RNA interference-mediated inhibition of catenin (cadherin-binding protein) beta 1 (CTNNB1) gene expression using small interfering nucleic acids (siNA)

Abstract

To provide agents capable of inhibiting CTNNB1 gene expression and drugs capable of treating diseases linked to CTNNB1 expression, such as cancer.SOLUTION: 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 β-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 mediate RNA interference (RNAi) against CTNNB1 gene expression.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Sequence Listing Sequence listings submitted via EFS pursuant to 37 CFR §1.52(e)(5) are not citations. The text file of the sequence listing submitted via EFS is incorporated herein by reference. , the file "Se" with a size of 2,173,912 bytes created on July 25, 2011 Includes "quenceListingSIRONC2". [Background technology]

[0002] Beta-catenin (also known as cadherin-binding protein and β-catenin) β-catenin is a member of the catenin family of cytosolic proteins. It is encoded by the CTNNB1 gene.

[0003] β-catenin is a key player in the Wnt / Wg signaling pathway and has been implicated in several In the absence of Wnt, the serine / threonine The protein kinase glycogen synthase kinase 3 (GSK-3β) binds β-catenin. The frizzled receptor (F) is constitutively phosphorylated by the phosphokinin protein. z) as disheveled (Dsh) Known intracellular signaling proteins are recruited to the membrane and phosphorylated. 3β is inhibited by the activation of Dsh, resulting in the loss of β-catenin receptors in the cytosol. The signal level increases, and it is translocated to the nucleus where it performs various functions. Nin acts together with the transcription factors TCF and LEF and is involved in various processes. Activates target genes.

[0004] β-catenin is phosphorylated upon growth factor stimulation, leading to reduced cell adhesion. This results in multiple pathways mediating cell adhesion, cell-cell communication, and cytoskeletal anchoring. It functions as a component of the protein complex, the adhesive junction apparatus (Non-patent Document 1).

[0005] According to Thompson et al., β-catenin is involved in various aspects of liver biology, e.g. development (both embryonic and postnatal), liver regeneration after partial hepatectomy, hepatocyte growth factor (HGF) ) plays an important role in the pathogenesis of induced hepatomegaly, liver zonation, and liver cancer It has been suggested that this is the case (Non-Patent Document 2).

[0006] Wang et al. (2008) reported that β-catenin functions as an oncogene. It has been shown that this may be effective in treating patients with basal cell carcinoma. In these patients, there is an increase in β-catenin levels, leading to an associated increase in tumor growth. Mutations in the tenin gene are associated with colorectal cancer (CRC), pilorheoblastoma (PTR), and medulloblastoma. (MDB), a cause of germinal tumors and ovarian cancer.

[0007] The role of β-catenin in colorectal cancer development is related to the tumor suppressor APC (colon adenomatous ductal carcinoma). It has been shown that the expression of the ovarian tumorigenesis (ovarian tumorigenesis) gene is regulated by the expression product of the ovarian tumorigenesis (ovarian tumorigenesis) gene (Non-Patent Document 1). APC protein normally binds to β-catenin together with TCF / LEF. Morin et al. (Non-Patent Document 6) reported that APC binds to ATP and forms a transcription factor complex. Protein inhibits transcriptional activity mediated by β-catenin and Tcf-4 in colon cancer It has been reported that β-catenin downregulates AP activation. C is crucial for the tumor suppressor effect, and this regulation is mediated by either APC or β-catenin. It has been shown that this can be avoided by mutations in either

[0008] Mutations in the β-catenin gene result in deletion of part of the N-terminus of β-catenin Serine and threonine residues that are cleaved or targeted by GSK3α / β or CKIα These mutant β-catenin proteins are either point mutations affecting the β-catenin residues or Proteins are refractory to phosphorylation and therefore avoid proteasomal degradation. β-catenin accumulates in affected cells. Stabilized and nuclear-localized β-catenin It is a hallmark of almost all cases of intestinal cancer (Non-Patent Document 7). Mutations in β-catenin that alter the phosphorylation site allow cells to selectively target β-catenin in the APC mediator. This leads to insensitivity to mediated downregulation of IL-1 and disruption of this mechanism contributes to colorectal tumorigenesis. It has been shown that this is an urgent issue (Non-Patent Document 8).

[0009] Other studies have also reported the detection of β-catenin mutations in various cancer cell lines. It has been reported (e.g., Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11; Non-Patent Document 1 2) Furthermore, β-catenin levels have been found to be abnormally high in melanoma cell lines. (See, for example, Non-Patent Document 13).

[0010] Similarly, other cancers, such as hepatocellular carcinoma (HCC), are also associated with the Wnt / β-catenin pathway. HCC is a complex and heterogeneous disease with over 660,000 new cases reported worldwide each year. Numerous reports have demonstrated that Wnt signaling components are involved in human HCC patients. It has been shown that activated Wnt signaling and Nuclear β-catenin correlates with disease recurrence and poor prognosis (Non-Patent Document 14). Intracranial β-catenin staining has been documented in 17–66% of HCC patients (non-patented). Reference 15; Non-patent document 16). Approximately 300 cases of H were created in a joint study with the University of Hong Kong. Merck's internal dataset of CC patient tumors shows that 50% of HCC patients External data indicate that Wnt signaling components are activated by β-carboxylates. Activating mutations in tenin were present in 13–40% of HCC patients, whereas Axin 1 or showed that inactivating mutations in 2 were present in only approximately 10% of HCC patients ( Non-patent document 17).

[0011] Preclinical studies have demonstrated that activation of the Wnt / β-catenin pathway is important in the development and maintenance of HCC. Liver-targeted disruption of APC in mice leads to the production of β -catenin signaling is activated, leading to the formation of HCC (Non-Patent Document 18) Overexpression of mutant β-catenin that lacks the GSK-3β phosphorylation site alone does not cause hepatocellular carcinoma. Although overexpression of tumorigenic mutant β-catenin is not sufficient for survival (Non-patent Document 19), Mice were tested for HC induced by DEN (diethylnitrosamine), a known carcinogen. It has been shown that HIV-1 increases susceptibility to C. Overexpression of the human Met receptor in mice (Tre-Met transgenic mouse model) 95% of HCC tumors generated by overexpression have β-catenin activating mutations (non-specific This finding is reflected in human disease, where the Wnt pathway mediates the Met signaling pathway during hepatocellular carcinogenesis. Furthermore, a high rate of β-catenin activating mutations is associated with H This is also seen in other transgenic mouse models of CC (16% in FGF19). β-catenin mutations, 55% in c-Myc, and H-Ras transgenic mice 41%) (Non-Patent Documents 22 and 23).

[0012] Preclinical studies have also shown that β-catenin is a valid target for HCC. β-catenin siRNA inhibits the proliferation and viability of human HCC cell lines Similarly, human HCC cell lines were treated with anti-Wnt-1 antibody or TCF (Zeng et al. 2007). Treatment with β-catenin antagonists inhibits apoptosis, c-Myc, cyclin D1, and This induces a reduction in survivin and inflammatory cytokine expression, resulting in the suppression of tumor growth in vivo (Non-Patent Reference 24; Non-patent reference 25).

[0013] Hepatocellular carcinoma (HCC) is a common invasive cancer for which there is no effective treatment. HCC cases The Wnt / β-catenin pathway is activated in a high proportion (approximately 50%) of cases. β-catenin (i.e., CTNNB1) or the β-catenin destruction complex (e.g., Ax Furthermore, the Wnt pathway as a target is a challenging area. Currently, there is no small molecule inhibitor available for drug treatment, and β-catenin is being investigated using RNAi-based therapeutic approaches. It has been shown that these proteins are attractive targets for further research (Non-Patent Document 26).

[0014] Gene expression by RNA interference (hereinafter referred to as "RNAi"), particularly CTNN Altering B1 gene expression is one approach to meet this need. by single-stranded RNA ("ssRNA") or double-stranded RNA ("dsRNA") molecules The short dsRNA molecules are also called "small interfering nucleic acids ("siNA") or " These siNs are called "small interfering RNAs" or "siRNAs" or "RNAi inhibitors." Silencing the expression of messenger RNA ("mRNA") that shares sequence homology with A This is commonly referred to as the RNA-induced silencing complex (RISC). by cleavage of the mRNA mediated by a siNA-containing endonuclease complex Cleavage of the target RNA is typically achieved by cleaving the target RNA using a sequence complementary to the guide sequence of the siNA duplex. RNA interference occurs in the center of the region (Non-Patent Document 27). For example, it may involve microRNA (or miRNA)-mediated gene silencing. This is probably due to translation inhibition or modulation of chromatin structure, which This is due to a cellular mechanism that either inhibits transcription of the target gene sequence or inhibits transcription of the target gene sequence (e.g., See, for example, Non-Patent Document 28; Non-Patent Document 29; Non-Patent Document 30; and Non-Patent Document 31. Despite considerable progress in the field of RNAi, the CTNNB1 gene Agents that can inhibit expression and diseases associated with CTNNB1 expression, such as cancer There is a need for agents that can treat [Prior art documents] [Non-patent literature]

[0015] [Non-Patent Document 1] Willert et al.,1998,Curr.Opin.Genet.Dev.8:95-102 [Non-patent document 2] Thompson MD.,2007,Hepatology May;45(5):1298-305 [Non-licensed document 3] Wang et al.,2008,Cancer Epidemiol.Biomarkers Prev.17(8):2101-8

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[0016] The present invention provides novel small interfering nucleic acid (siNA) molecules that modulate CTNNB1 expression. The challenge is to treat diseases that respond to modulation of CTNNB1 gene expression using Provide a solution to the problem.

[0017] The present invention relates to a method for detecting the expression of the CTNNB1 gene, particularly the CTNNB1 gene associated with cancer. and RNA interference (RNAi) using small nucleic acid molecules useful for modulation of The present invention provides compounds, compositions and methods useful for treating such conditions by

[0018] In particular, the present invention relates to small nucleic acid molecules, i.e., small interfering nucleic acid (siNA) molecules, e.g. For example, but not limited to, small interfering RNA (siRNA), double-stranded RNA (dsRNA), MicroRNAs (miRNAs), short hairpin RNAs (shRNAs), and circular RNAs A molecule, and the expression of the CTNNB1 gene and / or the CTNNB1 gene or used to modulate the expression of other genes involved in the pathway of activity. It features a method.

[0019] In one aspect, the present invention provides a method for detecting the expression of the CTNNB1 gene in a cell or a mammal. The present invention provides a double-stranded small interfering nucleic acid (siNA) molecule that inhibits It contains a sense strand and an antisense strand. The antisense strand is the CTNNB1 gene It contains a sequence complementary to at least a portion of the RNA associated with expression. The strand comprises a sequence complementary to the antisense strand. At least one of the strands is selected from the group of sequences consisting of SEQ ID NOs: 1 to 6374. In certain embodiments, the antisense oligonucleotide comprises a sequence of 15 nucleotides. The sense strand contains at least 15, 16 sequences complementary to the target sequences shown in Table 1a. In other embodiments, the amino acid sequence may contain 17, 18 or 19 nucleotides. In the same embodiment, the antisense strand is one of the antisense sequences shown in Table 1b. and a sequence of at least 15, 16, 17, 18, or 19 nucleotides of one of In some embodiments, the sense strand has at least one of the sense strand sequences shown in Table 1b. All contain sequences of 15, 16, 17, 18 or 19 nucleotides.

[0020] In some specific embodiments of this aspect of the invention, the antisense strand is a double-stranded small molecule comprising a modified sequence shown in Table 1c that has sequence complementarity to the sequence Also provided are interfering nucleic acid (siNA) molecules. In some embodiments, the sense strand is selected from the group consisting of: The modified sequence includes the sequence shown in

[0021] In certain embodiments, the present invention provides a method for the preparation of siNA comprising the steps of: each strand independently being 15 to 30 nucleotides in length; and the antisense strand : 5'-CUGUUGGAUUGAUUCGAAA-3' (SEQ ID NO: 5); 5'-ACGACUAGUUCAGUUGCUU-3' (SEQ ID NO: 194); 5'-GGAUGAUCCUAGCUAUCGU-3' (SEQ ID NO: 196); or 5'-CCAGGAUGAUCCUAGCUAU-3' (SEQ ID NO: 151) at least 15, 16, 17, 18, or 19 nucleotides having a complementary sequence to any of Double-stranded small interfering nucleic acids that modulate the expression of CTNNB1, which contain an oxidase (siNA) molecules are provided.

[0022] In some embodiments of the invention, the antisense strand of the siNA molecule is: 5'-UUUCGAAUCAAUCCAACAG-3' (SEQ ID NO: 4918); 5'-AAGCAACUGAACUAGUCGU-3' (SEQ ID NO: 5107); 5'-ACGAUAGCUAGGAUCAUCC-3' (SEQ ID NO: 5109); or 5'-AUAGCUAGGAUCAUCCUGG-3' (SEQ ID NO: 5064) and a sequence of at least 15, 16, 17, 18 or 19 nucleotides of do.

[0023] In some embodiments, the sense strand of a siNA molecule of the invention is: 5'-CUGUUGGAUUGAUUCGAAA-3' (SEQ ID NO: 5); 5'-ACGACUAGUUCAGUUGCUU-3' (SEQ ID NO: 194); 5'-GGAUGAUCCUAGCUAUCGU-3' (SEQ ID NO: 196); or 5'-CCAGGAUGAUCCUAGCUAU-3' (SEQ ID NO: 151) and a sequence of at least 15, 16, 17, 18 or 19 nucleotides of do.

[0024] In some embodiments, the siNA molecule of the invention comprises: 5'-CUGUUGGAUUGAUUCGAAA-3' (SEQ ID NO: 5) and 5'-UUU CGAAUCAAUCCAACAG-3' (SEQ ID NO: 4918); or 5'-ACGACUAGUUCAGUUGCUU-3' (SEQ ID NO: 194) and 5'-A AGCAACUGAACUAGUCGU-3' (SEQ ID NO: 5107); or 5'-GGAUGAUCCUAGCUAUCGU-3' (SEQ ID NO: 196) and 5'-A CGAUAGCUAGGAUCAUCC-3' (SEQ ID NO: 5109); or 5'-CCAGGAUGAUCCUAGCUAU-3' (SEQ ID NO: 151) and 5'-A UAGCUAGGAUCAUCCUGG-3' (SEQ ID NO: 5064) It includes any of the following.

[0025] In some embodiments, the siNA molecule of the invention comprises SEQ ID NOs: 6372 and 6374. It is something that

[0026] In some embodiments, the siNA molecule of the invention comprises SEQ ID NOs: 6370 and 6369. It is something that

[0027] In some embodiments, the siNA molecule of the invention comprises SEQ ID NOs: 2021 and 2068. It is something that

[0028] In some embodiments, the siNA molecule of the invention comprises SEQ ID NOs: 6372 and 6373. It is something that

[0029] In some embodiments, the siNA molecule of the invention comprises SEQ ID NOs: 2147 and 6368. It is something that

[0030] In some embodiments, the present invention provides a method for treating a skin condition comprising: (a) a double-stranded small interfering nucleic acid (siNA) of the present invention; (b) a cationic lipid having any one of Compounds Nos. 1 to 46 or any combination thereof compound; (c) cholesterol; (d) DSPC; and (e) PEG-DMG The present invention features a composition comprising:

[0031] In some embodiments, the present invention provides a method for treating a skin condition comprising: (a) a double-stranded small interfering nucleic acid (siNA) of the present invention; (b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene phen-1-amine; (c) cholesterol; (d) DSPC; and (e) PEG-DMG The present invention features a composition comprising:

[0032] In some embodiments, the present invention provides a method for treating a skin condition comprising: (a) a double-stranded small interfering nucleic acid (siNA) having SEQ ID NOs: 6372 and 6374; (b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene phen-1-amine; (c) cholesterol; (d) DSPC; and (e) PEG-DMG The present invention features a composition comprising:

[0033] In some embodiments, the present invention provides a method for treating a skin condition comprising: (a) a double-stranded small interfering nucleic acid (siNA) having SEQ ID NOs: 6370 and 6369; (b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene phen-1-amine; (c) cholesterol; (d) DSPC; and (e) PEG-DMG The present invention features a composition comprising:

[0034] In some embodiments, the present invention provides a method for treating a skin condition comprising: (a) a double-stranded small interfering nucleic acid (siNA) having SEQ ID NOs: 2021 and 2068; (b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene phen-1-amine; (c) cholesterol; (d) DSPC; and (e) PEG-DMG The present invention features a composition comprising:

[0035] In some embodiments, the present invention provides a method for treating a skin condition comprising: (a) a double-stranded small interfering nucleic acid (siNA) having SEQ ID NOs: 6372 and 6373; (b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene phen-1-amine; (c) cholesterol; (d) DSPC; and (e) PEG-DMG The present invention features a composition comprising:

[0036] In some embodiments, the present invention provides a method for treating a skin condition comprising: (a) a double-stranded small interfering nucleic acid (siNA) having SEQ ID NOs: 2147 and 6368; (b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene phen-1-amine; (c) cholesterol; (d) DSPC; and (e) PEG-DMG The present invention features a composition comprising:

[0037] In some embodiments, the composition of the present invention comprises any one of Compounds Nos. 1 to 46 in the following form: Molar ratio of: 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 ; and Cationic lipid / Cholesterol / PEG-DMG / DSPC 58 / 30 / 2 / 10 The present invention includes any cationic lipid having the formula:

[0038] In some embodiments, the compositions of the present invention comprise (13Z,16Z)-N,N-dimethyl -3-nonyldocosa-13,16-dien-1-amine, cholesterol, DSPC, and and PEG-DMG in a molar ratio of 50:30:10:2, respectively.

[0039] In some embodiments, the compositions of the present invention further comprise a cryoprotectant. In an embodiment, the cryoprotectant is sucrose, trehalose, raffinose, stachyrose, sugar, verbascose, mannitol, glucose, lactose, maltose, maltodextrin Ose-heptaose, dextran, hydroxyethyl, starch, insulin, sol Vitol, glycerol, arginine, histidine, lysine, proline, dimethyl sulfonate In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant is trehalose. The agent is a combination of sucrose and trehalose.

[0040] In some embodiments of the invention, all of the nucleotides of the siNA of the invention are unmodified. In another embodiment, one or more independent strands of either or both strands of the siNA molecule are Above (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 1, 29 or 30) of the nucleotide positions are modified. Modifications include the sugar chain of the nucleic acid. modifications, base modifications, backbone (internucleotide bond) modifications, non-nucleotide modifications, and / or In some specific cases, purine nucleotides and pyrimidine nucleotides may be used. nucleotides are differently modified. For example, purine nucleotides and pyrimidine nucleotides are differently modified. The nucleotides can be differently modified at the 2' position of the sugar chain (i.e., At least one purine at the 2' position of the glycan of one or a different chain is (Pyrimidines have different modifications). In some specific cases, purines may have one or both The first strand is unmodified, but the pyrimidines of one or both strands are modified. In certain other cases, the pyrimidines are unmodified in one or both strands, but The purines of one or both strands are modified. In some cases, at least one modified nucleotide The nucleotides are 2'-deoxy-2'-fluoronucleotides, 2'-deoxynucleotides In some cases, one or both of the At least five or more of the pyrimidine nucleotides in the first strand are all 2'-deoxyribonucleic acid. with either 2'-fluoro or all 2'-O-methyl pyrimidine nucleotides In some cases, at least five or more of the purine nucleotides on one or both strands may be More than that, all 2'-deoxy-2'-fluoro or all 2'-O-methylprop In certain instances, the siNA molecule may be any of the nucleotides described herein. 1 at the 5' end of the guide (antisense) strand, if it contains one or more of the modifications described , the nucleotides at positions 2 and 3 are unmodified.

[0041] In certain embodiments, the siNA molecules of the invention comprise one or both strands of a siNA molecule having one or more amino acids selected from the group consisting of 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, In another embodiment, the siNA has a 3' overhang of 2, 3, or 4 nucleotides. Preferably, the siNA molecule has no overhanging ends (i.e., has blunt ends). The antisense strand has a 3' overhang of two nucleotides on both the sense and antisense strands. The overhanging nucleotides may be modified or unmodified. Examples of modified nucleotides at the overhanging ends include Not specified, but may be 2'-O-alkyl nucleotides, 2'-deoxy-2'-fluoro nucleotides nucleotides, locked nucleic acid (LNA) nucleotides, or 2'-deoxynucleotides The overhanging nucleotides of the antisense strand are nucleotides within the CTNNB1 target sequence. Similarly, the overhanging end of the sense strand may contain a nucleotide complementary to the C It may contain nucleotides present within the TNNB1 target sequence. In the present invention, the siNA molecules have a 2'-O-alkyl (e.g., 2'- O-methyl nucleotides and two 3'-overhanging nucleotides on the sense strand. have two 3' overhanging nucleotides that are oxynucleotides. In the present invention, the siNA molecules have 2'-O-alkyl groups in both the antisense and sense strands. have two 3' overhanging nucleotides that are (e.g., 2'-O-methyl) nucleotides In certain embodiments, the 2'-O-alkyl nucleotides are 2'-O -methyluridine nucleotides. In some specific cases, the overhangs are It contains one or more phosphorothioate bonds between the nucleotides.

[0042] In some embodiments, the siNA molecules of the invention comprise a cap (referred to herein as a "terminal cap"). The cap may be present at the 5' end (also called a 5'-cap). ) at the 3' end (3'-cap) or at both ends (siN (e.g., the 5' and 3' ends of the sense strand of A).

[0043] In some embodiments, the siNA molecules of the invention comprise a 5'-terminal phosphodiester of the antisense strand. The phosphate group is oxidized to phosphate, diphosphate, or triphosphate. It could be.

[0044] When double-stranded, the siNA molecules of the invention can be symmetric or asymmetric. Each strand of such a double-stranded siNA independently contains a nucleoside of 3 to 30 nucleotides. Generally, each strand of a siNA molecule of the invention can range in length from about 15 to 30 (i.e., i.e., about 19, 20, 21, 22, 23, or 24 nucleotides in length.

[0045] The siNA molecules of the present invention, which are double-stranded or have a double-stranded structure, can be independently and 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, Generally, the siNs of the invention contain 27, 28, 29 or 30 base pairs. The double-stranded structure of A is 15-30, more commonly 18-25, and even more commonly 19- 24, most commonly 19–21 base pairs in length.

[0046] In certain embodiments, the compound has a sense strand and an antisense strand and has the formula (A):

[0047] [ka] a double-stranded small interfering nucleic acid (siNA) molecule comprising: wherein the upper strand is the sense strand of the double-stranded nucleic acid molecule and the lower strand is the The antisense strand is SEQ ID NO: 4918, SEQ ID NO: 5107 , SEQ ID NO: 5109, or at least 15, 16, 17, 18 of SEQ ID NO: 5064 or a sequence of 19 nucleotides, wherein the sense strand is comprising a sequence complementary to each N is independently an unmodified or chemically modified nucleotide or a non-nucleotide; each B is an end cap that is present or absent; (N) represents an overhanging nucleotide (each independently unmodified or chemically modified); [N] represents a nucleotide that is a ribonucleotide; X1 and X2 are independently an integer of 0 to 4; X3 is an integer between 15 and 30; X4 is an integer from 9 to 30; X5 is an integer between 0 and 6, and the sum of X4 and X5 is between 15 and 30.

[0048] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (a)N X4 one or more pyrimidine nucleotides at positions 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, 30, 31, 2'-Fluoronucleotides, 2'-O-Alkylnucleotides, 2'-Deoxynucleotides nucleotides, ribonucleotides, or any combination thereof; (b)N X4 one or more purine nucleotides at positions 1 and 2 are independently 2'-deoxy-2' -Fluoronucleotides, 2'-O-alkylnucleotides, 2'-deoxynucleotides nucleotides, ribonucleotides, or any combination thereof; (c)N X3 one or more pyrimidine nucleotides at positions 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, 30, 31, 2'-Fluoronucleotides, 2'-O-Alkylnucleotides, 2'-Deoxynucleotides nucleotides, ribonucleotides, or any combination thereof; and (d)N X3 one or more purine nucleotides at positions 1 and 2 are independently 2'-deoxy-2' -Fluoronucleotides, 2'-O-alkylnucleotides, 2'-deoxynucleotides , which is a ribonucleotide, The invention features a double-stranded small interfering nucleic acid (siNA) of formula (A).

[0049] Furthermore, the present invention provides a method for the preparation of a nucleic acid molecule comprising the double-stranded nucleic acid molecules described herein, in a pharmaceutically acceptable carrier. The composition may also include a carrier or diluent.

[0050] Administration of the composition may be performed in vitro or in vivo to introduce the nucleic acid into the desired target cells. This can be done by known methods.

[0051] Commonly used techniques for introducing the nucleic acid molecules of the present invention into cells, tissues and organisms include These include the use of a variety of carrier systems, reagents and vectors. Non-limiting examples of such carrier systems include conjugates, nucleic acid-lipid particles, lipid nanoparticles, and the like. Lipoproteins (LNPs), liposomes, lipoplexes, micelles, virosomes, virus-like particles (V LP), nucleic acid complexes, and mixtures thereof.

[0052] The compositions of the present invention may be formulated as aerosols, dispersions, solutions (e.g., injectable solutions), creams, or the like. Such compositions may be in the form of an ointment, an ointment, a tablet, a powder, a suspension, or the like. It can be administered in any manner, for example, orally, sublingually, bucally, parenterally, nasally or topically. In some embodiments, the composition is aerosolized and delivered by inhalation.

[0053] The molecules and compositions of the present invention have utility in a wide range of therapeutic applications. Thus, another aspect of the present invention relates to the use of the compounds and compositions of the present invention in treating a subject. Therefore, the present invention provides a method for treating cancer caused by the action of CTNNB1 or by the reduction of said action. A method of treating a subject (such as a human) suffering from a disease condition mediated by a virus, comprising administering to the subject a and administering an effective amount of a double-stranded small interfering nucleic acid (siNA) molecule of the invention to a subject. In certain embodiments, the condition is cancer.

[0054] These and other aspects of the present invention will become apparent upon reference to the following detailed description and accompanying drawings. Additionally, any of the methods or compositions described herein may be used in combination with any of the methods or compositions described herein. It should be understood that the present invention may be practiced with respect to any other method or composition, and that different embodiments may be combined. It is expected that this will also be possible.

[0055] Furthermore, throughout this specification, specific references are made to various aspects of the present invention to explain and more fully illustrate them. These references cited herein include patents, patent applications, and other documents. each of which is incorporated herein by reference in its entirety (including any drawings). [Brief explanation of the drawings]

[0056] [Figure 1] A diagram of a proposed, non-limiting mechanism of target RNA degradation involved in RNAi is shown. Double-stranded RNA (dsRNA) generated from foreign single-stranded RNA (e.g., viral, transposon, or other exogenous RNA) by RNA-dependent RNA polymerase (RdRP) activates the DICER enzyme, thereby generating siNA duplexes. Alternatively, synthetic or expressed siNAs can be directly introduced into cells by appropriate means. An active siNA complex is formed, which recognizes the target RNA and either leads to degradation of the target RNA by the RISC endonuclease complex or leads to synthesis of additional RNA by RNA-dependent RNA polymerase (RdRP), which activates DICER and generates additional siNA molecules, thereby amplifying the RNAi response. [Figure 2]Non-limiting examples of chemically modified siNA constructs of the present invention are shown using a generalized structure of a representative siNA duplex. The specific modifications shown can be used alone or in combination with other modifications shown, in addition to other modifications and features described herein for any siNA molecule of the present invention. In the diagram, N represents any nucleotide, which may be a non-nucleotide as described herein. The upper strand, having B-NX3-(N)X2B-3', is the sense (or passenger) strand of the siNA, and the lower strand, having B(N)X1-NX4-[N]X5-5', is the antisense (or guide) strand of the siNA. Nucleotides (or non-nucleotides) in the internal portion of the sense strand are designated NX3, and nucleotides (or non-nucleotides) in the internal portion of the antisense strand are designated NX4. Nucleotides (or non-nucleotides) in the internal portion are generally base-paired between the two strands, although in some embodiments they may not be base-paired (e.g., have a mismatch or gap). Overhanging nucleotides (or non-nucleotides) are indicated in parentheses (N). Nucleotides at the 5'-end of the antisense strand are indicated as [N]. Terminal caps may be present at the 5'- and / or 3'-end of the sense strand, and may also be 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 may vary depending on the presence of overhanging nucleotides. In certain embodiments, X1 and X2 are independently integers between 0 and 4; X3 is an integer between 15 and 30; X4 is an integer between 9 and 30; and X5 is an integer between 0 and 6, with the sum of X4 and X5 being 15 to 30. Various modifications to nucleotides in the sense and antisense strands of siNA constructs are shown. The (N) overhanging nucleotide positions may be chemically modified as described herein (e.g., 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-deoxy, LNA, universal base, etc.), either derived from the corresponding target nucleic acid sequence or not. Additionally, the illustrated constructs may contain phosphorothioate linkages as described herein.For example, a phosphorothioate bond can be present between any N, (N), and / or [N] positions. Such phosphorothioate incorporation can be used between purine "R" and pyrimidine "Y" positions, or for stabilization of pyrimidine bonds in general. Furthermore, although not shown, the illustrated constructs may contain a ribonucleotide at the 9th position from the 5' end of the sense strand or the 11th position relative to the 5' end of the guide strand (the 11th nucleotide position counting inward from the 5' end of the guide strand). Similarly, the antisense strand may contain a ribonucleotide at the 14th position from the 5' end, or alternatively, may be selected or designed such that a 2'-O-alkyl nucleotide (e.g., 2'-O-methyl purine) is not present at this position. Furthermore, although not shown, the 5'-terminal position of the antisense strand may contain a terminal phosphate group as described herein. The antisense strand generally contains a sequence complementary to any target nucleic acid sequence of the present invention (such as those set forth in Table 1a herein). [Figure 3] Non-limiting examples of specific combinations of modifications applied to the representative siNA duplex depicted in Figure 2 are provided. The table below the representative structure illustrates specific combinations of (N)X1, (N)X2, NX3, NX4, and / or [N]X5 nucleotide (and potentially non-nucleotide) positions. For example, five or more (e.g., 5, 6, 7, 8, 9, or 10 or more) NX3 and five or more (e.g., 5, 6, 7, 8, 9, or 10 or more) NX4 pyrimidine "Y" and purine "R" nucleotide combinations are illustrated, each of which may independently have a specific (N)X1 and / or (N)X2 substitution as shown, in addition to any phosphorothioate substitutions that may be present. The [N] nucleotide in the 5'-terminal antisense strand is generally a ribonucleotide, but may be modified or unmodified depending on whether it is a purine "R" nucleotide or a pyrimidine "Y" nucleotide. [Figure 4A]Non-limiting examples of various siNA constructs of the present invention are shown below. The criteria for the representative structures shown in Figures 2 and 3 can be applied to any of the structures shown in Figures 4A-C. The example shown in Figure 4A (constructs 1, 2, and 3) has 19 representative base pairs; however, various embodiments of the present invention can include any number of base pairs as described herein. The region in brackets represents a nucleotide overhang, e.g., 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 include a polynucleotide or non-nucleotide linker, which can be designed as a biodegradable linker. In one embodiment, the loop structure shown in construct 2 can include 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 make construct 2 using the same principles, where a linker is used to generate active siNA construct 2 in vivo and / or in vitro, and another biodegradable linker can be used to generate active siNA construct 1 in vivo and / or in vitro. Thus, the stability and / or activity of the siNA construct can be modulated based on the design of the siNA construct for in vivo or in vitro and / or in vitro use. [Figure 4B] Non-limiting examples of various siNA constructs of the present invention are shown. The criteria for the representative structures shown in Figures 2 and 3 can be applied to any of the structures shown in Figures 4A-C. The example shown in Figure 4B represents different types of double-stranded nucleic acid molecules of the present invention (e.g., microRNAs) that can include overhangs, bulges, loops, and stem-loops resulting from partial complementarity. Such motifs with bulges, loops, and stem-loops are generally characteristic of miRNAs. The bulges, loops, and stem-loops can result from any degree of partial complementarity (e.g., mismatches or bulges of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides within one or both strands of a double-stranded nucleic acid molecule of the present invention). [Figure 4C]Non-limiting examples of various siNA constructs of the invention are shown below. The criteria for the representative structures shown in Figures 2 and 3 can be applied to any of the structures shown in Figures 4A-C. The example shown in Figure 4C depicts a model double-stranded nucleic acid molecule of the invention, comprising a 19-base pair duplex of two sequences of 21 nucleotides with a two-nucleotide 3'-overhang. The top strand (1) represents the sense strand (passenger strand), the middle strand (2) represents the antisense strand (guide strand), and the bottom strand (3) represents the target polynucleotide sequence. The two-nucleotide overhang (NN) can comprise a sequence derived from the target polynucleotide. For example, the 3'-(NN) sequence of the guide strand can be complementary to the 5'-[NN] sequence of the target polynucleotide. Alternatively, 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 overhang (NN) is not derived from the target polynucleotide sequence; for example, the 3'-(NN) sequence of the guide strand is not complementary to the 5'-[NN] sequence of the target polynucleotide, and the 5'-(NN) sequence of the passenger strand may contain a sequence different from the 5'-[NN] sequence of the target polynucleotide. In further embodiments, the (NN) nucleotides (if any) are chemically modified, e.g., 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or other modifications described herein. Additionally, the passenger strand may contain a ribonucleotide at the N position of the passenger strand. In the exemplary 19-base-paired 21-mer duplex shown, the N position may be 9 nucleotides inward from the 3' end of the passenger strand. However, for duplexes of different lengths, the N position is determined by counting 11 nucleotide positions inward from the 5' end of the guide strand relative to the 5' end of the guide strand and selecting the corresponding base-paired nucleotide of the passenger strand. Cleavage by Ago2 occurs between positions 10 and 11, as indicated by the arrows. In a further embodiment, two ribonucleotides, NN, are present at positions 10 and 11 by counting 10 and 11 nucleotide positions inward from the 5' end of the guide strand relative to the 5' end of the guide strand and selecting corresponding base-pairing nucleotides in the passenger strand. [Figure 5] For example, various stabilizing chemistries (1-10) that can be used to stabilize the 5' and / or 3' ends of siNA sequences of the invention are shown, including, but not limited to, (1) [3-3']-inverted deoxyribose; (2) deoxyribonucleotides; (3) [5'-3']-3'-deoxyribonucleotides; (4) [5'-3']-ribonucleotides; (5) [5'-3']-3'-O-methylribonucleotides; (6) 3'-glyceryl; (7) [3'-5']-3'-deoxyribonucleotides; (8) [3'-3']-deoxyribonucleotides; (9) [5'-2']-deoxyribonucleotides; and (10) [5-3']-dideoxyribonucleotides (where X=O). In addition to the modified and unmodified backbone chemistries shown, such chemistries can be combined with various sugar and base nucleotide modifications described herein. [Figure 6] A non-limiting example of a strategy used to identify chemically modified siNA constructs of the present invention that are nuclease-resistant but retain the ability to mediate RNAi activity is shown below. Chemical modifications are introduced into the siNA construct based on informed design parameters (e.g., 2'-modifications, base modifications, backbone modifications, end-cap modifications, etc.). The modified constructs are tested in appropriate systems (e.g., human serum (as indicated) for nuclease resistance or animal models for PK / delivery parameters). In parallel, the siNA constructs are tested for RNAi activity, for example, in cell culture systems (luciferase reporter assay) and / or against endogenous mRNA. Lead siNA constructs with specific characteristics but retaining RNAi activity can then be identified, further modified, and assayed again. Using this same approach, siNA-conjugate molecules with improved pharmacokinetic profiles, delivery, and RNAi activity can be identified. [Figure 7] 1 shows non-limiting examples of phosphorylated siNA molecules of the invention, including linear double-stranded constructs as well as asymmetric derivatives thereof. [Figure 8] 1 shows non-limiting examples of chemically modified terminal phosphate groups of the present invention. [Figure 9] 1 shows non-limiting examples of cholesterol-linked phosphoramidites that can be used to synthesize cholesterol-conjugated siNA molecules of the invention. An example is shown in which the cholesterol moiety is linked to the 5' end of the sense strand of the siNA molecule. [Figure 10] 5' and 3' inverted abasic caps linked to a nucleic acid strand represent one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0057] A. Terms and Definitions As used in this application, the following terms and definitions apply.

[0058] The term "basic" as used herein has its art-recognized meaning. The term generally refers to a sugar moiety in which the 1' nucleobase is missing or A sugar moiety that has a hydrogen atom (H) or other non-nucleobase chemical group in place of a nucleobase. See, for example, Adamic et al., U.S. Patent No. 5,998,203. In certain embodiments, the abasic moiety of the present invention is ribose, deoxyribose, or dideoxyribose. It is a bose sugar chain.

[0059] The term "acyclic nucleotide" as used herein refers to a nucleotide that is generally accepted in the art. The term generally refers to any nucleoside having an acyclic ribose sugar chain. The carbon / carbon bond or carbon / oxygen bond (if any) of the ribose, e.g., Or it refers to a combination that does not exist in nucleotides.

[0060] The term "alkyl" as used herein has its art-recognized meaning. The term generally refers to saturated or unsaturated hydrocarbons, e.g., straight chain, branched chain, It refers to alkenyl, alkynyl and cyclic groups, but excludes aromatic groups. However, alkyl also refers to non-aromatic heterocyclic groups. Preferably, the alkyl group is It has 1 to 12 carbon atoms, more preferably 1 to 7 carbon atoms, and even more preferably is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted. If substituted, the substituent(s) are preferably hydroxyl alkoxy, ... or NR1R2 (wherein R1 and R2 are independently H or C1-C4 alkyl). (There is.)

[0061] The phrase "agents that interfere with cell cycle checkpoints" refers to agents that interfere with cell cycle checkpoints. inhibits protein kinases that transmit signals, thereby preventing cancer cells from responding to DNA-damaging agents A compound that sensitizes to

[0062] The phrase "agents that interfere with receptor tyrosine kinases (RTKs)" refers to agents that inhibit and Thus, it refers to compounds that inhibit mechanisms involved in carcinogenesis and tumor progression.

[0063] The term "androgen receptor modulator" refers to an androgen receptor modulator that modulates the receptor for androgens, regardless of mechanism. A compound that prevents or inhibits binding to the body.

[0064] The term "angiogenesis inhibitor" refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. .

[0065] The term "aryl" as used herein has its art-recognized meaning. The term generally refers to an aromatic compound having at least one ring with a conjugated π electron system. aromatic groups, including carbocyclic aryl, heterocyclic aryl and biaryl groups (all of which Preferred substituent(s) for the aryl group include: is halogen, trihalomethyl, hydroxyl, SH, OH, cyano, C1-C4 alkoxy oxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, NH2, etc. and NR1R2 groups (wherein R1 and R2 are independently H or C1-C4 alkyl). (It is a rule).

[0066] The term "alkylaryl" as used herein is an alkyl group generally accepted in the art. The term generally refers to a group that is covalently linked to an aryl group (as described above). A carbocyclic aryl group is an aromatic ring atom that is bonded to an alkyl group (as described above). are all carbon atoms. The carbon atoms may be substituted. The aromatic ring has 1 to 3 heteroatoms as ring atoms, and the remaining ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen; Examples of heterocyclic aryl groups containing such heteroatoms include furanyl, thienyl, pyridinyl, and the like. pyrrolyl, N-lower alkylpyrrolo, pyrimidyl, pyrazinyl, imidazolyl, etc. Preferably, the alkyl group is a C1 to C4 It is an alkyl group.

[0067] The term "amide" as used herein has its art-recognized meaning. The term generally refers to -C(O)-NH-R, where R is alkyl, The aryl group is either aryl, alkylaryl, or hydrogen.

[0068] The term "antisense region" as used herein means a region generally accepted in the art. With respect to exemplary nucleic acid molecules of the present invention, the term refers to a target nucleic acid sequence. The term "antisense oligonucleotide" refers to the nucleotide sequence of a siNA molecule that is complementary to the target nucleic acid. The sense region comprises a nucleic acid sequence having complementarity to the sense region of the siNA molecule. In one embodiment, the antisense region of the siNA molecule may be an antisense strand or Also called a guide chain.

[0069] The phrase "asymmetric hairpin" refers to a region having an antisense region and a nucleotide or non-nucleotide sequence. and a sense region, wherein the sense region is an antisense region. It has enough complementary nucleotides to base pair with the base region to form a double strand with a loop. A linear siNA molecule that contains fewer nucleotides than the antisense region, but does not exceed the target nucleotide range. For example, the asymmetric hairpin siNA molecules of the present invention can be used in cells or in vitro. A length sufficient to mediate RNAi (e.g., about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29If an antisense region having a length of about 30 nucleotides; About 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12) nuclei a loop region containing a nucleotide and about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 a sense region having 9, 20, 21, 22, 23, 24, or 25 nucleotides; The asymmetric hairpin siNA molecule may also contain a 5'-terminal phosphate group ( The asymmetric hairpin siNA molecule may contain a nucleotide sequence (which may be chemically modified). The loop portion may be a nucleotide, a non-nucleotide, a linker molecule, or a conjugate molecule. (described herein).

[0070] The term "biodegradable" as used herein means a material that is biodegradable and has the same meaning as generally accepted in the art. The term generally refers to degradation in biological systems, e.g., enzymatic degradation or refers to chemical decomposition.

[0071] The term "biodegradable linker" as used herein refers to a linker that is generally accepted in the art. With respect to exemplary nucleic acid molecules of the present invention, the term refers to the association of one molecule with another molecule. A linker molecule that is designed to link to a target molecule and is susceptible to degradation in biological systems. The linker may be a nucleic acid-based linker or a non-nucleic acid-based linker. For example, The biodegradable linker connects the ligand or biologically active molecule to the siNA molecule of the invention. Alternatively, biodegradable linkers can be used to attach siNA molecules of the invention. A biodegradable linker can be used to link the sense and antisense strands of a molecule. The stability of the molecule can be modulated for specific purposes (e.g., delivery to specific tissues or cell types). The stability of nucleic acid-based biodegradable linker molecules is determined by the ability to withstand various chemicals. ribonucleotides, deoxyribonucleotides, as well as chemically modified nucleotides. Reotide (2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2' -C-allyl, 2'-O-allyl, and other 2'- or base-modified nucleotides The biodegradable nucleic acid linker molecule can be modulated by using a combination of: Dimers, trimers, tetramers or longer nucleic acid molecules (e.g., about 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 2 0 nucleotide long oligonucleotides), and phosphorus-based linkages (e.g., phospho containing a single nucleotide with a phosphodiester bond (e.g., thiamidate or phosphodiester bond) The biodegradable nucleic acid linker molecule may be a nucleic acid backbone, a nucleic acid sugar chain, or a nucleic acid base. It may also include modifications.

[0072] The phrase "biologically active molecule" as used herein refers to a molecule that is generally accepted in the art. With respect to exemplary nucleic acid molecules of the present invention, the term refers to a biological may induce or improve the response and / or pharmacokinetic characteristics of other biologically active molecules. refers to compounds or molecules that can modulate the physiological and / or pharmacodynamic properties of a biological Examples of therapeutically active molecules include those that, alone or in combination with other molecules (such as, but not limited to, therapeutically active molecules). molecules, e.g., antibodies, cholesterol, hormones, antivirals, peptides, proteins Proteins, chemotherapy drugs, small molecules, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides Nucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, polymerases Polyamines, polyamides, polyethylene glycols, other polyethers, 2-5A chimeras, In combination with siNA, dsRNA, allozymes, aptamers, decoys and their analogues Examples of such siNA molecules include:

[0073] The term "biological system" as used herein means a biological system generally recognized in the art. The term generally refers to a biological source, such as, but not limited to, a human or animal. The term refers to a substance, in purified or unpurified form, derived from a system containing the components necessary for RNAi activity. Thus, the term includes, for example, cells, tissues, specimens, or other components. The term also includes any organism or extract thereof derived from a biological source. It also includes constituent substances.

[0074] The term "blunt end" as used herein has its generally accepted meaning in the art. With respect to exemplary nucleic acid molecules of the invention, the term refers to a nucleic acid molecule that does not have overhanging nucleotides. The terminus of a double-stranded siNA molecule. For example, the two ends of a blunt-ended double-stranded siNA molecule. The strands are aligned with each other and have matching base pairs, with no overhanging nucleotides at the ends The siNA duplex molecules of the present invention may be blunt-ended at one or both ends of the duplex (e.g., The 5' end of the antisense strand, the end located at the 5' end of the sense strand, or both ends of the duplex ) may be included.

[0075] The term "cap" (also referred to herein as "end cap") is used herein to refer to a When used, the meaning generally accepted in the art is indicated. Exemplary Nucleic Acid Molecules of the Invention With respect to the term, the term may be incorporated into one or more termini of one or more nucleic acid molecules of the present invention. A terminal modification refers to a moiety that may be a chemically modified nucleotide or a non-nucleotide. protects nucleic acid molecules from exonuclease degradation and facilitates intracellular delivery and / or The cap may be at the 5' end (5'-cap) or at the 3' end. (3'-cap), and may be present at both ends of any nucleic acid molecule of the present invention. The cap may be at the 5' end, 3' end and / or 5' end of the sense strand of the nucleic acid molecule of the present invention. Furthermore, caps may be present at the 3' and 4' ends of the nucleic acid molecules of the present invention. It may be present at the 3' end of the sense strand. In a non-limiting example, as a 5'-cap Examples include, but are not limited to, LNA; glyceryl; inverted deoxy abasic residues (moieties); 4',5 '-methylene nucleotide; 1-(β-D-erythrofuranosyl) nucleotide, 4'- Thionucleotides; Carbocyclic nucleotides; 1,5-anhydrohexitol nucleotides L-nucleotides; α-nucleotides; modified base nucleotides; phosphorodithioates threo-pentofuranosyl nucleotide; acyclic 3',4'-seconucleotide ;Acyclic 3,4-dihydroxybutyl nucleotide;Acyclic 3,5-dihydroxybutyl nucleotide nucleotide; 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3 '-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol Aminohexyl phosphate; 3'-phosphoramidate; Hexyl phosphate; Aminohexyl phosphate sulphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; or a bridging or non-bridging methylphosphonate moiety. Non-limiting examples of caps include, but are not limited to, LNA; glyceryl; inverted deoxyribonucleic acid; Base residue (part); 4',5'-methylene nucleotide; 1-(β-D-erythrofuran 4'-Thionucleotide; Carbocyclic nucleotide; 5'-Amino- Alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl Pyridyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate phosphate;hydroxypropyl phosphate;1,5-anhydrohexitol nucleotide ;L-nucleotides;α-nucleotides;Modified base nucleotides;Phosphorodithioates ;Threo-pentofuranosyl nucleotide;Acyclic 3',4'-seconucleotide;3, 4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5 '-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramid 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino ;Bridged and / or non-bridged 5'-phosphoramidates;Phosphorothioates bridged or non-bridged methylphosphonates; and 5'-mercapto moieties (see Beaucage and and Iyer, 1993, Tetrahedron 49, 1925; see citations (The caps are incorporated herein by reference in their entirety.) Figure 5 shows non-limiting examples of various caps. Here are a few:

[0076] The term "cell" as used herein has its art-recognized meaning. With respect to the exemplary nucleic acid molecules of the present invention, the term is used in its ordinary biological sense. It does not refer to the entire multicellular organism (e.g., it does not refer specifically to humans). ). Cells can be derived from organisms such as birds, plants, and mammals, e.g., humans, cattle, sheep, etc. The cells may be those present in mammals, apes, monkeys, pigs, dogs, and cats. systems (e.g., bacterial cells) or eukaryotic systems (e.g., mammalian or plant cells). The cells may be of somatic or germline origin, totipotent or pluripotent, dividing, Alternatively, the cells may be gametes or embryos, stem cells, or fully differentiated. The cell may be derived from or constituted by the cells.

[0077] The phrase "chemical modification" as used herein has its art-recognized meaning. With respect to exemplary nucleic acid molecules of the present invention, the term refers to common naturally occurring siRNA or refers to any modification of the chemical structure of a nucleotide that differs from that of RNA nucleotides. "Chemical modifications" include modifications such as sugar chains, salts, etc., as described herein or known in the art. Additions, substitutions, or modifications of naturally occurring siRNA or RNA at groups or internucleotide linkages In certain embodiments, the term "chemical modification" refers to a specific biological Certain forms of RNA that occur naturally in a system (e.g., 2'-O-methyl modified or It may also refer to a modified phospholipid.

[0078] The term "CTNNB1" refers to catenin (cadherin-binding protein) β1, which refers to CTNNB1 protein, CTNNB1 peptide, CTNNB1 polypeptide, CT NNB1 regulatory polynucleotides (e.g., CTNNB1 miRNA and siNA) The coding gene, mutant CTNNB1 gene, and CTNNB1 gene splat Is variants, and gene expression and / or activity involved in the CTNNB1 pathway Therefore, each of the genes described herein relating to the term "CTNNB1" Embodiments are encompassed by the term "CTNNB1" (as that term is defined herein). All proteins, peptides, polypeptides, and / or polynucleotide molecules In general, such gene targets are generally referred to herein as "targets." " sequences (e.g., target sequences shown in Table 1a).

[0079] The terms "complementary" or "complementary" as used herein are generally used in the art to refer to With respect to exemplary nucleic acid molecules of the present invention, the term generally refers to: Between one nucleic acid sequence and another, either traditional Watson-Crick or as described herein the formation or presence of hydrogen bond(s) through either the In reference to the nucleic acid molecules of the present invention, the term "binding of a nucleic acid molecule with its complementary sequence" refers to a binding of a nucleic acid molecule with its complementary sequence. The combined free energy indicates that the relevant function of the nucleic acid (e.g., RNAi activity) proceeds. Measurement of the binding free energy of nucleic acid molecules is well known in the art. It is well known (e.g., Turner et al., 1987, CSH Symp. Quan t.Biol.LII pp.123-133;Frier et al., 1986, Proc.N at. Acad. Sci. USA 83:9373-9377; Turner et al., 198 7, J. Am. Chem. Soc. 109:3783-3785). Complementary means that all of the contiguous residues of a nucleic acid sequence have the same number of contiguous residues in a second nucleic acid sequence. Partial complementarity means that the residues will hydrogen bond with the corresponding residues. base-pairing nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or any further mismatches, non-nucleotide linkers or non-base paired nucleotides) to the nucleic acid The sense strand or sense region of the nucleic acid molecule may be contained within the nucleic acid molecule. between the sense strand or antisense region, or between a nucleic acid molecule and the corresponding target nucleic acid molecule Bumps, loops, or overhangs generated between the sense and antisense strands may also be present. Such partial complementarity can be expressed as % complementarity, and can be expressed as the percentage of non-base-paired nucleotides. , i.e., approximately 50%, 60%, 70%, depending on the total number of nucleotides involved. 80%, 90%, etc. Such partial complementarity is desired for nucleic acid molecules (e.g., siNAs) is acceptable to the extent that it maintains its function (e.g., the ability to mediate sequence-specific RNAi). do.

[0080] The terms "composition" or "formulation" as used herein refer to any combination of compounds commonly accepted in the art. These terms generally refer to cells or subjects (e.g., humans) that are in a form suitable for administration (e.g., systemic or local administration) to a patient, e.g., a pharmaceutically acceptable carrier. Suitable forms include, in part, compositions or formulations with carriers or diluents that can be used in the preparation of pharmaceutical compositions. Dependent on the use or route of entry (e.g., oral, transdermal, inhalation, or injection). The composition or formulation is preferably administered to a target cell (i.e., a cell in which a negatively charged nucleic acid is desirable for delivery). For example, a composition injected into the bloodstream should Other factors are known in the art and may affect the toxicity and composition of the compound. As used herein, the term "anticoagulant" includes considerations such as the form of the substance or formulation that would prevent it from functioning. Pharmaceutical formulations include formulations for human and veterinary use. Non-limiting examples of agents suitable for use in the formulation include: lipid nanoparticles (e.g., Semple et al., 2010, Nat Biotechnol., Feb;28(2):172-6 ); P-glycoprotein inhibitors (e.g., Pluronic P85); biodegradable polymers (e.g., Poly(DL-lactide-co-glycolide) microspheres for broadcasting (Emery ch, DF et al., 1999, Cell Transplant, 8, 47-58); and and loaded nanoparticles (made of polybutylcyanoacrylate). Other non-limiting examples of delivery strategies for nucleic acid molecules include those described by Boado et al., 998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 199 9, FEBS Lett., 421, 280-284; Pardridge et al., 1995 ,PNAS USA.,92,5592-5596;Boado,1995,Adv.D rug Delivery Rev.,15,73-107;Aldrian-Herr ada et al., 1998, Nucleic Acids Res., 26, 4910-491 6; and Tyler et al., 1999, PNAS USA., 96, 7053-7058 The term "pharmaceutically acceptable composition" or "pharmaceutically acceptable "Possible formulations" refer to formulations that allow for effective distribution of the nucleic acid molecules of the present invention in the body location most suitable for their desired activity. It may refer to a composition or formulation that allows fabrication.

[0081] The term "cytotoxic / cytostatic" refers to drugs that act primarily by directly interfering with the function of cells. , compounds that cause cell death or inhibit cell proliferation, or inhibit cell mitosis ( compounds that inhibit or prevent tumor necrosis (mytosis), including alkylating agents, tumor necrosis factors, molecules, intercalators, hypoxia-activated compounds, microtubule inhibitors / microtubule stabilizers, mitotic molecules Inhibitors of cleaved kinesins and histone deacetylases, involved in mitotic progression Kinase inhibitors, antimetabolites; biological response modifiers; hormone / antihormonal therapy agents; Hematopoietic growth factors, monoclonal antibody-targeted therapeutic agents, topoisomerase inhibitors , proteasome inhibitors and ubiquitin ligase inhibitors.

[0082] The term "estrogen receptor modulator" refers to an agent that modulates the receptors of estrogens, regardless of mechanism. A compound that prevents or inhibits binding to the body.

[0083] The term "gene" or "target gene" as used herein refers to a gene that is commonly used in the art. The term generally refers to the coding sequences necessary to produce a polypeptide. It refers to a nucleic acid (e.g., DNA or RNA) sequence that includes the length of a portion or the entire length of the sequence. The target gene may also include UTRs or non-coding regions of the nucleic acid sequence. In addition, the gene or target gene may be a functional RNA (fRNA) or a non-coding RNA. (ncRNA), e.g., small temporal RNA (stRNA ), microRNA (miRNA), small nuclear RNA (snRNA), small interfering RNA A (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), It may encode transfer RNA (tRNA) and its precursor RNA. Such non-coding RNAs are RNAs that are involved in functional or regulatory cellular processes. A target of siNA-mediated RNA interference in modulating the activity of ncRNA Therefore, abnormal fRNAs that cause disease may be involved. The activity of the ncRNA or ncRNA can be modulated by the siNA molecules of the invention. siNA molecules targeting fRNA and ncRNA have been shown to printing, transcription, translation, or nucleic acid processing (e.g., transamination, methylation) by intervening in cellular processes such as transcription, transcription, and transcriptional regulation of the subject, organism, or cell. The target gene can be used to manipulate or modify the genetic or phenotype of a cell. Genes that cause the infection, endogenous genes, transgenes, or exogenous genes (those present in the cell after infection) The target gene may be a gene of a pathogen (e.g., a virus) that is causing the target gene. derived from any living organism, for example, a plant, animal, protozoan, virus, bacterium, or fungus Non-limiting examples of plants include monocotyledons, Plants include dicotyledons, or gymnosperms. Non-limiting examples of animals include vertebrates. Non-limiting examples of fungi include molds or yeasts. For an overview, see, for example, Snyder and Gerstein, 2003, See Science, 300, 258-260.

[0084] The term "HMG-CoA reductase inhibitors" refers to 3-hydroxy-3-methylglutarinin The term HMG-CoA reductase inhibitor refers to an inhibitor of HMG-CoA reductase. As used herein, refers to any pharmaceutically acceptable lactone and open-acid form. form (i.e., when the lactone ring is opened to form its free acid) and HMG and salt and ester forms of compounds having -CoA reductase inhibitory activity. Therefore, the use of such salts, esters, open acid and lactone forms is within the scope of the present invention. It can be enjoyed.

[0085] The term "homologous sequence" as used herein has its generally accepted meaning in the art. The term generally refers to one or more polynucleotide sequences, e.g., genes, Nucleotides shared by gene transcripts and / or non-coding polynucleotides, etc. For example, homologous sequences may be sequences that contain related but different proteins (gene families). Different members of the genome, different protein epitopes, different protein isoforms, etc. Nucleotides shared by two or more genes or completely different genes that code for Homologous sequences can be sequences of two or more non-coding polynucleotides, e.g. For example, non-coding DNA or RNA, regulatory sequences, introns, and control sites of transcription or The homologous sequence may be a nucleotide sequence shared by a regulatory site, etc. may contain sequence regions shared by more than one polynucleotide sequence Homology does not necessarily have to be perfect (100%) identity, and partially homologous sequences are also within the scope of the present invention. is contemplated and within the scope of the present invention (e.g., at least 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, (83%, 82%, 81%, 80%, etc.) The percentage of homology is the number of matches between the two sequences. The number of nucleotides in a sequence is calculated by dividing the total length of the sequence being compared and multiplying by 100.

[0086] The phrase "improved RNAi activity" refers to activity measured in vitro and / or in vivo. The term "enhanced RNAi activity" refers to the ability of siNA to mediate RNAi and This is a reflection of both the activity and the stability of the siNA of the present invention. The results were comparable to those of all-RNA siRNA or siRNA containing multiple ribonucleotides. , in vitro and / or in vivo. Some iNA molecules have reduced activity or stability (i.e., less than 10-fold). Although this may be the case, the overall activity of the siNA molecule may be improved in vitro and / or in vivo. There are.

[0087] The terms "inhibit," "downregulate," or "reduce" as used herein When used herein, the meaning generally accepted in the art is given. In general, the term refers to the expression of a gene or the production of one or more proteins. RNA molecules encoding proteins or protein subunits or equivalent RNA molecules The level of a protein or the activity of one or more proteins or protein subunits, a decrease from that observed in the absence of a nucleic acid molecule of the invention (e.g., siNA) Downregulation can also be caused by post-transcriptional silencing (such as RNAi-mediated cleavage, or associated with alterations in DNA methylation patterns or DNA chromatin structure Inhibition, down-regulation or reduction by siNA molecules may be achieved by inactive or weakly Toxic molecules, siNA molecules with scrambled sequences, or siNA molecules with mismatches It may relate to molecules or alternatively to systems in which nucleic acids are not present. It is okay The phrase "inhibitors of cell proliferation and survival signaling pathways" refers to inhibitors of cell surface receptors and It refers to a pharmaceutical agent that inhibits the signal transduction cascade downstream of the surface receptor.

[0088] The term "integrin blocker" refers to an integrin whose physiological ligand is α ω Binds to β3 integrin compounds that selectively antagonize, inhibit, or counteract the physiological ligand α ω β5 Intel compounds that selectively antagonize, inhibit, or oppose binding to erythrin, physiological ligands is α ω β3 integrin and α ω β5 integrin. The integrins expressed on capillary endothelial cells are either specific integrins or specific integrins that interact with the capillary endothelial cells. The term also refers to a compound that antagonizes, inhibits, or reverses the activity of a ω β .6, α ω β.8, α1β.1, α2β.1, α5β.1, α6β1, and α6β4 The term also refers to an antagonist of α-interferon. ω β3, α ω β5, α ω β.6, α ω β .8, α1β.1, α2β.1, α5β.1, α6β1, and α6β4 integrins It also refers to any combination of antagonists.

[0089] The terms "intermittent" or "intermittently" as used herein refer to a condition generally known in the art. The term generally refers to either regularly spaced or irregularly spaced intervals. This refers to periodically stopping and starting the system.

[0090] The terms "internucleoside linkage" or "internucleoside linker" or "nucleotide" "Internucleotide linkage" or "internucleotide linker" are used interchangeably herein; refers to any linker or bond between two nucleoside units known in the art, For example, but not limited to, phosphates, phosphate analogs, phosphonates, guanidinium amine, hydroxyl amine, hydroxyl hydrazinyl, amide, carbamate, alkyl and substituted alkyl linkages. Internucleoside linkages constitute the backbone of nucleic acid molecules. are.

[0091] The terms "mammalian" or "mammal" as used herein refer to any organism known in the art. The term generally refers to any warm-blooded vertebrate species, e.g., Humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, etc. say.

[0092] The term "metered dose inhaler" or MDI refers to a canister, a screw cap that closures the canister, and the This refers to a unit equipped with a valve that measures the dosage of the formulation located in the cap. Suitable channeling devices include: The device may, for example, comprise a valve actuator and a columnar or conical channel through which the drug is filled. and (b) a canister that can be delivered to the patient's nose or mouth by a metering valve. (mouthpiece actuator).

[0093] The term "microRNA" or "miRNA" as used herein refers to a molecule that is a protein of interest in the art. The term generally refers to a target messenger RNA. expression by either mRNA cleavage, translational repression / inhibition, or heterochromatic silencing. This refers to small double-stranded RNAs that regulate the expression of certain proteins (e.g., Ambros, 2004, Na ture,431,350-355;Bartel,2004,Cell,116,28 1-297;Cullen,2004,Virus Research.,102,3- 9;He et al., 2004, Nat. Rev. Genet., 5, 522-531; Ying et al., 2004, Gene, 342, 25-28; and Sethupathy et al., 200 6, RNA, 12:192-197).

[0094] The term "modulate," as used herein, refers to a compound commonly accepted in the art. With respect to exemplary nucleic acid molecules of the present invention, the term refers to the expression of a gene, or is at the level of one or more RNA molecules (coding or non-coding) or one or more R The activity of the NA molecule or protein or protein subunit can be determined by the expression, level, or the activity is greater than that observed in the absence of the modulating molecule. For example, the term "Modulate" in some embodiments refers, for example, to the inhibition of gene expression. In other embodiments, it may refer to an enhancement or upregulation thereof.

[0095] The phrase "modified nucleotides" as used herein refers to any nucleotide that is generally accepted in the art. The term generally refers to the unmodified (or unmodified) moiety commonly known in the art. Nucleotides containing modifications within the chemical structure of the base, sugar chain, and / or phosphate group of a (naturally occurring) nucleotide Non-limiting examples of modified nucleotides are described herein and in U.S. Patent Application No. 20050023344. It is described in issue 12 / 064,014.

[0096] The phrase "NSAID that is a selective COX-2 inhibitor" is used herein to mean a cell IC for COX-1 assessed by assay or microsomal assay 50 to IC for COX-2 50 COX-1 is more potent than COX-1 when measured by the ratio of An NSAID with at least 100-fold specificity for inhibiting X-2.

[0097] The term "non-base paired" refers to the difference between the sense strand or sense region and the antisense strand of a double-stranded siNA molecule. Nucleotides that are not base-paired between the sense strand or the antisense region These include, but are not limited to, mismatches, overhangs, single-stranded loops, etc. It is possible.

[0098] The term "non-nucleotide" refers to a non-nucleotide that is incorporated in place of one or more nucleotide units in a nucleic acid strand. Any group or compound that may be present (such as, but not limited to, an abasic moiety or an alkyl chain) The group or compound is a commonly recognized nucleotide base (adenosine, guanine, cytosine, uracil, or thymine) and therefore It is "abasic" in that it has no nucleobase at position 1.

[0099] The term "nucleotide" is used as commonly recognized in the art. Nucleotides generally consist of a nucleobase, a sugar chain, and an internucleoside linkage (e.g., phosphodiesterase). Bases can be natural (standard) or modified, or base analogs. and which are well known in the art. Such bases are generally is located at the 1' position of the sugar chain of the nucleotide. Furthermore, the nucleotide is unmodified. The sugar chain, the internucleoside bond, and / or the base moiety may be modified. (interchangeably referred to as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides) See, e.g., U.S. Patent Application Serial No. 12 / 064,014. and).

[0100] The term "protruding end" as used herein has its generally accepted meaning in the art. With respect to exemplary double-stranded nucleic acid molecules, the term generally refers to the two strands of a double-stranded nucleic acid molecule. The term "terminal portion of a nucleotide sequence" refers to a portion of a nucleotide sequence where the two strands are not base-paired (see, for example, Figure 4). Overhanging ends, if present, are typically located at one or both ends of the siNA duplex. It is located at the 3' end of the strand.

[0101] The term "parenteral" as used herein has its art-recognized meaning. The term generally refers to the delivery of a molecule, drug, agent or compound in a manner other than via the digestive tract. This refers to the method or technique of administration, such as epicutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or These include intrathecal injection or infusion techniques.

[0102] The phrase "pathway target" refers to any target that is involved in a pathway of gene expression or activity. For example, any given target may have related pathway targets within a biological pathway, Such pathway landmarks may include upstream genes, downstream genes, or modified genes. The target genes may provide additive or additive effects in the treatment of the diseases, conditions and traits herein. A multiplicative effect can result.

[0103] The term "phosphorothioate" refers to a nucleic acid that contains one or more sulfur atoms in place of an oxygen atom. The term phosphorothioate therefore refers to an interthiol phosphate bond. Both thioate and phosphorodithioate internucleotide linkages Also refers to.

[0104] "Prenyl-protein transferase inhibitors" are drugs that inhibit the action of prenyl-protein transferase. Farnesyl-protein transferase enzymes, such as farnesyl-protein transferase (FPTas e), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl-protein transferase type II (GGPTase-II Inhibits any one or any combination of Rab GGPTases A compound.

[0105] The term "retinoid receptor modulator" refers to a compound that acts on the receptor of a retinoid, regardless of mechanism. The term "antibody" refers to a compound that prevents or inhibits binding to an antigen.

[0106] The term "ribonucleotide" as used herein refers to a ribonucleotide that is generally accepted in the art. The term generally refers to a hydroxyl group at the 2' position of a β-D-ribofuranose moiety. A nucleotide having a xyl group.

[0107] The term "RNA" as used herein has its art-recognized meaning. In general, the term RNA refers to molecules that contain at least one ribofuranoside moiety. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA. RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and naturally occurring The RNA present is transformed by the addition, deletion, substitution and / or modification of one or more nucleotides. Such modifications can include different modified RNAs, such as at the ends (one or more) of the siNA. a non-nucleotide to one or more nucleotides of the RNA, e.g., multiple nucleotides) or internal The nucleotides in the RNA molecules of the present invention may also include those that are naturally occurring. nucleotides, chemically synthesized nucleotides, deoxynucleotides, etc. These modified RNAs may also contain non-standard nucleotides. They may be referred to as analogs of naturally occurring RNA.

[0108] The phrase "RNA interference" or the term "RNAi" refers to the ability of a molecule to inhibit or reduce gene expression in a cell. This refers to a biological process that regulates the level of a given molecule, which is generally known in the art and Molecular interference is mediated by nucleic acid molecules. See, e.g., Zamore and Haley, 200 5, Science, 309, 1519-1524; Vaughn and Martien ssen,2005,Science,309,1525-1526;Zamore et al., 2000,Cell,101,25-33;Bass,2001,Nature,411 ,428-429;Elbashir et al.,2001,Nature,411,494-4 98; and Kreutzer et al., PCT International Application Publication No. 00 / 44895; Zer Nicka-Goetz et al., PCT International Application Publication No. 01 / 36646; Fire, PC T International Application Publication No. 99 / 32619; Plaetinck et al., PCT International Application Publication No. 0 0 / 01846; Mello and Fire, PCT International Application Publication No. 01 / 29058 No. Deschamps-Depaillette, PCT International Application Publication No. 99 / 074 09; and Li et al., PCT International Application Publication No. 00 / 44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, S science,297,1833-1837;Jenuwein,2002,Science ce, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Sc ience, 297, 2056-60; McManus et al., 2002, RNA, 8, 84 2-850;Reinhart et al., 2002, Gene & Dev., 16, 1616 -1626; and Reinhart & Bartel, 2002, Science ,297,1831). Furthermore, the term RNAi refers to a sequence-specific RNA Other terms used to describe interference include post-transcriptional gene silencing and translational inhibition. For example, the siNAs of the present invention are intended to be equivalent to the siNAs of the present invention. The molecule mediates epigenetic gene silencing at either the post-transcriptional or pre-transcriptional level. In a non-limiting example, gene expression by siNA molecules of the invention can be used to Current epigenetic modulation involves altering chromatin structure or methylation to alter gene expression. This may be the result of siNA-mediated improvement of the ligation pattern (e.g., Verde et al., l et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004,Science,303,669-672;Allshire,2002,S science,297,1818-1819;Volpe et al.,2002,Science ,297,1833-1837;Jenuwein,2002,Science,297 , 2215-2218; and Hall et al., 2002, Science, 297, 223 In another non-limiting example, genetic modification of a gene by a siNA molecule of the invention can be achieved. Modulation of gene expression is mediated by RNA (including coding RNA) through RISC or translational inhibition. This may occur as a result of siNA-mediated cleavage of a specific RNA (either a coding or non-coding RNA). (as known in the art), or modulation occurs as a result of transcriptional inhibition. (e.g., Janowski et al., 2005, Nature Chemistry cal Biology, 1, 216-222).

[0109] The term "RNAi inhibitor" refers to an RNAi inhibitor that inhibits the function or activity of RNA interference in a cell or organism. RNAi inhibitors refer to any molecule that can downregulate, reduce, or inhibit RNAi (e.g., For example, RNAi-mediated cleavage, translational inhibition, or transcriptional silence of a target polynucleotide. ing) to any component of the RNAi pathway (e.g., a protein component such as RISC, or interacting with or functioning in a nucleic acid component such as a miRNA or siRNA RNAi inhibitors can downregulate, reduce, or inhibit the activity of a gene. , siNA molecules, antisense molecules, aptamers, or interacting with the function of RISC or a small molecule, miRNA, or siRNA or a cell or organism that interferes with said function The substance may be any other component of the RNAi pathway. by inhibiting RNAi-mediated cleavage of the nucleotide sequence, translational inhibition, or transcriptional silencing. Thus, the RNAi inhibitors of the present invention modulate (e.g., upregulate) the expression of a target gene. It can be used to regulate or downregulate a gene.

[0110] The phrase "sense region" as used herein means a region of interest generally accepted in the art. With respect to exemplary nucleic acid molecules of the invention, the term refers to the antisense The term "siNA" refers to the nucleotide sequence of the siNA molecule that has complementarity to the nucleotide sequence of the siNA molecule. The sense region of the A molecule comprises a nucleic acid sequence that has homology or sequence identity with a target nucleic acid sequence. In one embodiment, the sense region of the siNA molecule can be a sense strand or a package strand. Also called the Senger chain.

[0111] The terms "small interfering nucleic acid", "siNA", "small interfering RNA", "siRNA", "small interfering nucleic acid molecule," "small interfering oligonucleotide molecule," or "chemically modified Small interfering nucleic acid molecules (SIMs) are used to induce RNA interference (RNAi) or gene silencing. Inhibiting or down-regulating gene expression or viral replication by mediating in a specific manner These terms refer to any nucleic acid molecule capable of being synthesized. , or a pool of such nucleic acid molecules. The nucleic acid molecule may be a double-stranded nucleic acid molecule comprising a target nucleic acid molecule and an antisense strand, in which case the antisense strand is a target nucleic acid molecule. Contains a nucleotide sequence complementary to the nucleotide sequence of a nucleic acid molecule or a portion thereof The sense strand contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. siNAs can be double-stranded, asymmetric double-stranded, hairpin-type, or asymmetric hairpin-type secondary strands. It is a polynucleotide having a structure and having self-complementary sense and antisense regions. In this case, the antisense region may be a nucleic acid sequence of a separate target nucleic acid molecule or a portion thereof. the sense region comprises a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid; The siNA contains a nucleotide sequence corresponding to the sequence or a portion thereof. The above loop structure and a stem containing self-complementary sense and antisense regions are included. In this case, the antisense region may be a circular single-stranded polynucleotide that containing a nucleotide sequence complementary to the nucleotide sequence of a target nucleic acid molecule or a portion thereof and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. wherein the circular polynucleotide is processed either in vivo or in vitro. Upon swabbing, active siNA molecules capable of mediating RNAi can be generated. iNAs are nucleic acid molecules that contain nucleotide sequences complementary to the nucleotide sequence of a target nucleic acid molecule or a portion thereof. The nucleotide sequence may comprise a single-stranded polynucleotide having a sequence (e.g., such a siNA a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof within the siNA molecule; In this case, the single-stranded polynucleotide may further comprise a terminal phosphorylase. acid groups, e.g., 5'-phosphate groups (e.g., Martinez et al., 2002, Cell, 1 10, 563-574 and Schwarz et al., 2002, Molecular Cell l, 10, 537-568), or containing a 5',3'-diphosphate group, etc. It's fine.

[0112] The term "subject" as used herein has its art-recognized meaning. The term generally refers to an organism to which the nucleic acid molecules of the present invention can be administered. The cell may be a mammal or a mammalian cell (e.g., a human or a human cell). The term refers to an organism, which may be the donor or recipient of harvested cells or the cells themselves. This is itself.

[0113] The phrase "systemic administration" as used herein has its art-recognized meaning. The term generally refers to the in vivo systemic absorption or accumulation of a drug in the bloodstream, Next comes distribution throughout the body.

[0114] The term "target," when referring to CTNNB1, includes any CTNNB1 target protein. Proteins, peptides, or polypeptides (encoded by the Genbank accession numbers shown in Table 5) The term also refers to any target protein, peptide, or polypeptide. peptides (proteins encoded by sequences with GenBank accession numbers shown in Table 5) a nucleic acid sequence encoding a target polynucleotide (e.g., a protein, peptide, or polypeptide) The target of interest may be a target polypeptide, such as a target DNA or a target RNA. The term "target" may also refer to various isoforms, mutations, and the like. target genes, splice variants of target polynucleotides, target polymorphisms, and non-coding a gene sequence (e.g., ncRNA, miRNA, stRNA, sRNA) or a gene sequence described herein It is intended to encompass other sequences such as the other regulatory polynucleotide sequences described above.

[0115] The term "target site" as used herein has its art-recognized meaning. The term generally refers to, for example, siNA constructs (which contain a target molecule within their antisense region). a target nucleic acid that is "targeted" for cleavage mediated by a sequence complementary to the target nucleic acid Refers to a sequence within a molecule (e.g., RNA).

[0116] The phrase "therapeutically effective amount" as used herein means that amount generally accepted in the art. The term generally refers to a biological or medical application to a cell, tissue, system, animal or human. the amount of compound or composition that elicits a physiological response, and is used by researchers, veterinarians, physicians, or other A quantity sought by clinicians, e.g., to determine whether a given clinical treatment is associated with a disease or disorder. A treatment is considered effective when it produces at least a 25% reduction in the measurable parameter being studied. If so, a therapeutically effective amount of a drug for treating the disease or disorder will be at least Both are the amounts required to produce a 25% reduction.

[0117] The term "universal base" as used herein refers to a base that is generally accepted in the art. The term universal base generally refers to the bases found in natural DNA / RNA. Nucleotide base analogs that form base pairs with each base with little or no discrimination Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic bases. Aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives (3- Nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole and the like), and are known in the art (e.g., Loakes, 200 1, Nucleic Acids Research, 29, 2437-2447 (This refers to

[0118] The term "upregulate" as used herein means to upregulate a gene that is a protein or protein that is a ... With respect to exemplary nucleic acid molecules of the present invention, the term refers to the expression of a gene or RNA molecules that code for more than one protein or protein subunit or equivalent RNA molecules, or one or more RNA, protein or proteinases The activity of the protein subunit is compared to that observed in the absence of a nucleic acid molecule of the invention (e.g., siNA). In some specific cases, the gene expression of siNA molecules can be increased above the normal range. The upregulation or enhancement of gene expression is at a level lower than that observed in the presence of an inactive or attenuating molecule. In other cases, upregulation of gene expression by siNA molecules or or enhancement, for example, in the presence of siNA molecules with scrambled sequences or mismatches. In still other cases, the nucleic acid molecules of the invention may be capable of expressing a gene that is higher than the level observed in the original. The upregulation or promotion of gene expression by the nucleic acid molecule is greater in the presence than in its absence. In some cases, upregulation or promotion of gene expression is achieved through RNA-mediated Inhibition of gene silencing (downregulating, inhibiting or silencing the expression of an upregulated gene) RNAi-mediated cleavage or disruption of coding or non-coding RNA targets Downregulation of gene expression is associated with, for example, by the RNA or its encoded protein, e.g., through a negative feedback effect or Downregulation of gene expression can be induced by, for example, an antagonistic effect. Non-coding RNAs that have regulatory control over genes can cause, for example, translational inhibition, chromosomal Regulation of expression of the gene by transcriptional structure, methylation, RISC-mediated RNA cleavage, or translational inhibition can be induced by silencing, thus downregulating, suppressing or suppressing a gene of interest. Inhibition or downregulation of an illuminating target can be used to inhibit expression of a gene of interest for therapeutic use. It can be used to upregulate

[0119] The term "vector" as used herein has its art-recognized meaning. The term vector generally refers to a molecule used to deliver one or more nucleic acid molecules. It refers to any nucleic acid-based and / or viral-based expression system or technique used.

[0120] B. siNA Molecules of the Invention The present invention includes siNAs targeted to CTNNB1 and associated with CTNNB1 expression. Compositions and methods that can be used to treat diseases, such as malignancies and / or cancers. In particular aspects and embodiments of the invention, the nucleic acid molecules of the invention are containing a sequence of at least 15 nucleotides of the sequences shown in Table 1a and Table 1b The siNA can be provided in several forms. For example, the siNA can be one or more The siNA compound may be isolated as a transcription factor in a DNA plasmid. The siNA may be in the form of a set. Alternatively, the siNA may be chemically synthesized. For example, but not limited to, modifications such as those shown in Table 1c and Table 6 may be included. Thus, in various embodiments, at least one strand or strands of the nucleic acids of the invention The region is composed of at least 15 sequences selected from the group consisting of SEQ ID NOs: 1 to 6374. The siNA may be administered alone or in combination with other siNAs. In combination with conventional drugs for treating NA molecules or CTNNB1-related diseases or conditions. It may be administered.

[0121] The siNA molecules of the present invention may be used to target specific RNA transcripts by interacting with them or by targeting specific RNA transcripts. By interacting with gene sequences (in this case, such interactions may affect transcription levels or modulation of gene silencing at either the post-transcriptional level (e.g., limited RNAi) or altering the chromatin structure or methylation of the target. The target gene having the target nucleotide sequence is then transcribed. , gene silencing by the cellular processes by which silencing is mediated. , specifically CTNNB1. More specifically, the target can be C TNNB1 can be RNA, DNA, or mRNA.

[0122] In one aspect, the present invention provides a method for detecting the expression of the CTNNB1 gene in a cell or a mammal. The present invention provides a small interfering nucleic acid (siNA) molecule for inhibiting the expression of ribonucleotides (RIs). The siNA is single-stranded. If double-stranded, the siNA contains a sense strand and an antisense strand. The antisense strand is a small portion of the mRNA formed by the expression of the CTNNB1 gene. The sense strand contains the region complementary to the antisense strand. In a specific embodiment, the antisense strand is an antisense strand of the antisense sequence shown in Table 1b. It contains a sequence of at least 15 nucleotides. Generally, double-stranded siNAs are , a sequence of at least 15 nucleotides of the sense strand of Table 1b and an antisense sequence of Table 1b The nucleic acid sequence of the siNA of the present invention comprises at least 15 nucleotides of the first strand. One or more of the leucine moieties may be modified. In further embodiments having modifications, Some siNAs of the invention contain at least one nucleic acid selected from the group of sequences shown in Table 1c. In another embodiment, the siNA comprises a nucleotide sequence shown in Table 1c. The at least two sequences are selected from the group of sequences One of the sequences is complementary to another of the at least two sequences, and One of the two sequences corresponds to the mRNA sequence produced by expression of the CTNNB1 gene. Examples of specific modified siNAs of the invention are shown in Table 1c.

[0123] The double-stranded RNA molecules of the present invention comprise two complementary strands which may be symmetric or asymmetric. It may contain two separate, different strands, i.e., two single-stranded RNA molecules, The complementary portions (e.g., sense and antisense regions) are base-paired and These are covalently linked by a single-stranded "hairpin" region (i.e., loop) such as For example, a single-stranded short hairpin polynucleotide or a circular single-stranded polynucleotide It may comprise one single-stranded molecule comprising:

[0124] The linker may be a polynucleotide linker or a non-nucleotide linker. In some embodiments, the linker is a non-nucleotidic linker. In one embodiment, the hairpin or circular siNA molecules of the invention contain one or more loops. and wherein at least one of the loop portions of the siNA molecule is biodegradable. For example, the single-stranded hairpin siNA molecule of the present invention may have a loop portion of the siNA molecule. In vivo degradation of so that double-stranded siNA molecules with terminal ends (such as 3'-nucleotide overhangs) can be generated. Alternatively, the circular siNA molecules of the present invention are designed to In vivo degradation of the 3'-end protruding end (3'-end nucleotide) containing approximately 2 nucleotides The nucleotide overhangs and the like can be used to generate double-stranded siNA molecules.

[0125] In the symmetric siNA molecules of the invention, the sense (passenger) strand and the antisense Each strand of the (guide) strand may independently comprise from about 15 to about 30 (e.g., about 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) Generally, each strand of a symmetric siNA molecule of the invention is about 19-24 (e.g., nucleotides) in length. For example, about 19, 20, 21, 22, 23, or 24 nucleotides in length.

[0126] In asymmetric siNA molecules, the antisense region (or strand) of the molecule is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, or 30 nucleotides in length, and the sense region is 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) nucleotide length Generally, each strand of an asymmetric siNA molecule of the invention has about 19 to 24 (e.g., about 19 , 20, 21, 22, 23, or 24) nucleotides in length.

[0127] In yet another embodiment, the siNA molecule of the invention comprises a single-stranded hairpin siNA molecule. The siNA molecule comprises about 25 to about 70 (e.g., about 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 8, 29, 30, 31, 32, 33, 34, 35, 36, 40, 45, 50, 55, 60 , 65 or 70) nucleotides in length.

[0128] In yet another embodiment, the siNA molecule of the invention comprises a single-stranded, circular siNA molecule. The siNA molecule comprises about 38 to about 70 (e.g., about 38, 40, 45, 50 , 55, 60, 65 or 70 nucleotides in length.

[0129] In yet another embodiment, the siNA molecule of the invention is a single-stranded, non-circular siNA molecule. The siNA molecules may independently comprise about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and is 30) nucleotides long.

[0130] In various symmetrical embodiments, the siNA duplexes of the invention are independently from each other and comprise from about 15 to about 30 (For example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 2 Generally, the s of the present invention contain 6, 27, 28, 29 or 30 base pairs. The double-stranded structure of iNA is 15-30, more commonly 18-25, and even more commonly 1 They are 9 to 24, most commonly 19 to 21 base pairs in length.

[0131] In yet another embodiment, when the double-stranded siNA molecule of the invention is asymmetric, the si The NA molecules are about 3 to 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 Generally, the double-stranded structure of the siNA of the present invention contains 15 to 2 base pairs. 5, more commonly 18-25, even more commonly 19-24, most commonly 19- It is 21 base pairs long.

[0132] In yet another embodiment, the siNA molecule of the invention has a hairpin or circular structure. In this case, the siNA molecule is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) bases It includes pairs.

[0133] The sense and antisense strands, or the sense and antisense regions, of the siNA molecules of the invention The regions may be complementary to each other. The antisense strand or region may also be complementary to the CTNNB The nucleotide sequence of the siNA may be complementary to the nucleotide sequence of the target RNA or a portion thereof. The sense strand or sense region contains the nucleotide sequence of the CTNNB1 gene or a portion thereof. In certain embodiments, the sense region of the siNA molecule of the invention may comprise The antisense region or sense strand of the siNA molecule is the CTN of the antisense region or antisense strand. NB1 target polynucleotide sequences (such as, but not limited to, GENBANK recipient sequences listed in Table 5) It is complementary to a portion that is complementary to a sequence (such as a sequence represented by an accession number).

[0134] In some embodiments, the siNA molecule of the invention comprises a sense strand or Perfect complementarity between the sense and antisense strands or between the antisense regions. In another embodiment or in the same embodiment, the antisense strand of a siNA molecule of the invention is perfectly complementary to the corresponding target nucleic acid molecule.

[0135] In yet another embodiment, a siNA molecule of the invention comprises a sense strand or between the sense and antisense strands or between the antisense regions, or between the nucleotides of the siNA molecule There is partial complementarity (i.e., Therefore, in some embodiments, Thus, the double-stranded nucleic acid molecule of the present invention has in one strand a nucleotide that is complementary to a nucleotide in the other strand. Add about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22) , 23, 24, 25, 26, 27, 28, 29 or 30). In an embodiment, the molecule comprises a double-stranded nucleic acid molecule having a sense region and an antisense region nucleotide. About 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) In certain embodiments, the double-stranded nucleic acid molecule of the present invention is an antisense oligonucleotide. Each strand contains approximately 1 nucleotide complementary to the nucleotide sequence of its corresponding target nucleic acid molecule. 5 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29 or 30).

[0136] In another embodiment, the siNA molecule contains one or more nucleotide deletions, substitutions, or mistakes. Matches and / or additions may be included; however, the siNA molecule For example, it should maintain its activity in mediating RNAi. The deletions, substitutions, mismatches and / or additions may be in loops or bulges, or alternatively , wobble or other alternative (non-Watson-Crick) base pairs. Thus, in some embodiments, for example, the double-stranded The nucleic acid molecule may have one strand or region mismatched with the other strand or region, or One or more unbase-paired nucleotides (e.g., 1, 2, 3, 4, 5, or 6) In another embodiment, the double-stranded nucleic acid molecule of the present invention has each strand or each There are nucleotides within the region that are mismatched or unpaired with the other strand or region. Preferably, the nucleotide sequence has one or more (e.g., 1, 2, 3, 4, 5, or 6) octides. In a preferred embodiment, the siNA of the invention contains three or fewer mismatches. If the antisense strand of the siNA contains a mismatch to the target sequence, Preferably, the tack region is not located in the center of the complementary region.

[0137] In another embodiment, the siNA molecule contains one or more nucleic acids corresponding to the sequences shown in Table 1b. It may contain deletions, substitutions, mismatches and / or additions of nucleotides, The siNA molecule should retain its activity, for example, to mediate RNAi. In typical examples, the deletions, substitutions, mismatches and / or additions are in the form of loops or bulges. Alternatively, wobble or other alternative (non-Watson-Crick) base pairs may be present. It can be something that brings about change.

[0138] The present invention also provides a method for preparing a nucleic acid sequence comprising the steps of: (a) preparing a nucleic acid sequence comprising: a first strand and a second strand that are complementary to each other; A polynucleotide sequence of the sequence any nucleotide thereof may be unmodified or chemically modified but is otherwise identical to that described herein. The present invention also includes double-stranded nucleic acid (siNA) molecules as described above in the present specification.

[0139] Hybridization techniques are well known to those skilled in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, See Cold Spring Harbor, NY (1989). Stringent hybridization conditions include 50% formamide and 5xSS. C (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms Incubation overnight at 42°C in a solution containing 1000µL / ml sheared, denatured salmon sperm DNA. followed by washing of the filters in 0.1×SSC at about 65° C.

[0140] In a specific embodiment, the first strand contains nucleotides that are complementary to the nucleotides of the other strand. and at least one of the chains has about 15, 16, 17, 18, 19, 20, or 21 amino acids. In a more preferred embodiment, the polynucleotide sequences are hybridizable to the polynucleotide sequences of Table 1b. The first strand contains approximately 15, 16, 17 nucleotides that are complementary to the nucleotides of the other strand. , 18, 19, 20 or 21 amino acids, and at least one of the strands is SEQ ID NO: 1, SEQ ID NO: 1049, SEQ ID NO: 43, SEQ ID NO: 1091, SEQ ID NO: 51, SEQ ID NO: 1099 , SEQ ID NO: 53, or SEQ ID NO: 1101 under high stringency conditions. Any nucleotide thereof may be unmodified or chemically modified. do.

[0141] In certain embodiments, the siNA molecules of the invention comprise from about 1 to about 4 (e.g., about 1 , 2, 3 or 4) nucleotide overhangs. may be the same or different nucleotides. In this case, the overhanging end is present at the 3' end of one or both strands of the double-stranded nucleic acid molecule. The double-stranded nucleic acid molecule of the present invention has a 3' end of the antisense strand / region of the double-stranded nucleic acid molecule, a 3' end of the sense strand / region, The 3' end of the antisense strand / region or both the antisense and sense strands / regions The overhang may be nucleotide or non-nucleotide.

[0142] In some embodiments, the nucleotides comprising the overhanging end of a siNA molecule of the invention comprises a sequence based on a CTNNB1 target polynucleotide sequence, The nucleotides constituting the overhanging portion of the antisense strand / region of the siNA molecule of the present invention are , which may be complementary to a nucleotide within the CTNNB1 target polynucleotide sequence, and and / or the nucleotides comprising the overhanging end of the sense strand / region of the siNA molecule of the invention are The CTNNB1 target polynucleotide sequence may include a nucleotide within the CTNNB1 target polynucleotide sequence. Thus, in some embodiments, the overhanging end is The overhangs consist of two partially complementary nucleotides. In this embodiment, the overhanging ends are not complementary to a portion of the CTNNB1 target polynucleotide sequence. In certain embodiments, the overhang comprises: CTNNB1 constructs a 3'-UU overhang that is not complementary to a portion of the target polynucleotide sequence In another embodiment, the overhang comprises a UU overhang at the 3' end of the antisense strand. and a TT overhang at the 3' end of the sense strand. The starting ends include the nucleotides set out in the Examples, Tables and Figures herein.

[0143] Any embodiment wherein the siNA molecule described herein has a 3' terminal nucleotide overhang. In the method, the protruding end is formed by chemically modifying one or more sugar chain positions, base positions or main chain positions of the nucleic acid. Modified nucleotides at the overhanging ends of the double-stranded nucleic acid (siNA) molecules of the present invention may be used. Representative, but non-limiting examples include: 2'-O-alkyl (e.g., 2'-O-methyl ), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-deoxy-2'-fluoro FANA, 4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl 2'-O-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, universal salt In a more preferred embodiment, the nucleotides are 5-C-methyl, acyclic, or 5-C-methyl nucleotides. wherein each overhanging nucleotide is independently a 2'-O-alkyl nucleotide, a 2' -O-methyl nucleotides, 2'-deoxy(dexoy)-2-fluoro nucleotides , or 2'-deoxyribonucleotides. In some cases, overhanging nucleotides are linked by one or more phosphorothioate bonds.

[0144] In yet another embodiment, the siNA molecules of the invention have blunt ends (i.e., and double-stranded nucleic acid molecules (without nucleotide overhangs), in which both ends are blunt. Alternatively, one end is blunt. The iNA molecule may be, for example, a molecule in which the 5' end of the antisense strand and the 3' end of the sense strand are completely overhanging. If the siNA has no nucleotides, it may contain one blunt end. The molecule may be, for example, a nucleotide sequence in which the 3' end of the antisense strand and the 5' end of the sense strand are completely overhanging. In another embodiment, the siNA molecules of the invention comprise one blunt end that does not have a tail. The molecule may be, for example, the 3' end of the antisense strand and the 5' end of the sense strand, and the antisense strand. The 5' end of the sense strand and the 3' end of the sense strand contain two blunt ends with no overhanging nucleotides. It is something that

[0145] In any embodiment or aspect of the siNA molecule of the invention, the sense strand and / or The antisense strand may further comprise a cap (as described herein or known in the art). (e.g., those prepared by the method described above) at the 3' end, 5' end, or Alternatively, it may have a hairpin siNA at both the 3' and 5' ends. As in the case of a polynucleotide, the cap may be located at any one of the terminal nucleotides of the polynucleotide. In some embodiments, the cap may be present on one or both of the One or both of the ends of the sense strand of the double-stranded siNA molecule In another embodiment, the cap is present at the antisense (gain end of the In a preferred embodiment, the cap is located at the 3' end of the sense strand. It is present at the 5' end of the sense strand and at the 5' end of the sense strand.

[0146] Representative, but non-limiting, examples of such end caps include inverted abasic nucleotides, inverted Deoxy abasic nucleotide, inverted nucleotide moiety, groups shown in Figure 5, glyceryl modified a ring, an alkyl or cycloalkyl group, a heterocycle, or a group commonly known in the art Any other caps may be used.

[0147] All embodiments of the siNA molecules of the present invention have a 5' phosphate terminal group. In some embodiments, the siNA molecule lacks a terminal phosphate group.

[0148] Any siNA molecule or construct of the present invention may contain one or more chemical modifications. Modifications may be made to improve in vitro or in vivo properties, such as stability, activity, toxicity, Immune responses (e.g., interferon responses, inflammatory or pro-inflammatory cytokine responses) response, or inhibition of stimulation of Toll-like receptor (TIF) responses) and / or bioavailability It can be used to improve irritability, etc.

[0149] Applicants herein demonstrate that siNA molecules exhibit increased RNAi activity compared to the corresponding unmodified siNA molecules. Chemically modified siNA molecules with improved activity and / or stability are described herein. The various chemically modified siNA motifs disclosed herein allow for the production of unmodified or minimally modified siNAs with active This results in the ability to maintain RNAi activity substantially similar to that of conventional siRNA (e.g., E See Ibashir et al., 2001, EMBO J., 20:6877-6888 ) while also possessing nuclease resistance and pharmacokinetic properties suitable for use in therapeutic applications. is brought about.

[0150] In various embodiments, the siNA molecules of the invention comprise modifications, such as: Any (e.g., one or more or all) of the amino acids present in the sense and / or antisense strands ) is a modified nucleotide (e.g., one nucleotide is modified) In some cases, several nucleotides (i.e., multiple or more than one) are modified. In some embodiments, some or all of the nucleotides are modified nucleotides. The siNA molecules of the invention are partially modified by chemical modification (e.g., about 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4 5, 46, 47, 48, 49, 50, 55, or 59 nucleotides are modified In some embodiments, the siNA molecules of the invention contain at least one nucleotide that is a modified nucleotide. All sizes are approximately 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 , 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 In another embodiment, the siNA of the invention comprises 60 nucleotides. The molecule is fully modified by chemical modification (e.g., 100% modified). ), i.e., the siNA molecule does not contain any ribonucleotides. In embodiments, one or more of the nucleotides in the sense strand of a siNA molecule of the invention are modified. In this or other embodiments, the antisense oligonucleotides of the siNA molecules of the invention may be used. One or more of the nucleotides in the base strand are modified.

[0151] The chemical modifications within a single siNA molecule can be the same or different. In another embodiment, at least one strand has at least one chemical modification. In the method, each strand contains at least one chemical modification (e.g., Modifications of the sugar chain, base, or backbone (i.e., internucleotide linkage) are also possible. In embodiments, the siNA molecules of the invention comprise at least 2, 3, 4, 5 or more siNA molecules. These include different chemical modifications of:

[0152] Non-limiting examples of chemical modifications suitable for use in the present invention are described in U.S. patent application Ser. No. 10 / 444 ,853; 10 / 981,966; 12 / 064,014 and the following: The sugar chains, base and phosphate groups, non-nucleotide modifications are disclosed in the cited references. decoration, and / or any combination thereof.

[0153] In certain specific embodiments of the invention, at least one modified nucleotide is '-deoxy-2-fluoronucleotide, 2'-deoxynucleotide, 2'-O-a alkyl (e.g., 2'-O-methyl) nucleotides, or locked nucleic acid (LNA) nucleotides The compound is leotide (as it is commonly known in the art).

[0154] In yet another embodiment of the invention, at least one nucleotide is a ribo-Nor and those having a helical or A-form configuration (e.g., Saenger ,Principles of Nucleic Acid Structure,Sp (See Ringer-Verlag, ed., 1984). Non-limiting examples of nucleotides include locked nucleic acid (LNA) nucleotides (e.g., 2 -0,4'-C-methylene-(D-ribofuranosyl)nucleotide); 2'-methoxyethoxy 2'-methyl-thio-ethyl nucleotide; 2'-deoxy (MOE) nucleotide; 2'-Deoxy-2'-fluoronucleotide; 2'-Deoxy-2'-chloronucleotide; 2' -Azidonucleotides; 2'-O-trifluoromethylnucleotides; 2'-O-ethyl -trifluoromethoxynucleotide; 2'-O-difluoromethoxy-ethoxynucleotide nucleotides; 4'-thionucleotides and 2'-O-methyl nucleotides.

[0155] In various embodiments, the pyrimidine nucleotides present in the double-stranded siNA molecule The majority (e.g., more than 50%) of the nucleotides contain glycosylation. or in other embodiments, purine nucleotides present in the double-stranded siNA molecule The majority (eg, more than 50%) contain glycosylation modifications.

[0156] In some embodiments, the pyrimidine nucleotides in the antisense strand are 2'-O-methyl 2'-deoxy-2'-fluoropyrimidine nucleotides, and antisense The purine nucleotides present in the base strand are either 2'-O-methyl nucleotides or 2'- In another embodiment, pyrimidine nucleotides in the sense strand are deoxynucleotides. The nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides present in the sense strand. The purine nucleotides present are 2'-O-methyl or 2'-deoxypurine nucleotides. It's Chido.

[0157] In certain embodiments of the present invention, pyrimidine nucleotides within the complementary region on the sense strand All nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides. In certain embodiments, all pyrimidine nucleotides in the complementary region of the antisense strand are All are 2'-deoxy-2'-fluoropyrimidine nucleotides. In an embodiment, all purine nucleotides in the complementary region on the sense strand are 2'-deoxyribonucleotides. In certain embodiments, the complementary All purines within the region are 2'-O-methylpurine nucleotides. In embodiments, all pyrimidine nucleotides in the complementary region on the sense strand are 2'-deoxyribonucleotides. oxy-2'-fluoropyrimidine nucleotides; in the complementary region of the antisense strand All pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides. all purine nucleotides in the complementary region on the sense strand are 2'-deoxynucleotides. phosphorus nucleotides, and all purines in the complementary region on the antisense strand are 2'-O- It is a methylpurine nucleotide.

[0158] In some embodiments, at least one of the pyrimidine nucleotides of one or both strands Five or more are 2'-deoxy-2'-fluoro pyrimidine nucleotides. In some embodiments, at least 5 of the pyrimidine nucleotides of one or both strands In some embodiments, one or more of the nucleotides are 2'-O-methylpyrimidine nucleotides. In one or both strands, at least five or more purine nucleotides are 2 In some embodiments, the nucleotide is 2'-deoxy-2'-fluoropurine nucleotide. At least five or more of the purine nucleotides on one or both strands are 2'-O-methyl It is a chirupurin nucleotide.

[0159] In certain embodiments, the purines and pyrimidines are different at the 2' position of the sugar chain. modified (i.e., at least one 2'-position of a glycan on the same or a different chain) (One purine has a different modification than at least one pyrimidine). For example, in some cases In this case, at least five or more pyrimidine nucleotides on one or both strands is a 2'-deoxy-2'-fluoropyrimidine nucleotide, and one or both strands At least five or more purine nucleotides of In other cases, at least one of the pyrimidine nucleotides on one or both strands is five or more of the nucleotides are 2'-O-methylpyrimidine nucleotides, and one or both of the nucleotides are 2'-O-methylpyrimidine nucleotides. At least five or more purine nucleotides on one strand are 2'-deoxy-2'- It is a fluoropurine nucleotide.

[0160] The sense and antisense strands of such siNA molecules with various modifications and modification patterns were Further non-limiting examples of sequence strands are shown in Figures 2 and 3.

[0161] Any of the above modifications or combinations thereof (including those in the cited references) are included in the present invention. It can be applied to any siNA molecule.

[0162] The modified siNA molecules of the invention can include modifications at various positions within the siNA molecule. In some embodiments, the double-stranded siNA molecules of the invention comprise modified nucleotides, such as: In another embodiment, the siNA duplex of the present invention comprises a base paired site within the siNA duplex. The stranded siNA molecule may incorporate modified nucleotides in the non-base paired regions or overhanging regions of the siNA molecule. In yet another embodiment, the double-stranded siNA molecule of the invention comprises a modified nucleotide. For example, such terminal regions may include the s the 3' and / or 5' ends of the sense and / or antisense strands (or regions) of the iNA molecule; Furthermore, in any of the modified siNA molecules of the present invention, the modification is at the 5' position. One or both oligonucleotide strands of the NA duplex (e.g., sense strand, antisense strand) In addition, chemical modifications of the siNA molecules of the invention may be With respect to the above, each strand of the double-stranded siNA molecule of the invention may have one or more chemical modifications. , resulting in each strand containing a different chemical modification pattern.

[0163] In certain embodiments, each strand of a double-stranded siNA molecule of the invention comprises a different chemically modified A decorative pattern, such as any of the Stab modified chemical structures described herein (see Table 9) or or any combination thereof, i.e., defined stabilization chemical structures. chemistry) (Stab) containing different combinations of sense and antisense strands Additionally, non-limiting examples of modification schemes that can result in different modification patterns are shown in Table 9. The stabilizing chemistry, designated Stab in Table 9, is used for any of the sense / antisense chemistries. It can be combined in the following combinations (Stab 7 / 8, Stab 7 / 11, Sta b 8 / 8, Stab 18 / 8, Stab 18 / 11, Stab 12 / 13, St ab 7 / 13, Stab 18 / 13, Stab 7 / 19, Stab 8 / 19, S tab 18 / 19, Stab 7 / 20, Stab 8 / 20, Stab 18 / 20 , Stab 7 / 32, Stab 8 / 32, or Stab 18 / 32, etc., any other stabilizing chemical structure combination).

[0164] In any siNA of the invention, the guide strand or guide region (amplifier) of the siNA molecule one or more (e.g., For example, 1, 2, 3, 4, or 5) of the nucleotides are ribonucleotides.

[0165] In certain embodiments, the present invention provides a method for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of a compound that modulates the expression of CTNNB1. A double-stranded small interfering nucleic acid (siNA) molecule, the siNA comprising a sense strand and an antisense strand. each strand independently having a length of 15 to 30 nucleotides; The chain: 5'-CUGUUGGAUUGAUUCGAAA-3' (SEQ ID NO: 5); 5'-ACGACUAGUUCAGUUGCUU-3' (SEQ ID NO: 194); 5'-GGAUGAUCCUAGCUAUCGU-3' (SEQ ID NO: 196); or 5'-CCAGGAUGAUCCUAGCUAU-3' (SEQ ID NO: 151) at least 15, 16, 17, 18, or 19 nucleotides having a complementary sequence to any of Double-stranded small interfering nucleic acid (siNA) molecules are provided that contain a nucleotide.

[0166] In some embodiments, the antisense strand of a siNA molecule of the invention: 5'-UUUCGAAUCAAUCCAACAG-3' (SEQ ID NO: 4918); 5'-AAGCAACUGAACUAGUCGU-3' (SEQ ID NO: 5107); 5'-ACGAUAGCUAGGAUCAUCC-3' (SEQ ID NO: 5109); or 5'-AUAGCUAGGAUCAUCCUGG-3' (SEQ ID NO: 5064) and a sequence of at least 15, 16, 17, 18 or 19 nucleotides of do.

[0167] In some embodiments, the sense strand of a siNA molecule of the invention is: 5'-CUGUUGGAUUGAUUCGAAA-3' (SEQ ID NO: 5); 5'-ACGACUAGUUCAGUUGCUU-3' (SEQ ID NO: 194); 5'-GGAUGAUCCUAGCUAUCGU-3' (SEQ ID NO: 196); or 5'-CCAGGAUGAUCCUAGCUAU-3' (SEQ ID NO: 151) and a sequence of at least 15, 16, 17, 18 or 19 nucleotides of do.

[0168] In some embodiments, the siNA molecule of the invention comprises: 5'-CUGUUGGAUUGAUUCGAAA-3' (SEQ ID NO: 5) and 5'-UUU CGAAUCAAUCCAACAG-3' (SEQ ID NO: 4918); or 5'-ACGACUAGUUCAGUUGCUU-3' (SEQ ID NO: 194) and 5'-A AGCAACUGAACUAGUCGU-3' (SEQ ID NO: 5107); or 5'-GGAUGAUCCUAGCUAUCGU-3' (SEQ ID NO: 196) and 5'-A CGAUAGCUAGGAUCAUCC-3' (SEQ ID NO: 5109); or 5'-CCAGGAUGAUCCUAGCUAU-3' (SEQ ID NO: 151) and 5'-A UAGCUAGGAUCAUCCUGG-3' (SEQ ID NO: 5064) It includes any of the following.

[0169] Any of the above modifications or combinations thereof (including those in the cited references) may be used in these This can be applied to any embodiment.

[0170] In certain embodiments, SEQ ID NO:5, SEQ ID NO:4918, SEQ ID NO:19 4, SEQ ID NO: 5107, SEQ ID NO: 196, SEQ ID NO: 5109, SEQ ID NO: 151, or said at least 15, 16, 17, 18 or 19 nucleotides of SEQ ID NO: 5064 The nucleotides of the nucleotide sequence form a continuous nucleotide chain.

[0171] In some embodiments, the siNA molecule comprises SEQ ID NO:5, SEQ ID NO:4918, SEQ ID NO:5919, SEQ ID NO:6020, SEQ ID NO:6021, SEQ ID NO:6022, SEQ ID NO:6023, SEQ ID NO:6024, SEQ ID NO:6025, SEQ ID NO:6026, SEQ ID NO:6027, SEQ ID NO:6028, Sequence number: 194, sequence number: 5107, sequence number: 196, sequence number: 5109, sequence number No.: 151, or at least 15, 16, 17, 18 or 19 of SEQ ID NO: 5064 deletion, substitution, mismatch of one or more nucleotides in the nucleotide sequence and and / or additions may be included; however, the siNA molecule may be, for example, In a non-limiting example, the deletion, substitution, or The replacement, mismatch and / or addition may result in a loop or ridge, or alternatively, a wobble. or may result in other alternative (non-Watson-Crick) base pairs.

[0172] In certain embodiments of the present invention, the compound has a sense strand and an antisense strand and is of the formula: A):

[0173] [ka] providing a double-stranded siNA molecule comprising wherein the upper strand is the sense strand of the double-stranded nucleic acid molecule and the lower strand is the antisense strand. The antisense strand is SEQ ID NO: 4918, SEQ ID NO: 5107, SEQ ID NO: 510 9, or at least 15, 16, 17, 18 or 19 nucleotides of SEQ ID NO:5064 The sense strand has a sequence complementary to the antisense strand. a sequence comprising: each N is independently an unmodified or chemically modified nucleotide or a non-nucleotide; each B is an end cap that is present or absent; (N) represents an overhanging nucleotide (each independently unmodified or chemically modified); [N] represents a nucleotide that is a ribonucleotide; X1 and X2 are independently an integer of 0 to 4; X3 is an integer between 15 and 30; X4 is an integer from 9 to 30; X5 is an integer between 0 and 6, and the sum of X4 and X5 is between 15 and 30.

[0174] In certain embodiments, SEQ ID NO: 4918, SEQ ID NO: 5107, SEQ ID NO: 5109, or at least 15, 16, 17, 18 or 1 of SEQ ID NO: 5064 The nucleotides of the nine nucleotide sequence form a continuous nucleotide chain.

[0175] In some embodiments, the siNA molecule of Formula A is SEQ ID NO:4918, SEQ ID NO:5 107, SEQ ID NO: 5109, or at least 15, 16, 17 of SEQ ID NO: 5064 , deletion or substitution of one or more nucleotides in the 18 or 19 nucleotide sequence, Mismatches and / or additions may be contained; however, the siNA molecule The gene is intended to retain its activity, e.g., to mediate RNAi. wherein the deletions, substitutions, mismatches and / or additions are in loops or bulges, or Also, those that result in wobble or other alternative (non-Watson-Crick) base pairs. could be.

[0176] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (a)N X4 one or more pyrimidine nucleotides at positions 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, 30, 31, 2'-Fluoronucleotides, 2'-O-Alkylnucleotides, 2'-Deoxynucleotides nucleotides, ribonucleotides, or any combination thereof; (b)N X4 one or more purine nucleotides at positions 1 and 2 are independently 2'-deoxy-2' -Fluoronucleotides, 2'-O-alkylnucleotides, 2'-deoxynucleotides nucleotides, ribonucleotides, or any combination thereof; (c)N X3 one or more pyrimidine nucleotides at positions 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, 30, 31, 2'-Fluoronucleotides, 2'-O-Alkylnucleotides, 2'-Deoxynucleotides nucleotides, ribonucleotides, or any combination thereof; and (d)N X3 one or more purine nucleotides at positions 1 and 2 are independently 2'-deoxy-2' -Fluoronucleotides, 2'-O-alkylnucleotides, 2'-deoxynucleotides nucleotides, ribonucleotides, or any combination thereof; The invention features a double-stranded small interfering nucleic acid (siNA) of formula (A).

[0177] In some specific embodiments, the present invention provides (a)N X4 1, 2, 3, 4, 5 or more pyrimidine nucleotides at positions is a 2'-deoxy-2'-fluoronucleotide; (b)N X4 1, 2, 3, 4, 5 or more purine nucleotides at positions 2'- O-alkyl nucleotides; (c)N X3 1, 2, 3, 4, 5 or more pyrimidine nucleotides at positions 2 '-deoxy-2'-fluoronucleotide; and (d)N X3 2'-deoxypurine nucleotides at positions 1, 2, 3, 4, 5 or more is an oxynucleotide, The invention features a double-stranded small interfering nucleic acid (siNA) molecule of formula (A):

[0178] In some specific embodiments, the present invention provides (a)N X4 1, 2, 3, 4, 5 or more pyrimidine nucleotides at positions 2 '-O-alkylnucleotides; (b)N X4 1, 2, 3, 4, 5 or more purine nucleotides at positions 1, 2, 3, 4, 5 or more ribonucleoside It is a cleotide; (c)N X3 1, 2, 3, 4, 5 or more pyrimidine nucleotides at positions 2 '-O-alkyl nucleotide; and (d)N X3 1, 2, 3, 4, 5 or more purine nucleotides at positions 1, 2, 3, 4, 5 or more ribonucleoside It is a nucleotide The invention features a double-stranded small interfering nucleic acid (siNA) molecule of formula (A):

[0179] In some specific embodiments, the present invention provides (a)N X41, 2, 3, 4, 5 or more pyrimidine nucleotides at positions 2 '-deoxy-2'-fluoronucleotides; (b)N X4 1, 2, 3, 4, 5 or more purine nucleotides at positions 2'- O-alkyl nucleotides; (c)N X3 1, 2, 3, 4, 5 or more pyrimidine nucleotides at positions 2 '-O-alkyl nucleotide; and (d)N X3 1, 2, 3, 4, 5 or more purine nucleotides at positions 2'- deoxy-2'-fluoronucleotides, The invention features a double-stranded small interfering nucleic acid (siNA) molecule of formula (A):

[0180] In certain embodiments, the present invention further comprises one or more phosphorothioate nucleic acids. The present invention features a double-stranded small interfering nucleic acid (siNA) molecule of formula (A) comprising an internucleotide linkage.

[0181] In some embodiments, the siNA molecule having Formula A is the antisense strand of the nucleic acid molecule. or containing a terminal phosphate group at the 5' end of the antisense region.

[0182] In various embodiments, the siNA molecule having Formula A has X5=0, 1, 2 or 3; Each X1 and X2 = 1 or 2; X3 = 18, 19, 20, 21, 22 or 23, and and X4=17, 18, 19, 20, 21, 22 or 23.

[0183] In certain embodiments, the siNA molecule having Formula A comprises X5=3. In another embodiment, the siNA molecule having Formula A comprises X5=0.

[0184] In certain embodiments, the siNA molecule having Formula A has X1=2 and X2=2 It includes:

[0185] In various embodiments, the siNA molecule having Formula A has X5=0, X1=2, and In another embodiment, the siNA molecule having Formula A comprises X5=3. , X1=2, and X2=2.

[0186] In one specific embodiment, the siNA molecule having Formula A has X5=3; each X1 and X2=2; X3=19, and X4=16.

[0187] In another specific embodiment, the siNA molecule having Formula A has X5=0; each X1 and X 2=2; X3=19, and X4=19.

[0188] In certain embodiments, the siNA molecule having Formula A is a sense strand or a sense It contains caps (B) at the 3' and 5' ends of the region.

[0189] In certain embodiments, the siNA molecule having Formula A is an antisense strand or It contains a cap (B) at the 3' end of the antisense region.

[0190] In various embodiments, the siNA molecule having Formula A is Caps (B) at the 3' and 5' ends and 3' ends of the antisense strand or antisense region 'It contains a cap (B) at the end.

[0191] In yet another embodiment, the siNA molecule having Formula A is a siNA molecule having a sense (upper) amino acid sequence of the double-stranded nucleic acid molecule. It contains a cap (B) only at the 5' end of the (side) strand.

[0192] In some embodiments, the siNA molecule having Formula A further comprises one internucleotide sequence. Some specific embodiments include one or more phosphorothioate internucleotide linkages. In one embodiment, a siNA molecule having Formula A comprises a first end (N) and a sense strand of the nucleic acid molecule, The adjacent nucleotides on the 3' end of the antisense strand or both the sense and antisense strands It contains one or more phosphorothioate internucleotide bonds between the nucleotides. For example, The double-stranded nucleic acid molecule comprises X1 and / or X2=2, and is a phosphorothioate nucleotide. An overhanging nucleotide position having an internucleotide bond (e.g., (NsN) (where "s" is It may have a structure (showing a phosphorothioate).

[0193] In some embodiments, one or more of the nucleotides of the siNA molecule having Formula A is univalent. It has a universal base.

[0194] In certain embodiments, the siNA molecule having Formula A is 5' of the antisense strand. If the nucleotide at the 14th position from the end is a purine, the nucleotide at the 14th position is a ribonucleotide. In another embodiment, the siNA molecule having Formula A has the following structure: When the nucleotide at the 14th position from the 5' end is a pyrimidine nucleotide, the 14th position is nucleotides, 2'-deoxy-2'-fluoronucleotides or 2'-O-methyl It has nucleotides.

[0195] In some embodiments, the siNA molecule having Formula A comprises the antisense strand (bottom strand): Within the CTNNB1 target polynucleotide sequence (also the antisense (lower) strand N and and (N) nucleotides complementary to the nucleotides in It contains nucleotides.

[0196] In certain embodiments, one or more siNA molecules of the invention comprise USSN61 / 408,428 and USSN 61 / 408,303 (both of which are incorporated herein by reference). The product will be qualified according to the qualification criteria illustrated and described in the table below.

[0197] Any of the modifications discussed above as applicable to the siNAs of the invention, or The combinations (including those in the cited references) may be used in any embodiment of the siNA molecules of the invention. It is applicable to various situations.

[0198] C. Creation / Synthesis of siNA Molecules The siNAs of the present invention can be obtained using several techniques known to those skilled in the art. For example, the siNA may be chemically synthesized or may be encoded on a plasmid. It may be a double stranded molecule (e.g., a molecule that automatically folds into a double stranded molecule with a hairpin loop). The siNA is also a long dsRNA (e.g., about 2 dsRNA (longer than 5 nucleotides) is cleaved by E. coli RNase II or Dicer. The dsRNA may be prepared by cleaving it with these enzymes. is processed into biologically active siRNA (see, e.g., Yang et al., PN AS USA 99:9942-9947(2002); Calegari et al. PNAS USA 99:14236(2002)Byron et al.Ambion Tech No tes;10(1):4-6(2009);Kawaski et al., Nucleic Aci ds Res.,31:981-987(2003),Knight and Bass,S. Science, 293: 2269-2271 (2001) and Roberston et al. , J. Biol. Chem 243:82 (1969).

[0199] 1.Chemical synthesis Preferably, the siNA of the present invention is chemically synthesized. For example, some specific modified oligonucleotides or oligonucleotides lacking ribonucleotides (part of the octide) can be found, for example, in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., PCT International Application Publication No. 99 / 54459, Wincott et al., 1995, Nucleic Acids R es.23, 2677-2684, Wincott et al., 1997, Methods Mo l.Bio.,74,59, Brennan et al., 1998, Biotechnol Bi oeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311 The oligonucleotides are synthesized using protocols known in the art, such as those described in US Pat. In the synthesis of nucleotides, common nucleic acid protecting groups and coupling groups (dimethoxy at the 5' end) are used. trityl, and phosphoramidites at the 3' end) are used.

[0200] Unmodified siNA molecules were prepared as described by Usman et al., 1987, J. Am. Chem. Soc. 109,7845;Scaringe et al., 1990, Nucleic Acids Re The synthesis is carried out using the procedure described in s., 18, 5433. Protecting and coupling groups are utilized (dimethoxytrityl at the 5' end and dimethoxytrityl at the 3' end). Terminal phosphoramidites, etc., are used in some specific siNA molecules of the invention. (possibly).

[0201] In certain embodiments, the siNA molecules of the invention are those disclosed in U.S. Pat. No. 59, No. 6,686,463, No. 6,673,918, No. 6,649,75 Nos. 1, 6,989,442, and U.S. patent application Ser. No. 10 / 190,359. are synthesized, deprotected, and analyzed according to the method of

[0202] In one non-limiting example of synthesis, small scale synthesis is performed using the method of 394 Applied Biosystems. 2' was synthesized using a 0.2 μmol scale protocol on a synthesizer manufactured by Stems, Inc. For -O-methylated nucleotides, the coupling step was 2.5 min, and for 2'-deoxynucleotides, 45 seconds of coupling for nucleotides or 2'-deoxy-2'-fluoronucleotides Table 10 shows the amounts of reagents and contact times used in the synthesis cycle. An outline is shown below.

[0203] Alternatively, the siNA molecules of the invention can be synthesized separately and subsequently combined, e.g., by ligation. by ion (Moore et al., 1992, Science 256, 9923; Dra Per et al., PCT International Application Publication No. 93 / 23569; Shabarova et al., 1991 ,Nucleic Acids Research 19,4247;Bellon et al. 1997,Nucleosides & Nucleotides,16,951;Be Illon et al., 1997, Bioconjugate Chem. 8, 204), or They may be linked together by hybridization after synthesis and / or deprotection. .

[0204] Various siNA molecules of the present invention can also be synthesized using methods described in Scaringe et al., U.S. Pat. No. 5,889, 136; 6,008,400; and 6,111,086. It can also be synthesized.

[0205] 2. Vector Expression Alternatively, it interacts with and downregulates the gene encoding the target CTNNB1 molecule. The siNA molecules of the invention can be inserted into a DNA or RNA vector as a transcription unit (e.g., (see Couture et al., 1996, TIG., 12, 510) The recombinant vector may be a DNA plasmid or a viral vector. siNA-expressing viral vectors include, but are not limited to, adeno-associated viruses, retroviruses, They may be constructed based on viruses, adenoviruses, or alphaviruses.

[0206] In some embodiments, pol III-based constructs are used to express the nucleic acid molecules of the invention. Transcription of the siNA molecule sequence is initiated by eukaryotic RNA polymerase I (pol I), RNase I. A polymerase II (pol II), or RNA polymerase III (pol II I) promoters (see, for example, Thompson, US Pat. No. 5,929,139). See Patent Nos. 5,902,880 and 6,146,886. (See also I zant and Weintraub, 1985, Science, 229, 345; Mc Garry and Lindquist, 1986, Proc. Natl. Acad. Sc i.,USA 83,399;Scanlon et al.,1991,Proc.Natl.Ac ad.Sci.USA,88,10591-5;Kashani-Sabet et al.,199 2,Antisense Res.Dev.,2,3-15;Dropulic et al.,19 92, J. Virol., 66, 1432-41; Weerasinghe et al., 1991 ,J.Virol.,65,5531-4;Ojwang et al.,1992,Proc.Na Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nu Cleic Acids Res.,20,4581-9;Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nuc leic Acids Res.,23,2259;Good et al.,1997,Gene See also Therapy, 4, 45. Pol II or Pol III promoter Transcripts from the gene are expressed at high levels in all cells; The level of a given pol II promoter depends on the gene regulatory sequences (encoding It depends on the properties of the RNA polymerases (e.g., promoters, silencers, etc.). A promoter is also used, but the prokaryotic RNA polymerase enzyme is required in the appropriate cells. (Elroy-Stein and Moss, 1990, Proc .Natl.Acad.Sci.USA,87,6743-7;Gao and Huang 1993,Nucleic Acids Res.,21,2867-72;Lieb er et al., 1993, Methods Enzymol, 217, 47-66; Zhou et al. ,1990,Mol.Cell.Biol.,10,4529-37). Several researchers Thus, nucleic acid molecules expressed by such promoters are functional in mammalian cells. (e.g., Kashani-Sabet et al., 1992, Antisense Res.Dev.,2,3-15;Ojwang et al.,1992,P roc.Natl.Acad.Sci.USA,89,10802-6;Chen et al.,1 992, Nucleic Acids Res., 20, 4581-9; Yu et al., 199 3,Proc.Natl.Acad.Sci.USA,90,6340-4;L'Hui llier et al., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 8000- 4;Thompson et al., 1995, Nucleic Acids Res.,23,2 259;Sullenger & Cech,1993,Science,262,15 66). More specifically, U6 small nuclear RNA (snRNA), transfer RNA (tR NA) and those derived from the gene encoding adenovirus VA RNA Transcription units are useful for generating high concentrations of desired RNA molecules (e.g., siNA) within cells (Thompson et al., supra; Couture and Stinchcomb, 1999 96, supra; Noonberg et al., 1994, Nucleic Acid Res., 2 2,2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et al., 1 997, Gene Ther., 4, 45; Beigelman et al., PCT International Application Publication The above siNA transcription units are suitable for introduction into mammalian cells. Among the various vectors are, for example, but not limited to, plasmid DNA vectors, viruses DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vector (e.g., retrovirus or alphavirus vector) (For a review, see Couture and Stinchcomb, 1996 See above).

[0207] The vectors used to express the siNA molecules of the invention contain one of the siNA duplexes. The siNA molecule may encode one or both strands and may self-hybridize to form the siNA molecule. The siNA molecule of the present invention may encode a single self-complementary strand that becomes double-stranded. and a nucleic acid sequence encoding the siNA molecule are operably linked in a manner that allows for expression of the siNA molecule. (See, e.g., Paul et al., 2002, Nature Biotechnology ,19,505;Miyagishi and Taira,2002,Nature Bi otechnology, 19, 497; Lee et al., 2002, Nature Biot echnology, 19, 500; and Novina et al., 2002, Nature Medicine, published online in advance doi:10.1038 / nm725 reference).

[0208] D. Carriers / Delivery Systems The siNA molecules of the invention can be added directly to target cells or tissues or can be added to cationic The siNA molecule may be complexed with a lipid or encapsulated in a liposome, and the siNA molecule may be expressed. These may be delivered as recombinant plasmid or viral vectors or in other ways. Methods for delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell B io.,2,139;Delivery Strategies for Antise nse Oligonucleotide Therapeutics, edited by Akhtar. ,1995,Maurer et al.,1999,Mol.Membr.Biol.,16,12 9-140; Hofland and Huang, 1999, Handb. Exp. Pha rmacol., 137, 165-192; and Lee et al., 2000, ACS Sy mp. Ser., 752, 184-192. US Patent No. 6,395,713 and Sullivan et al., PCT Publication No. WO 94 / 02 No. 595 further describes general methods for delivery of nucleic acid molecules. The protocol can be used to deliver virtually any nucleic acid molecule. , by a variety of methods known to those skilled in the art, including, but not limited to, intracellular transport within liposomes. Encapsulation, iontophoresis, or other vehicles (biodegradable polymers, hydrogels, cyclodextrins, etc.) (e.g., Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., PCT International Application Publication No. 03 / 47518 and and 03 / 46185), poly(lactic-co-glycolic acid) (PLGA ) and PLCA microspheres (see, e.g., U.S. Patent 6,447,796 and U.S. Patent See Application Publication No. US2002130430), biodegradable nanocapsules, etc. and bioadhesive microspheres) or by incorporating proteinaceous vectors. (O'Hare and Normand, PCT International Application Publication No. 00 / 53722) It can be administered accordingly.

[0209] In one aspect, the present invention provides a carrier system incorporating the siNA molecules described herein. In some embodiments, the carrier system is a lipid-based carrier system, a cationic lipid, or a lipid-based carrier system. liposome-nucleic acid complexes, liposomes, micelles, virosomes, lipid nanoparticles or mixtures thereof In other embodiments, the carrier system is a polymer-based carrier system (cationic polymer-nucleic acid In a further embodiment, the carrier system is a cyclodextrin-based carrier system. (e.g., cyclodextrin polymer-nucleic acid complexes). The system is a protein-based carrier system (such as a cationic peptide-nucleic acid complex). In the present invention, the carrier system is a lipid nanoparticle ("LNP") formulation.

[0210] In certain embodiments, the siNA molecules of the invention are those disclosed in U.S. Patent Application Serial No. 11 / 35 No. 3,630, No. 11 / 586,102, No. 61 / 189,295, No. 61 / No. 204,878, No. 61 / 235,476, No. 61 / 249,807, No. 6 1 / 298,022, 61 / 351373, 61 / 347640, 6 Nos. 1 / 345754, 61 / 322054, 12 / 640342, and No. 12 / 617079, and PCT Patent Application No. PCT / US10 / 020013 and lipid nanoparticle compositions such as those described in PCT / US09 / 053336. In certain preferred embodiments, the siNA molecules of the invention are formulated as Lipid / Cholesterol / PEG-C-DMA / DSPC 40 / 48 / 2 / 10 or cationic lipid / cholesterol / PEG-DMG / DSPC in a ratio of 40 / 48 In some specific embodiments, the lipid nanoparticle composition is formulated with a lipid nanoparticle composition containing 100% glycerol and 20% glycerol in a ratio of 100% to 200%. In the formulation, the cationic lipid is DLinDMA (see Table 12) and the PEG is PEG-D MG and the formulation has an N / P ratio of 2.8. The soluble lipid is DLinDMA (see Tables 11 and 12).

[0211] In various embodiments, the lipid nanoparticle formulations shown in Table 11 can be any of the lipid nanoparticle formulations described herein. In some embodiments, the present invention is applied to any siNA molecule or combination of siNA molecules. The present invention relates to the formulations LNP-051; LNP-053; LNP-054; LNP-069; LNP-073;LNP-077;LNP-080;LNP-082;LNP-083; LNP-060;LNP-061;LNP-086;LNP-097;LNP-098; LNP-099;LNP-100;LNP-101;LNP-102;LNP-103; or LNP-104 (see Table 11). It features a composition including a child.

[0212] In certain other embodiments, the present invention provides the method of claim 1, further comprising: No. 61 / 204,878, No. 61 / 235,476, No. 61 / 249,8 07, and any of the cationic lipid formulations described in US Pat. No. 61 / 298,022. The present invention features compositions containing siNA molecules of the invention formulated using

[0213] In another embodiment, the present invention provides conjugates and / or siNA molecules of the present invention. Such conjugates and / or complexes are characterized by their ability to interact with biological systems. The present invention can be used to facilitate delivery of the siNA molecule to a target cell. The conjugates and complexes provided are capable of transporting therapeutic compounds across cell membranes. and to alter the pharmacokinetic properties and / or modulate the localization of the nucleic acid molecules of the invention. Such conjugates may be conferred therapeutic activity by incorporating the conjugate. Illustrative examples are U.S. Patent Application Publication Nos. US2008 / 0152661A1 and US20 04 / 0162260A1 (e.g., CDM-LBA, CDM-Pip-LBA, CDM -PEG, CDM-NAG, etc.) and U.S. Patent Application No. 10 / 427,160, Nos. 10 / 201,394, 61 / 322422, and 61 / 315223 and U.S. Patent Nos. 6,528,631; 6,335,434; and 6,23 Nos. 5,886; 6,153,737; 5,214,136; and 5, 138,045.

[0214] In various embodiments, polyethylene glycol (PEG) is incorporated into the siNA compounds of the invention. The PEG that is attached can be of any molecular weight (preferably about 10 0 to approximately 50,000 Daltons (Da).

[0215] In yet another embodiment, the present invention provides poly(ethylene glycol) lipids (PEG-modified long-circulating liposomes or stealth liposomes) A composition or product comprising a surface-modified liposome encapsulating a siNA molecule of the present invention. (See, e.g., PCT International Application Publication No. 96 / 10391; Ansell et al., PCT International Application Publication No. 96 / 10390; Holland et al., PCT International Application Publication No. 9 (such as that disclosed in No. 6 / 10392).

[0216] Additionally, in some embodiments, the siNA molecules of the invention comprise polyethyleneimine and its Derivatives such as polyethyleneimine-polyethylene glycol-N-acetylgalactose Polyethyleneimine (PEI-PEG-GAL) or Polyethyleneimine-Polyethylene Glycol -tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives, etc. In one embodiment, the nucleic acid molecules of the present invention can be formulated or complexed with It is formulated as described in Patent Application Publication No. 20030077829.

[0217] In another embodiment, the siNA molecules of the invention are administered in combination with a membrane disruptive agent (see U.S. Patent Application Publication No. 2004 / 0129994). In yet another embodiment, the compound is complexed with a hydroxybenzoate (such as those described in US Pat. No. 5,666,010). In this embodiment, the membrane disrupting agent(s) and the siNA molecule are coupled to a cationic lipid or a helix. complexed with a lipid molecule (such as the lipids described in U.S. Pat. No. 6,235,310) do.

[0218] In certain embodiments, the siNA molecules of the invention are those disclosed in U.S. Patent Application Publication No. 2003 / 0129994. No. 003077829; No. 20050287551; No. 20050164220 ; Same No. 20050191627; Same No. 20050118594; Same No. 2005015 No. 3919; No. 20050085486; and No. 20030158133; Also described in PCT International Application Publication Nos. 00 / 03683 and 02 / 087541 The compound is complexed with a delivery system such as

[0219] In some embodiments, the liposomal formulations of the present invention are prepared as described in U.S. Pat. No. 6,858,224 No. 6,534,484; No. 6,287,591; No. 6,835,395 ; Same No. 6,586,410; Same No. 6,858,225; Same No. 6,815,432; Same No. 6,586,001; Same No. 6,120,798; Same No. 6,977,223; Same No. 6,977,223; Same No. 6,977,223; No. 6,998,115; No. 5,981,501; No. 5,976,567; No. 5,976,567; No. 5,705,385; and U.S. Patent Application Publication No. 2006 / 0019912; Same No. 2006 / 0019258; Same No. 2006 / 0008909; Same No. 2005 / 0 No. 255153; No. 2005 / 0079212; No. 2005 / 0008689; Same No. 2003 / 0077829, Same No. 2005 / 0064595, Same No. 2005 / 0 No. 175682, No. 2005 / 0118253; No. 2004 / 0071654; No. 2005 / 0244504; No. 2005 / 0265961 and No. 2003 / 00 The compounds and compositions of the present invention are formulated or complexed with the compounds and compositions described in 77829. Includes iNA molecules (e.g., siNA).

[0220] Alternatively, a recombinant plasmid as discussed above that expresses the siRNA of the invention and viral vectors may be used to deliver the molecules of the invention. Delivery of the drug can be systemic (e.g., intravenous or intramuscular), or by extraction from the subject. by administering to the target cells released and then reintroducing them into the subject, or by administering to the desired target cells. or by any other means which may allow for introduction into target cells (see for review (See Couture et al., 1996, TIG., 12, 510). The recombinant plasmid may be administered directly or in combination with an appropriate delivery reagent, e.g., Miru Transit LT1 Lipophilic Reagent; Lipofectin; Lipofectamine; Self- polycations (e.g., polylysine) or liposomal lipid-based carrier systems, cationic with carboxylic lipids, or liposome-nucleic acid complexes, micelles, virosomes, lipid nanoparticles, etc. It may also be administered to

[0221] E. Kit The present invention also provides nucleic acids in the form of a kit. The kit may include a container. The package typically includes a nucleic acid of the invention along with instructions for its administration. In certain cases, the nucleic acid may have a targeting moiety attached. Methods for binding antibodies (e.g., antibodies, proteins) are known to those skilled in the art. In some cases, the nucleic acid is chemically modified. The kit includes a siNA molecule of the invention. The kit may further comprise an excipient.

[0222] F. Therapeutic Uses / Pharmaceutical Compositions The current body of knowledge in CTNNB1 research has led to the development of assays for CTNNB1 activity. and the need for methods for analyzing CTNNB1 expression for research, diagnostic, and therapeutic uses. As described below, the nucleic acids of the present invention provide compounds that can modulate The molecule is used in an assay to diagnose a disease state associated with CTNNB1 levels. The nucleic acid molecule and pharmaceutical composition can be used for detecting CTNNB1 RNA levels and can be used to treat associated disease states.

[0223] 1. Disease conditions associated with CTNNB1 Specific disease states that may be associated with modulation of CTNNB1 expression include: Examples of such cancers include various cancers, such as solid tumors. Non-limiting examples of such cancers include: bile duct Cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, osteosarcoma, brain cancer, glioma, astrocytoma, breast cancer , metaplastic cancer, cervical cancer, squamous cell carcinoma of the cervix, rectal cancer, colorectal cancer, colon cancer, hereditary non- Polyposis colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, Endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, intraocular melanoma, uveal melanoma, Gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms' tumor cancer, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia AML, chronic lymphocytic leukemia (CLL), chronic myelogenous myeloma (CML), chronic myelomonocytic leukemia (CML), MML), liver cancer, liver cancer, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, embryonal tumor, lung cancer, non-small cell lung cancer Lung cancer (NSCLC), mesothelioma, B-cell lymphoma, non-Hodgkin's lymphoma, diffuse large cell B-cell lymphoma, mantle cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NP) C), neuroblastoma, oropharyngeal cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudomyeloma Pseudopapillary neoplasms, acinic cell carcinoma, prostate cancer, prostate adenocarcinoma , skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, gastric cancer, gastric cancer, gastrointestinal stromal tumor ( GIST), uterine cancer, and uterine sarcoma.

[0224] The siNA molecules of the present invention degrade the target CTNNB1 mRNA (and thus inhibit the It will be understood that disease inhibition can be defined as the slowing down of disease progression in a subject. The disease can be assessed by directly measuring the blood pressure. This may be inferred by observing a change or reversal. The nucleic acid molecules and pharmaceutical compositions of the present invention may be used as prophylactic agents. and the use of steroids to ameliorate, treat, or prevent these and other diseases associated with modulation of CTNNB1 gene expression. It can be used to prevent and / or cure.

[0225] 2. Pharmaceutical Compositions The siNA molecules of the present invention can be used in a wide variety of therapeutic, prophylactic, cosmetic, veterinary, and diagnostic applications. Reagents and methods useful for target validation, genome enlightenment, genetic engineering, and pharmacogenomic applications A method is provided.

[0226] a. Formulation Thus, the present invention also provides, in one aspect, pharmaceutical compositions of the siNA molecules described, namely That is, compositions in a pharmaceutically acceptable carrier or diluent are provided. The composition may be a salt, an ester, or a salt of such an ester of the above compound, e.g., an acid addition salt, Examples include salts of hydrochloric acid, hydrobromic acid, hydroiodic acid, acetic acid, and benzenesulfonic acid. Other salts include, for example, sodium, potassium, manganese, ammonium and Such formulations or compositions include salts of niacin, ... Commonly known pharmaceutically acceptable carriers or diluents may be included.

[0227] In one embodiment, the present invention provides a nucleic acid sequence of at least 15 nucleotides of SEQ ID NO:5. In another embodiment, the invention features a pharmaceutical composition comprising a siNA molecule comprising a sequence. A pharmaceutical composition comprising a siNA molecule comprising a sequence of at least 15 nucleotides of sequence number 4918. In another embodiment, the present invention features a pharmaceutical composition comprising at least one of the sequences set forth in SEQ ID NO:194. The present invention also features a pharmaceutical composition comprising a siNA molecule comprising a sequence of at least 15 nucleotides. In another embodiment, the present invention provides a method for detecting at least 15 nucleotides of SEQ ID NO:5107. In another embodiment, the present invention features a pharmaceutical composition comprising a siNA molecule comprising the sequence , including siNA molecules comprising a sequence of at least 15 nucleotides of SEQ ID NO: 196. In another embodiment, the present invention features a pharmaceutical composition comprising at least one of the sequences of SEQ ID NO:5109. Also featured is a pharmaceutical composition comprising a siNA molecule comprising a sequence of 15 nucleotides. In an embodiment, the present invention provides a method for the preparation of a nucleic acid comprising a sequence of at least 15 nucleotides of SEQ ID NO: 151. In another embodiment, the invention features a pharmaceutical composition comprising a siNA molecule comprising the A pharmaceutical composition comprising a siNA molecule comprising a sequence of at least 15 nucleotides of sequence number 5064. In yet another embodiment, the present invention features a pharmaceutical composition comprising a siNA molecule comprising Formula (A): The present invention features a pharmaceutical composition comprising the compound.

[0228] The siNA molecules of the invention are preferably purified by methods known in the art prior to administration to a subject. The pharmaceutical composition of the present invention is formulated as a pharmaceutical composition according to the method described above. The pharmaceutical composition of the present invention is characterized by being pyrogen-free. Methods are within the skill of the art and are described, for example, in Remington's Pharmaceuticals. maceutical Science, 17th edition, Mack Publishing Company, Easton, Pa. (1985).

[0229] In some embodiments, the pharmaceutical compositions of the invention (e.g., siNA and / or The LNP formulation may further comprise conventional pharmaceutical excipients and / or additives. Pharmaceutical excipients include preservatives, flavoring agents, stabilizers, antioxidants, and osmolality adjusters. Suitable additives include physiologically active agents, such as phosphate buffers, buffers, and pH adjusters. Compatible buffers (e.g., trimethylamine hydrochloride), chelating agents (e.g., DTPA or DTPA-bisamide, etc.) or calcium chelate complexes (e.g., calcium Examples include the addition of calcium DTPA, calcium DTPA-bisamide, etc., or calcium DTPA calcium or sodium salts (e.g., calcium chloride, calcium ascorbate, glutamic acid, Calcium citrate or calcium lactate) may be added. A suspending agent may also be used.

[0230] Non-limiting examples of various types of formulations for topical administration include ointments, lotions, creams, and the like. creams, gels, foams, preparations for delivery by transdermal patches, powders, sprays formulations, aerosols, capsules or cartridges for use in inhalers or insufflators tablets or drops (e.g., eye drops or nasal drops), nebulized solutions / suspensions, suppositories, vaginal Suppositories, retention enemas, and chewable or suckable tablets or pellets (e.g. , for the treatment of aphthous ulcers) or liposomal or microencapsulated preparations. can be done.

[0231] Ointments, creams and gels may be prepared using, for example, an aqueous or oily base and a suitable thickening agent. The composition may be formulated with the addition of a gelling agent and / or a solvent. Non-limiting examples of such bases include water and / or oils, e.g., liquid Paraffin or vegetable oil (such as peanut oil or castor oil), or solvent (polyethylene glycol) Depending on the nature of the base, various thickeners and gels may be included. Non-limiting examples of such agents include soft paraffin, stearin, Aluminum acetate, cetostearyl alcohol, polyethylene glycol, wool fat, beeswax, Carboxypolymethylene and cellulose derivatives and / or glyceryl monostearate These may include phosphates and / or non-ionic emulsifiers.

[0232] In one embodiment, the lotion may be formulated with an aqueous or oily base, or They also generally contain one or more emulsifiers, stabilizers, dispersants, suspending agents or thickeners. do.

[0233] In one embodiment, the powder for topical application may be prepared using any suitable powder base, for example, talc, lactose, or the like. Drops may be formulated with the aid of corn starch or starch. Drops may be formulated with an aqueous or non-aqueous base. The pharmaceutical composition may also contain one or more dispersing agents, solubilizing agents, suspending agents or preservatives. It can be enjoyed.

[0234] Compositions intended for oral use may be prepared using any of the methods known in the art for the manufacture of pharmaceutical compositions. The compositions may be prepared according to any method, and such compositions may have a pharmaceutically elegant and palatable taste. Contains one or more sweeteners, flavoring agents, coloring agents or preservatives to obtain the preparation Tablets may contain the active ingredient in non-toxic pharmaceutically acceptable carriers suitable for the manufacture of tablets. It is included in a state where it is mixed with an excipient. Such excipients include, for example, an inert diluent; calcium carbonate; calcium, sodium carbonate, lactose, calcium phosphate or sodium phosphate, etc. granulating and disintegrating agents, such as corn starch, or alginic acid; binders, such as starch, gelatin, or acacia; and lubricants, such as magnesium stearate The tablets may be coated or uncoated. In some cases, such coating may be performed by known techniques. The drug delays disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period. For example, glyceryl monostearate ( A time delay material such as glyceryl distearate or glyceryl distearate may be used. It is possible.

[0235] Also, formulations for oral use may contain the active ingredient in an inert solid diluent (e.g., calcium carbonate). As a hard gelatin capsule mixed with calcium phosphate or kaolin Alternatively, the active ingredient may be dissolved in water or an oil medium (e.g., peanut oil, liquid paraffin, or oleic acid). It may be presented as a soft gelatin capsule mixed with leaven oil.

[0236] Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents, e.g., sodium carboxymethylcellulose, methyl Cellulose, hydropropyl methylcellulose, sodium alginate, polyvinylpyrrolidone dispersing or wetting agents are naturally occurring cellulose gums, such as linoleic acid, ... phosphatides, such as lecithin, or condensation products of alkylene oxides with fatty acids. products (e.g., polyoxyethylene stearate); or ethylene oxide and long-chain fatty acids Condensation products with aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), Condensation products of ethylene oxide with partial esters obtained from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or ethylene oxide and , condensation products with partial esters obtained from fatty acids and hexitol anhydrides (e.g., The aqueous suspension may contain one or more One or more of the above preservatives (e.g., ethyl or n-propyl p-hydroxybenzoate) coloring agents, one or more flavoring agents, and one or more sweetening agents (sucrose or sucrose) It may contain sorbitan, sorbitan, etc.

[0237] Oily suspensions are suspensions in which the active ingredient is dissolved in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, etc.). Formulated by suspending in a mineral oil (e.g., liquid paraffin) or coconut oil. Oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents may be included to provide a palatable oral preparation. Such compositions may be supplemented with antioxidants such as ascorbic acid. and can be stored.

[0238] The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil or Suitable emulsifiers include naturally occurring gums (e.g., cellulose gum ... gum acacia or gum tragacanth), naturally occurring phosphatides, e.g., dimethicone Lecithin, and esters or moieties derived from fatty acids and hexitol anhydrides Esters, such as sorbitan monooleate, and the partial esters and ethylene oxides thereof. and condensation products with sorbitan oxides (e.g., polyoxyethylene sorbitan monooleate). The emulsions may also contain sweetening and flavoring agents.

[0239] Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, or the like. The formulation may be with ethanol, sorbitol, glucose or sucrose. The formulation may also contain a demulcent, a preservative and flavoring and coloring agents. The products may be in the form of a sterile injectable aqueous or oleaginous suspension. According to known art, a dispersion may be prepared using suitable dispersing or wetting agents and suspending agents which have been mentioned above. In addition, the sterile injection preparation can be formulated into a non-toxic parenteral preparation. ly) a sterile injectable solution or suspension in an acceptable diluent or solvent (e.g., Among the acceptable vehicles that can be used are: The solvents are water, Ringer's solution, and isotonic sodium chloride solution. Fixed oils are also conventionally employed as a solvent or suspending medium. Any bland fixed oil may be employed, including synthetic mono- or diglycerides. Fatty acids such as oleic acid find use in the preparation of injectables.

[0240] The nucleic acid molecules of the present invention may also be administered in the form of suppositories, for example, for rectal administration of the drug. Such compositions can be prepared by mixing the drug with suitable non-irritating excipients. The excipient is solid at normal temperatures but liquid at rectal temperatures, and therefore These materials melt in the intestine and release the drug. ethylene glycol.

[0241] The nucleic acid molecules of the present invention may be administered parenterally in a sterile medium. Either suspended or dissolved in the vehicle depending on the vehicle and concentration Advantageously, adjuvants such as a local anesthetic, preservatives and buffering agents can be dissolved in the vehicle. It's better to solve it.

[0242] In other embodiments, the compositions provided herein for use in pulmonary delivery are The iNA and LNP compositions and formulations further comprise one or more surfactants. As a suitable surfactant or surfactant component to enhance uptake of the compositions of the invention are, inter alia, synthetic and natural, as well as intact and truncated surfactant proteins. A, surfactant protein B, surfactant protein C, surfactant protein D, and Contains surfactant protein E, di-saturated phosphatidylcholine (other than dipalmitoyl), di Palmitoylphosphatidylcholine, Phosphatidylcholine, Phosphatidylglycerol Phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine Phosphorus; phosphatidic acid, ubiquinone, lysophosphatidylethanolamine, lysophos Phatidylcholine, palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone Tetron, dolichol, sulfatidic acid, glycerol-3- Phosphate, dihydroxyacetone phosphate, glycerol, glycero-3-phosphocholine acetone, dihydroxyacetone, palmitate, cytidine diphosphate (CDP) diacylglycerol chol, CDP choline, choline, choline phosphate; as well as natural carrier vehicles for surfactant components Natural and artificial lamellar structures, omega-3 fatty acids, polyenoic acids, polyenoic acids, lecithin Palmitic acid, ethylene or propylene oxide, polyoxypropylene, monomer Monomeric and polymeric polyoxyethylene, monomeric and polymeric poly(vinyl Non-ionic block copolymers of dextran and / or alkanoyl side chains Polymer, Brij 35, Triton X-100 and synthetic surfactant ALEC , Exosurf, Survan, and Atovaquone. The surfactant may be used in the formulation alone or as part of a multi-component surfactant. As an adduct covalently attached to the 5' and / or 3' termini of a nucleic acid component in a pharmaceutical composition It can be used in either

[0243] b. Use in combination The siNA and pharmaceutical formulations of the present invention may be administered to a subject alone or in combination with one or more other agents. It may be used in combination with other therapeutic agents (e.g., anticancer agents), and the other therapeutic agents Therefore, the combination of compounds of the present disclosure with other anti-cancer or chemotherapeutic agents may Examples of such agents are included within the scope of the present invention. nd Practice of Oncology by VTDevita an d S. Hellman (editor), 6th edition (February 15, 2001), Lippinc See ott Williams & Wilkins Publishers. Those skilled in the art will be able to determine which drug combinations may be useful depending on the specific drugs and cancers involved. Such anti-cancer agents may be recognized based on their properties. : Estrogen receptor modulator, androgen receptor modulator, retinoid receptor Protein kinase inhibitors, cytotoxic / cytostatic agents, antiproliferative agents, prenyl-protein transcripts spherase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, inducers of apoptosis and cell cycle regulation The siNA of the present invention can be used in the treatment of HCC. It is also effective in combination with any therapeutic agent used in the treatment of The compounds of the invention are particularly useful when co-administered with radiotherapeutic agents.

[0244] In a further embodiment, therefore, the present invention provides a siNA molecule of the invention (e.g., Although not specified, SEQ ID NO: 5, SEQ ID NO: 4918, SEQ ID NO: 194, SEQ ID NO: 51 07, SEQ ID NO: 196, SEQ ID NO: 5109, SEQ ID NO: 151, or SEQ ID NO: a sequence of at least 15 nucleotides of 5064; or a siNA molecule comprising formula (A). etc.) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof Combinations comprising the compound of formula (I) together with one or more anti-cancer or chemotherapeutic agents are provided.

[0245] In certain embodiments, the siNA molecules of the invention are directed to known anti-cancer agents, such as Below: Estrogen receptor modulators, androgen receptor modulators, retinoic acid Idiopathic receptor modulators, cytotoxic agents, antiproliferative agents, prenyl-protein transferases HMG-CoA reductase inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors It is also useful in combination with angiogenesis inhibitors and other angiogenesis inhibitors.

[0246] Examples of estrogen receptor modulators that may be used in combination with the compounds of the present invention include , including but not limited to tamoxifen, raloxifene, idoxifene, LY35338 1, LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethylaminomethyl) 1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy] [phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropane Lopanoate, 4,4'-dihydroxybenzophenone-2,4-dinitrophenyl- Drazone, and SH646.

[0247] Examples of androgen receptor modulators that may be used in combination with the compounds of the present invention include , including but not limited to, finasteride and other 5α-reductase inhibitors, nilutamide, These include flutamide, bicalutamide, liarozole, and abiraterone acetate.

[0248] Examples of such retinoid receptor modulators that may be used in combination with the compounds of the present invention include: Examples include, but are not limited to, bexarotene, tretinoin, 13-cis-retinoic acid, 9- cis-retinoic acid, α-difluoromethylornithine, ILX23-7553, trans N-(4'-hydroxyphenyl)retinamide, and N-4-carboxyphenyl Luretinamide is one example.

[0249] Examples of cytotoxic agents that may be used in combination with compounds of the present invention include, but are not limited to, cerebrospinal fluid (e.g., cerebrospinal fluid), ... Lutenef, Cachectin, Ifosfamide, Tasonermin, Lonidamine, Carboplatin, Altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine oxaliplatin, temozolomide, heptaplatin, estramustine fluoxetine, improsulfan tosylate, trofosfamide, nimustine, dibromospiroyl chloride Pharmacist, pumitapa, lobaplatin, satraplatin, profilomycin, cisplatin, ibuprofen Rofulven, dexyphosphamide, cis-aminedichloro(2-methyl-pyridine)platinum , benzylguanine, glufosfamide, GPX100, (trans, trans, trans (Hexane-1,6-diamine)-μ-[diamine-platinum(II)]bis [diammine(chloro)platinum(II)] tetrachloride, diarylididinyl spermine, trichloroplatinum(II) Arsenic oxide, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethyl Tilxanthin, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxan Thoron, pirarubicin, pinafide, valrubicin, amrubicin, antineoplast 3'-deamino-3'-morpholino-13-deoxo-10-hydroxycarbaminomer Isin, annamycin, galarubicin, elinafide, MEN10755, and 4- Demethoxy-3-deamino-3-aziridinyl-4-methylsulfonyl-daunorubicin (See International Publication No. 00 / 50032).

[0250] An example of a hypoxia activatable compound that may be used in combination with the compounds of the present invention is tirapazamine. be.

[0251] Examples of proteasome inhibitors that may be used in combination with the compounds of the present invention include, but are not limited to: However, lactacystin and bortezomib are not included.

[0252] Examples of microtubule inhibitors / microtubule stabilizing agents that may be used in combination with the compounds of the present invention include: Non-limiting examples include paclitaxel, vindesine sulfate, 3',4'-didehydro-4'-de Oxy-8'-norvincaleukoblastine, docetaxel, rhizoxin, dolastatin , mibobulin isethionate, auristatin, cemadotin, RPR109881, BMS 184476, Vinflunine, Cryptophycin, 2,3,4,5,6-pentafluoro -N-(3-fluoro-4-methoxyphenyl)benzenesulfonamide, anhydrobiphenyl Imblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl- L-prolyl-L-proline-t-butylamide, TDX258, epothilones (e.g., See U.S. Patent Nos. 6,284,781 and 6,288,237) and BMS 188797 is one example.

[0253] An example of a topoisomerase inhibitor that may be used in combination with the compounds of the present invention is Although not used, topotecan, hycaptamine, irinotecan, Rubitecan, 6-ethoxypropionyl-3',4'-O-exo-benzimidazole Den-Schaertrusin, 9-Methoxy-N,N-dimethyl-5-nitropyrazolo [3 ,4,5-kl]acridine-2-(6H)propanamine, 1-amino-9-ethyl- 5-Fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo [de]pyrano[3',4':b,7]-indolizino[1,2b]quinoline-10,13 (9H,15H)dione, raltotecan, 7-[2-(N-isopropylamino)ethyl ]-(20S)camptothecin, BNP1350, BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2'-dimethylamino -2'-Deoxy-etoposide, GL331, N-[2-(dimethylamino)ethyl]- 9-Hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxylate Ruboxamide, Asulaculin, (5a,5aB,8aa,9b)-9-[2-[N-[2 -(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy -3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3 ',4':6,7) naphtho(2,3-d)-1,3-dioxol-6-one, 2,3-( (methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]phena 6,9-bis[(2-aminoethyl)amino]benzo[g]isopropyl (guinoline)-5,10-dione, 5-(3-aminopropylamino)-7 ,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[ 4,5,1-de]acridin-6-one, N-[1-[2(diethylamino)ethylamine 7-Methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide N-(2-(dimethylamino)ethyl)acridine-4-carboxamide, 6-[[ 2-(Dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1- c]quinolin-7-one, and dimesna.

[0254] Inhibitors of mitotic kinesins, particularly human mitotic kinesins, that may be used in combination with the compounds of the present invention Examples of kinesin KSP include, but are not limited to, those described in PCT Publication No. 01 / 30768; Same No. 01 / 98278, Same No. 03 / 050,064, Same No. 03 / 050,122, Same No. 03 / 049,527, Same No. 03 / 049,679, Same No. 03 / 049,678 No. 04 / 039774, No. 03 / 079973, No. 03 / 099211 , No. 03 / 105855, No. 03 / 106417, No. 04 / 037171, Same No. 04 / 058148, Same No. 04 / 058700, Same No. 04 / 126699, Same No. No. 05 / 018638, No. 05 / 019206, No. 05 / 019205, No. 05 / 018547, 05 / 017190, U.S. Patent Application Publication No. 2005 / 01 In one embodiment, inhibitors of mitotic kinesins include those described in US Pat. No. 76776. Anti-inflammatory drugs include, but are not limited to, KSP inhibitors, MKLP1 inhibitors, CENP-E inhibitors, inhibitors, MCAK inhibitors, Kifl4 inhibitors, Mphosphl inhibitors and Ra Examples include inhibitors of b6-KIFL.

[0255] Examples of "histone deacetylase inhibitors" that can be used in combination with the compounds of the present invention include include, but are not limited to, TSA, oxamflatin, PXD101, MG98, valproic acid and Scriptaid. Further investigations into other histone deacetylase inhibitors are The references are the following papers: Miller, TA et al. J. Med. Chem. 46 ( 24):5097-5116(2003).

[0256] Inhibition of kinases involved in mitotic progression that may be used in combination with the compounds of the present invention Examples of drugs include, but are not limited to, inhibitors of Aurora kinase, Polo-like kinase (P LK) inhibitors (especially inhibitors of PLK-1), inhibitors of bub-1 and bub-R1 Inhibitors of

[0257] Examples of antiproliferative agents that may be used in combination with compounds of the present invention include, but are not limited to, anthracyclines, cyclosporines, cyclosporine ... Antisense RNA and DNA oligonucleotides (G3139, ODN698, RVAS KRAS, GEM231, and INX3001), and antimetabolites (enoshita Bin, Carmofur, Tegafur, Pentostatin, Doxifluridine, Trimetrexa fosfate, fludarabine, capecitabine, gallocitabine, cytarabine ocfosfate, fosfate Fosteabine sodium hydrate, raltitrexed, paltitrexed Cid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, ne Ruzarabine, 2'-deoxy-2'-methylidenecytidine, 2'-fluoromethylene-2 '-Deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]- N'-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2( E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-BL-ma [N-heptopyranosyl]adenine, aplidine, ecteinascidin, troxacitabine , 4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[ 5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl- L-glutamic acid, aminopterin, 5-fluorouracil, alanosine, 11-acetyl 8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa- 1,11-Diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trimethylsilyl En-9-yl acetate, swainsonine, lometrexol, dexrazoxane, Methioninase, 2'-cyano-2'-deoxy-N4-palimitoyl-1-BD-a Rabinofuranosylcytosine and 3-aminopyridine-2-carboxaldehyde thiosemiconjugate carbazones, etc.

[0258] Monoclonal antibody-targeted therapeutic agents that may be used in combination with the compounds of the present invention Examples include cells bound to cancer cell-specific or target cell-specific monoclonal antibodies. and therapeutic agents containing damaging agents or radioisotopes (e.g., Bexxar, etc.). do.

[0259] that may be used in combination with the compounds of the present invention Examples of HMG-CoA reductase inhibitors include, but are not limited to: However, lovastatin (MEVACOR®; U.S. Patent No. 4,231,938 4,294,926 and 4,319,039), simvastatin (ZOCOR®; U.S. Patent Nos. 4,444,784 and 4,820,850 and 4,916,239), pravastatin (PRAVACHOL ( Registered trademark); U.S. Patent Nos. 4,346,227, 4,537,859, 4,4 10,629, 5,030,447, and 5,180,589), Rubastatin (LESCOL®; U.S. Patent No. 5,354,772, U.S. Patent No. 4, No. 911,165, No. 4,929,437, No. 5,189,164, No. 5,1 (See US Patent Nos. 18,853, 5,290,946 and 5,356,896) and atorvastatin (LIPITOR®; U.S. Patent No. 5,273,995 , Nos. 4,681,893, 5,489,691 and 5,342,952 These and additional HMG-Co receptors that can be used in the methods of the present invention include: Structural formula of A reductase inhibitors M. Yalpani, "Cholesterol Low ering Drugs”, Chemistry & Industry, pp.85- 89 (February 5, 1996) at page 87 and U.S. Pat. Nos. 4,782,084 and No. 4,885,314.

[0260] Prenyl-protein transferase inhibitors that may be used in combination with the compounds of the present invention Examples of drugs include, but are not limited to, the following publications and patents: WO 96 / 3034 3, 97 / 18813, 97 / 21701, 97 / 23478, 97 / 23478, No. 97 / 38665, No. 98 / 28980, No. 98 / 29119, No. 95 / 32987, U.S. Patent Nos. 5,420,245, 5,523,430, and 5, No. 532,359, No. 5,510,510, No. 5,589,485, No. 5,6 02,098, European Patent Publication Nos. 0618221, 0675112, and 060 4181, 0696593, WO 94 / 19357, WO 95 / 085 No. 42, No. 95 / 11917, No. 95 / 12612, No. 95 / 12572, No. 95 / 10514, U.S. Patent No. 5,661,152, WO 95 / 1051 No. 5, No. 95 / 10516, No. 95 / 24612, No. 95 / 34535, No. 95 / 10516, No. 95 / 24612, No. 95 / 34535, No. No. 95 / 25086, No. 96 / 05529, No. 96 / 06138, No. 96 / No. 06193, No. 96 / 16443, No. 96 / 21701, No. 96 / 2145 6, 96 / 22278, 96 / 24611, 96 / 24612, 96 / 22278, 96 / 24611, 96 / 24612, Nos. 96 / 05168, 96 / 05169, and 96 / 00736, U.S. Pat. No. 5,571,792, International Publication No. 96 / 17861, International Publication No. 96 / 33159, International Publication No. 96 / 34850, 96 / 34851, 96 / 30017, 96 / 30 No. 018, No. 96 / 30362, No. 96 / 30363, No. 96 / 31111 , No. 96 / 31477, No. 96 / 31478, No. 96 / 31501, No. 9 7 / 00252, 97 / 03047, 97 / 03050, 97 / 04 No. 785, No. 97 / 02920, No. 97 / 17070, No. 97 / 23478 , No. 97 / 26246, No. 97 / 30053, No. 97 / 44350, No. 9 No. 8 / 02436, and U.S. Pat. No. 5,532,359. The role of prenyl-protein transferase inhibitors in angiogenesis For an example, see European Journal of Cancer, Vol. 35, No. 9 , pp. 1394-1401 (1999).

[0261] Examples of angiogenesis inhibitors that may be used in combination with compounds of the present invention include, but are not limited to, , tyrosine kinase inhibitors, such as tyrosine kinase receptor Flt-1 (VEGFR1 ) and Flk-1 / KDR (VEGFR2) inhibitors, epithelial-derived, fibroblast-derived or Platelet-derived growth factor inhibitors, MMP (matrix metalloproteinase) inhibitors, Integrin blockers, interferon-α, interleukin-12, pentosan polysulfate , cyclooxygenase inhibitors, e.g., nonsteroidal anti-inflammatory drugs (NSAIDs) (Ascorbic Acid pyridine and ibuprofen) and selective cyclooxygenase-2 inhibitors ( lecoxib and rofecoxib) (PNAS, Vol. 89, p. 7 384(1992);JNCI,Vol.69,p.475(1982);Arch.O pthalmol,Vol.108,p.573(1990);Anat.Rec,Vo l.238,p.68(1994);FEBS Letters,Vol.372,p. 83(1995);Clin,Orthop.Vol.313,p.76(1995); J Mol.Endocrinol,Vol.16,p.107(1996);Jpn. J.Pharmacol,Vol.75,p.105(1997);Cancer Re s.,Vol.57,p.1625(1997);Cell,Vol.93,p.705 (1998);Intl.J.Mol.Med.,Vol.2,p.715(1998) ;J Biol. Chem., Vol. 274, p. 9116 (1999)), steroids Anti-inflammatory drugs (corticosteroids, mineralocorticoids, dexamethasone, prednisone) , prednisolone, methylpred, betamethasone, etc.), carboxyamidotriazole , combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)- Fumagillol, thalidomide, angiostatin, troponin-1, angiotensin I I antagonists (Fernandez et al., J Lab. Clin. Med. 105:141-1 45 (1985)), and antibodies against VEGF (Nature Biotech nology, Vol. 17, pp. 963-968 (October 1999); Kim et al. Nature, 362, 841-844 (1993); International Publication No. 00 / 44777; and Ref. No. 00 / 61186).

[0262] Other therapeutic agents that modulate or inhibit angiogenesis may also be used in combination with the compounds of the present invention. These include drugs that modulate or inhibit the coagulation and fibrinolytic systems. (Clin. Chem. La. Med. 38:679-692 (2000) (See discussion below.) Such coagulation pathway and fibrinogen inhibitors that may be used in combination with the compounds of the present invention Examples of agents that modulate or inhibit the phospholytic pathway include, but are not limited to, heparin (See Thromb. Haemost. 80:10-23 (1998)), low molecular weight helix Verocycline and carboxypeptidase U inhibitors (active thrombin-activatable fibrinolysis Also known as an inhibitor of TAFIa (Thrombosis Res 101:329-354 (2001). TAFIa inhibitors are Publication No. 03 / 013,526 and U.S. Patent Application No. 60 / 349,925 (200 (filed January 18, 2012).

[0263] Agents that interfere with cell cycle checkpoints that may be used in combination with the compounds of the present invention Examples include, but are not limited to, inhibition of ATR, ATM, Chkl and Chk2 kinases. and Cdk and cdc kinase inhibitors, particularly 7-hydroxystaurate. Rosporin, flavopiridol, CYC202 (Cyclacel), and BMS-38 7032 is an example.

[0264] Interfering with receptor tyrosine kinases (RTKs) may be used in combination with the compounds of the present invention. Examples of agents include, but are not limited to, c-Kit, Eph, PDGF, Flt3, and C Further agents include Bume-Jensen and and Hunter, Nature, 411:355-365, 2001. Harmful drugs include:

[0265] Inhibitors of cell proliferation and survival signaling pathways that may be used in combination with the compounds of the present invention Examples of inhibitors include, but are not limited to, EGFR inhibitors (e.g., gefitinib and EGFR inhibitors). rulotinib), ERB-2 inhibitors (e.g., trastuzumab), IGFR inhibitors, Inhibitors of cytokine receptors, CTNNB1 inhibitors, PI3K inhibitors (e.g., LY2 94002), serine / threonine kinases (such as, but not limited to, those described in WO 02 / 044449), Nos. 083064, 02 / 083139, and 02 / 083140, U.S. Patent Applications Publication No. 2004-0116432, International Publication No. 02 / 083138, U.S. Patent Application Publication No. Patent Publication No. 2004-0102360, International Publication No. 03 / 086404, International Publication No. 03 / 086 No. 279, No. 03 / 086394, No. 03 / 084473, No. 03 / 0864 No. 03, No. 2004 / 041162, No. 2004 / 096131, No. 2004 / 096129, 2004 / 096135, 2004 / 096130, same Akt inhibitors described in Patent Applications Nos. 2005 / 100356 and 2005 / 100344, etc. ), Raf kinase inhibitors (e.g., BAY-43-9006), MEK inhibitors (e.g., CI-1040 and PD-098059) and inhibitors of mTOR (e.g., Wyeth CCI-779). Such agents include small molecule inhibitor compounds and antibody antagonists.

[0266] Examples of apoptosis inducers that may be used in combination with the compounds of the present invention include, but are not limited to, However, some of these drugs are activators of TNF receptor family members (e.g., TRAIL receptors). can be.

[0267] Examples of NSAIDs that are selective COX-2 inhibitors that may be used in combination with compounds of the present invention Examples include, but are not limited to, U.S. Patent Nos. 5,474,995 and 5,861,419 No. 6,001,843, No. 6,020,343, No. 5,409,944 , No. 5,436,265, No. 5,536,752, No. 5,550,142, US No. 5,604,260, US No. 5,698,584, US No. 5,710,140 , International Publication No. 94 / 15932, U.S. Patent No. 5,344,991, U.S. Patent No. 5,134, No. 142, No. 5,380,738, No. 5,393,790, No. 5,466,8 Nos. 23, 5,633,272, and 5,932,598 (all of which are , incorporated herein by reference).

[0268] COX-2 inhibitors that are particularly useful in combination with the compounds of the present invention include: 3-phenyl-4 -(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and 5-chloro -3-(4-methylsulfonyl)-phenyl-2-(2-methyl-5-pyridinyl)pyridin or a pharmaceutically acceptable salt thereof.

[0269] Compounds that are reported to be specific inhibitors of COX-2 and are therefore useful in the present invention are These include, but are not limited to: parecoxib, CELEBREX®, and BEXT RA® or a pharmaceutically acceptable salt thereof.

[0270] Examples of angiogenesis inhibitors that may be used in combination with compounds of the present invention include, but are not limited to, , endostatin, ukrain, ranpirnase, IM862, 5- Methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1- Oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldi Nanarin, 5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)- [phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide, CM101 , squalamine, combretastatin, RPI4610, NX31838, sulfated mannoproteins Pentanolic acid, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolidone Isocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-( 1,3-naphthalenedisulfonate), and 3-[(2,4-dimethylpyrrol-5-yl) [1-methyl]-2-indolinone (SU5416).

[0271] Examples of tyrosine kinase inhibitors that may be used in combination with the compounds of the present invention include, but are not limited to, However, N-(trifluoromethylphenyl)-5-methylisoxazole-4-carbohydrate Voxamide, 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indoline- 2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3-chloro- 3-(4-morpholinyl)-7-methoxy-6-[(4-methyl-4-fluorophenylamino)-2-methyl-4-methyl- quinazoline, N-(3-ethynylphenyl)-6,7-bis(2-methoxyphenyl)quinazoline, (Chiethoxy)-4-quinazolinamine, BIBX1382, 2,3,9,10,11,1 2-Hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9, 12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyro rho[3,4-i][1,6]benzodiazocin-1-one, SH268, genistein, Imatinib (STI571), CEP2563, 4-(3-chlorophenylamino)-5 ,6-Dimethyl-7H-pyrrolo[2,3-d]pyrimidine methanesulfonate, 4-(3 -bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 4-(4 '-Hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, ST I571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazine Min, and EMD121974.

[0272] Combinations with compounds other than anti-cancer compounds are also encompassed by the compositions and methods of the present invention. For example, the compounds claimed in the present invention may be used in combination with PPAR-γ (i.e., PPAR-γ receptors). Concomitant use of PPAR-δ (i.e., PPAR-delta) agonists and PPAR-δ (i.e., PPAR-δ) agonists is not recommended for certain PPAR-γ and PPAR-δ are useful in the treatment of malignant diseases such as encephalopathy. The expression of PPAR-γ and its receptors in endothelial cells The involvement of α-glucan in angiogenesis has been reported in the literature (J Cardiovasc. Phar macol.31:909-913(1998);J Biol.Chem.274:9 116-9121(1999);Invest.Ophthalmol Vis.Sci 41:2309-2317 (2000). More recently, PPAR-γ agonists have been Troglitazone and VEGF have been shown to inhibit the angiogenic response to VEGF in vitro. Both rosiglitazone maleate and rosiglitazone maleate inhibited the development of retinal neovascularization in mice. (Arch. Ophthamol. 119:709-717(2001)). PPAR-γ agonists and PPAR-γ / α agonists that may be used in combination with the compounds of the invention Examples of thiazolidinediones include, but are not limited to, thiazolidinediones (DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, Gemfibrozil, Clogemfibrozil, GW2570, SB219994, AR-H 039242, JTT-501, MCC-555, GW2331, GW409544, N N2344, KRP297, NP0110, DRF4158, NN622, GI2625 70, PNU182716, DRF552926, 2-[(5,7-dipropyl-3- Trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylprop propionic acid (disclosed in USSN 09 / 782,856), and 2(R)-7-(3-( 2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-ethyl Rhoman-2-carboxylic acid (USSN 60 / 235,708 and 60 / 244,697 disclosure).

[0273] Another embodiment of the present invention relates to the use of compounds of the present disclosure in combination with gene therapy for the treatment of cancer. For a review of genetic strategies for treating cancer, see Hall et al. m J Hum Genet 61:785-789(1997)) and Kufe et al. ( Cancer Medicine, 5th edition, pp 876-889, B.C. Decker , Hamilton, 2000). Gene therapy can be used to target any tumor suppressor gene. Examples of such genes include, but are not limited to, p53 ( It can be delivered by recombinant virus-mediated gene transfer (see, e.g., U.S. Patent No. 6, 069,134)), uPA / uPAR antagonists (see "Adenovirus-Med" iated Delivery of a uPA / uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and Dissemination in Mice”, Gene T herapy, August 5(8):1105-13(1998)), and Examples include γ-feron (J Immunol 164:217-222(2000)). do.

[0274] The compounds of the present invention can also be used as inhibitors of intrinsic multidrug resistance (MDR), particularly transporter proteins. These drugs may also be administered in combination with inhibitors of MDR, which are associated with high levels of expression of MD. R inhibitors include inhibitors of p-glycoprotein (P-gp), such as LY335979 , XR9576, OC144-093, R101922, VX853 and PSC833 (Valspodar) is one example.

[0275] The compounds of the present invention may be used alone or in combination with radiation therapy to treat or prevent the development of inflammatory bowel diseases. To treat possible nausea or vomiting, including acute, delayed, late and anticipatory vomiting For the prevention or treatment of emesis, the compounds of the invention may be used in combination with: Other antiemetics, in particular neurokinin-1 receptor antagonists, 5HT3 receptor antagonists, e.g. Ondansetron, granisetron, tropisetron and zatisetron, GABAB receptors receptor agonists, e.g., baclofen; corticosteroids, e.g., dextromethorphan; Metasone, Kenalog, Aristocort, Nasalid, Preferid , Benecorten, or others (U.S. Patent No. 2,789, No. 118, No. 2,990,401, No. 3,048,581, No. 3,126,3 No. 75, No. 3,929,768, No. 3,996,359, No. 3,928,32 6 and 3,749,712), antidopaminergic agents, e.g., Phenothiazines (e.g., prochlorperazine, fluphenazine, thioridazine, and methazine) It may also be used in conjunction with solidazine, metoclopramide, or dronabinol. In its form, it acts as a neurokinin-1 receptor antagonist, a 5HT3 receptor antagonist and a corticosteroid. Treatment of emesis can be by administration of a compound of the present invention in which an antiemetic agent selected from the group consisting of steroids. Or administered as an adjuvant for prophylaxis.

[0276] Neurokinin-1 receptor antagonists for use in conjunction with the compounds of the present invention are described, for example, in U.S. Pat. No. 5,162,339, No. 5,232,929, No. 5,242,930, No. 5,242,930, No. No. 5,373,003, No. 5,387,595, No. 5,459,270, No. No. 5,494,926, No. 5,496,833, No. 5,637,699, No. 5 ,719,147; European Patent Publication Nos. 0360390, 0394989, and 0 No. 428434, No. 0429366, No. 0430771, No. 0436334 , No. 0443132, No. 0482539, No. 0498069, No. 0499 No. 313, No. 0512901, No. 0512902, No. 0514273, No. 0512901, No. 0512902, No. 0514273, No. No. 0514274, No. 0514275, No. 0514276, No. 0515681 No. 0517589, No. 0520555, No. 0522808, No. 052 8495, 0532456, 0533280, 0536817, 0532456, 0533280, 0536817, No. 0545478, No. 0558156, No. 0577394, No. 058591 No. 3, No. 0590152, No. 0599538, No. 0610793, No. 0634 No. 402, No. 0686629, No. 0693489, No. 0694535, No. 0694535, No. 0694535, No. 0694535, No. No. 0699655, No. 0699674, No. 0707006, No. 0708101 No. 0709375, No. 0709376, No. 0714891, No. 072 3959, 0733632 and 0776893; PCT International Patent Publication No. 90 / 05525, 90 / 05729, 91 / 09844, 91 / 1 No. 8899, No. 92 / 01688, No. 92 / 06079, No. 92 / 12151 No. 92 / 15585, No. 92 / 17449, No. 92 / 20661, No. 92 / 15585, No. 92 / 17449, No. 92 / 20661, No. 92 / 20676, 92 / 21677, 92 / 22569, 93 / 0 No. 0330, same as No. 93 / 00331, same as No. 93 / 01159, same as No. 93 / 01165 No., same as No. 93 / 01169, same as No. 93 / 01170, same as No. 93 / 06099, same as No. No. 93 / 09116, same as No. 93 / 10073, same as No. 93 / 14084, same as No. 93 / 1 No. 4113, same as No. 93 / 18023, same as No. 93 / 19064, same as No. 93 / 21155 No. 93 / 21181, No. 93 / 23380, No. 93 / 24465, No. No. 94 / 00440, same as No. 94 / 01402, same as No. 94 / 02461, same as No. 94 / 0 No. 2595, same as No. 94 / 03429, same as No. 94 / 03445, same as No. 94 / 04494 No. 94 / 04496, No. 94 / 05625, No. 94 / 07843, No. No. 94 / 08997, same as No. 94 / 10165, same as No. 94 / 10167, same as No. 94 / 1 No. 0168, same as No. 94 / 10170, same as No. 94 / 11368, same as No. 94 / 13639 No. 94 / 13663, No. 94 / 14767, No. 94 / 15903, No. No. 94 / 19320, same as No. 94 / 19323, same as No. 94 / 20500, same as No. 94 / 2 No. 6735, same as No. 94 / 26740, same as No. 94 / 29309, same as No. 95 / 02595 No. 95 / 04040, No. 95 / 04042, No. 95 / 06645, No. No. 95 / 07886, same as No. 95 / 07908, same as No. 95 / 08549, same as No. 95 / 1 No. 1880, same as No. 95 / 14017, same as No. 95 / 15311, same as No. 95 / 16679 No. 95 / 17382, No. 95 / 18124, No. 95 / 18129, No. No. 95 / 19344, same as No. 95 / 20575, same as No. 95 / 21819, same as No. 95 / 2 No. 2525, No. 95 / 23798, No. 95 / 26338, No. 95 / 28418 No. 95 / 30674, No. 95 / 30687, No. 95 / 33744, No. 95 / 30674, No. 95 / 30687, No. 95 / 33744, No. 96 / 05181, 96 / 05193, 96 / 05203, 96 / 0 No. 6094, No. 96 / 07649, No. 96 / 10562, No. 96 / 16939 No. 96 / 18643, No. 96 / 20197, No. 96 / 21661, No. 96 / 29304, 96 / 29317, 96 / 29326, 96 / 2 No. 9328, No. 96 / 31214, No. 96 / 32385, No. 96 / 37489 No. 97 / 01553, No. 97 / 01554, No. 97 / 03066, No. 97 / 01553, No. 97 / 01554, No. 97 / 03066, No. 97 / 08144, 97 / 14671, 97 / 17362, 97 / 1 No. 8206, No. 97 / 19084, No. 97 / 19942 and No. 97 / 21702 and UK Patent Applications Nos. 2266529, 2268931, and 2269 No. 170, No. 2269590, No. 2271774, No. 2292144, No. 2293168, 2293169, and 2302689. The preparation of such compounds is described in the aforementioned patents and publications, which are incorporated herein by reference. are fully described in the

[0277] In one embodiment, a neurokinin-1 receptor antagonist for use in conjunction with a compound of the invention The antidote is: 2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)-phenyl )ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H ,4H-1,2,4-triazolo)methyl)morpholine, or a pharmaceutically acceptable salt thereof The salts are selected from the group consisting of hydroxybenzoates, ...

[0278] The compounds of the present invention are also bisphosphonates (bisphosphonates, diphosphonates, biphosphonates, and diphosphonic acids) in combination with cancer (e.g., bone marrow endothelial cells, e.g., osteoporosis ... Examples of bisphosphonates include, but are not limited to, No: etidronate (Didronel), pamidronate (Aredia), endronate (Fosamax), risedronate (Actonel), zoledronate (Zometa), ibandronate (Boniva), incadronate or Simad Ionate, Clodronate, EB-1053, Minodronate, Neridronate, Pyridine tiludronate and tiludronate (any and all pharmaceutically acceptable salts, derivatives thereof, (including hydrates and mixtures thereof).

[0279] The compounds of the present invention may also be administered with agents useful in the treatment of anemia. The agent may be, for example, an erythropoiesis receptor activator (epoet). (such as tin alpha).

[0280] The compounds of the present invention may also be administered with agents useful in the treatment of neutropenia. Agents for treating neutropenia include, for example, hematopoietic growth factors that regulate neutrophil production and function, e.g., For example, human granulocyte colony-stimulating factor (G-CSF). These include filgrastim and PEG-filgrastim.

[0281] The compounds of the present invention may also be used in combination with immunological enhancers such as levamisole, isoprinosine, and zadazol. It may also be administered with other drugs (e.g., steroids).

[0282] The compounds of the present invention are also useful in the treatment or prevention of breast cancer in combination with aromatase inhibitors. Examples of aromatase inhibitors include, but are not limited to: anastrozole, These include letrozole and exemestane.

[0283] The compounds of the invention are also useful in the treatment or prevention of cancer in combination with other siNA therapeutics. It could be.

[0284] The compounds of the present invention can also be used as γ-secretase inhibitors and / or NOTCH signal inhibitors. Such inhibitors may be administered in combination with inhibitors of signal transduction. No. 084, No. 02 / 30912, No. 01 / 70677, No. 03 / 013506 No. 02 / 36555, No. 03 / 093252, No. 03 / 093264, Same No. 03 / 093251, Same No. 03 / 093253, Same No. 2004 / 039800 , No. 2004 / 039370, No. 2005 / 030731, No. 2005 / 01 4553, USSN 10 / 957,251, International Publication No. 2004 / 089911, No. 02 / 081435, No. 02 / 081433, No. 03 / 018543, No. 2004 / 031137, 2004 / 031139, 2004 / 03113 No. 8, No. 2004 / 101538, No. 2004 / 101539 and No. 02 / 47671 (eg, LY-450139).

[0285] The compounds of the present invention are also useful in the treatment or prevention of cancer in combination with PARP inhibitors. It is possible.

[0286] The compounds of the present invention are also useful in treating the following diseases: abarelix (Plenaxis de pot®); aldesleukin (Prokine®); aldeslo Ikin (Proleukin®); alemtuzumab (Campath®); alitretinoin (Panretin®); allopurinol (Zyl oprim®); altretamine (Hexalen®); amiphos cin (Ethyol®); anastrozole (Arimidex®) arsenic trioxide (Trisenox®); asparaginase (Elspar® azacitidine (Vidaza®); bendamustine hydrochloride (Tr eanda®); bevacizumab (Avastin (registered trademark); bexarotene capsules (Targretin(R)); Bexa Rotengell (Targretin®); Bleomycin (Blenoxan e(R); bortezomib (Velcade(R)); brefeldin A intravenous busulfan (Busulfex®); oral busulfan (Myle ran®); calusterone (Methosarb®); capecitabine carboplatin (Paraplatin®); ); Carmustine (BCNU®, BiCNU®); Carmustine ( Gliadel®; Carmustine and Porifeprosan 20 Implant (Gliadel Wafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil (Leuke cisplatin (Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine (Cl olar®); cyclophosphamide (Cytoxan®, Neos ar®); cyclophosphamide (Cytoxan Injection®) cyclophosphamide (Cytoxan Tablets®); Cytara cytarabine (Cytosar-U®); liposomal cytarabine (DepoCyt®); Decarbazine (DTIC-Dome®); Dactinomycin, Cutinomycin D (Cosmegen®); Dalteparin Sodium Injection ( Fragmin®); darbepoetin alfa (Aranesp®); Satinib (Sprycel®); liposomal daunorubicin (DanuoXo me (registered trademark); Daunorubicin, Daunomycin (registered trademark) Daunorubicin, daunomycin (Cerubidine®); degarelix (Firmagon®); denileukin diftitox (Onta k(R)); dexrazoxane (Zinecard(R)); dexrazoxane hydrochloride Zoxan (Totect®); Didemnin B; 17-DMAG; Docetaxel (Taxotere®); doxorubicin (Adriamycin PFS ( doxorubicin (Adriamycin®, Rubex®) Doxorubicin (Adriamycin PFS Injection (Registered Trademark)) liposomal doxorubicin (Doxil®); dromedine propionate Stanolone (Dromostanolone®); dromostanolone propionate Masterone Injection (registered trademark); Eculizumab injection ( Soliris®; Elliott's B Solution ion®); eltrombopag (Promacta®); Epirubi Epoetin (Ellence®); epoetin alfa (epogen®); Rulotinib (Tarceva®); estramustine (Emcyt®) )); ethinyl estradiol; etoposide phosphate (Etopophos® etoposide, VP-16 (Vepesid®); everolimus tablets (Af initor®); exemestane (Aromasin®); phen Lumoxytol (Feraheme Injection®); Filgra stim (Neupogen®); floxuridine (intra-arterial) (FUDR( fludarabine (Fludara®); fluorouracil, 5- FU (Adrucil®); fulvestrant (Faslodex®) )); gefitinib (Iressa®); geldanamycin; gemcitabine ( Gemzar®; gemtuzumab ozogamicin (Mylotarg®) )); goserelin acetate (Zoladex Implant®); goserelin acetate Histrelin acetate (Histrelin impl ant®); hydroxyurea (Hydrea®); ibritumomab Tiuxetan (Zevalin®); Idarubicin (Idamycin®) ifosfamide (IFEX®); imatinib mesylate (Gleev®) ec®); interferon alpha 2a (Roferon A®); Interferon alfa-2b (Intron A®); iobenguane I 123 Injection (AdreView®); irinotecan (Camptosar®) ixabepilone (Ixempra®); lapatinib tablets (Tyker®) b®); lenalidomide (Revlimid®); letrozole (F emara®); leucovorin (Wellcovorin®, Le ucovorin®); leuprolide acetate (Eligard®); Levamisole (Ergamisol®); lomustine, CCNU (CeeBU (registered trademark); mechlorethamine, nitrogen mustard (Mustarg megestrol acetate (Megace®); melphalan, LP AM (Alkeran®); mercaptopurine, 6-MP (Purineth ol(R); Mesnex(R); Mesnex t abs®); methotrexate®; Methoxsalen (Uvadex®); 8-methoxypsoralen; Mitomycin C ( Mutamycin®; mitotane (Lysodren®); mitotane Xantrone (Novantrone®); mithramycin; nandrolone propionate (Durabolin-50®); nelarabine (Arran on®); nilotinib (Tasigna®); nofetumomab (V erluma®); ofatumumab (Arzerra®); Opre Rubequin (Neumega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel (Taxol (R); paclitaxel protein-bound particles (Abraxane®) palifermin (Kepivance®); pamidronate (Aredia (R); panitumumab (Vectibix®); pazopanib tablets (V Otrient™; pegademase (Adagen e Bovine®); pegaspargase (Oncaspar®) pegfilgrastim (Neulasta®); pemetrexed sodium methicillin (Alimta®); pentostatin (Nipent®); Pobroman (Vercyte®); plerixafor (Mozobil®) plicamycin, mithramycin (Mithracin®); mal sodium (Photofrin (registered trademark)); pralatrexate injection ( Folotyn®; procarbazine (Matulane®); Nacrine (Atabrine®); rapamycin; rasburicase (Elit ek®); raloxifene hydrochloride (Evista®); rituximab (Rituxan®); romidepsin (Istodax®); Prostim (Nplate®); Sargramostim (Leukine®) sargramostim (Prokine®); sorafenib (Nexav®) ar®); streptozocin (Zanosar®); streptozocin (Saccharin®); Nitinib (Sutent®); talc (Sclerosol®); Tamoxifen (Nolvadex®); temozolomide (Temodar®); (Trademark); Temsillosim (Torisel®); Teniposide, VM-26 (Vumon®); testolactone (Teslac®); thioguaia Thioguanine, 6-TG (Thioguanine®); thiopurine; thiotepa (Th ioplex®); topotecan (Hycamtin®); Toremif fluphen (Fareston®); tositumomab (Bexxar®); Tositumomab / I-131 tositumomab (Bexxar®); trans-retin trastuzumab (Herceptin®); tretinoin, ATR A (Vesanoid®); triethylenemelamine; uracil mustard (U racil Mustard Capsules®); valrubicin (Val star®); vinblastine (Velban®); vincristine vinorelbine (Navelbine®); vorinostat (Zolinza®); wortmannin; and zoledrone It may also be useful in the treatment of cancer in combination with steroids (Zometa®).

[0287] The present invention also relates to SEQ ID NO: 5, SEQ ID NO: 4918, SEQ ID NO: 194, SEQ ID NO: 5107, SEQ ID NO: 196, SEQ ID NO: 5109, SEQ ID NO: 151 or SEQ ID NO: :5064 a sequence of at least 15 nucleotides; or a sequence of the invention comprising formula (A) NA molecule and / or its pharmaceutically acceptable salts, solvates or physiologically functional The present invention provides a combination comprising a CTNNB1 inhibitor and a CTNNB1 derivative thereof together with another CTNNB1 inhibitor.

[0288] The above combinations may be conveniently presented for use in the form of pharmaceutical preparations, and thus A pharmaceutical composition comprising the combination of the compounds of the present invention together with a pharmaceutically acceptable diluent or carrier is also provided. This represents an embodiment comprising:

[0289] The individual compounds of such combinations may be administered either sequentially or simultaneously in separate pharmaceutical formulations or in combination. In one embodiment, the individual compounds may be administered in a combined pharmaceutical formulation. are administered simultaneously.

[0290] Thus, the described molecules may be used in combination with other CTNNB1 inhibitors known in the art. and the like, to prevent or treat the diseases, disorders, conditions and traits described herein in a subject or organism. It may be used in combination with one or more known compounds, therapeutic agents or procedures for treatment.

[0291] 3. Therapeutic applications The current body of findings in CTNNB1 research supports the development of CTNNB1 for therapeutic use. These findings demonstrate the need for methods by which expression of .ALPHA. 1 can be regulated.

[0292] Therefore, one aspect of the present invention is to provide a method for treating a cancer that is caused by the action or loss of action of CTNNB1 gene expression. Methods for treating a subject (e.g., without limitation, a human) suffering from a pathology mediated by administering to said subject an effective amount of a double-stranded siNA molecule of the invention. In one embodiment of this aspect, the siNA molecule comprises SEQ ID NO:5, SEQ ID NO:49 18, SEQ ID NO: 194, SEQ ID NO: 5107, SEQ ID NO: 196, SEQ ID NO: 5109 , SEQ ID NO: 151 or SEQ ID NO: 5064 A sequence of at least 15 nucleotides or comprising formula (A). In another embodiment of this aspect, the condition is cancer. or caused by cancer. In this regard, the molecules and compositions of the present invention are useful in methods for treating cancer. Cancers treatable by this embodiment include cholangiocarcinoma, bladder cancer, transitional cell carcinoma, urothelial carcinoma, Osteosarcoma, brain cancer, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma epithelial cancer, rectal cancer, colorectal cancer, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal glandular cancer Cancer, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, squamous esophageal cancer Skin cancer, Esophageal adenocarcinoma, Intraocular melanoma, Uveal melanoma, Gallbladder cancer, Gallbladder adenocarcinoma, Renal cell carcinoma, Clear cell renal Cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms' tumor, leukemia, acute lymphocytic leukemia (A LL), acute myeloid leukemia (AML), chronic lymphocytic (CLL), chronic myeloid (CML) ), chronic myelomonocytic myeloma (CMML), liver cancer, liver cancer, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, embryonic Blastocyst, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphoma, non-Hodgkin's lymphoma lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, T-cell lymphoma, non- Hodgkin's lymphoma, precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma, multiple Myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer , pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary neoplasm, acinar cell carcinoma, prostate cancer, prostate adenocarcinoma, skin cancer, Melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, gastric cancer, gastrointestinal stromal tumor (GIST) , uterine cancer, and uterine sarcoma.

[0293] In one embodiment, the siNA molecules of the invention are used to treat: brain cancer, breast cancer, cervical cancer, colorectal cancer, Cancer, renal cell carcinoma, leukemia, hepatocellular carcinoma, lung cancer, B-cell lymphoma, multiple myeloma, ovarian cancer, pancreatic and a method for treating or preventing cancer selected from pancreatic cancer, prostate cancer, melanoma, and gastric cancer. In certain embodiments, the compounds of the present invention are useful in treating breast cancer, colorectal cancer, liver cancer, and the like. In a particular embodiment, the compounds of the present invention are useful in the treatment of cancer, including ovarian cancer, ... The compounds are useful in the treatment of hepatocellular carcinoma.

[0294] In another embodiment, the siNA molecules of the invention are useful in preventing or monitoring cancer cells and cancer metastasis. In particular, the siNA molecules of the present invention are useful in methods for the treatment of brain cancer, breast cancer, Cancer, cervical cancer, colorectal cancer, renal cell carcinoma, leukemia, hepatocellular carcinoma, lung cancer, B-cell lymphoma, Prevent or modulate metastasis of multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, melanoma, and gastric cancer This is useful in methods for evaluating

[0295] In certain embodiments, the siNA molecule is administered locally or systemically. In another embodiment, the present invention provides a method for treating a subject or organism with siN of the present invention. A molecule and by local administration to the relevant tissue or cells (e.g., lung cells and tissues). In another embodiment, the contacting is performed by, for example, pulmonary delivery. The present invention relates to the treatment of a subject or organism with a siNA molecule of the present invention and a corresponding siNA molecule of the subject or organism. By systemic administration to target tissues or cells (cancerous tissues or cells) (intracellular administration of the siNA) It is characterized by contact (such as by intravenous or subcutaneous administration).

[0296] The siNA molecules of the invention may also be used as reagents in ex vivo applications. For example, the siNA reagents may be introduced into tissues or cells that are then transplanted into a subject for therapeutic effect. The cells and / or tissues are then transferred to the organism or subject receiving the explant. It may be derived from another organism or subject prior to transplantation. The siNA molecule may inhibit the expression of one or more genes in a cell or tissue by inhibiting the expression of the gene in the cell or tissue. These are used to ensure that the tissue has a desired phenotype or function once transplanted in vivo. In one embodiment, specific CT from a patient can be used to modulate NNB1 target cells are extracted. The extracted cells are subjected to a specific nucleotide sequence analysis. CTNNB1 siNA targeting CTNNB1 and conditions suitable for uptake of the siNA by the cells. contacting the cells under conditions of solubility (e.g., using a delivery agent such as a cationic lipid, liposome, or the like); or use techniques such as electroporation to facilitate delivery of siNA into cells. The cells are then reintroduced back into the same or another patient.

[0297] In therapeutic applications, a pharmaceutically effective dose of a siNA molecule or pharmaceutical composition of the invention is administered to a subject. A pharmaceutically effective dose is a dose that prevents, inhibits the occurrence of, or reduces the severity of a disease state. is needed to treat (alleviate to some extent, preferably all of the symptoms) the condition. Those skilled in the art will be able to determine the dosage based on the size and weight of the subject, the degree of progression or penetrance of the disease, the Age, health and sex of the body, route of administration and whether the administration is local or systemic By considering these factors, the therapeutic efficacy of the siNA of the present invention administered to a given subject can be determined. Dosages may be readily determined. Generally, dosages are in the range of 0.1 to 100 mg / kg depending on the potency of the negatively charged polymer. The active ingredient is administered in an amount of μg / kg to 100 mg / kg body weight / day. The dose can be calculated from measurements of drug accumulation in the patient's body. It may be administered in a single dose or in multiple doses.

[0298] The siNA molecules of the invention may be administered once a month, once a week, or once a day (QD). , multiple times per month, week, or day (e.g., but not limited to, twice a day (BID) The dose may be divided into separate doses (e.g., three times a day (TID), once every two weeks, etc.). Recovery rates are easily estimated based on measured residence times and drug concentrations in body fluids or tissues It may be possible.

[0299] Administration can be continuous (i.e., every day) or intermittent. For example, the intermittent administration of the compounds of the present invention may be 1 to 6 days per week, and may be administered in cycles. (e.g., daily administration for 2 to 8 consecutive weeks, followed by This may mean dosing every other day (with a drug-free rest period of up to one week).

[0300] G. Administration The composition or formulation can be administered in a variety of ways. Examples include oral, buccal, sublingual, and parenteral (i.e., intra-articular, intravenous, intraperitoneal, and subcutaneous) In one embodiment, administration includes administration by topical administration, intramuscular administration, or intramuscular administration, local rectal administration, or other local administration. In this case, the compositions of the present invention can be administered by insufflation and inhalation. In some embodiments, the pharmaceutical composition may be administered as a bolus injection or in divided doses. The drug is administered intravenously or intraperitoneally by injection (see, e.g., U.S. Pat. No. 5,286,634). The lipid-nucleic acid particles may be administered by direct injection into the diseased area. It may also be administered by injection at a site distal to the site (see, e.g., Culver, HUMAN GENE THERAPY,MaryAnn Liebert,Inc.,Publi (See Shers, New York, pp. 70-71 (1994)). In some embodiments, the siNA molecules of the invention and formulations or compositions thereof may be prepared in any of the manners described herein. and administered to a cell, subject, or organism by methods generally known in the art.

[0301] 1. In Vivo Administration In any of the treatment methods of the invention, the siNA is administered to a subject as described herein. or as known in the art, alone as a monotherapy, or Any combination with additional therapeutic agents described herein or known in the art. Systemic administration can be achieved by, for example, administering the compound in a manner generally known in the art. pulmonary (inhalation, nebulization, etc.), intravenous, subcutaneous, intramuscular, catheterization, nasopharyngeal, This may include dermal, or oral / gastrointestinal administration.

[0302] In any of the treatment or prevention methods of the invention, the siNA is administered to a subject in a manner consistent with the present invention. as monotherapy, as described herein or as known in the art. either alone or in combination with additional therapeutic agents known in the art, It can be administered locally or to a local tissue. Local administration can be, for example, by administering Commonly known methods include inhalation, nebulization, catheterization, implantation, direct injection, transdermal / transdermal application, and patch application. medications, stent insertion, ear / eye drops, or portal vein administration to the appropriate tissue, or any other local The desired administration procedure, method or treatment may be mentioned.

[0303] In one embodiment, siNA molecules and formulations or compositions thereof are administered to the liver: It is administered as commonly known in the art (see, e.g., Wen et al., 200 4,World J Gastroenterol.,10,244-9;Murao et al. ,2002,Pharm Res.,19,1808-14;Liu et al.,2003,ge ne Ther,10,180-7; Hong et al.,2003, J Pharm Phar macol.,54,51-8;Herrmann et al.,2004,Arch Virol .,149,1611-7; and Matsuno et al., 2003, gene Ther. ,10,1559-66).

[0304] In one embodiment, the present invention provides a method for administering a serotonin-modifying agent of the present invention to hematopoietic cells, such as monocytes and lymphocytes. This method features the use of iNA molecule delivery methods. Such methods are described by Hartmann et al., 998, J. Phamacol. Exp. Ther., 285(2), 920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862;Fil ion and Phillips, 1997, Biochim. Biophys. Acta .,1329(2),345-356;Ma and Wei,1996,Leuk.Res .,20(11 / 12),925-930; and Bongartz et al.,1994,N Details are given in Nucleic Acids Research, 22(22), 4681-8. It is listed.

[0305] In one embodiment, the siNA molecules and formulations or compositions thereof of the invention are administered to the dermis or The vesicles may be administered either directly or topically (e.g., topically) using methods commonly known in the art. (e.g., Brand, 2001, Curr. Opin. Mo l.Ther.,3,244-8;Regnier et al., 1998, J.Drug Tar get,5,275-89;Kanikkannan,2002,BioDrugs,1 6, 339-47; Wraight et al., 2001, Pharmacol. Ther., 9 0, 89-104; and Preat and Dujardin, 2001, STP P HarmaSciences, 11, 57-68). In one embodiment, The siNA molecules of the present invention and formulations or compositions thereof can be administered either directly or topically to alcohol-containing (e.g., ethanol or isopropanol), water, and an additional agent (myristyl Aqueous alcohols that may contain isopropyl phosphate and Carbomer 980, etc. In another embodiment, the siNA is administered topically using an alcohol gel formulation. Topical preparations are formulated for administration by application to the affected area one or more times daily. an occlusive dressing of the entire skin area may be advantageously used. This can be done by an adhesive reservoir system.

[0306] In one embodiment, the siNA molecules of the invention are iontophoretically delivered, e.g., to a specific organ or or compartments (e.g., eye, fundus, heart, liver, kidney, bladder, prostate, tumor, CNS, etc.) Non-limiting examples of iontophoretic delivery are described, for example, in WO 03 / 04368 No. 9 and No. 03 / 030989, which are incorporated herein by reference in their entireties. It is listed in the

[0307] In one embodiment, the siNA molecules and formulations or compositions thereof are administered to the lung. Administered as described herein and as generally known in the art In another embodiment, the siNA molecules and formulations or compositions thereof of the present invention are administered to the lung. For tissues and cells, see U.S. Patent Application Publication Nos. 2006 / 0062758; 014289; and 2004 / 0077540. .

[0308] 2. Aerosols and Delivery Devices Aerosol formulations The compositions of the present invention can be administered in the form of aerosols, either alone or in combination with other suitable components. It can be formulated (i.e., "nebulized") and administered by inhalation (e.g., intranasally or intravenously). Intratracheal administration can be used (Brigham et al., Am. J. Sci., 298:278 (1999) 89). Aerosol formulations may be dispensed in pressurized acceptable propellants (e.g., dichloromethane). difluoromethane, propane, nitrogen, etc.

[0309] In one embodiment, the siNA molecules and formulations thereof of the present invention are delivered via pulmonary delivery, e.g. For example, aerosol formulations or spray-dried formulations administered by inhalation devices or nebulizers. The formulation is administered by inhalation, resulting in rapid local uptake of the nucleic acid molecule into the relevant pulmonary tissue. The solid particulate composition comprising respirable dry particles of the micronized nucleic acid composition is a dry or The lyophilized nucleic acid composition is ground and then the micronized composition is passed through, for example, a 400 mesh filter. The present invention can be prepared by passing the granules through a micro-sieve to break up or disaggregate large aggregates. A solid particulate composition containing the siNA composition is provided with a compound that functions to promote the formation of an aerosol. A dispersing agent such as lactose may be included as well as other therapeutic compounds. This can be blended with the nucleic acid compound in any suitable ratio (e.g., a 1:1 ratio by weight). can be input.

[0310] Spray compositions containing siNA molecules or compositions of the invention can be administered, for example, in pressurized packs ( Aqueous solutions or suspensions delivered from a metered dose inhaler or other device with the use of a suitable liquefied propellant. It may be formulated as a suspension or as an aerosol. In one embodiment, it is suitable for inhalation. The aerosol compositions of the present invention may be either suspensions or solutions and generally comprise: SEQ ID NO: 5, SEQ ID NO: 4918, SEQ ID NO: 194, SEQ ID NO: 5107, SEQ ID NO: SEQ ID NO:196, SEQ ID NO:5109, SEQ ID NO:151, or SEQ ID NO:5064 or a siNA molecule comprising a sequence of at least 15 nucleotides; or formula (A) and an appropriate injection agents (e.g., fluorocarbons or hydrogen-containing chlorofluorocarbons or mixtures thereof) , in particular hydrofluoroalkanes, in particular 1,1,1,2-tetrafluoroethane, 1, 1,1,2,3,3,3-heptafluoro-n-propane, or mixtures thereof The aerosol composition may contain surfactants, such as surfactants, which are well known in the art. Non-limiting examples include oleic acid, lecithin, or oligolactic acid or its derivatives (described in W094 / 21229 and W098 / 34596) In one embodiment, the solvent may be a solvent such as those described above, as well as a co-solvent (e.g., ethanol). The pharmaceutical aerosol formulation of the present invention comprises a compound of the present invention and a fluorocarbon as a propellant. or hydrogen-containing chlorofluorocarbons or mixtures thereof (surfactants and / or co- (which may be combined with a solvent).

[0311] The aerosol formulations of the present invention may be buffered by the addition of a suitable buffering agent. stomach.

[0312] Aerosol preparations may contain additives such as preservatives if the preparation is not prepared in a sterile condition. Non-limiting examples include methylhydroxybenzoate, antioxidants, flavorings, and the like. The ingredients may include but are not limited to: barium carbonate, volatile oil, buffering agents and emulsifiers, and other formulation surfactants. In this state, the non-liquid particulate suspension of the present invention is used to reduce degradation and to provide a safer biocompatible solution. To provide a suspension composition (e.g., siNA and / or its LNP formulation), In another embodiment, a nebulizer is used. The device that makes up the device uses fluorine-containing chemicals (which are bacteriostatic and therefore compatible The compositions of the present invention (e.g., which reduce the likelihood of microbial growth within a sexual device) , siNA and / or its LNP formulation).

[0313] Capsules and cartridges containing the compositions of the present invention for use in an inhaler or insufflator The totridge may be made, for example, from gelatin, and may contain the compound of the present invention and a suitable powder base (lactose). In one embodiment, the composition may be formulated to include a powder mix for inhalation of a soluble fiber (such as cellulose or starch). In this case, each capsule or cartridge contains SEQ ID NO: 5, SEQ ID NO: 4918, Sequence number: 194, sequence number: 5107, sequence number: 196, sequence number: 5109, sequence number SEQ ID NO:151, or a sequence of at least 15 nucleotides of SEQ ID NO:5064; or or a siNA molecule comprising Formula (A) and one or more excipients. The compounds of the present invention may be presented without excipients such as lactose.

[0314] The aerosol compositions of the present invention are intended to deliver respirable particles (e.g., particles that are inhaled) into the respiratory system. as a formulation containing particles small enough to pass through the nose, mouth and larynx when They can be administered via the bronchi and alveoli. Generally, respirable particles are between about 0.5 and 1 mm in size. In one embodiment, the fine range may be 1 to 5 microns. In another embodiment, the fine particle range may be 2 to 3 microns. The non-respirable particles tend to be deposited in the throat and swallowed, and therefore The amount of non-respirable particles in the aerosol is minimized. For nasal administration, retention in the nasal cavity is improved. To ensure this, the particle size is preferably in the range of 10 to 500 μm.

[0315] In some embodiments, the siNA compositions of the invention are administered to the nose, e.g., for the treatment of rhinitis. topically administered by pressurized aerosol formulation, aqueous formulation, by pressurized pump or nebulization Suitable formulations include water as a diluent or carrier for this purpose. In certain embodiments, a method for administering the compositions of the present invention to the lungs or nose is provided. Aqueous formulations may be provided with conventional excipients, such as buffers, tonicity modifiers, and the like.

[0316] b. Device The siNA molecules of the invention can be administered as particulate and / or aerosol formulations as discussed above. and can be dispensed from a variety of aerosolization devices known to those skilled in the art. obtain.

[0317] Liquid or non-liquid particle aerosols containing siNA molecules or formulations of the invention can be prepared in any suitable form. by any suitable means, for example, a nebulizer (see, e.g., U.S. Pat. No. 4,501,729). (e.g., ultrasonic or air-jet nebulizers) It can be made by

[0318] A solid particle aerosol comprising a siNA molecule or formulation of the invention and a surfactant may optionally be The aerosols used for the siNA molecules of the present invention can be produced using a solid particulate aerosol generator. An example of a type of solid particle aerosol generator that can be used is an insufflator. Illustrative Aerosol Generators The second type of device comprises a metered dose inhaler ("MDI"). MDIs containing the siNA molecules or formulations taught herein can be prepared by state-of-the-art methods. (See, for example, Byron (supra) and WO 96 / 32099).

[0319] The siNA molecules can also be dispensed into a fluid dispenser (see WO 05 / 044354). or as a fluid formulation for delivery from a good.

[0320] In certain embodiments of the present invention, conscious, spontaneously breathing subjects and controlled-ventilation subjects In this application to subjects of all ages, a nebulizer device is used. The Lyza device is used for targeted drug delivery to the lungs and systemically. In one embodiment, the device constituting the nebulizer comprises a siNA molecule of the present invention. In another embodiment, the neb is used for localized delivery of the formulation to the lung or pulmonary tissue. The device comprising the disseminator can be used for systemic delivery of the siNA molecules or formulations of the present invention. It is used.

[0321] H. Other Applications / Uses of the siNA Molecules of the Invention The siNA molecules of the present invention may also be used in diagnostic applications, research applications and / or medicines. It can also be used for the production of

[0322] In one aspect, the present invention provides a method for diagnosing a disease, trait, or condition in a subject, comprising: administering a composition of the present invention to the body under conditions suitable for diagnosing said disease, trait or condition in said subject. The present invention features a method including:

[0323] In one embodiment, the siNA molecules of the invention are used to identify a trait, disease or condition in a subject or organism. The authors analyzed the expression of CTNNB1 protein caused by haplotype polymorphisms associated with pathology. It is used to downregulate or inhibit the CTNNB1 gene or CTNNB1 tandem Analysis of protein or RNA levels may be performed on subjects with such polymorphisms or those described herein. can be used to identify subjects at risk for developing the trait, condition, or disease. Such subjects may be candidates for treatment, e.g., administration of siNA molecules of the invention and target genes. The cells are amenable to treatment with any other composition useful in treating diseases associated with the expression of the gene. Therefore, analysis of CTNNB1 protein or RNA levels can be used to determine whether treatment is appropriate for the subject. The type of treatment and course of treatment can be determined. Monitoring levels can be used to predict treatment outcomes, as well as to identify traits, disorders, conditions, or The present invention modulates the level and / or activity of specific CTNNB1 proteins associated with disease. The present invention can be used to determine the effectiveness of compounds and compositions that inhibit the growth of tumors.

[0324] In another embodiment, the present invention provides a double-stranded nucleic acid molecule according to the present invention for use in the manufacture of a medicament. In one embodiment, the medicament is a compound that inhibits the action or loss of action of CTNNB1. In one embodiment, the pharmaceutical is for use in the treatment of cancer. In one embodiment, the medicament is for use in the treatment of brain cancer, breast cancer , cervical cancer, colorectal cancer, renal cell carcinoma, leukemia, hepatocellular carcinoma, lung cancer, B-cell lymphoma, multi- for use in the treatment of myeloma, ovarian cancer, pancreatic cancer, prostate cancer, melanoma and gastric cancer In one particular embodiment, the compounds of the invention are useful in the treatment of hepatocellular carcinoma.

[0325] In certain embodiments, at least one strand is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:49 18, SEQ ID NO: 194, SEQ ID NO: 5107, SEQ ID NO: 196, SEQ ID NO: 5109 , SEQ ID NO: 151 or SEQ ID NO: 5064 A sequence of at least 15 nucleotides or those comprising formula (A) are useful in treating cancers, including but not limited to, brain cancer, breast cancer, Cancer, cervical cancer, colorectal cancer, renal cell carcinoma, leukemia, hepatocellular carcinoma, lung cancer, B-cell lymphoma, Use in methods of treating multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, melanoma, and gastric cancer, etc. It is for use.

[0326] I. Working Example The present invention will now be illustrated by the following non-limiting examples. Those skilled in the art will appreciate that essentially the same results can be achieved. Various non-critical parameters can be easily changed or modified to achieve the desired results. Let's be recognized. [Example]

[0327] Design, synthesis and identification of siNA active compounds against CTNNB1 Synthesis of CTNNB1 siNA A series of siNA molecules were designed, synthesized, and evaluated for their efficacy in inhibiting CTNNB1 gene expression. Specific CTNNB1 sequences were designed and evaluated using the methods described in U.S. Patent Application No. 60 / 182,604. Other sequences were designed and selected using proprietary algorithms. The main criteria for the design of specific CTNNB1 sequences for human siNAs were: i) homology between two species (human and rhesus macaque), and (ii) proprietary High efficacy scores were measured by rhythm. Effect of siNA. The target sequences of selected siNAs are shown in Table 1a (target sequences). The sense and antisense strands of the siNA sequences corresponding to the target sequences are shown in Table 1b. The various chemically modified siNAs are shown in Table 1c.

[0328] [Table 1] TIFF0007720384000004.tif245135 TIFF0007720384000005.tif245135 TIFF0007720384000006.tif245135 TIFF0007720384000007.tif245135 TIFF0007720384000008.tif245135 TIFF0007720384000009.tif245135 TIFF0007720384000010.tif245135 TIFF0007720384000011.tif245135 TIFF0007720384000012.tif245135 TIFF0007720384000013.tif245135 TIFF0007720384000014.tif245135 TIFF0007720384000015.tif245135 TIFF0007720384000016.tif245135 TIFF0007720384000017.tif245135 TIFF0007720384000018.tif245135 TIFF0007720384000019.tif245135 TIFF0007720384000020.tif245135 TIFF0007720384000021.tif245135 TIFF0007720384000022.tif245135 TIFF0007720384000023.tif245135 TIFF0007720384000024.tif245135 TIFF0007720384000025.tif245135 TIFF0007720384000026.tif245135 TIFF0007720384000027.tif245135 TIFF0007720384000028.tif245135 TIFF0007720384000029.tif245135 TIFF0007720384000030.tif245135 TIFF0007720384000031.tif245135 TIFF0007720384000032.tif245135 TIFF0007720384000033.tif168167

[0329]

Table 2

[0330] Briefly, single-stranded oligonucleotides are synthesized using automated solid phase synthesizers, as is commonly known in the art. The compounds were synthesized using phosphoramidite chemistry according to known procedures (see, e.g., U.S. Pat. No. 6,413,999). (See U.S. Pat. No. 12 / 064,014). The synthesis column contains the first nucleoside residue ( The solid support was loaded with a derivatized carboxyl group (either native or chemically modified). Detritylation of the 5'-O-dimethoxytrityl group releases the 5'-hydroxyl. The reaction was initiated by reacting a suitably protected phosphoramidite with a suitable activating agent (acetone). nitrile) to the synthesis column simultaneously, amidite formation to the 5'-hydroxyl The column was then washed with a solvent (e.g., acetonitrile) An oxidizing solution (such as an iodine solution) was pumped through the column to form a phosphite triester bond. The complex P(III) was oxidized to its phosphotriester P(V) analogue. The hydroxyl groups were converted to acetic anhydride in the presence of 2,6-lutidine and N-methylimidazole. The resulting product was capped using a reagent such as HCl. The product was detritylated for subsequent phosphoramidite incorporation. The elongation cycle was then resumed with the addition step. This process was continued until the desired sequence was synthesized. The synthesis was repeated until the final 5'-terminal protecting group (trityl or 5'-O-dimethoxytrimethylsilyl) was added. The reaction was terminated with chilling.

[0331] Once synthesis is complete, the solid support and bound oligonucleotides are removed under argon or vacuum. Aqueous base was added and the mixture was heated until cleavage of the succinyl bond occurred. The cyanoethyl phosphate protecting group was removed, and the exocyclic amine was deprotected.

[0332] The following process was carried out on a single strand containing no ribonucleotides: After treatment with aqueous base, the mixture is filtered and the solid support is freed from the deprotected crude synthetic material. The solid support was then rinsed with water and combined with the filtrate. By obtaining the above, the 5'-O-dimethoxytrityl group is retained and the 5'-terminal position is It is allowed to remain (trityl-on).

[0333] For single strands containing ribonucleotides, the following process was carried out: The solid support was immersed in an aqueous base After treatment with HCl, the mixture was filtered and the solid support was separated from the deprotected crude synthetic material. The solid support was then rinsed with dimethyl sulfoxide (DMSO), which was then combined with the filtrate. To this mixture, add a fluoride reagent such as triethylamine trihydrofluoride. The reaction mixture was quenched with an appropriate buffer, and a 5′-nucleotide was inserted at the final 5′-end position. A solution of crude single chains bearing '-O-dimethoxytrityl groups was obtained.

[0334] Each crude single-chain trityl-on solution was chromatographically purified (SPE RPC purified) The hydrophobic nature of the trityl group allowed the desired full-length oligos to be purified. This allows for stronger retention than non-tritylated truncated truncated truncated sequences. was selectively washed off the resin with a small proportion of an appropriate solvent such as acetonitrile. The retained oligonucleotides were then detritonated on the column using trifluoroacetic acid. The acid-labile trityl group was removed by washing the remaining acid from the column. The full-length oligos were purified using aqueous organic solvents. The final product was then analyzed for purity (HPLC), identity (Maldi-TOF) and purity. F MS) and yield (UV A260 The oligos were freeze-dried or Concentrated to dryness in vacuo Annealing: Based on the analysis of the product, the dried oligos are dissolved in an appropriate buffer. After incubation, equimolar amounts (calculated using theoretical extinction coefficients) of sense and antisense oligonucleotides were added. The nucleotide strands were mixed. Then, this solution was analyzed for double-strand purity (chromatographic method). The analysis showed that either strand was in excess. If so, additional strands, not in excess, were titrated in until duplex formation was complete. If the desired product purity is demonstrated, the material is delivered and ready for use. It was decided.

[0335] The following have been synthesized using this protocol or have been synthesized using this protocol: 1 is a table showing various modified siNAs that can be synthesized using methods known in the art.

[0336] [Table 3] TIFF0007720384000067.tif230169 TIFF0007720384000068.tif230169 TIFF0007720384000069.tif230169 TIFF0007720384000070.tif230169 TIFF0007720384000071.tif230169 TIFF0007720384000072.tif230169 TIFF0007720384000073.tif230169 TIFF0007720384000074.tif230169 TIFF0007720384000075.tif230169 TIFF0007720384000076.tif230169 TIFF0007720384000077.tif230169 TIFF0007720384000078.tif230169 TIFF0007720384000079.tif230169 TIFF0007720384000080.tif230169 TIFF0007720384000081.tif230169 TIFF0007720384000082.tif230169 TIFF0007720384000083.tif230169 TIFF0007720384000084.tif230169 TIFF0007720384000085.tif230169 TIFF0007720384000086.tif230169 TIFF0007720384000087.tif230169 TIFF0007720384000088.tif230169 TIFF0007720384000089.tif230169 TIFF0007720384000090.tif230169 TIFF0007720384000091.tif230169 TIFF0007720384000092.tif230169 TIFF0007720384000093.tif230169 TIFF0007720384000094.tif230169 TIFF0007720384000095.tif230169 TIFF0007720384000096.tif230169 TIFF0007720384000097.tif230169 TIFF0007720384000098.tif230169 TIFF0007720384000099.tif230169 TIFF0007720384000100.tif230169 TIFF0007720384000101.tif230169 TIFF0007720384000102.tif230169 TIFF0007720384000103.tif230169 TIFF0007720384000104.tif230169 TIFF0007720384000105.tif230169 TIFF0007720384000106.tif230169 TIFF0007720384000107.tif230169 TIFF0007720384000108.tif230169 TIFF0007720384000109.tif230169 TIFF0007720384000110.tif230169 TIFF0007720384000111.tif230169 TIFF0007720384000112.tif230169 TIFF0007720384000113.tif230169 TIFF0007720384000114.tif230169 TIFF0007720384000115.tif230169 TIFF0007720384000116.tif230169 TIFF0007720384000117.tif230169 TIFF0007720384000118.tif230169 TIFF0007720384000119.tif230169 TIFF0007720384000120.tif230169 TIFF0007720384000121.tif230169 TIFF0007720384000122.tif230169 TIFF0007720384000123.tif230169 TIFF0007720384000124.tif230169 TIFF0007720384000125.tif230169 TIFF0007720384000126.tif230169 TIFF0007720384000127.tif230169 TIFF0007720384000128.tif230169 TIFF0007720384000129.tif230169 TIFF0007720384000130.tif230169 TIFF0007720384000131.tif230169 TIFF0007720384000132.tif230169 TIFF0007720384000133.tif230169 TIFF0007720384000134.tif230169 TIFF0007720384000135.tif230169 TIFF0007720384000136.tif230169 TIFF0007720384000137.tif230169 TIFF0007720384000138.tif230169 TIFF0007720384000139.tif230169 TIFF0007720384000140.tif230169 TIFF0007720384000141.tif230169 TIFF0007720384000142.tif230169 TIFF0007720384000143.tif230169 TIFF0007720384000144.tif230169 TIFF0007720384000145.tif230169 TIFF0007720384000146.tif230169 TIFF0007720384000147.tif230169 TIFF0007720384000148.tif230169 TIFF0007720384000149.tif230169 TIFF0007720384000150.tif230169 TIFF0007720384000151.tif230169 TIFF0007720384000152.tif230169 TIFF0007720384000153.tif230169 TIFF0007720384000154.tif230169 TIFF0007720384000155.tif230169 TIFF0007720384000156.tif230169 TIFF0007720384000157.tif230169 TIFF0007720384000158.tif230169 TIFF0007720384000159.tif230169 TIFF0007720384000160.tif230169 TIFF0007720384000161.tif230169 TIFF0007720384000162.tif230169 TIFF0007720384000163.tif230169 TIFF0007720384000164.tif230169 TIFF0007720384000165.tif230169 TIFF0007720384000166.tif230169 TIFF0007720384000167.tif230169 TIFF0007720384000168.tif230169 TIFF0007720384000169.tif230169 TIFF0007720384000170.tif230169 TIFF0007720384000171.tif230169 TIFF0007720384000172.tif230169 TIFF0007720384000173.tif230169 TIFF0007720384000174.tif230169 TIFF0007720384000175.tif230169 TIFF0007720384000176.tif230169 TIFF0007720384000177.tif230169 In the table: A, C, G and U = ribose A, C, G or U a, g, c and u = 2'-deoxy-2'-fluoro A, G, C or U A , U , C and G = 2'-O-methyl (2'-OMe) A, U, C, or G A, U, C, and G = deoxy A, U, C, or G B=inverted abasic T=thymidine I = inosine s = phosphorothioate linkage.

[0337] Further synthetic steps of commercially available preparations If analysis after the annealing step indicates that the target product is pure, The material is transferred to a tangential flow filtration (TFF) system for concentration and desalting. (As opposed to doing this before the annealing step).

[0338] Ultrafiltration: The annealed product solution is filtered through a TFF system containing a suitable molecular weight cut-off membrane. After concentration, the product solution is purified by diafiltration. The filtrate is desalted with Milli-Q water until the conductivity is that of water.

[0339] Freeze-drying: The concentrated solution is transferred to a bottle, flash-frozen, and then placed in a freeze-dryer. The product is then freeze-dried into a powder. The bottle is removed from the freeze dryer. When you press the

[0340] Initial screening protocol (96-well plate transfection) Preparation of cell culture: Human hepatoma cell line HepG2, rhesus monkey kidney epithelial cell line, LLC-MK2 derivative and Huh7 cell lines were cultured in modified Eagle's medium. All culture media contained 10% Fetal bovine serum, 100 μg / mL streptomycin, 100 U / mL penicillin, and and 1% sodium bicarbonate. Transfection and screening Cells were placed in all wells of a tissue culture treated 96-well plate at 3500 (Hep G2 and LLC-MK2 derivatives and Huh7) cells / well at a final count of 100 μL After plating, cells were incubated in a 5% CO atmosphere at 4°C for 1 hour in the appropriate culture medium. The mixture was cultured overnight at 37°C in the presence of .

[0341] The next day, the complex containing siNA and RNAiMax (Invitrogen) was incubated in the following manner. A 33-fold diluted solution of RNAiMax was prepared in OPTI-MEM. In parallel, a solution of test siNA was prepared in OPTI-MEM to a final concentration of 120 nM. Incubate the RNAiMax / OPTI-MEM solution for 5 minutes at room temperature. After annealing, equal volumes of siNA solution and RNAiMax solution were added together for each siNA. Ta.

[0342] Upon mixing, a siNA / RNAiMax solution was formed with a siNA concentration of 60 nM. This solution was incubated at room temperature for 20 minutes. After incubation, 20 μL The final concentration of siNA in each well was 10 nM. The final volume of RNAiMax in each well was 0.3 ul.

[0343] For low-concentration screening, siNA was added at 200, 150, 100, or 75 pM / µL. For a 12-point dose-response curve study, a series of siNAs were transfected at 30 Start with 40 nM and dilute 6-fold serially or 40 nM and dilute 4-fold serially. All experiments were performed in biological replicates.

[0344] The incubation time with the RNAiMax-siNA complex was 24 hours. The medium was not changed between infection and harvest for screening and dose-response curve studies. In the persistence assay, the RNAiMax-siNA complex was incubated The time intervals between transfection and 24 hours and 72 hours were 24, 72, and 120 hours. The medium was not changed between harvests at the time points between the 120-hour transfection and the 120-hour transfection. After 72 hours, the medium was replaced with fresh medium.

[0345] Cells-to-Ct and reverse transcription At the desired time points, the culture medium was aspirated and discarded from the wells of the culture plate. The cells were washed once with 50 μL of DPBS solution per well. 1 / well of TaqMan® Gene Expression Cells s-to-CT™ Kit (Applied Biosystems, catalog no. Lysis solution (supplemented with DNase I) from 4399002) was added directly to the plate, and the cells were Add 5 microliters / well of the stop solution provided with the kit to the plate. After 5 minutes, DNase I was inactivated. The lysis plate was incubated at room temperature for at least 2 minutes. The plate can be stored at 4°C for 2 hours or at -80°C for 2 months. It is possible.

[0346] Each well of the reverse transcription plate contains 10 uL of 2x reverse transcriptase buffer, 1 uL of 20 2x reverse transcription buffer and 2 uL of nuclease-free water were required. 1. Prepare the reverse transcriptase by mixing 1000kJ of the 20x reverse transcriptase mix and nuclease-free water. A transcription master mix was prepared. 13 uL of the reverse transcription master mix was added to the reverse transcription plate. A separate reverse transcription assay was performed for each cell plate. A separate reverse transcription plate was prepared for each cell plate. 7 uL of each lysate from the cell lysis procedure was added to each well of the reverse transcription plate. The plate was sealed and spun in a centrifuge (1000 rpm for 30 seconds) and the contents were reverse transcribed. The plate was placed in a thermocycler at 37°C for 60 minutes, 95°C for 60 minutes, and The plate was placed at 4°C for 5 minutes and then at 4°C until removed from the thermocycler. If not used immediately, freeze the plates at -20°C.

[0347] For persistence assays, a similar protocol was followed, but after transfecting the cells, Lysis was performed after 3 or 5 days. cDNA was prepared using the ELISA kit (Biosystems).

[0348] Quantitative RT-PCR (Taqman) A series of probes and primers were used to identify the CTNNB1 and GAPDH genes. Various mRNA transcripts were detected. All primers and primers were prepared in advance by Applied Biosystems, Inc. It was supplied as a verified set (see Table 2).

[0349] [Table 4] The assay was performed on an ABI 7900 instrument according to the manufacturer's instructions. aqMan Gene Expression Master Mix(Cells-t o-CT™ Kit, Applied Biosystems, Catalog No. 439 The PCR reaction was carried out at 50°C for 2 minutes and at 95°C for 10 minutes. After 15 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute were performed.

[0350] In each experiment, a baseline was set in the logarithmic growth phase of the amplification curve, and the baseline and amplification Based on the intersection of the curves, a Ct (cycle number) value was assigned by the instrument.

[0351] calculation The expression level of the target gene and the % inhibition of gene expression (%KD) were determined using the comparative Ct method. I calculated it.

[0352] ΔCt=Ct 標的 -Ct GAPDH ΔΔCt(log2(fold change))=ΔCt (標的siNA) -ΔCt (NTC) Relative expression level = 2 -ΔΔCt %KD = 100 × (1-2 -ΔΔCt ) Unless otherwise stated, a non-targeting control siNA was used, as it is the most relevant control. It was chosen as the comparative value for calculating percent inhibition (knockdown) of gene expression.

[0353] Furthermore, normalization data (which reflects the general health of the cells and the quality of the RNA extraction) This is because we only examined two different mRNAs in treated cells (the first one is the target This is done by examining the levels of one mRNA and the second mRNA normalizer. This allows for the production of potentially toxic compounds to cells, rather than simply knocking down the gene of interest. This allowed us to eliminate potentially harmful siNAs. , by comparing with the Ct of GAPDH across the plate.

[0354] I C 50 All calculations were performed using R.2.9.2 software. The analysis was performed using a sigmoidal dose-response (variable slope) equation for pure ligand binding. For all percent knockdown calculations, calculations are based on non-targeted control unless otherwise stated. Relative to the normalized expression level of the gene of interest in the control (Ctrl siNA)-treated sample And so he went.

[0355] Protein levels were measured using a Bio-Rad VersaDoc Imager. Pixel counts were calculated by dividing the area by the same size in each lane. Each sample was then compared to the appropriate control treatment sample to determine the residual tannins compared to the control. The data were converted to percent protein.

[0356] The effect of the lead siNA on CTNNB1 protein levels was compared with that of the universal control. The results were compared using Student's two-tailed T-test, and P values were obtained. P<0.05 was considered significant. It was considered that.

[0357] result: CTNNB1 siNA was designed and synthesized as previously described. A was screened in HepG2, MK2D, and Huh7 cells. CTNNB1 gene expression data when treated with various modified CTNNB1 siNAs in The log2 (fold change) of the data is shown in Table 3a. Each screening was performed at 24 hours. Quantitative RT-PCR was used to assess CTNNB1 mRNA levels, and the data were analyzed using GAP Normalization was performed to the expression level of DH (a ubiquitously expressed "housekeeping" gene). Each treatment was then normalized to a non-CTNNB1-targeted control.

[0358] [Table 5] TIFF0007720384000180.tif231167 TIFF0007720384000181.tif231167 TIFF0007720384000182.tif231168 TIFF0007720384000183.tif140167 A subset of siNAs in Table 3a that had large log2 (fold change) in the primary screen The kits were rescreened in Huh7 cells, and the results are shown in Table 3b.

[0359] [Table 6] TIFF0007720384000185.tif161168 CTNNB1 siNA was designed and synthesized as previously described. A was screened in MK2D cells. Various modified CTNNBs were screened in human cells. 1 Log2 (fold change) of CTNNB1 gene expression data when treated with siNA The results are shown in Table 3c. Each screening was performed at 24 hours. Quantitative RT-PCR was used to CTNNB1 mRNA levels were assessed and the data were analyzed using GAPDH (ubiquitously expressed Each treatment was then normalized to the expression level of the non-CTNN gene ("keeping" gene). Normalized to B1 targeting control.

[0360] [Table 7] TIFF0007720384000187.tif229167 TIFF0007720384000188.tif229166 TIFF0007720384000189.tif230167 TIFF0007720384000190.tif229167 TIFF0007720384000191.tif228165 TIFF0007720384000192.tif229166 TIFF0007720384000193.tif131167 Selected highly ranked siNAs in Tables 3a and 3b were compared for efficacy and potency. The results of these siNAs were further analyzed in 7 cells (using a dose-response curve). Potency 50 is the number of siNA transactivators that result in 50% knockdown of target mRNA. The calculated transfection concentration was calculated as IC after 24 hours of exposure. 50 asked for.

[0361] [Table 8] TIFF0007720384000195.tif60167 Additional siNAs in Tables 3a and 3b were tested for efficacy and potency in MK2D cells. These siNAs were further analyzed using dose-response curves. The results are shown in Table 5. 0 indicates siNA transfection resulting in 50% knockdown of target mRNA. The calculated IC concentration was 0.1% after 24 hours of exposure. 50 asked for.

[0362] [Table 9] [Example]

[0363] Determination of in vitro serum stability of siNA siNA was reconstituted in HO as a 50 μM-100 μM stock solution and diluted in human serum. (pre-warmed to 37°C) to a final concentration of 20 μg / mL. The mixture is then heated at 37°C. Incubate for 0, 1, and 2 hours at RT. At the end of each time point, add an equal volume of Phenomenon The reaction is stopped by mixing with the next Lysis-Loading Buffer. Oligonucleotides were plated onto Phenomenex Solid Phase plates in a 96-well format. Purified by Hase Extraction and Labconco Triad Lys The freeze-dried sample was then added to 150 μL of 1 mM NaCl solution. Reconstitute in EDTA (prepared with RNase-free H2O). The solution was analyzed by liquid chromatography / mass spectrometry on a ThermoFisher Orbitrap. For analytical (LC / MS) analysis, dilute 5-fold with 1 mM EDTA. The metabolites were examined based on their measured molecular weights. [Example]

[0364] Cytokine induction assay Lipid nanoparticles (DLinDMA / cholesterol / S-PEG-C-DMA / DSPC The immunostimulatory effects of various siNAs of the present invention loaded into the IgG1-containing ... To evaluate the efficacy of LNP-formulated siNA, C57B1 / 6 mice were injected with a single 3 mpk dose into the tail. Administered by intravenous injection. Serum or plasma samples are collected 3 and 24 hours after administration. Cytokine and chemokine levels in these samples were analyzed using SearchLight IR Cytokine Array (Aushon Biosciences) Measurements were performed using a kit according to the manufacturer's instructions. The cytokines and chemokines measured were: , IL-1α, IL-1β, IL-6, KC, IL-10, IFNγ, TNF, GMCS F, MIP-1β, MCP-1 / JE, and RANTES. [Example]

[0365] Efficacy studies in mice Mice were injected with LNP-encapsulated siNA or vehicle control by tail vein injection at two different sites. A 3-week dosing scheme: a single 1 mg / kg dose for 3 consecutive days or a single 6 mg / kg dose In some experiments, mice were administered IV at 100 mg / kg BID. The total tumor tissue was analyzed by micro-CT scanning. Animals were then cultured 5 days after the last siNA injection (day 23) to measure the number of siNA-positive cells. Upon sacrifice, normal liver tissue and tumor tissue from each animal are collected for RNA purification. was purified using the RNeasy 96 kit (Qiagen, Cat. No. 74182). cDNA is generated from total RNA using the High Capacity cDNA Reverse Prepared using the e Transcription Kit (Cat. No. 4368813) Quantitative PCR reactions were performed using TaqMan Universal PCR Master. The Taq DNA fragment of human CTNNB1 was purified using Taq DNA fragment r Mix (Cat. No.: 4304437). Man Gene Expression Assay(Hs00355045_m1) and human GAPDH TaqMan Gene Expression Assay The mRNA levels of both transcripts are monitored in tumor tissue using mouse CTN. TaqMan Gene Expression Assay of NB1 (Mm00483 033_m1) and TaqMan Gene Expression of mouse GAPDH The NIH Assay was used to monitor the mRNA levels of both transcripts in liver tissue. The expression levels of CTNNB1 were normalized to GAPDH to minimize technical variations. do. [Example]

[0366] Pharmacodynamic studies in non-human primates Rhesus monkeys were administered a single 2.5 mpk dose of siNA-loaded lipid nanoparticles by intravenous injection. To monitor target mRNA knockdown, the species were administered before and after administration. At various time points, approximately 20 mg of the sample was removed from each animal using a 16T gauge Menghini needle. Liver biopsies of the tissues will be performed. Whole blood and serum / plasma samples will also be collected at different time points before and after administration. All procedures will be conducted in accordance with USDA Animal Health Guidelines. outlined in the Mal Welfare Act (9 CFR, Parts 1, 2, and 3) and The Guide for Care and Use of Lab oratory Animals(ILAR publication,1996,Na This work complies with the terms and conditions set forth in the National Academy Press. Total RNA from this liver biopsy tissue was purified using the RNeasy 96 kit (Qiagen, Cat. No. 10011001). cDNA was purified from total RNA using High Capacitance PCR (Protein No. 74182). ity cDNA Reverse Transcription Kit (Catalog No. Quantitative PCR reactions were performed using TaqMan Univ. Using Standard PCR Master Mix (Cat. No. 4304437) TaqMan Gene Expression Assay for Human CTNNB1 y(Hs00355045_m1) and TaqMan Gen for rhesus GAPDH e Expression Assay (Rh02621745_g1) was used to The mRNA levels of both transcripts in the biopsied tissues were monitored. Levels are normalized to GAPDH to minimize technical variation.

[0367] LNP formulation containing siNA (DLinDMA / cholesterol / S-PEG-C-DM A / DSPC (ratio of 40 / 48 / 2 / 10)) was examined. The fold change in og2 was determined on days 3, 7, 14, and 28 after administration. NB1 expression levels are measured 7 days before the first administration. [Example]

[0368] Pharmacodynamic studies in mice Mice received a single 0.3 mL injection of LNP-encapsulated siNA or vehicle control via tail vein injection. Five animals were administered IV at 3 mg / kg for each treatment. Animals were sacrificed after 7, 14, and 21 days, and liver tissue was collected from each animal for RNA purification. Total RNA was purified using the RNeasy 96 kit (Qiagen, Cat. No. 74182). cDNA was purified from total RNA using High Capacity cDNA R everse Transcription Kit (Catalog Number: 4368813) Quantitative PCR reactions were performed using TaqMan Universal PCR. This was performed using Master Mix (Cat. No.: 4304437). TaqMan Gene Expression Assay of NB1 (Mm00483 033_m1) and TaqMan Gene Expression of mouse GAPDH The mRNA levels of both transcripts were monitored using the CTNN assay. Expression levels of B1 were normalized to GAPDH to minimize technical variations. Shown in Table 6.

[0369] [Table 10] [Example]

[0370] Pharmacodynamic studies in non-human primates Rhesus monkeys, single dose of 3.34 mg / m 2 Body surface area dose of siNA-loaded lipid nanoparticles The drug was administered by intravenous infusion. To monitor the knockdown of target mRNA, Each animal was punctured with a 16T gauge Menghini needle at various time points before and after administration. Approximately 20 mg of tissue was biopsied from the liver before and after administration. and serum / plasma will be collected to monitor potential toxicity associated with the treatment. , USDA Animal Welfare Act (9 CFR, Parts 1, 2 and 3) and the provisions outlined in The Guide for Care and se of Laboratory Animals(ILAR publicatio (N, 1996, National Academy Press) Total RNA from this liver biopsy tissue was purified using the RNeasy 96 kit (Qiagen) cDNA was purified from total RNA using High C apacity cDNA Reverse Transcription Kit Quantitative PCR reactions were performed using TaqMan PCR kits (catalog number: 4368813). Universal PCR Master Mix (Cat. No. 4304437) TaqMan Gene Expression of Human CTNNB1 Assay (Hs00355045_m1) and TaqMan for rhesus monkey GAPDH Gene Expression Assay (Rh02621745_g1) was used and monitored the mRNA levels of both transcripts in liver biopsy tissue. Expression levels of α-glucan were normalized to GAPDH to minimize technical variations. Data are shown in Table 7. The LNP formulation containing siNA (DLinDMA / cholesterol / S-PEG-C -DMA / DSPC (ratio of 40 / 48 / 2 / 10)) was tested. The Log2 (fold change) of the CTNNB1 activity was calculated on the 2nd and 7th days after administration. Expression levels are measured 6 days before the first administration.

[0371] [Table 11] [Example]

[0372] Small interfering nucleic acid lipid nanoparticle (LNP) formulations A. General Description of the LNP Process for LNP Formulations: Lipid nanoparticles were prepared by an impinging jet process. The particles were dissolved in alcohol. The lipids were mixed with siNA dissolved in citrate buffer to form a soluble phospholipid. The lipid solution contains cationic lipids, helper lipids (cholesterol), PEG (e.g., P EG-C-DMA, PEG-DMG) lipids, and DSPC were mixed with an alcohol (e.g., The concentration was 5-15 mg / mL (target: 9-12 mg / mL) in ethanol. The lipid ratio was cationic lipids in the molar percentage range of 25-98 (target 35-65). and the helper lipids have a mole percent range of 0 to 75 (target 30 to 50). wherein the PEG lipid has a mole percent range of 1 to 15 (target 1 to 6); The DSPC had a mole percent range of 0-15 (target 0-12). Solution A contains one or more siNA sequences in a sodium citrate buffered saline solution (3.5-5%). 0.3-0.6 mg / mL (target 0.3-0.9 mg / mL) These two solutions were stored at 15-40°C (target temperature: 30-40°C). When heated to a temperature in the range of 0.1 to 1.5°C and then mixed in an impinging jet mixer, LNPs are formed immediately. The T-junction inner diameter (tee ID) was in the range of 0.25 to 1.0 mm, and the total flow rate was 10 to 100 mm. The flow rate and inner diameter of the tube were set to 600 mL / min. The LNP suspension was then mixed with a buffer solution at a high pH, 1:1 to 1: Mix ratios in the range of 3 (vol:vol) (but aiming for 1:2 (vol:vol)) The buffer solution was brought to a temperature in the range of 15-40°C (target 30-40°C). The LNP suspension was further diluted with a buffer solution at a high pH in a ratio of 1:1 to 1:3 (vol:vol) The buffer solutions were mixed at a ratio ranging from 1:2 (vol:vol) to 15 The temperature was adjusted to a range of 10-40°C (target 30-40°C). The mixed LNP was kept for 30 minutes to 2 hours. After that, an anion exchange filtration step was carried out. The incubation temperature was 15 to 40°C (target After incubation, the LNP suspension was filtered through a 0.8 μm filter. The mixture was filtered through a 1 mm filter (including an anion exchange separation step). Use tubes with ID ranging from 5 mm ID and flow rates of 10 to 2000 mL / min. The LNPs were concentrated and diafiltered by an ultrafiltration process, during which , the alcohol is removed and the citrate buffer is the final buffer solution (phosphate buffered saline). The ultrafiltration process involves tangential flow filtration (T In this process, a nominal molecular weight cutoff range of 30-500 KD was used. The membranes used were either hollow fiber or flat sheet cassettes. The TFF process with a cutoff ensures that the LNPs are retained in the retentate and not in the filtrate or permeate. The filtrate contained alcohol; citrate buffer; and final buffer waste. The FF process is a multi-step process that increases the initial concentration of siNA to a concentration of 1–3 mg / mL. After concentration, the LNP suspension is diafiltered with 10–20 volumes of the final buffer. The material was then further diluted with HCl to remove the alcohol and perform a buffer exchange. The final step in the LNP process is the sterile filtration and purification of the concentrated LNP solution. The resulting product was sealed in a vial.

[0373] Analysis steps: 1) siNA concentration The concentration of the siNA duplex was determined by strong anion exchange high performance liquid chromatography (SAX-HP LC) on a Waters 2695 Alliance system (Water Co poration, Milford MA) with a 2996 PDA detector. LNP (also referred to as RNAi delivery vehicle (RDV)) was added to 0.5% T Total siNA was released by treatment with riton X-100, and then purified by SAX isolation. ex BioLC DNAPac PA 200 (4 x 250 mm) column (254 nm The mobile phase was A: 25 mM NaClO4, 10 mM T B: 20% EtOH, pH 7.0, and C: 250 mM NaClO4, 10 mM T ris, 20% EtOH, pH 7.0, with a linear gradient from 0 to 15 min and a flow rate of The amount of siNA was determined by comparison with a siNA standard curve.

[0374] 2) Encapsulation speed The fluorescent reagent SYBR Gold was used for RNA quantification to monitor the kinetics of RDV incorporation. RDV with or without Triton X-100 was used to detect free siNA. The assay was performed using a SpectraMax M5e microplate reader. Rate spectrophotometer (Molecular Devices, Sunnyvale, CA) The samples were excited at 485 nm and the fluorescence emission was measured at 530 nm. The amount of siNA is determined by comparison with a siNA standard curve.

[0375] Encapsulation rate = (1 - free siNA / total siNA) x 100% 3) Particle size and polydispersity RDV containing 1 μg of siNA was diluted to a final volume of 3 ml with 1× PBS. The particle size and polydispersity of the samples were determined by dynamic light scattering using a ZetaPALS instrument (Brook haven Instruments Corporation,Holtsville ,NY). The scattering intensity was measured at 25°C with a He-Ne laser at a scattering angle of 90°. Measured at the corners.

[0376] 4) Zeta potential analysis RDV containing 1 μg of siNA was added to 1 mM Tris buffer (pH 7.4 ) to a final volume of 2 ml. The electrophoretic mobility of the samples was measured using a ZetaPALS instrument (Br ookhaven Instruments Corporation,Holtsvi The study was carried out using a 1000-kV FET (1000 kV, NY) with a He-Ne laser as the electrode and light source. The Smoluchowski limit was assumed in the calculation of the zeta potential.

[0377] 5) Lipid analysis Individual lipid concentrations were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). TERS 2695 Alliance System (Water Corporation , Milford MA) to the Corona Charged Aerosol Detector (CAD) (ESA Bios RD was measured using a 100% RI* meter with a 100% RI* meter. Individual lipids within V were separated into Agilent Zorbax SB-CI8 (50 × 4.6 mm The analysis was carried out using a 1.8 μm particle size column with CAD at 60°C. The mobile phase was A:0 A: 0.1% TFA in H2O and B: 0.1% TFA in IPA. The gradient was 60 % mobile phase A and 40% mobile phase B from time 0 to 40% mobile phase A and 60% mobile phase B for 1 4.00 min; 40% mobile phase A and 60% mobile phase B from 1.00 to 5.00 min; 4 0% mobile phase A and 60% mobile phase B to 5.00 min to 25% mobile phase A and 75% mobile phase B until 10:00 min; 25% mobile phase A and 75% mobile phase B from 10:00 min to 5% Mobile phase A and 95% mobile phase B until 15.00 min; and 5% mobile phase A and 95% mobile phase B until 15.00 min. Phase B from 15:00 to 60% mobile phase A and 40% mobile phase B at 20:00 min. The flow rate was 1 ml / min. The individual lipid concentrations were determined by comparison with a standard curve. All lipid components were fitted to a quadratic curve. The mole fraction of each lipid was calculated based on its molecular weight. This was calculated.

[0378] B. Typical LNP preparations for various formulations in Table 11 The siNA nanoparticle suspensions in Table 11 were prepared by adding siNA and / or carrier molecules to 20 mM quencher. Dissolve in sodium phosphate buffer (pH 5.0) at a concentration of approximately 0.40 mg / mL. The lipid solution was prepared by adding a cationic lipid (e.g., (13Z,16Z)-N,N -dimethyl-3-nonyldocosa-13,16-dien-1-amine, see structure in Table 12) A mixture of DSPC, cholesterol, and PEG-DMG (ratios shown in Table 11) was used. It was prepared by dissolving it in aqueous ethanol at a concentration of approximately 8 mg / mL. The nitrogen to sucrose ratio was estimated to be 6:1.

[0379] Approximately equal volumes of siNA / carrier solution and lipid solution were used with two FPLC pumps at the same flow rate. The mixture was delivered to a mixing T-connector using a back pressure valve (Wais). The resulting milky mixture was collected in a sterile glass bottle. This mixture was then diluted with an equal volume of Dilute with an equal volume of citrate buffer, followed by an equal volume of PBS (pH 7.4) and perform ion exchange. The mixture was filtered through a membrane to remove any free siNA / carrier. 4) Ultrafiltration was used to remove the ethanol and exchange the buffer. The final LNP was obtained by concentrating to volume and sterile filtering through a 0.2 μm filter. The obtained LNPs were analyzed for particle size, zeta potential, alcohol content, total lipid content, encapsulated nucleic acid, and The samples were characterized for their nucleic acid content and total nucleic acid concentration.

[0380] LNP Manufacturing Process In a non-limiting example, LNPs were prepared in bulk as follows: (1) Preparation of lipid solution; (2) Preparation of siNA / carrier solution; (3) Mixing / particle formation (4) incubation; (5) dilution; and (6) ultrafiltration and concentration. Ta.

[0381] 1. Lipid Solution Preparation A 2 L glass reagent bottle and a measuring cylinder were depyrogenated. The temperature was raised. 8.0 g of (13Z,16Z)-N,N-dimethyl Pipette in 1.2 g of 3-nonyldocosa-13,16-dien-1-amine. DSPC, 3.5 g of cholesterol, and 0.9 g of PEG-DMG were added to this mixture. Add 1 L of ethanol to the mixture. Place the reagent bottle in a heated water bath (temperature not exceeding 50°C). The lipid suspension was stirred with a stir bar. A thermocouple probe was inserted into one end of the suspension using a sealed adapter. It was heated to 40° C. The solution was allowed to cool to room temperature.

[0382] 2. Preparation of siNA / carrier solution In a sterile container (Corning storage bottle), add 0.4 times the water correction factor (approximately 1.2). g of siNA powder was weighed into a 2 L glass tube that had been depyrogenated. The weighing vessel was then diluted with citrate buffer (20 mM, pH 5.0) for 3 minutes. Rinse the container several times, pour the rinse solution into the 2L glass bottle, and add the citric acid buffer solution until it reaches 1L. The concentration of the siNA solution was measured by UV spectroscopy using the following procedure. 20 μL of the solution was taken out and diluted to 1000 μL (50 times the original volume). UV readings were recorded at A260 nm after a blank measurement with citrate buffer. Repeat. Note: If the readings for two samples are consistent, take the average and use the siNA digestion data. The concentration can be calculated based on the diffusion coefficient. The final concentration is in the range of 0.40 ± 0.01 mg / mL. If the concentration is higher than 100 ppm, add more siNA / carrier powder or add more citrate buffer. The concentration can be adjusted by adding a second The antibody may be repeated with siNA.

[0383] The siNA / carrier solution may not be a cocktail of two or more siNA duplexes and / or carriers. When the siNA was composed of a single siNA duplex, the siNA / carrier was diluted in 20 mM citrate buffer. (pH 5.0) to give a final concentration of 0.4 mg / mL.

[0384] The lipid solution and ethanol solution were then mixed in Pall Acropak 20 0.8 / 0. Sterile filter through a 2 μm sterile filter PN 12203 and store in Master Flex Depyrogenation using Peristaltic Pump Model 7520-40 The mixture was placed in a sterilized glass vessel to provide a sterile starting material for the encapsulation process. is 20cm 2 The experiment was carried out on an 80 mL scale with a membrane area of 1000 m / s. The flow rate was 280 mL / min. The process can be scaled up by increasing the tube diameter and filtration area.

[0385] 3. Particle formation-mixing process Two-barrel syringe-driven pump (Harvard 33 Twin Syringe ) and sterile lipid / ethanol solution and sterile siNA / carrier or siNA / carrier capsule. Add 0.5 mL of 20 mM citrate buffer (20 mM citrate buffer, pH 5.0) solution to 0.5 mL of 20 mM citrate buffer. Mixed in a 5mm ID T-mixer (mixing stage I) at equal or nearly equal flow rates The resulting discharged LNP suspension contained 40–50 vol% ethanol. To obtain a 45 vol% ethanol suspension, sterile lipid / ethanol and sterile silane were added. A / carrier or siNA / carrier cocktail / citrate buffer solution was added to 54 ml of each L / min and 66 mL / min flow rates, so that the total flow rate of the discharge mixture was 120 mL / min Mixed.

[0386] 4. Dilution The discharge stream of mixing stage I goes directly into a 4mm ID T-mixer (mixing stage II). A high pH buffer solution (20 mM sodium citrate, 300 mM sodium chloride) was used. The buffer was diluted 1:1 (vol:vol%) with thorium hydroxide (pH 6.0). The buffer solution was at a temperature ranging from 30 to 40 °C and was pumped through a 4 mm T-mixer using a peristaltic pump (Cole Parmer MasterFlex L / S (600 RPM) 120mL The fluid was delivered at a flow rate of 1 / min.

[0387] The discharge stream of Mixing Stage II is directly fed into a 6mm ID T-mixer (Mixing Stage III). where it is diluted 1:1 (vol: The buffer solution was diluted at a ratio of 15 to 25°C and 6 ml m T-mixer with a peristaltic pump (Cole Parmer MasterFlex L / S The fluid was delivered at a flow rate of 240 mL / min by a flowmeter (600 RPM).

[0388] 5. Incubation and Removal of Free siNA Mixing Stage III output stream held after mixing for 30 min incubation Incubation was carried out at a temperature of 35-40°C, and the in-process suspension was protected from light. After incubation, free (unencapsulated) siNA was added to the Mustang The ions were removed by anion exchange using a Q chromatography filter (capsule). Before use, the chromatography filter was washed with 1N NaOH, 1M NaCl, and The cells were pretreated with a final flush of 12.5 vol% ethanol solution (in PBS). The pH of the final flush solution was confirmed to be <8. The suctioned LNP flow was passed through a Mustang Q filter and then pumped through a peristaltic pump (Cole Parmer MasterFlex L / S 600 RPM) approximately 10 The filtrate was filtered at a flow rate of 0 mL / min. The filtrate was transferred to a sterile glass filter for ultrafiltration and concentration as follows. It was received in a container.

[0389] 6. Ultrafiltration, concentration and sterile filtration The ultrafiltration process is a timed process and the flow rate must be carefully monitored. This is a two-step process; first, the diluted material is taken and diluted to approximately 0.3-0. This is a concentration step in which the siNA is concentrated approximately 8-fold to a concentration of 6 mg / mL.

[0390] In the first step, an ultrafiltration membrane, 100 kDa PES (Spectrum Labs), was used. The attached ring stand was connected to a peristaltic pump (Spectrum KrosFlo II) Add 9.2 L of sterile distilled water to the reservoir; drain 3 L. Drain to waste, drain the remainder into the permeate and discard. 5.3 L of 0.25 N water Add sodium hydroxide to the reservoir, drain 1.5 L to waste, and add 3.1 L to the permeate. The remaining sodium hydroxide was drained into the filtrate and discarded, and kept in the system for disinfection. The pump was then allowed to drain (at least 10 minutes) and 9.2 L of 70 (v / v) Add 1.5 L of 100% isopropyl alcohol to the reservoir and drain to waste. The remainder was drained into the permeate and discarded. Add 1.5 L of 12.5% ethanol in phosphate-buffered saline (PBS) and drain. The remaining part was drained into the permeate until the effluent reached a neutral pH (7-8). The outflow value was recorded and then the pump was drained.

[0391] The diluted LNP solution was filled into the reservoir up to the 1.1 L mark. The pump was operated at 2.3 L / min. After 5 minutes of recirculation, the permeate pump was turned on at 62.5 mL / min to bring the liquid level to The reservoir was kept at approximately 950 mL. The diluted LNP solution was reduced from 9.8 L to 1.1 L. Concentrate for 140 minutes at 4°C, and pause the pump when all the diluted LNP solution has been transferred to the reservoir. did.

[0392] The second step is a dialysis step to exchange the ethanol / aqueous buffer for phosphate buffered saline. During this process, approximately 10 to 20 diafiltration steps were performed. After diafiltration, a second concentration was performed using 1000 volumes of phosphate buffered saline. The LNP suspension was concentrated three-fold to approximately 1-1.5 mg / mL of siRNA. The resulting suspension was collected in a sterile plastic PETG bottle. The final suspension was then , Pall 0.45um PES filter and Pall 0.2um PES filter The solution was filtered through a filter for terminal sterilization and then filled into vials.

[0393] The obtained LNPs were analyzed for particle size, zeta potential, alcohol content, total lipid content, encapsulated nucleic acid, and The total nucleic acid concentration was characterized.

[0394] C. Synthesis of Novel Cationic Lipids The synthesis of novel cationic lipids is a linear process starting from lipid acids (i). Coupling to N,O-dimethylhydroxylamine affords Weinreb amide ii Grignard addition gives ketone III. Titanium-mediated reductive amine Conversion gives final products of type iv.

[0395] General Scheme 1

[0396] [ka] The synthesis of single-carbon homologated cationic lipid v begins with lipid ketone (iii). The conversion of the ketone to the nitrile (iv) is a linear process. The reduction of the nitrile to a primary amine is achieved by treatment with sodium tert-butoxide. After deprotection, reductive amination gives the final cationic lipid v.

[0397] General Scheme 2

[0398] [ka] The synthesis of two-carbon homologated cationic lipid viii was carried out starting from lipid ketone (iii). The conversion of the ketone to the α,β-unsaturated amide vi is a linear process as described by Peter The conjugate reduction of the α,β-unsaturated hydroxyl group is carried out using LS-selectride. The amide vii is reduced with lithium aluminum hydride to give: The final cationic lipid viii is obtained.

[0399] General Scheme 3

[0400] [ka] Cyclopropyl-containing lipids are prepared according to General Scheme 4. When cyclopropyl-containing cyclopropanation of methyl methyl ether II was carried out under Simmons-Smith cyclopropanation conditions, the cyclopropyl-containing cyclopropanol was obtained. This affords the levamide ix, which can be further reacted with the final amide ix as outlined in General Schemes 1-3. To produce a product.

[0401] General Scheme 4

[0402] [ka] The synthesis of allylic amine cationic lipid Xv was carried out by linear protonation starting from aldehyde X. Addition of t-butyl acetate yields the β-hydroxyester XI. After converting the hydroxyl functionality to a fluoro group, acid treatment produces the β-fluoro acid x Conversion of the acid to a Weinreb amide followed by Grignard addition gives ii. , affording the β-fluoroketone xiv. Reductive amination leads to the concomitant elimination , the desired allylic amine xv is produced.

[0403] General Scheme 5

[0404] [ka] 20,23-Nonacosadiene-10-amine, N,N-dimethyl-, (20Z, 23Z )(Compound 1)

[0405] [ka] 11,14-Eicosadienoic acid, (11Z,14Z)-(50g, 162mmol), N,O-Dimethylhydroxylamine hydrochloride (31.6 g, 324 mmol), HOAt (44.1 g, 324 mmol), EtN (45.2 mL, 324 mmol), and EDC (62.1 g, 324 mmol) was mixed in DCM (810 mL) and stirred at ambient temperature. The reaction was then washed with 5 x 700 mL of water and then 1 x 600 mL of water. Wash with 1M NaOH, dry with sodium sulfate, filter through Celite, and evaporate. 53.06 g (93%) of 11,14-eicosadienamide, N-methoxy- N-Methyl-, (11Z,14Z) was obtained as a clear golden oil. 1 H NMR(4 00MHz, CDCl3)δ 5.35(m,4H),3.68(s,3H),3.18 (s,3H),2.77(m,2H),2.41(t,J=7Hz,2H),2.05( m,4H),1.63(m,2H),1.40-1.26(m,18H),0.89(t ,J=7Hz,3H).

[0406] [ka] 11,14-Eicosadienamide, N-methoxy-N-methyl-, (11Z,14Z )-1 (4 g, 11.38 mmol) was dissolved in dry THF (50. 0 ml), and then 1 M nonyl magnesium bromide (22.76 ml, 22.76 mmol) was added under nitrogen at ambient temperature. After 10 min, excess saturated aqueous NH The reaction mixture was slowly quenched with Cl. The reaction mixture was washed with hexane and water in a separatory funnel. The lower aqueous layer was discarded, and the upper layer was dried over sodium sulfate, filtered, and evaporated. The crude ketone was obtained as a golden oil. HF (2M) (14.22 ml, 28.4 mmol) was added, followed by Ti(Oi-Pr ) 4 (6.67 ml, 22.76 mmol) was added and the mixture was stirred overnight. OH (50 ml) was added, followed by NaBH4 (0.646 g, 17.07 mmol). After stirring for 5 minutes, the entire reaction mixture was transferred to a 40 g silica column that was combined with a 330 g silica column. The mixture was then infused with 100% DCM for 10 minutes, followed by 0 to 15% MeOH for the next 30 minutes. Elution with DCM yielded 20,23-nonacosadiene-10-amine, N,N-dimethyl (20Z,23Z) (1) (2.45 g, 5.47 mmol, 48.1% yield) Collected as a faintly golden oil. 1 H NMR (400 MHz, CDCl3) δ 5.35(m,4H),2.78(m,2H),2.23(m,1H),2.21(s ,6H),2.05(m,4H),1.45-1.16(m,38H),0.89(m, 6H). HRMS calculated for C31H61N: 448.4877, found: 448.4872 .

[0407] Compounds 2-30 are novel cationic lipids, prepared according to General Scheme 1 above. Ta.

[0408] [Table 12] TIFF0007720384000207.tif220170 TIFF0007720384000208.tif226170 TIFF0007720384000209.tif118170 (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-diene- 1-Amine (Compound 31)

[0409] [ka] Ketone III (4.0 g, 9.55 mmol), Tosmic (2.4 g, 12.4 mmol), A solution of potassium tert-butyl ether (45 mL) in dimethoxyethane was cooled to 0°C and t-butoxide (19.1 mmol, 19.1 mL of 1 M tBuOH solution) After 90 minutes, the reaction mixture was partitioned between hexane and water. The organic portion was washed with water and diluted with sodium sulfate. The material was dried over ice, filtered, and evaporated in vacuo. Purification by chromatography (0 to 5% EtOAc / hexanes) gave the desired product ( This mixture was carried to the next step as is. Carried over. LC / MS (M+H) = 430.6.

[0410] [ka] Lithium aluminum hydride (23.9 mmol, 23.9 mL of a 1 M solution in THF) was added directly to nitrile iv (3.42 g, 8 mmol) at ambient temperature and the reaction was stirred for 20 min. The reaction mixture was diluted with 100 mL of THF, cooled to 0°C, and added with sodium sulfate. The mixture was carefully quenched with hydrate solution. The solid was filtered off and washed with THF. Evaporated in vacuo and carried directly crude to the next reaction. LC / MS (M+H )=434.6.

[0411] [ka] A solution of the primary amine (3.45 g, 6.2 mmol) in dichloroethane (100 mL) After treatment with formaldehyde (1.6 mL, 21.7 mmol), triacetoxy hydrogen After 5 minutes, the reaction mixture was treated with sodium borohydride (6.6 g, 31 mmol). Partitioned between methane and 1N NaOH. Dry the organic portion over sodium sulfate and filter. The crude mixture was purified by reversed-phase preparative chromatography (C8 column). ) and purified by (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12 15-Diene-1-amine was obtained. HRMS calculated value: 462.5033, found value: 462 .5026. 1 H NMR(400MHz,CDCl3)δ 5.35(m,4H),2 .78(2H,t,J=5.6Hz),2.18(s,6H),2.05(m,6H), 1.3(m,39H),0.89(m,6H).

[0412] (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-diene-1 -amine (compound 32)

[0413] [ka] Silylamide Peterson reagent (3.1 g, 16.7 mmol) was dissolved in THF (35 mL ) and cooled to -63°C. This solution was added to nBuLi (16.7 mmol, 6. 7 mL of a 2.5 M solution) was added. The reaction was allowed to warm to ambient temperature for 30 minutes. (5.0 g, 11.9 mmol) was dissolved in THF (25 mL) in a second flask The Peterson reagent was transferred to the ketone solution at -60°C. The reaction mixture was warmed to -40°C for 1 hour. The reaction was then allowed to warm to 0° C. for 30 minutes. The organic portion was washed with brine and diluted with sulfuric acid. The extract was dried over sodium hydroxide, filtered, and evaporated in vacuo. Purified by filtration (0 to 40% MTBE / hexane) and Got mid vi. 1 H NMR(400MHz,CDCl3)δ 5.75(s,1H) ,5.36(m,4H),3.01(s,3H),2.99(s,3H),2.78(t ,2H),2.28(t,2H),2.05(m,6H),1.35(m,34H),0 .89(m,6H).

[0414] [ka] α,β-Unsaturated amide VI (1 g, 2.1 mmol) and LS-Selectride (4.1 mmol, 4.1 mL of 1 M solution) in a sealed tube and heated to 60 °C for 24 hours. The reaction was cooled to ambient temperature and partitioned between ammonium chloride solution and heptane. The organic portion was dried over sodium sulfate, filtered, and evaporated in vacuo to give amide vii. This intermediate was carried directly crude to the next reaction.

[0415] [ka] A solution of amide vii (2.85 g, 5.8 mmol) was added to lithium aluminum hydride. (8.7 mmol, 8.7 mL of a 1 M solution) was added. The reaction was stirred at ambient temperature for 10 minutes. Stir and then quench by slow addition of sodium sulfate decahydrate solution. The solid was filtered, washed with THF, and the filtrate was evaporated in vacuo. Purification by reversed-phase preparative chromatography (C8 column) afforded (13Z,16Z)-N ,N-Dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32) Obtained as an oil. HRMS (M+H) calculated 476.5190, found 476.518 9. 1 H NMR(400MHz,CDCl3)δ 5.37(m,4H),2.78( t,2H),2.42(m,8H),2.05(q,4H),1.28(m,41H), 0.89(m,6H).

[0416] N,N-dimethyl-1-(2-octylcyclopropyl)heptadecan-8-amine( Compound 33)

[0417] [ka] A solution of oleic acid (1 g, 3.5 mmol) in DCM (500 mL) cooled to 0 °C was CDI (0.63 g, 3.9 mmol) was added, and the reaction was allowed to warm to ambient temperature for 30 minutes. After heating, the mixture was cooled to 0°C and first treated with triethylamine (0.39 g, 3.9 mmol). , and then treated with dimethylhydroxylamine hydrochloride (0.38 g, 3.9 mmol) After 1 hour, the reaction was partitioned between water and heptane. The organic portion was dried over magnesium sulfate. The mixture was then filtered and evaporated in vacuo to give crude Weinreb amide ii, which was then directly Carried over to the next reaction.

[0418] [ka] Diet...

Claims

1. A double-stranded small interfering nucleic acid (siNA) molecule that inhibits expression of cadherin-binding protein β1 (CTNNB1), comprising: (a) the siNA comprises a sense strand and an antisense strand; (b) each strand is independently 15 to 30 nucleotides in length; (c) the sense strand comprises the nucleotide sequence 5'-GCCACAAGAUUACAAGAAA-3' (SEQ ID NO: 101), and the antisense strand comprises the nucleotide sequence 5'-UUUCUUGUAAUCUUGUGGC-3' (SEQ ID NO: 5014); Double-stranded small interfering nucleic acid (siNA) molecules.

2. The double-stranded small interfering nucleic acid (siNA) molecule of claim 1, wherein at least one nucleotide is a chemically modified nucleotide.

3. The double-stranded short interfering nucleic acid (siNA) molecule of claim 1, further comprising at least one non-nucleotide.

4. The double-stranded short interfering nucleic acid (siNA) molecule of claim 1, wherein at least one nucleotide comprises a universal base.

5. The double-stranded small interfering nucleic acid (siNA) molecule of claim 1, having at least one phosphorothioate internucleotide linkage.

6. The double-stranded short interfering nucleic acid (siNA) molecule of claim 1, comprising a cap at the 3'-end, the 5'-end, or both the 3'- and 5'-ends of at least one strand.

7. 10. The double-stranded short interfering nucleic acid (siNA) molecule of claim 1, comprising one or more 3'-overhanging nucleotides on one or both strands.

8. The double-stranded small interfering nucleic acid (siNA) molecule of claim 1, wherein the 5' end of the antisense strand is phosphorylated.

9. 8. The double-stranded short interfering nucleic acid (siNA) molecule of claim 7, wherein the 3'-overhanging nucleotides of at least one strand are 2'-O-methyl nucleotides.

10. 10. The double-stranded short interfering nucleic acid (siNA) molecule of claim 9, wherein the 2'-O-methyl nucleotides are linked by phosphorothioate internucleotide linkages.

11. The double-stranded small interfering nucleic acid (siNA) molecule of claim 2, wherein the chemically modified nucleotide is a 2'-deoxy-2'-fluoro nucleotide.

12. The double-stranded small interfering nucleic acid (siNA) molecule of claim 2, wherein the chemically modified nucleotides are 2'-deoxynucleotides.

13. The double-stranded short interfering nucleic acid (siNA) molecule of claim 2, wherein the chemically modified nucleotide is a 2'-O-alkyl nucleotide.

14. 10. The double-stranded short interfering nucleic acid (siNA) molecule of claim 1, wherein five or more pyrimidine nucleotides in one or both strands are 2'-deoxy-2'-fluoro pyrimidine nucleotides.

15. The double-stranded short interfering nucleic acid (siNA) molecule of claim 1, wherein five or more pyrimidine nucleotides in one or both strands are 2'-O-methyl pyrimidine nucleotides.

16. 10. The double-stranded short interfering nucleic acid (siNA) molecule of claim 1, wherein five or more purine nucleotides in one or both strands are 2'-deoxy-2'-fluoropurine nucleotides.

17. The double-stranded short interfering nucleic acid (siNA) molecule of claim 1, wherein five or more purine nucleotides in one or both strands are 2'-O-methyl purine nucleotides.

18. 15. The double-stranded short interfering nucleic acid (siNA) molecule of claim 14, wherein five or more purine nucleotides in one or both strands are 2'-O-methyl purine nucleotides.

19. 16. The double-stranded short interfering nucleic acid (siNA) molecule of claim 15, wherein five or more purine nucleotides in one or both strands are 2'-deoxy-2'-fluoro nucleotides.

20. A double-stranded small interfering nucleic acid (siNA) molecule comprising SEQ ID NOs: 2005 and 2004.

21. A double-stranded small interfering nucleic acid (siNA) molecule comprising SEQ ID NOs: 1655 and 1654.

22. 22. The double-stranded short interfering nucleic acid (siNA) molecule of any of claims 1, 20 and 21, comprising one or more phosphorothioate internucleotide linkages.

23. A composition comprising a double-stranded small interfering nucleic acid (siNA) of any one of claims 1, 20 and 21 in a pharmaceutically acceptable carrier or diluent.

24. (a) a double-stranded small interfering nucleic acid (siNA) having SEQ ID NOs: 2005 and 2004; (b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine; (c) cholesterol; (d) DSPC; and (e) PEG-DMG A composition comprising:

25. (a) a double-stranded small interfering nucleic acid (siNA) having SEQ ID NOs: 1655 and 1654; (b) (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine; (c) cholesterol; (d) DSPC; and (e) PEG-DMG A composition comprising:

26. 26. The composition of claim 24 or 25, wherein (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, cholesterol, DSPC, and PEG-DMG have a molar ratio of 50:30:10:2, respectively.

27. 26. The composition of claim 24 or 25, further comprising sucrose, trehalose, or any combination thereof.

28. A pharmaceutical composition for use in a method for treating a human subject suffering from a pathology mediated by the action or loss of action of CTNNB1, the pharmaceutical composition comprising an effective amount of a double-stranded small interfering nucleic acid (siNA) molecule described in any of claims 1, 20 and 21.

29. 29. The pharmaceutical composition of claim 28, wherein the condition is cancer.

30. Use of a double-stranded small interfering nucleic acid (siNA) molecule according to any one of claims 1, 20 and 21 in the preparation of a medicament for inhibiting the expression of cadherin-binding protein beta 1 (CTNNB1) in a cell.

31. The use described in claim 30, wherein the cells are a subject.

32. The use described in claim 31, wherein the subject is a human.

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