu ucacuucac 19

<210> 163

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 163

cgcuggacaa caggcucga 19

<210> 164

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 164

ucgagccugu uguccagcg 19

<210> 165

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 165

cuggacaaca ggcucgacu 19

<210> 166

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 166

agucgagccu guuguccag 19

<210> 167

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 167

guccucagca ccccguacu 19

<210> 168

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 168

aguacggggu gcugaggac 19

<210> 169

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 169

uccucagcac cccguacuu 19

<210> 170

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 170

aaguacgggg ugcugagga 19

<210> 171

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 171

agcaccccgu acuucccca 19

<210> 172

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 172

uggggaagua cggggugcu 19

<210> 173

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 173

cagcuacuac ucgccccaa 19

<210> 174

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 174

uuggggcgag uaguagcug 19

<210> 175

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 175

agcuacuacu cgccccaaa 19

<210> 176

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 176

uuuggggcga guaguagcu 19

<210> 177

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 177

acuacucgcc ccaaaccca 19

<210> 178

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 178

uggguuuggg gcgaguagu 19

<210> 179

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 179

ucgccccaaa cccacugcu 19

<210> 180

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 180

agcagugggu uuggggcga 19

<210> 181

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 181

gcuccuggca ccucacggu 19

<210> 182

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 182

accgugaggu gccaggagc 19

<210> 183

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 183

cccucucugg acuacggcu 19

<210> 184

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 184

agccguaguc cagagaggg 19

<210> 185

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 185

acggcuuggc ccucugguu 19

<210> 186

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 186

aaccagaggg ccaagccgu 19

<210> 187

<211> 19

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 187

cggcttggcc ctctggttt 19

<210> 188

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 188

aaaccagagg gccaagccg 19

<210> 189

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 189

gcuuggcccu cugguuuga 19

<210> 190

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 190

ucaaaccaga gggccaagc 19

<210> 191

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 191

ccucugguuu gaugccuau 19

<210> 192

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 192

auaggcauca aaccagagg 19

<210> 193

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 193

cagaaguaug auuugccgu 19

<210> 194

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 194

acggcaaauc auacuucug 19

<210> 195

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 195

aaguaugauu ugccgugca 19

<210> 196

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 196

ugcacggcaa aucauacuu 19

<210> 197

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 197

augauuugcc gugcaccca 19

<210> 198

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 198

ugggugcacg gcaaaucau 19

<210> 199

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 199

acccagggcc aguggacga 19

<210> 200

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 200

ucguccacug gcccugggu 19

<210> 201

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 201

agggccagug gacgaucca 19

<210> 202

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 202

uggaucgucc acuggcccu 19

<210> 203

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 203

ggccagugga cgauccaga 19

<210> 204

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 204

ucuggaucgu ccacuggcc 19

<210> 205

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 205

gccaguggac gauccagaa 19

<210> 206

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 206

uucuggaucg uccacuggc 19

<210> 207

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 207

cagcccuacg ccgagagga 19

<210> 208

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 208

uccucucggc guagggcug 19

<210> 209

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 209

cggugugcgg gugcacuau 19

<210> 210

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 210

auagugcacc cgcacaccg 19

<210> 211

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 211

gugcgggugc acuauggcu 19

<210> 212

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 212

agccauagug cacccgcac 19

<210> 213

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 213

ugcgggugca cuauggcuu 19

<210> 214

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 214

aagccauagu gcacccgca 19

<210> 215

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 215

gggugcacua uggcuugua 19

<210> 216

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 216

uacaagccau agugcaccc 19

<210> 217

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 217

ugcacuaugg cuuguacaa 19

<210> 218

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 218

uuguacaagc cauagugca 19

<210> 219

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 219

ugcccuggag aguuccucu 19

<210> 220

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 220

agaggaacuc uccagggca 19

<210> 221

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 221

uggaugagag aaacugcgu 19

<210> 222

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 222

acgcaguuuc ucucaucca 19

<210> 223

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 223

ggacagcaca ugcaucuca 19

<210> 224

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 224

ugagaugcau gugcugucc 19

<210> 225

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 225

acagcacaug caucucacu 19

<210> 226

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 226

agugagaugc augugcugu 19

<210> 227

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 227

ccaaggucug ugaugggca 19

<210> 228

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 228

ugcccaucac agaccuugg 19

<210> 229

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 229

augggcagcc ugauugucu 19

<210> 230

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 230

agacaaucag gcugcccau 19

<210> 231

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 231

ggcagccuga uugucucaa 19

<210> 232

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 232

uugagacaau caggcugcc 19

<210> 233

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 233

ccugauuguc ucaacggca 19

<210> 234

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 234

ugccguugag acaaucagg 19

<210> 235

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 235

ucucaacggc agcgacgaa 19

<210> 236

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 236

uucgucgcug ccguugaga 19

<210> 237

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 237

ugccaggaag gggugccau 19

<210> 238

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 238

auggcacccc uuccuggca 19

<210> 239

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 239

ggggugccau gugggacau 19

<210> 240

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 240

augucccaca uggcacccc 19

<210> 241

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 241

gugccaugug ggacauuca 19

<210> 242

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 242

ugaauguccc acauggcac 19

<210> 243

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 243

caugugggac auucaccuu 19

<210> 244

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 244

aaggugaaug ucccacaug 19

<210> 245

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 245

cagugugagg accggagcu 19

<210> 246

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 246

agcuccgguc cucacacug 19

<210> 247

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 247

ugaagaagcc caacccgca 19

<210> 248

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 248

ugcggguugg gcuucuuca 19

<210> 249

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 249

gaagcccaac ccgcagugu 19

<210> 250

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 250

acacugcggg uugggcuuc 19

<210> 251

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 251

ccccuccagc cgcauuguu 19

<210> 252

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 252

aacaaugcgg cuggagggg 19

<210> 253

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 253

cagguucggg gucgacaca 19

<210> 254

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 254

ugugucgacc ccgaaccug 19

<210> 255

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 255

agguucgggg ucgacacau 19

<210> 256

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 256

augugucgac cccgaaccu 19

<210> 257

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 257

guucgggguc gacacaucu 19

<210> 258

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 258

agaugugucg accccgaac 19

<210> 259

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 259

cgcugaccgc ugggugaua 19

<210> 260

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 260

uaucacccag cggucagcg 19

<210> 261

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 261

gcugaccgcu gggugauaa 19

<210> 262

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 262

uuaucaccca gcggucagc 19

<210> 263

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 263

ugaccgcugg gugauaaca 19

<210> 264

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 264

uguuaucacc cagcgguca 19

<210> 265

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 265

ccgcugggug auaacagcu 19

<210> 266

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 266

agcuguuauc acccagcgg 19

<210> 267

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 267

ugcuguggac cguguuccu 19

<210> 268

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 268

aggaacacgg uccacagca 19

<210> 269

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 269

guguggcaga acucgcgcu 19

<210> 270

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 270

agcgcgaguu cugccacac 19

<210> 271

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 271

uccugcaccc guaccacga 19

<210> 272

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 272

ucgugguacg ggugcagga 19

<210> 273

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 273

ccugcacccg uaccacgaa 19

<210> 274

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 274

uucgugguac gggugcagg 19

<210> 275

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 275

ugcacccgua ccacgaaga 19

<210> 276

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 276

ucuucguggu acgggugca 19

<210> 277

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 277

cgcgcuccca cuucuucga 19

<210> 278

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 278

ucgaagaagu gggagcgcg 19

<210> 279

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 279

ggccugcacu gcuggauua 19

<210> 280

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 280

uaauccagca gugcaggcc 19

<210> 281

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 281

cacugcugga uuacgggcu 19

<210> 282

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 282

agcccguaau ccagcagug 19

<210> 283

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 283

ggaugugcag uugauccca 19

<210> 284

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 284

ugggaucaac ugcacaucc 19

<210> 285

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 285

ugcagcgagg ucuaucgcu 19

<210> 286

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 286

agcgauagac cucgcugca 19

<210> 287

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 287

gcagcgaggu cuaucgcua 19

<210> 288

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 288

uagcgauaga ccucgcugc 19

<210> 289

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 289

gcgaggucua ucgcuacca 19

<210> 290

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 290

ugguagcgau agaccucgc 19

<210> 291

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 291

gucuaucgcu accagguga 19

<210> 292

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 292

ucaccuggua gcgauagac 19

<210> 293

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 293

aggugacgcc acgcaugcu 19

<210> 294

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 294

agcaugcgug gcgucaccu 19

<210> 295

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 295

gugacgccac gcaugcugu 19

<210> 296

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 296

acagcaugcg uggcgucac 19

<210> 297

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 297

gacgccacgc augcugugu 19

<210> 298

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 298

acacagcaug cguggcguc 19

<210> 299

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 299

ggcuguggcc ggccuaacu 19

<210> 300

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 300

aguuaggccg gccacagcc 19

<210> 301

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 301

gcuguggccg gccuaacua 19

<210> 302

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 302

uaguuaggcc ggccacagc 19

<210> 303

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 303

uguggccggc cuaacuacu 19

<210> 304

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 304

aguaguuagg ccggccaca 19

<210> 305

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 305

ggccuaacua cuucggcgu 19

<210> 306

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 306

acgccgaagu aguuaggcc 19

<210> 307

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 307

ccuaacuacu ucggcgucu 19

<210> 308

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 308

agacgccgaa guaguuagg 19

<210> 309

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 309

cuaacuacuu cggcgucua 19

<210> 310

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 310

uagacgccga aguaguuag 19

<210> 311

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 311

aacuacuucg gcgucuaca 19

<210> 312

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 312

uguagacgcc gaaguaguu 19

<210> 313

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 313

acacccgcau cacaggugu 19

<210> 314

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 314

acaccuguga ugcgggugu 19

<210> 315

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 315

cgcaucacag gugugauca 19

<210> 316

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 316

ugaucacacc ugugaugcg 19

<210> 317

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 317

gcuggaucca gcaaguggu 19

<210> 318

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 318

accacuugcu ggauccagc 19

<210> 319

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 319

ggaacugccc cccugcaaa 19

<210> 320

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 320

uuugcagggg ggcaguucc 19

<210> 321

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 321

aggagguggc aucuugucu 19

<210> 322

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 322

agacaagaug ccaccuccu 19

<210> 323

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 323

agguggcauc uugucucgu 19

<210> 324

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 324

acgagacaag augccaccu 19

<210> 325

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 325

ggcaucuugu cucgucccu 19

<210> 326

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 326

agggacgaga caagaugcc 19

<210> 327

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 327

caucuugucu cgucccuga 19

<210> 328

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 328

ucagggacga gacaagaug 19

<210> 329

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 329

aucuugucuc gucccugau 19

<210> 330

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 330

aucagggacg agacaagau 19

<210> 331

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 331

cagcuggggg ucaagacgu 19

<210> 332

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 332

acgucuugac ccccagcug 19

<210> 333

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 333

gggggucaag acguccccu 19

<210> 334

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 334

aggggacguc uugaccccc 19

<210> 335

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 335

gggucaagac guccccuga 19

<210> 336

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 336

ucaggggacg ucuugaccc 19

<210> 337

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 337

ccacugcugc cuaaugcaa 19

<210> 338

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 338

uugcauuagg cagcagugg 19

<210> 339

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 339

ugcugccuaa ugcaaggca 19

<210> 340

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 340

ugccuugcau uaggcagca 19

<210> 341

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 341

cuaaugcaag gcaguggcu 19

<210> 342

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 342

agccacugcc uugcauuag 19

<210> 343

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 343

cagcaagaau gcugguucu 19

<210> 344

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 344

agaaccagca uucuugcug 19

<210> 345

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 345

gaggugcgcc ccacucugu 19

<210> 346

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 346

acagaguggg gcgcaccuc 19

<210> 347

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 347

cuucggaagc cccuggucu 19

<210> 348

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 348

agaccagggg cuuccgaag 19

<210> 349

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 349

ucggaagccc cuggucuaa 19

<210> 350

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 350

uuagaccagg ggcuuccga 19

<210> 351

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 351

ggaagccccu ggucuaacu 19

<210> 352

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 352

aguuagacca ggggcuucc 19

<210> 353

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 353

gaagccccug gucuaacuu 19

<210> 354

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 354

aaguuagacc aggggcuuc 19

<210> 355

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 355

cccuggucua acuugggau 19

<210> 356

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 356

aucccaaguu agaccaggg 19

<210> 357

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 357

cuggucuaac uugggaucu 19

<210> 358

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 358

agaucccaag uuagaccag 19

<210> 359

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 359

cuaacuuggg aucugggaa 19

<210> 360

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 360

uucccagauc ccaaguuag 19

<210> 361

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 361

ccaucggagg ggacccuca 19

<210> 362

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 362

ugaggguccc cuccgaugg 19

<210> 363

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 363

ugggccugcu gccacugua 19

<210> 364

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 364

uacaguggca gcaggccca 19

<210> 365

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 365

gggccugcug ccacuguaa 19

<210> 366

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 366

uuacaguggc agcaggccc 19

<210> 367

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 367

gcugccacug uaagccaaa 19

<210> 368

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 368

uuuggcuuac aguggcagc 19

<210> 369

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 369

ccacuguaag ccaaaaggu 19

<210> 370

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 370

accuuuuggc uuacagugg 19

<210> 371

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 371

agguggggaa guccugacu 19

<210> 372

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 372

agucaggacu uccccaccu 19

<210> 373

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 373

gaauaaagcu gccugauca 19

<210> 374

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 374

ugaucaggca gcuuuauuc 19

<210> 375

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 375

aauaaagcug ccugaucaa 19

<210> 376

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 376

uugaucaggc agcuuuauu 19

<210> 377

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 377

agcugccuga ucaaaaaaa 19

<210> 378

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 378

uuuuuuugau caggcagcu 19

<210> 379

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 379

cuacagggcc gaguacgaa 19

<210> 380

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 380

uucguacucg gcccuguag 19

<210> 381

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 381

ugugaugggg ucaaggacu 19

<210> 382

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 382

aguccuugac cccaucaca 19

<210> 383

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 383

cuggagaggu guccuucaa 19

<210> 384

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 384

uugaaggaca ccucuccag 19

<210> 385

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 385

ggaccgacug gccauguau 19

<210> 386

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 386

auacauggcc agucggucc 19

<210> 387

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 387

aguugauccc acaggaccu 19

<210> 388

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 388

agguccugug ggaucaacu 19

<210> 389

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 389

aaaccgccaa agcccagaa 19

<210> 390

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 390

uucugggcuu uggcgguuu 19

<210> 391

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 391

ugugugccgg cuaccgcaa 19

<210> 392

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 392

uugcgguagc cggcacaca 19

<210> 393

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 393

auuccacgcu ggguuguua 19

<210> 394

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 394

uaacaaccca gcguggaau 19

<210> 395

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 395

ucgcugaccg cugggugau 19

<210> 396

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 396

aucacccagc ggucagcga 19

<210> 397

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 397

caagcagggg gacaaguau 19

<210> 398

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 398

auacuugucc cccugcuug 19

<210> 399

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 399

ugaugucugc uccagugau 19

<210> 400

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 400

aucacuggag cagacauca 19

<210> 401

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 401

gagguguccu ucaagguga 19

<210> 402

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 402

ucaccuugaa ggacaccuc 19

<210> 403

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 403

aagcaggggg acaaguauu 19

<210> 404

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 404

aauacuuguc ccccugcuu 19

<210> 405

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 405

cuugggaucu gggaaugga 19

<210> 406

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 406

uccauuccca gaucccaag 19

<210> 407

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 407

gguauuuccu aggguacaa 19

<210> 408

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 408

uuguacccua ggaaauacc 19

<210> 409

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 409

ggcuaccgca agggcaaga 19

<210> 410

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 410

ucuugcccuu gcgguagcc 19

<210> 411

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 411

gcagggggac aaguauucu 19

<210> 412

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 412

agaauacuug ucccccugc 19

<210> 413

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 413

gcucagcagc aagaaugcu 19

<210> 414

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 414

agcauucuug cugcugagc 19

<210> 415

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 415

uugggaucug ggaauggaa 19

<210> 416

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 416

uuccauuccc agaucccaa 19

<210> 417

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 417

ccaaagccca gaagaugcu 19

<210> 418

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 418

agcaucuucu gggcuuugg 19

<210> 419

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 419

gcuaccgcaa gggcaagaa 19

<210> 420

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 420

uucuugcccu ugcgguagc 19

<210> 421

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 421

gcucagcugc ccuuuggaa 19

<210> 422

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 422

uuccaaaggg cagcugagc 19

<210> 423

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 423

ugguggcagg agguggcau 19

<210> 424

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 424

augccaccuc cugccacca 19

<210> 425

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 425

cccacucugu acagaggcu 19

<210> 426

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 426

agccucugua cagaguggg 19

<210> 427

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 427

cucacagccc agacccuca 19

<210> 428

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 428

ugagggucug ggcugugag 19

<210> 429

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 429

ccucucugga cuacggcuu 19

<210> 430

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 430

aagccguagu ccagagagg 19

<210> 431

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 431

guggcaggag guggcaucu 19

<210> 432

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 432

agaugccacc uccugccac 19

<210> 433

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 433

uucggaagcc ccuggucua 19

<210> 434

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 434

uagaccaggg gcuuccgaa 19

<210> 435

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 435

agcucagcug cccuuugga 19

<210> 436

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 436

uccaaagggc agcugagcu 19

<210> 437

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 437

ggccuggaug agagaaacu 19

<210> 438

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 438

aguuucucuc auccaggcc 19

<210> 439

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 439

uggcaggagg uggcaucuu 19

<210> 440

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 440

aagaugccac cuccugcca 19

<210> 441

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 441

acugugacug uggccucca 19

<210> 442

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 442

uggaggccac agucacagu 19

<210> 443

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 443

ccccuggucu aacuuggga 19

<210> 444

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 444

ucccaaguua gaccagggg 19

<210> 445

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 445

ucagcugccc uuuggaaua 19

<210> 446

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 446

uauuccaaag ggcagcuga 19

<210> 447

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 447

ucggggucga cacaucugu 19

<210> 448

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 448

acagaugugu cgaccccga 19

<210> 449

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 449

gucccugaug ucugcucca 19

<210> 450

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 450

uggagcagac aucagggac 19

<210> 451

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 451

ucaucgcuga ccgcugggu 19

<210> 452

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 452

acccagcggu cagcgauga 19

<210> 453

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 453

ggggugcuac ucugguauut t 21

<210> 454

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 454

aauaccagag uagcacccct t 21

<210> 455

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 455

ucuucugguu cauucuccat t 21

<210> 456

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 456

uggagaauga accagaagat t 21

<210> 457

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 457

acgcuggguu guuaccgcut t 21

<210> 458

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 458

agcgguaaca acccagcgut t 21

<210> 459

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 459

cagaaguaug auuugccgut t 21

<210> 460

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 460

acggcaaauc auacuucugt t 21

<210> 461

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 461

cgcugaccgc ugggugauat t 21

<210> 462

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 462

uaucacccag cggucagcgt t 21

<210> 463

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 463

ucugguauuu ccuaggguat t 21

<210> 464

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 464

uacccuagga aauaccagat t 21

<210> 465

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 465

ccuacagggc cgaguacgat t 21

<210> 466

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 466

ucguacucgg cccuguaggt t 21

<210> 467

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 467

cgcuggguug uuaccgcuat t 21

<210> 468

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 468

uagcgguaac aacccagcgt t 21

<210> 469

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 469

ggccagugga cgauccagat t 21

<210> 470

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 470

ucuggaucgu ccacuggcct t 21

<210> 471

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 471

ugaccgcugg gugauaacat t 21

<210> 472

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 472

uguuaucacc cagcggucat t 21

<210> 473

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 473

ggucagccag guguacucat t 21

<210> 474

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 474

ugaguacacc uggcugacct t 21

<210> 475

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 475

cuacagggcc gaguacgaat t 21

<210> 476

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 476

uucguacucg gcccuguagt t 21

<210> 477

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 477

uuauuccaaa gggcagcugt t 21

<210> 478

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 478

cuggguuguu accgcuacat t 21

<210> 479

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 479

uguagcggua acaacccagt t 21

<210> 480

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 480

ugcacuaugg cuuguacaat t 21

<210> 481

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 481

uuguacaagc cauagugcat t 21

<210> 482

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 482

ccuggagagg uguccuucat t 21

<210> 483

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 483

ugaaggacac cucuccaggt t 21

<210> 484

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 484

agccaggugu acucaggcat t 21

<210> 485

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 485

ugccugagua caccuggcut t 21

<210> 486

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 486

acagggccga guacgaagut t 21

<210> 487

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 487

acuucguacu cggcccugut t 21

<210> 488

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 488

gguuguuacc gcuacagcut t 21

<210> 489

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 489

agcuguagcg guaacaacct t 21

<210> 490

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 490

ugugaugggg ucaaggacut t 21

<210> 491

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 491

aguccuugac cccaucacat t 21

<210> 492

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 492

cuggagaggu guccuucaat t 21

<210> 493

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 493

uugaaggaca ccucuccagt t 21

<210> 494

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 494

uccgcaguga aaccgccaat t 21

<210> 495

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 495

uuggcgguuu cacugcggat t 21

<210> 496

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 496

gggccgagua cgaaguggat t 21

<210> 497

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 497

uccacuucgu acucggccct t 21

<210> 498

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 498

ggaccgacug gccauguaut t 21

<210> 499

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 499

auacauggcc agucggucct t 21

<210> 500

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 500

caacggccug gaugagagat t 21

<210> 501

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 501

ucucucaucc aggccguugt t 21

<210> 502

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 502

aguugauccc acaggaccut t 21

<210> 503

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 503

agguccugug ggaucaacut t 21

<210> 504

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 504

ccgcagugaa accgccaaat t 21

<210> 505

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 505

uuuggcgguu ucacugcggt t 21

<210> 506

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 506

ccgagggccu agugauccut t 21

<210> 507

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 507

aggaucacua ggcccucggt t 21

<210> 508

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 508

uccucagcac cccguacuut t 21

<210> 509

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 509

aaguacgggg ugcugaggat t 21

<210> 510

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 510

cagguucggg gucgacacat t 21

<210> 511

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 511

ugugucgacc ccgaaccugt t 21

<210> 512

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 512

aggugacgcc acgcaugcut t 21

<210> 513

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 513

agcaugcgug gcgucaccut t 21

<210> 514

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 514

aaaccgccaa agcccagaat t 21

<210> 515

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 515

uucugggcuu uggcgguuut t 21

<210> 516

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 516

cagugugaaa gacauagcut t 21

<210> 517

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 517

agcuaugucu uucacacugt t 21

<210> 518

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 518

cccucucugg acuacggcut t 21

<210> 519

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 519

agccguaguc cagagagggt t 21

<210> 520

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 520

guucgggguc gacacaucut t 21

<210> 521

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 521

agaugugucg accccgaact t 21

<210> 522

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 522

ugugugccgg cuaccgcaat t 21

<210> 523

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 523

uugcgguagc cggcacacat t 21

<210> 524

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 524

gcuucuucug guucauucut t 21

<210> 525

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 525

agaaugaacc agaagaagct t 21

<210> 526

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 526

auuccacgcu ggguuguuat t 21

<210> 527

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 527

uaacaaccca gcguggaaut t 21

<210> 528

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 528

acggcuuggc ccucugguut t 21

<210> 529

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 529

aaccagaggg ccaagccgut t 21

<210> 530

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 530

ucgcugaccg cugggugaut t 21

<210> 531

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 531

aucacccagc ggucagcgat t 21

<210> 532

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 532

aguggugacc ugaggaacut t 21

<210> 533

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 533

aguuccucag gucaccacut t 21

<210> 534

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 534

caagcagggg gacaaguaut t 21

<210> 535

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 535

auacuugucc cccugcuugt t 21

<210> 536

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 536

ugaugucugc uccagugaut t 21

<210> 537

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 537

aucacuggag cagacaucat t 21

<210> 538

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 538

cuaacuuggg aucugggaat t 21

<210> 539

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 539

uucccagauc ccaaguuagt t 21

<210> 540

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 540

ugguauuucc uaggguacat t 21

<210> 541

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 541

uguacccuag gaaauaccat t 21

<210> 542

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 542

gagguguccu ucaaggugat t 21

<210> 543

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 543

ucaccuugaa ggacaccuct t 21

<210> 544

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 544

aagcaggggg acaaguauut t 21

<210> 545

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 545

aauacuuguc ccccugcuut t 21

<210> 546

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 546

cagcuggggg ucaagacgut t 21

<210> 547

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 547

acgucuugac ccccagcugt t 21

<210> 548

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 548

cuugggaucu gggaauggat t 21

<210> 549

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 549

uccauuccca gaucccaagt t 21

<210> 550

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 550

gguauuuccu aggguacaat t 21

<210> 551

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 551

uuguacccua ggaaauacct t 21

<210> 552

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 552

ggcuaccgca agggcaagat t 21

<210> 553

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 553

ucuugcccuu gcgguagcct t 21

<210> 554

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 554

gcagggggac aaguauucut t 21

<210> 555

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 555

agaauacuug ucccccugct t 21

<210> 556

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 556

gcucagcagc aagaaugcut t 21

<210> 557

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 557

agcauucuug cugcugagct t 21

<210> 558

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 558

uugggaucug ggaauggaat t 21

<210> 559

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 559

uuccauuccc agaucccaat t 21

<210> 560

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 560

ccaaagccca gaagaugcut t 21

<210> 561

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 561

agcaucuucu gggcuuuggt t 21

<210> 562

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 562

gcuaccgcaa gggcaagaat t 21

<210> 563

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 563

uucuugcccu ugcgguagct t 21

<210> 564

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 564

ugguggcagg agguggcaut t 21

<210> 565

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 565

augccaccuc cugccaccat t 21

<210> 566

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 566

cccacucugu acagaggcut t 21

<210> 567

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 567

agccucugua cagagugggt t 21

<210> 568

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 568

cucacagccc agacccucat t 21

<210> 569

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 569

ugagggucug ggcugugagt t 21

<210> 570

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 570

ccucucugga cuacggcuut t 21

<210> 571

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 571

aagccguagu ccagagaggt t 21

<210> 572

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 572

guggcaggag guggcaucut t 21

<210> 573

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 573

agaugccacc uccugccact t 21

<210> 574

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 574

uucggaagcc ccuggucuat t 21

<210> 575

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules S Synthesis of oligonucleotides

<400> 575

uagaccaggg gcuuccgaat t 21

<210> 576

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 576

agcucagcug cccuuuggat t 21

<210> 577

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 577

uccaaagggc agcugagcut t 21

<210> 578

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 578

ggccuggaug agagaaacut t 21

<210> 579

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 579

aguuucucuc auccaggcct t 21

<210> 580

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 580

uggcaggagg uggcaucuut t 21

<210> 581

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 581

aagaugccac cuccugccat t 21

<210> 582

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 582

ucggaagccc cuggucuaat t 21

<210> 583

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 583

uuagaccagg ggcuuccgat t 21

<210> 584

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 584

gcucagcugc ccuuuggaat t 21

<210> 585

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 585

uuccaaaggg cagcugagct t 21

<210> 586

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 586

acugugacug uggccuccat t 21

<210> 587

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 587

uggaggccac agucacagut t 21

<210> 588

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 588

aggagguggc aucuugucut t 21

<210> 589

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 589

agacaagaug ccaccuccut t 21

<210> 590

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 590

ccccuggucu aacuugggat t 21

<210> 591

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 591

ucccaaguua gaccaggggt t 21

<210> 592

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 592

ucagcugccc uuuggaauat t 21

<210> 593

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 593

uauuccaaag ggcagcugat t 21

<210> 594

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 594

ucggggucga cacaucugut t 21

<210> 595

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 595

acagaugugu cgaccccgat t 21

<210> 596

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 596

gucccugaug ucugcuccat t 21

<210> 597

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 597

uggagcagac aucagggact t 21

<210> 598

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 598

cccuggucua acuugggaut t 21

<210> 599

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 599

aucccaaguu agaccagggt t 21

<210> 600

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 600

cagcugcccu uuggaauaat t 21

<210> 601

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 601

uuauuccaaa gggcagcugt t 21

<210> 602

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 602

ucaucgcuga ccgcugggut t 21

<210> 603

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of Artificial sequences Synthesis of oligonucleotides

<220>

<223> description of the Combined DNA/RNA molecules Synthesis of oligonucleotides

<400> 603

acccagcggu cagcgaugat t 21

<210> 604

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 604

ccuggagagg uguccuuca 19

<210> 605

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 605

ugaaggacac cucuccagg 19

<210> 606

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 606

caacggccug gaugagaga 19

<210> 607

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 607

ucucucaucc aggccguug 19

<210> 608

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 608

aguggugacc ugaggaacu 19

<210> 609

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 609

aguuccucag gucaccacu 19

<210> 610

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 610

cagcugcccu uuggaauaa 19

<210> 611

<211> 19

<212> RNA

<213> Intelligent (Homo sapiens)

<400> 611

uuauuccaaa gggcagcug 19