RNA INTERFERENCE MEDIATED INHIBITION OF CATENIN (CADHERIN-ASSOCIATED PROTEIN), BETA 1 (CTNNB1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (SINA)
United States Patent Application
The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of CTNNB1 gene expression and/or activity, and/or modulate a beta-catenin gene expression pathway. Specifically, the invention relates to double-stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules that are capable of mediating or that medium RNA interference (RNAi) against CTNNB1 gene expression.
Inventors:
Brown, Duncan (Berkeley, CA, US)
Cunningham, James J. (Wayne, PA, US)
Gindy, Marian (North Wales, PA, US)
Pickering, Victoria (San Mateo, CA, US)
Stanton, Matthew G. (Marlton, NJ, US)
Stirdivant, Steven M. (Cary, NC, US)
Strapps, Walter R. (Doylestown, PA, US)
Application Number:
Publication Date:
02/02/2017
Filing Date:
08/01/2016
Export Citation:
Sirna Therapeutics, Inc. (Cambridge, MA, US)
Primary Class:
International Classes:
C12N15/113
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Related US Applications:
February, 2010WeiMay, 2002Ratsimamanga et al.January, 2008Kinet et al.January, 2009Eriksen et al.September, 2005Morita et al.January, 2009Antoncic et al.January, 2009Garcia-pareja et al.December, 2008GroenewoudMay, 2009Gordon et al.December, 2007Gubler et al.February, 2006Qiu et al.
Primary Examiner:
GIBBS, TERRA C
Attorney, Agent or Firm:
MCCARTER & ENGLISH, LLP / ALNYLAM PHARMACEUTICALS (265 FRANKLIN STREET
BOSTON MA )
An isolated double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of cadherin-associated protein, beta 1 (CTNNB1), wherein (a) the siNA comprises a sense strand and (b) each strand is independently 15 to 30 n and (c) at least one strand comprises at least a 15 nucleotide sequence of SEQ ID NO:5.
A double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of cadherin-associated protein, beta 1 (CTNNB1), wherein (a) the siNA comprises a sense strand and (b) each strand is independently 15 to 30 n and (c) the antisense strand comprises at least 15 nucleotides having sequence complementary to 5′-CUGUUGGAUUGAUUCGAAA-3′ (SEQ ID NO:5).
A double-stranded short interfering nucleic acid (siNA) molecule that inhibits the expression of cadherin-associated protein, beta 1 (CTNNB1), wherein (a) the siNA comprises a sense strand and (b) each strand is independently 15 to 30 n and (c) the antisense strand comprises at least a 15 nucleotide sequence of 5′-UUUCGAAUCAAUCCAACAG-3′ (SEQ ID NO:4918); and wherein one of more of the nucleotides are optionally chemically modified.
4. 4.-5. (canceled)
The double-stranded short interfering nucleic acid (siNA) molecule according to any one of claims 1-3, wherein at least one nucleotide is a chemically modified nucleotide.
The double-stranded short interfering nucleic acid (siNA) molecule according to any one of claims 1-3 further comprising at least one non-nucleotide.
The double-stranded short interfering nucleic acid (siNA) molecule according to any one of claims 1-3, wherein at least one nucleotide comprises a universal base.
The double-stranded short interfering nucleic acid (siNA) molecule according to any one of claims 1-3, having at least one phosphorothioate internucleotide linkage.
The double-stranded short interfering nucleic acid (siNA) molecule according to any one of claims 1-3, comprising a cap on the 3′-end, 5′-end or both 3′ and 5′ ends of at least one strand.
The double-stranded short interfering nucleic acid (siNA) molecule according to any one of claims 1-3, comprising one or more 3′-overhang nucleotides on one or both strands.
12. 12.-24. (canceled)
The double-stranded short interfering nucleic acid (siNA) molecule of any one of claims 1-3, comprising SEQ ID NOS: 6370 and 6369.
The double-stranded short interfering nucleic acid (siNA) molecule of any one of claims 1-3, comprising SEQ ID NOS: 2021 and 2068.
27. 27.-29. (canceled)
A composition comprising the double-stranded short interfering nucleic acid (siNA) according to any one of claims 1-3 in a pharmaceutically acceptable carrier or diluent.
(canceled)
A composition comprising: (a) a double-stranded short interfering nucleic acid (siNA) having SEQ ID NOS: 6370 and 6369; (b) (13Z, 16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1- (c) (d) DSPC; and (e) PEG-DMG.
A composition comprising: (a) a double-stranded short interfering nucleic acid (siNA) having SEQ ID NOS: 2021 and 2068; (b) (13Z, 16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1- (c) (d) DSPC; and (e) PEG-DMG.
34. 34.-37. (canceled)
A method of treating a human subject suffering from a condition which is mediated by the action, or by loss of action, of CTNNB1, which comprises administering to said subject an effective amount of the double-stranded short interfering nucleic acid (siNA) molecule of claim 25 or 26.
(canceled)
Description:
SEQUENCE LISTINGThe sequence listing submitted via EFS, in compliance with 37 CFR §132(e)(5), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file “SequenceListingSIRONC2”, created on Jul. 25, 2011 which is 2,173,912 bytes inBACKGROUND OF THE INVENTIONBeta catenin (also known as cadherin-associated protein and β-catenin), is a member of the catenin family of cystololic proteins. β-catenin is encoded by the CTNNB1 gone.β-catenin is a pivotal player in the Wnt/Wg signaling pathway, mediators of several developmental processes. In the absence of Wnt, glycogen synthase kinase 3 (GSK-3β), a serine/threonine protein kinase constitutively phosphorylates the β-catenin protein. When Wnt is present and binds to any of the family members of the frizzled receptors (Fz), an intracellular signaling protein known as dishevelled (Dsh) is recruited to the membrane and phosphorylated. GSK-3β is inhibited by the activation of Dsh. As a result, β-catenin levels increase in the cytosol and are translocated into the nucleus to perform a variety of functions. β-catenin acts together with the transcription factors TCF and LEF to activate specific target genes involved in different processes.β-catenin undergoes phosphorylation upon growth factor stimulation resulting in reduced cell adhesion, thereby functioning as a component of adherin junctions which are multiprotein completes that mediate cell adhesion, cell-cell communication and cytoskeletal anchoring. (Willert et al., 1998, Curr. Opin. Genet. Dev. 8:95-102).Thompson et al. suggest that β-catenin plays an important role in various liver biology including liver development (both embryonic and postnatal), liver regeneration following partial hepatectomy, hepatocyte growth factor (HGF)-induced hepatomegaly, liver zonation, and pathogenesis of liver cancer. (Thompson M D., 2007, Hepatology M 45(5):).Wang et al. (2008) have shown that β-catenin can function as an oncogene. (Wang et al, 2008, Cancer Epidemiol. Biomarkers Prev. 17 (8):2101-8). In patients with basal cell carcinoma an increased level in β-catenin is present and leads to the increase in proliferation of related tumors. Mutations in the β-catenin gene are a cause of colorectal cancer (CRC), pilomatrixoma (PTR), medulloblastoma (MDB), hepatoblastoma, and ovarian cancer.The role of β-catenin in the development of colorectal cancer has been shown to be regulated by the expression product of the APC (adenomatous polyposis of the colon) gene, a tumor suppressor (Korinck et al., Science, 84-1787; Morin et al., Science, 87-1790). The APC protein normally binds β-catenin in conjunction with TCF/LEF forming a transcription factor complex. Morin et al. (Morin et al., Science, 87-1790) report that APC protein down-regulates the transcriptional activation mediated by β-catenin and Tcf-4 in colon cancer. Their results indicate that the regulation of β-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or β-catenin.Mutations in the β-catenin gene are either truncations that lead to deletion of part of the N-terminus of β-catenin, or point mutations that affect the serine and threonine residues that arc targeted by GSK3α/β or CKIα. These mutant β-catenin proteins are refractory to phosphorylation and thus escape proteasomal degradations. Consequently, β-catenin accumulates within affected cells. Stabilized and nuclear-localized β-catenin is a hallmark of nearly all cases of colon cancer. (Clevers, H., 2006, Cell 127:469-480). Morin et al. demonstrated that mutations of β-catenin that altered phosphorylation sites rendered the cells insensitive to APC-mediated down-regulation of β-catenin and that this disrupted mechanism was critical to colorectal tumorigenesis. (Morin et al., 1997, Science 275:).Other studies also report on the detection of mutations in β-catenin in various cancer cell lines (see e.g., Chan et al., 1999, Nature Genet. 21:410-413; Blaker et al., 1999, Genes Chromosomes Cancer 25:399-402; Sagae et al., 1999, Jpn. J. Cancer Res 90:510-515; Wang et al., 2008, Cancer Epidemiol. Biomarkers Prev. 17(8):2101-8). Additionally, abnormally high amounts of β-catenin have also been found in melanoma cell lines (see e.g., Rubinfeld et al., 1997, Science, 275:).Likewise other cancers, such as hepatocellular carcinoma (HCC), have also been associated with the Wnt/beta-catenin pathway. HCC is a complex and heterogeneous disease accounting for more than 660,000 new cases per year worldwide. Multiple reports have shown that Wnt signaling components are activated in human HCC patients. Activated Wnt signaling and nuclear beta-catenin correlate with recurrence of disease and poor prognosis (Takigawa et al. 2008, Curr Drug Targets N 9(11):1013-24). Elevated nuclear beta-catenin staining has been documented in 17-66% of HCC patients (Zulchner et al. 2010, Am J Pathol. J 176(1):472-81; Yu et al. 2009. J Hepatol. M 50(5)948-57). Merck's internal dataset on ~300 HCC patient tumors generated in collaboration with the Hong Kong University indicates Wnt signaling components are activated in 50% of HCC patients. External data have shown activating beta-catenin mutations in 13-40% of HCC patients, while inactivating Axin 1 or 2 mutations were present in ~10% of HCC patients (Lee et al. 2006, Frontiers in Bioscience May 1:11:).Preclinical studies provide evidence that activation of the Wnt/beta-catenin pathway is important in the generation and maintenance of HCC. Liver-targeted disruption of APC in mice activates beta-catenin signaling and leads to the formation of HCC (Colnot et al. 2004, Proc Natl Acad Sci U S A. December 7; 101(49):17216-21). Although overexpression of a beta-catenin mutant lacking the GSK-3beta phosphorylation sites alone is not sufficient for hepatocarcinogenesis (Harada et al. 2002, Cancer Res. April 1; 62(7):1971-7.), overexpression of tumorigenic mutant beta-catenin has been shown to make mice susceptible to HCC induced by (diethylnitrosamine), a known carcinogen (Nejak-Bowen et al. 2010, Hepatology 2010 M 51(5):1603-13. Interestingly, 95% of HCC tumors initiated by overexpression of the human Met receptor in mice (tre-Met transgenic mouse model) harbor beta-catenin activating mutations (Tward et al. 2007, Proc Natl Acad Sci U S A. September 11; 104(37):14771-6). This finding reflects the human disease and suggests that the Wnt pathway cooperates with Met signaling during hepatocarcinogenesis. High rates of beta-catenin activating mutations are also found in other transgenic mouse models for HCC (16% beta-catenin mutations in FGF19, 55% in c-Myc and 41% in H-Ras transgenic mice) (Nicholes et al. 2002, Am J Pathol. J 160(6): de la Coste et al. 1998, Proc Natl Acad Sci U S A. July 21; 95(15):8847-51).Preclinical studies have also shown that beta-catenin is a valid target for HCC. Beta-catenin siRNAs inhibit proliferation and viability of human HCC cell lines (Zeng et al. 2007). Similarly, treatment of human HCC cell lines with an anti-Wnt-1 antibody or TCF4/beta-catenin antagonists induce apoptosis, reduction of c-Myc, cyclin D1 and survivin expression as well as suppress tumor growth in vivo (Wei et al. 2009, Mol Cancer September 24; 8:76; Wei et al. 2010, Int J Cancer. May 15; 126(10):0).Hepatocellular carcinoma (HCC) is a common and aggressive cancer for which effective therapies are tacking. The Wnt beta-catenin pathway is activated in a high proportion of HCC cases (~50%), frequently owing to mutations in beta-catenin (i.e. CTNNB1) or in the beta-catenin destruction complex (e.g. Axin1). Moreover, the Wnt pathway as a target has proven to be challenging and is currently undruggable by small molecule inhibitors, making beta-catenin an attractive large) for an RNAi-based therapeutic approach (Llovet et al. 2008, Hepatology O 48: ).Alteration of gene expression, specifically CTNNB1 gene expression, through RNA interference (hereinafter “RNAi”) is one approach for meeting this need. RNAi is induced by short single-stranded RNA (“ssRNA”) or double-stranded RNA (“dsRNA”) molecules. The short dsRNA molecules, called “short interfering nucleic acids (“siNA”)” or “short interfering RNA” or “siRNA” or “RNAi inhibitors” silence the expression of messenger RNAs (“mRNAs”) that share sequence homology to the siNA. This can occur via cleavage of the mRNA mediated by an endonuclease complex containing a siNA. commonly referred to as an RNA-induced silencing complex (RISC). Cleavage of the larger RNA typically takes place in the middle of the region complementary to the guide sequence of the siNA duplex (Elbashir et al., 2001, Genes Dev., 15:188). In addition, RNA interference can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing, presumably through cellular mechanisms that either inhibit translation or that regulate chromatin structure and thereby prevent transcription of target gene sequences (see for example Allshire, 2002, Science, 297:; Volpe et al., 2002, Science, 297:; Jenuwein, 2002, Science, 297:; and Hall et al., 2002, Science, 297:). Despite significant advances in the field of RNAi, there remains a need for agents that can inhibit CTNNB1 gene expression and that can treat disease associated with CTNNB1 expression such as cancer.SUMMARY OF THE INVENTIONThe invention provides a solution to the problem of treating diseases that respond to the modulation of the CTNNB1 gene expression using novel short interfering nucleic acid (siNA) molecules to modulate CTNNB1 expression.The present invention provides compounds, compositions, and methods useful for modulating the expression of CTNNB1 genes, specifically those CTNNB1 genes associated with cancer and for treating such conditions by RNA interference (RNAi) using small nucleic acid molecules.In particular, the instant invention features small nucleic acid molecules, i.e., short interfering nucleic acid (siNA) molecules including, but not limited to, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA) and circular RNA molecules and methods used to modulate the expression of CTNNB1 genes and/or other genes involved in pathways of CTNNB1 gene expression and/or activity.In one aspect, the invention provides double-stranded short interfering nucleic acid (siNA) molecules that inhibit the expression of a CTNNB1 gene in a cell or mammal, wherein the double-stranded siNAs comprise a sense and an antisense stand. The antisense strand comprises a sequence that is complementary to at least a part of an RNA associated with the expression of the CTNNB1 gene. The sense strand comprises a sequence that is complementary to the antisense strand. In various embodiments, at least one strand comprises at least a 15 nucleotide sequence selected from the group of sequences consisting of SEQ ID NOS:1-6374. In certain embodiments, the antisense strand comprises at least 15, 16, 17, 18, or 19 nucleotides having sequence complementarity to a target sequence set forth in Table 1a. In other and/or in the same embodiments, the antisense strand comprises at least a 15, 16, 17, 18, or 19 nucleotide sequence of one of the antisense sequences set forth in Table 1b. In some embodiments, the sense strand comprises at least a 15, 16, 17, 18, or 19 nucleotide sequence of a sense strand sequence as set forth in Table 1b.In certain embodiments of this aspect of the invention, double-stranded short interfering nucleic acid (siNA) molecules are provided wherein the antisense stand comprises a modified sequence as set forth in Table 1c that has sequence complementarity to a target sequence of the invention. In some embodiments, the sense strand also comprises a modified sequence as set forth in Table 1c.In certain embodiments, the present invention provides a double-stranded short interfering nucleic acid (siNA) molecule that modulates the expression of CTNNB1, wherein the siNA comprises a sense strand and each strand is independently 15 to 30 n and the antisense strand comprises at least 15, 16, 17, 18, or 19 nucleotides having sequence complementary to any of:(SEQ ID NO: 5)5′-CUGUUGGAUUGAUUCGAAA-3′; (SEQ ID NO: 194)5′-ACGACUAGUUCAGUUGCUU-3′; (SEQ ID NO: 196)5′-GGAUGAUCCUAGCUAUCGU-3′; or (SEQ ID NO: 151)5′-CCAGGAUGAUCCUAGCUAU-3′. In some embodiments of the invention, the antisense strand of a siNA molecule comprises at least a 15, 16, 17, 18, or 19 nucleotide sequence of:(SEQ ID NO: 4918)5′-UUUCGAAUCAAUCCAACAG-3′; (SEQ ID NO: 5107)5′-AAGCAACUGAACUAGUCGU-3′; (SEQ ID NO: 5109)5′-ACGAUAGCUAGGAUCAUCC-3′; or (SEQ ID NO: 5064)5′-AUAGCUAGGAUCAUCCUGG-3′. In some embodiments, the sense strand of a siNA molecule of the invention comprises at least a 15, 16, 17, 18, or 19 nucleotide sequence of:(SEQ ID NO: 5)5′-CUGUUGGAUUGAUUCGAA-3′; (SEQ ID NO: 194)5′-ACGACUAGUUCAGUUGCUU-3′; (SEQ ID NO: 196)5′-GGAUGAUCCUAGCUAUCGU-3′; or (SEQ ID NO: 151)5′-CCAGGAUGAUCCUAGCUAU-3′. In some embodiments, a siNA molecule of the invention comprises any of:(SEQ ID NO: 5)5′-CUGUUGGAUUGAUUCGAAA-3′ and
(SEQ ID NO: 4918)5′-UUUCGAAUCAAUCCAACAG-3′; or (SEQ ID NO: 194)5′-ACGACUAGUUCAGUUGCUU-3′ and
(SEQ ID NO: 5107)5′-AAGCAACUGAACUAGUCGU-3′; or (SEQ ID NO: 196)5′-GGAUGAUCCUAGCUAUCGU-3′ and
(SEQ ID NO: 5109)5′-ACGAUAGCUAGGAUCAUCC-3′; or (SEQ ID NO: 151)5′-CCAGGAUGAUCCUAGCUAU-3′ and
(SEQ ID NO: 5064)5′-AUAGCUAGGAUCAUCCUGG-3′. In some embodiments, a siNA molecule of the invention comprises SEQ ID NOS: 6372 and 6374.In some embodiments, a siNA molecule of the invention comprises SEQ ID NOS: 6370 and 6369.In some embodiments, a siNA molecule of the invention comprises SEQ ID NOS: 2021 and 2068.In some embodiments, a siNA molecule of the invention comprises SEQ ID NOS: 6372 and 6373.In some embodiments, a siNA molecule of the invention comprises SEQ ID NOS: 2147 and 6368In some embodiments, the invention features a composition comprising:
(a) a double-stranded short interfering nucleic acid (siNA)(b) a cationic lipid compound having any of compound numbers 1-46 or any(c)(d) DSPC; and(c) PEG-DMG.
In some embodiments, the invention features a composition comprising:
(a) a double-stranded short interfering nucleic acid (siNA)(b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-(c)(d) DSPC; and(e) PEG-DMG.
In some embodiments, the invention features a composition comprising:
(a) a double-stranded short interfering nucleic acid (siNA) having SEQ ID NOS: 6372 and 6374;(b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-(c)(d) DSPC; and(e) PEG-DMG.
In some embodiments, the invention features a composition comprising:
(a) a double-stranded short interfering nucleic acid (siNA) having SEQ ID NOS: 6370 and 6369;(b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-(c)(d) DSPC; and(c) PEG-DMG.
In some embodiments, the invention features a composition comprising:
(a) a double-stranded short interfering nucleic acid (siNA) having SEQ ID NOS: 2021 and 2068;(b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-(c)(d) DSPC; and(e) PEG-DMG.
In some embodiments, the invention features a composition comprising:
(a) a double-stranded short interfering nucleic acid (siNA) having SEQ ID NOS: 6372 and 6373;(b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-(c)(d) DSPC; and(e) PEG-DMG.
In some embodiments, the invention features a composition comprising:
(a) a double-stranded short interfering nucleic acid (siNA) having SEQ ID NOS: 2147 and 6368;(b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-(c)(d) DSPC; and(E) PEG-DMG.
In some embodiments, a composition of the invention comprises any Cationic Lipid having any of compound numbers 1-46 in the following molar ratios:
Cationic Lipid/Cholesterol/PEG-DMG 56.6/38/5.4;Cationic Lipid/Cholesterol/PEG-DMG 60/38/2;Cationic Lipid/Cholesterol/PEG-DMG 67.3/29/3.7;Cationic Lipid/Cholesterol/PEG-DMG 49.3/47/3.7;Cationic Lipid/Cholesterol/PEG-DMG 50.3/44.3/5.4;Cationic Lipid/Cholesterol/PEG-C-DMA/DSPC 40/48/2/10;Cationic Lipid/Cholesterol/PEG-DMG/DSPC 40/48/2/10; andCationic Lipid/Cholesterol/PEG-DMG/DSPC 58/30/2/10.
In some embodiments, a composition of the invention comprises (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, cholesterol, DSPC, and PEG-DMG, having a molar ratio of 50:30:10:2 respectively.In some embodiments, a composition of the invention further comprises a cryo-protectant. protectant. In some embodiments, the cryoprotectant is Sucrose, Trehalose, Raffinose, Stachyose, Verbascose, Mannitol, Glucose, Lactose, Maltose, Maltotriose-heptaose, Dextran, Hydroxyethyl Starch, Insulin, Sorbitol, Glycerol, Arginine, Histidine, Lysine, Proline, Dimethylsulfoxide or any combination thereof. In some embodiments, the cryoprotectant is Sucrose. In some embodiments, the cryoprotectant is Trehalose. In some embodiments, the cryoprotectant is a combination of Sucrose and Trehalose.In some embodiments of the invention, all of the nucleotides of siNAs of the invention are unmodified. In other embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) of the nucleotide positions independently in either one or both strands of an siNA molecule are modified. Modifications include nucleic acid sugar modifications, base modifications, backbone (internucleotide linkage) modifications, non-nucleotide modifications, and/or any combination thereof. In certain instances, purine and pyrimidine nucleotides are differentially modified. For example, purine and pyrimidine nucleotides can be differentially modified at the 2′-sugar position (i.e., at least one purine has a different modification from at least one pyrimidine in the same or different strand at the 2′-sugar position). In certain instances the purines are unmodified in one or both strands, while the pyrimidines in one or both strands are modified. In certain other instances, the pyrimidines are unmodified in one or both strands while the purines in one or both strands are modified. In some instances, at least one modified nucleotide is a 2′-deoxy-2′-fluoro nucleotide, a 2′-deoxy nucleotide, or a 2′-O-alkyl nucleotide. In some instances, at least 5 or more of the pyrimidine nucleotides in one or both stands are either all 2′-deoxy-2′-fluoro or all 2′-O-methyl pyrimidine nucleotides. In some instances, at least 5 or more of the purine nucleotides in one or both stands are either all 2′-deoxy-2′-fluoro or all 2′-O-methyl purine nucleotides. In certain instances, wherein the siNA molecules comprise one or more modifications as described herein, the nucleotides at positions 1, 2, and 3 at the 5′ end of the guide (antisense) strand are unmodified.In certain embodiments, the siNA molecules of the invention have 3′ overhangs of one, two, three, or four nucleotides) on one or both of the strands. In other embodiments, the siNA molecules lack overhangs (i.e., have blunt ends). Preferably, the siNA molecule has 3′ overhangs of two nucleotides on both the sense and antisense strands. The overhangs can be modified or unmodified. Examples of modified nucleotides in the overhangs include, but are not limited to, 2′-O-alkyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, locked nucleic acid (LNA) nucleotides, or 2′-deoxy nucleotides. The overhang nucleotides in the antisense strand can comprise nucleotides that are complementary to nucleotides in the CTNNB1 target sequence. Likewise, the overhangs in the sense stand can comprise nucleotides that are in the CTNNB1 target sequence. In certain instances, the siNA molecules of the invention have two 3′ overhang nucleotides on the antisense stand that are 2′-O-alkyl (e.g., 2′-O-methyl) nucleotides and two 3′ overhang nucleotides on the sense stand that are 2′-deoxy nucleotides. In other instances, the siNA molecules of the invention have two 3′ overhang nucleotides that are 2′-O-alkyl (e.g., 2′-O-methyl) nucleotides on both the antisense stand and on the sense stand. In certain embodiments, the 2′-O-alkyl nucleotides are 2′-O-methyl uridine nucleotides. In certain instances, the overhangs also comprise one or more phosphorothioate linkages between nucleotides of the overhang.In some embodiments, the siNA molecules of the invention have caps (also referred to herein as “terminal caps.” The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present on both termini, such as at the 5′ and 3′ termini of the sense strand of the siNA.In some embodiments, the siNA molecules of the invention are phosphorylated at the 5′ end of the antisense strand. The phosphate group can be a phosphate, a diphosphate or a triphosphate.The siNA molecules of the invention when double stranded can be symmetric or asymmetric. Each strand of these double stranded siNAs independently can range in nucleotide length between 3 and 30 nucleotides. Generally, each strand of the siNA molecules of the invention is about 15 to 30 (i.e., about 19, 20, 21, 22, 23 or 24) nucleotides in length.The siNA molecules of the invention, which are double stranded or have a duplex structure, independently comprise about 3 to about 30 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs. Generally, the duplex structure of siNAs of the invention is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length.In certain embodiments, double-stranded short interfering nucleic acid (siNA) molecules are provided, wherein the molecule has a sense strand and an antisense strand and comprises formula (A):(A)B-NX3-(N)X2 B-3′ B(N)X1-NX4-[N]X5-5′
wherein, the upper strand is the sense strand and the lower strand is the antisense strand of the double-stranded n wherein the antisense strand comprises at least a 15, 16, 17, 18, or 19 nucleotide sequence of SEQ ID NO: 4918, SEQ ID NO: 5107, SEQ ID NO: 5109, or SEQ ID NO: 5064, and the sense strand comprises a sequence having complementarity to each N is independently a nucleotide which is unmodified or chemically modified or a non-each B is a terminal cap that (N) represents overhanging nucleotides, each of which is independently unmodified or[N] represents nucleotides that are ribonucleotides:X1 and X2 are independently integers from 0 to 4;X3 is an integer from 15 to 30;X4 is an integer from 9 to 30; andX5 is an integer from 0 to 6, provided that the sum of X4 and X5 is 15-30.
In one embodiment, the invention features a double-stranded short interfering nucleic acid (siNA) of formula (A); wherein
(a) one or more pyrimidine nucleotides in NX4 positions are independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl nucleotides, 2′-deoxy nucleotides. ribonucleotides, or any(b) one or more purine nucleotides in NX4 positions are independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any(c) one or more pyrimidine nucleotides in NX3 positions are independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl nucleotides, 2′-deoxy nucleotides, ribonucleotides, or any and(d) one or more purine nucleotides in NX1 positions are independently 2′-deoxy-2′-fluoro nucleotides, 2′-O-alkyl nucleotides, 2′-deoxy nucleotides, ribonucleotides.
The present invention further provides compositions comprising the double-stranded nucleic acid molecules described herein with optionally a pharmaceutically acceptable carrier or diluent.The administration of the composition can be carried out by known methods, wherein the nucleic acid is introduced into a desired target cell in vitro or in vivo.Commonly used techniques for introduction of the nucleic acid molecules of the invention into cells, tissues, and organisms include the use of various carrier systems, reagents and vectors. Non-limiting examples of such carrier systems suitable for use in the present invention include conjugates, nucleic-acid-lipid particles, lipid nanoparticles (LNP), liposomes, lipoplexes, micelles, virosomes, virus like particles (VLP), nucleic acid complexes, and mixtures thereof.The compositions of the invention can be in the form of an aerosol, dispersion, solution (e.g., an injectable solution), a cream, ointment, tablet, powder, suspension or the like. These compositions may be administered in any suitable way, e.g. orally, sublingually, buccally, parenterally, nasally, or topically. In some embodiments, the compositions are aerosolized and delivered via inhalation.The molecules and compositions of the present invention have utility over a broad range of therapeutic applications. Accordingly another aspect of this invention relates to the use of the compounds and compositions of the invention in treating a subject. The invention thus provides a method for treating a subject, such as a human, suffering from a condition which is mediated by the action, or by the loss of action, of CTNNB1, wherein the method comprises administering to the subject an effective amount of a double-stranded short interfering nucleic acid (siNA) molecule of the invention. In certain embodiments, the condition is cancer.These and other aspects of the invention will be apparent upon reference to the following detailed description and attached figures. Moreover, it is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.Additionally, patents, patent applications, and other documents are cited throughout the specification to describe and more specifically set forth various aspects of this invention. Each of these references cited herein is hereby incorporated by reference in its entirety, including the drawings.BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 shows a non-limiting proposed mechanistic representation of target RNA degradation involved in RNAi. Double-stranded RNA (dsRNA), which is generated by RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for example viral, transposon, or other exogenous RNA, activates the DICER enzyme that in turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be introduced directly into a cell by appropriate means. An active siNA complex forms that recognizes a target RNA, resulting in degradation of the target RNA by the RISC endonuclease complex or in the synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which can activate DICER and result in additional siNA molecules, thereby amplifying the RNAi response.FIG. 2 shows non-limiting examples of chemically modified siNA constructs of the present invention using a generalized structure of a representative siNA duplex. The specific modifications shown in the figure can be utilized alone or in combination with other modifications of the figure, in addition to other modifications and features described herein with reference to any siNA molecule of the invention. In the figure, N stands for any nucleotide or optionally a non-nucleotide as described here. The upper strand, having B—NX3—(N)X2—B-3′ is the sense (or passenger) strand of the siNA, whereas the lower strand, having B(N)X1—NX4—[N]X5-5′ is the antisense (or guide) strand of the siNA. Nucleotides (or optional non-nucleotides) of internal portions of the sense strand are designated NX3 and nucleotides (or optional non-nucleotides) of internal portions of the antisense strand are designated NX4. Nucleotides (or optional non-nucleotides) of the internal portions are generally base paired between the two strands, but can optionally lack base pairing (e.g. have mismatches or gaps) in some embodiments. Nucleotides (or optional non-nucleotides) of overhang regions are designated by parenthesis (N). Nucleotides of the 5′-terminal portion of the antisense strand are designated [N]. Terminal caps are optionally present at the 5′ and/or 3′ end of the sense strand and further optionally present at the 3′-end of the antisense strand. Generally, each strand can independently range from about 15 to about 30 nucleotides in length, but can vary depending on the presence of any overhang nucleotides. In certain embodiments, X1 and X2 are independently integers from 0 to 4; X3 is an integer from 15 to 30; X4 is an integer from 9 to 30; X5 is an integer from 0 to 6, provided that the sum of X4 and X5 is 15-30. Various modifications are shown for the nucleotides of the sense and antisense strands of the siNA constructs. The (N) overhang nucleotide positions can be chemically modified as described herein (e.g., 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-deoxy, LNA, universal bases etc.) and can be either derived from a corresponding target nucleic acid sequence or not. The constructs shown in the figure can also comprise phosphorothioate linkages as described herein for example, phosphorothioate linkages can exist between any N, (N), and/or [N] positions. Such phosphorothioate incorporation can be utilized between purine “R” and pyrimidine “Y” positions, or for stabilization of pyrimidine linkages in general. Furthermore, although not depicted on the Figure, the constructs shown in the figure can optionally include a ribonucleotide at the 9th position from the 5′-end of the sense strand or the 11th position based on the 5′-end of the guide strand by counting 11 nucleotide positions in from the 5′-terminus of the guide strand. Similarly, the antisense strand can include a ribonucleotide at the 14th position from the 5′-end, or alternately can be selected or designed so that a 2′-O-alkyl nucleotide (e.g., a 2′-O-methyl purine) is not present at this position. Furthermore, although not shown in the Figure, the 5′-terminal position of the antisense strand can comprise a terminal phosphate group as described herein. The antisense strand generally comprises sequence complementary to any target nucleic acid sequence of the invention, such as those set forth in Table 1a herein.FIG. 3 shows non-limiting examples of certain combinations of modifications applied to the representative siNA duplex described in FIG. 2. The table shown below the representative structure provides specific combinations of (N)X1, (N)X2, NX3, NX4, and/or [N]X5 nucleotide (and optional non-nucleotide) positions. For example, combinations of 5 or more (e.g., 5, 6, 7, 8, 9, or 10 or more) NX3 and 5 or more (e.g., 5, 6, 7, 8, 9, or 10 or more) NX4 pyrimidine “Y” and purine “R” nucleotides are specified, each of which can independently have specific (N)X1, and/or (N)X2, substitutions as shown in the figure, in addition to optional phosphorothioate substitutions. The 5′-terminal antisense strand [N] nucleotides are generally ribonucleotides, but can also be modified or unmodified depending on if they are purine “R” or pyrimidine “Y” nucleotidesFIGS. 4A-C shows non-limiting examples of different siNA constructs of the invention. The criteria of the representative structures shown in FIGS. 2 and 3 can be applied to any of the structures shown in FIGS. 4A-C.The examples shown in FIG. 4A (constructs 1, 2, and 3) have 19 repre however, different embodiments of the invention include any number of base pairs described herein. Bracketed regions represent nucleotide overhangs, for example, comprising about 1, 2, 3, or 4 nucleotides in length, preferably about 2 nucleotides. Constructs 1 and 2 can be used independently for RNAi activity. Construct 2 can comprise a polynucleotide or non-nucleotide linker, which can optionally be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and/or in vitro. In another example, construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active siNA construct 2 in vivo and/or in vitro, which can optionally utilize another biodegradable linker to generate the active siNA construct 1 in vivo and/or in vitro. As such, the stability and/or activity of the siNA constructs can be modulated based on the design of the siNA construct for use in vivo or in vitro and/or in vitro.The examples shown in FIG. 4B represent different variations of double-stranded nucleic acid molecule of the invention, such as microRNA, that can include overhangs, bulges, loops, and stem-loops resulting from partial complementarity. Such motifs having bulges, loops, and stem-loops are generally characteristics of miRNA. The bulges, loops, and stem-loops can result from any degree of partial complementarity, such as mismatches or bulges of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in one or both strands of the double-stranded nucleic acid molecule of the invention.The example shown in FIG. 4C represents a model double-stranded nucleic acid molecule of the invention comprising a 19 base pair duplex of two 21 nucleotide sequences having dinucleotide 3′-overhangs. The top strand (1) represents the sense strand (passenger strand), the middle strand (2) represents the antisense (guide strand), and the lower strand (3) represents a target polynucleotide sequence. The dinucleotide overhangs (NN) can comprise a sequence derived from the target polynucleotide. For example, the 3′-(NN) sequence in the guide strand can be complementary to the 5′-[NN] sequence of the target polynucleotide. In addition, the 5′-(NN) sequence of the passenger strand can comprise the same sequence as the 5′-[NN] sequence of the target polynucleotide sequence. In other embodiments, the overhangs (NN) are not derived from the target polynucleotide sequence, for example where the 3′-(N,N) sequence in the guide strand are not complementary to the 5′-[NN] sequence of the target polynucleotide and the 5′-(NN) sequence of the passenger strand can comprise different sequence from the 5′-[NN] sequence of the target polynucleotide sequence. In additional embodiments, any (NN) nucleotides are chemically modified, e.g., as 2′-O-methyl, 2′-deoxy-2-40 -fluoro, and/or other modifications herein. Furthermore, the passenger strand can comprise a ribonucleotide position N of the passenger strand. For the representative 19 base pair 21 mer duplex shown, position N can be 9 nucleotides in from the 3′ end of the passenger strand. However, in duplexes of differing length, the position N is determined based on the 5′-end of the guide strand by counting 11 nucleotide positions in from the 5′-terminus of the guide strand and picking the corresponding base paired nucleotide in the passenger strand. Cleavage by Ago2 takes place between positions 10 and 11 as indicated by the arrow. In additional embodiments, there are two ribonucleotides, NN, at positions 10 and 11 based on the 5′-end of the guide strand by counting 10 and 11 nucleotide positions in from the 5′-terminus of the guide strand and picking the corresponding base paired nucleotides in the passenger strand. FIG. 5 shows non-limiting examples of different stabilization chemistries (1-10) that can be used, for example, to stabilize the 5′ and/or 3′-ends of siNA sequences of the invention, including (1) [3-3′]- (2) (3) [5′-3′]-3′- (4) [5′-3′]- (5) [5′-3′]-3′-O-m (6) 3′- (7) [3′-5′]-3′- (8) [3′-3′]- (9) [5′-2′]- and (10) [5-3′]dideoxyribonucleotide (when X=O). In addition to modified and unmodified backbone chemistries indicated in the figure, these chemistries can be combined with different sugar and base nucleotide modifications as described herein.FIG. 6 shows a non-limiting example of a strategy used to identify chemically modified siNA constructs of the invention that are nuclease resistant while preserving the ability to mediate RNAi activity. Chemical modifications are introduced into the siNA construct based on educated design parameters (e.g. introducing 2′-modifications, base modifications, backbone modifications, terminal cap modifications etc). The modified construct is tested in an appropriate system (e.g., human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters). In parallel, the siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciverase reporter assay and/or against endogenous mRNA). Lead siNA constructs are then identified which possess a particular characteristic while maintaining RNAi activity, and can be further modified and assayed once again. This same approach can be used to identify siNA-conjugate molecules with improved pharmocokinetic profiles, delivery, and RNAi activity.FIG. 7 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear and duplex constructs and asymmetric derivatives thereof.FIG. 8 shows non-limiting examples of chemically modified terminal phosphate groups of the invention.FIG. 9 shows a non-limiting example of a cholesterol linked phosphoramidite that can be used to synthesize cholesterol conjugated siNA molecules of the invention. An example is shown with the cholesterol moiety linked to the 5′-end of the sense strand of an siNA molecule.FIG. 10 depicts an embodiment of 5′ and 3′ inverted abasic cap linked to a nucleic acid strand.DETAILED DESCRIPTION OF THE INVENTIONA. Terms and DefinitionsThe following terminology and definitions apply as used in the present application.The term “abasic” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to sugar moieties lacking a nucleobase or having a hydrogen atom (H) or other non-nucleobase chemical groups in place of a nucleobase at the 1′ position of the sugar moiety, see for example Adamic et al., U.S. Pat. No. 5,998,203. In one embodiment, an abasic moiety of the invention is a ribose, deoxyribose, or dideoxy ribose sugar.The term “acyclic nucleotide” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to any nucleotide having an acyclic ribose sugar, for example where any of the ribose carbon, carbon or carbon/oxygen bonds are independently or in combination absent from the nucleotide.The term “alkyl” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a saturated or unsaturated hydrocarbons, including straight-chain, branched-chain, alkenyl, alkynyl groups and cyclic groups, but excludes aromatic groups. Notwithstanding the foregoing, alkyl also refers to non-aromatic heterocyclic groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons The alkyl group can be substituted or unsubstituted. When substituted, the substituted group(s) is preferably, hydroxyl, halogen, cyano, C1-C4 alkoxy, ═O, ═S, NO2, SH, NH2, or NR1R2, where R1 and R2 independently are H or C1-C4 alkyl.The phrase “agents that interfere with cell cycle checkpoints” refers to compounds that inhibit protein kinases that transduce cell cycle checkpoint signals, thereby sensitizing the cancer cell to DNA damaging agents.The phrase “agents that interfere with receptor tyrosine kinases (RTKs)” refers to compounds that inhibit RTKs and therefore inhibit mechanisms involved in oncogenesis and tumor progression.The phrase “androgen receptor modulators” refers to compounds that interfere or inhibit the binding of androgens to the receptor, regardless of mechanism.The phrase “angiogenesis inhibitors” refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism.The term “aryl” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which can be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, C1-C4 alkoxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, NH2, and NR1R2 groups, where R1 and R2 independently are H or C1-C4 alkyl.The term “alkylaryl” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to an alkyl group (as described above) covalently joined to an aryl group (as described above). Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and examples of heterocyclic aryl groups having such heteroatoms include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. Preferably, the alkyl group is a C1-C4 alkyl group.The term “amide” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to an —C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen.The phrase “antisense region” as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to a nucleotide sequence of an siNA molecule having complementarity to a target nucleic acid sequence. In addition, the antisense region of an siNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the siNA molecule. In one embodiment, the antisense region of the siNA molecule is referred to as the antisense strand or guide strand.The phrase “asymmetric hairpin” refers to a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprising about 4 to about 12 (e.g. about 4, 5, 6, 7, 8, 9, 10, 11, or 12) nucleotides, and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region. The asymmetric hairpin siNA molecule can also comprise a 5′-terminal phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.The term “biodegradable” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to degradation in a biological system, for example, enzymatic degradation or chemical degradation.The term “biodegradable linker” as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to a linker molecule that is designed to connect one molecule to another molecule, and which is susceptible to degradation in a biological system. The linker can be a nucleic acid or non-nucleic acid based linker. For example, a biodegradable linker can be used to attach a ligand or biologically active molecule to an siNA molecule of the invention. Alternately, a biodegradable linker can be used to connect the sense and antisense strands of an siNA molecule of the invention. The biodegradable linker is designed such that its stability can be modulated for a particular purpose, such as delivery to a particular tissue or cell type. The stability of a nucleic acid-based biodegradable linker molecule can be modulated by using various chemistries, for example combinations of ribonucleotides, deoxyribonucleotides, and chemically modified nucleotides, such as 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified or base modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can comprise a single nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or phosphodiester linkage. The biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.The phrase “biologically active molecule” as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system and/or are capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules. Examples of biologically active molecules, include siNA molecules alone or in combination with other molecules including, but not limited to therapeutically active molecules such as antibodies, cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucteotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, polyamines, polyamides, polyethylene glycol, other polyethers, 2-5A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof.The phrase “biological system” as used herein refers to its meaning as is generally accepted in the art. The term generally refers to material, in a purified or unpurified form, from biological sources including, but not limited to, human or animal, wherein the system comprises the components required for RNAi activity. Thus, the phrase includes, for example, a cell, tissue, subject, or organism, or extract thereof. The term also includes reconstituted material from a biological source.The phrase “blunt end” as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to termini of a double-stranded siNA molecule having no overhanging nucleotides. For example, the two strands of a double-stranded siNA molecule having blunt ends align with each other with matched base-pairs without overhanging nucleotides at the termini. A siNA duplex molecule of the invention can comprise blunt ends at one or both termini of the duplex, such as termini located at the 5′-end of the antisense strand, the 5′-end of the sense strand, or both termini of the duplex.The term “cap” also referred to herein as “terminal cap,” as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to a moiety, which can be a chemically modified nucleotide or non-nucleotide that can be incorporated at one or more termini of one or more nucleic acid molecules of the invention. These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminal (3′-cap) or can be present on both termini of any nucleic acid molecule of the invention. A cap can be present at the 5′-end, 3-end and/or 5′ and 3′-ends of the sense strand of a nucleic acid molecule of the invention. Additionally, a cap can optionally be present at the 3′-end of the antisense strand of a nucleic acid molecule of the invention. In non-limiting examples, the 5′-cap includes, but is not limited to, LNA; inverted deoxy abasic residue (moiety); 4′,5′- 1-(beta-D-erythrofuranosyl) nucleotide, 4′- ca 1,5-unhyd L- alpha- modi phosph threo-pento acyclic 3′,4′- acyclic 3,4-dihyd acyclic 3,5-dihydr 3′-3′-invert 3′-3′-in 3′-2′-invert 3′-2′-in 1,4- 3′-
3′- 3′- or bridging or non-bridging methylphosphonate moiety. Non-limiting examples fl the 3′-cap include, but are not limited to, LNA; inverted deoxy abasic residue (moiety); 4′, 5′- 1-(beta-D-erythrofuranosyl) 4′- ca 5′-amino- 1,3-diamino-2- 3-a 6- 1,2-am hyd 1,5-anhyd L- alpha- modi threo-pento acyclic 3′,4′- 3,4-dihyd 3,5-dihydroxypentyl nucleotide, 5′-5′-invert 5′-5′-in 5′- 5′- 1,4- 5′- bridging and/or non-bridging 5′- phosphorothioate and/o bridging or non bridgi and 5′-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein). FIG. 5 shows some non-limiting examples of various caps.The term “cell” as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term is used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human being. The cell can be present in an organism, e.g., birds, plants and mammals, such as humans, cows, sheep, apes, monkeys, swine, dogs, and cuts. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line origin, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell.The phrase “chemical modification” as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the term refers to any modifications of the chemical structure of the nucleotides that differs from nucleotides of native siRNA or RNA in general. The term “chemical modification” encompasses the addition, substitution, or modification of native siRNA or RNA at the sugar, base, or internucleotide linkage, as described herein or as is otherwise known in the art. In certain embodiments, the term “chemical modification” can refer to certain forms of RNA that are naturally occurring in certain biological systems, for example 2′-O-methyl modifications or inosine modifications.The term “CTNNB1” refers to catenin (cadherin-associated protein), beta 1 which is gene that encodes CTNNB1 proteins, CTNNB1 peptides, CTNNB1 polypeptides, CTNNB1 regulatory polynucleotides (e.g., CTNNB1 miRNAs and siNAs), mutant CTNNB1 genes, and splice variants of a CTNNB1 genes, as well as other genes involved in CTNNB1 pathways of gene expression and/or activity. Thus, each of the embodiments described herein with reference to the term “CTNNB1” are applicable to all of the protein, peptide, polypeptide, and/or polynucleotide molecules covered by the term “CTNNB1”. as that term is defined herein. Comprehensively, such gene targets are also referred to herein generally as “target” sequences (including the target sequences listed in Table 1a).The term “complementarity” or “complementary” as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the invention, the terms generally refer to the formation or existence of hydrogen bond(s) between one nucleic acid sequence and another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types of bonding as described herein. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:; Turner et al., 1987. J. Am. Chem. Soc. 109:). Perfect complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of continuous residues in a second nucleic acid sequence. Partial complementarity can include various mismatches or non-based paired nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches, non-nucleotide linkers, or non-based paired nucleotides) within the nucleic acid molecule, which can result in bulges, loops, or overhangs that result between the sense strand or sense region and the antisense strand or antisense region of the nucleic acid molecule or between the antisense strand or antisense region of the nucleic acid molecule and a corresponding target nucleic acid molecule. Such partial complementarity can be represented by a % complementarity that is determined by the number of non-base paired nucleotides, i.e., about 50%, 60%, 70%, 80%, 90% etc. depending on the total number of nucleotides involved. Such partial complementarity is permitted to the extent that the nucleic acid molecule (e.g., siNA) maintains its function, for example the ability to mediate sequence specific RNAi.The terms “composition” or “formulation” as used herein refer to their generally accepted meaning in the art. These terms generally refer to a composition or formulation, such as in a pharmaceutically acceptable carrier or diluent, in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including, for example, a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, inhalation, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivers). For example, compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect. As used herein, pharmaceutical formulations include formulations for human and veterinary use. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include: Lipid Nanoparticles (see for example Semple et al., 2010, Nat Biotechnol., F 28(2):172-6.); P-glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, D F et al, 1990, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of polybutylcyanoacrylate. Other non-limiting examples of delivers strategies for the nucleic acid molecules of the instant invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, ; Tyler et al., 1999, FEBS Lett., 421, 280-284; Partridge et al., 1995, PNAS USA., 92, ; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, ; and Tyler et al., 1999, PNAS USA., 96, . A “pharmaceutically acceptable composition” or “pharmaceutically acceptable formulation” can refer to a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention to the physical location most suitable for their desired activity.The phrase “cytotoxic/cytostatic agents” refer to compounds that cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell mytosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, inhibitors of historic deacetylase, inhibitors of kinases involved in mitotic progression, biologica hormonal/anti-hormonal therapeutic agents, hematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteasome inhibitors and ubiquitin ligase inhibitors.The phrase “estrogen receptor modulators” refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism.The term “gene” or “target gene” as used herein refers to their meaning as is generally accepted in the art. The terms generally refer a nucleic acid (e.g., DNA or RNAi sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide. The target gene can also include the UTR or non-coding region of the nucleic acid sequence. A gene or target gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Such non-coding RNAs can serve as target nucleic acid molecules for siNA mediated RNA interference in modulating the activity of fRNA or ncRNA involved in functional or regulatory cellular processes. Aberrant fRNA or ncRNA activity leading to disease can therefore be modulated by siNA molecules of the invention. siNA molecules targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of a subject, organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.). The target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof. The cell containing the target gene can be derived from or contained in any organism, for example a plant, animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrates or invertebrates. Non-limiting examples of fungi include molds or yeasts. For a review, see for example Snyder and Gerstein, 2003, Science, 300, 258-260.The phrase “HMG-CoA reductase inhibitors” refers to inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds that have HMG-CoA reductase inhibitory activity, and therefore the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention.The phrase “homologous sequences” as used herein refers to its meaning as is generally accepted in the art. The term generally refers a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect identity (100%), as partially homologous sequences are also contemplated by and within the scope of the instant invention (e.g., at least 95%, 94%, 93%, 92%, 91%, 90%, 895, 88%, 87%, 86}