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Patent US6066500 - Antisense modulation of Beta catenin expression - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsAntisense compounds, compositions and methods are provided for modulating the expression of Beta catenin. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Beta catenin. Methods of using these compounds for modulation of Beta catenin...http://www.google.com/patents/US6066500?utm_source=gb-gplus-sharePatent US6066500 - Antisense modulation of Beta catenin expressionAdvanced Patent SearchPublication numberUS6066500 APublication typeGrantApplication numberUS 09/344,519Publication dateMay 23, 2000Filing dateJun 25, 1999Priority dateJun 25, 1999Fee statusPaidAlso published asEP1190094A1, WO2001000872A1Publication number09344519, 344519, US 6066500 A, US 6066500A, US-A-6066500, US6066500 A, US6066500AInventorsC. Frank Bennett, Lex M. CowsertOriginal AssigneeIsis Pharmaceuticals Inc.Export CitationBiBTeX, EndNote, RefManNon-Patent Citations (34), Referenced by (14), Classifications (18), Legal Events (4) External Links: USPTO, USPTO Assignment, EspacenetAntisense modulation of Beta catenin expression
US 6066500 AAbstract
Antisense compounds, compositions and methods are provided for modulating the expression of Beta catenin. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding Beta catenin. Methods of using these compounds for modulation of Beta catenin expression and for treatment of diseases associated with expression of Beta catenin are provided.
1. An antisense compound 8 to 30 nucleobases in length targeted to a 3'-untranslated region, a start codon, a coding region from nucleobases 276-2450, a stop codon or a 5'-untranslated region of human Beta catenin, wherein said antisense compound specifically hybridizes with and inhibits the expression of human Beta catenin.
3. An antisense compound up to 30 nucleobases in length comprising at least an 8-nucleobase portion of SEQ ID NO: 8, 9, 11, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 33, 34, 36, 38, 39, 40, 41, 42, 43, 44, 46, 47, 10, 12, 13, 17, 25, 35, 37 or 45 which inhibits the expression of human Beta catenin.
6. The antisense compound of claim 4 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.
11. A method of inhibiting the expression of human Beta catenin in human cells or tissues comprising contacting said cells or tissues in vitro with the antisense compound of claim 1 so that expression of human Beta catenin is inhibited.
12. The antisense compound of claim 3 which is an antisense oligonucleotide.
20. A method of inhibiting the expression of human Beta catenin in human cells or tissues comprising contacting said cells or tissues in vitro with the antisense compound of claim 3 so that expression of human Beta catenin is inhibited.
The present invention provides compositions and methods for modulating the expression of Beta catenin. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human Beta catenin. Such oligonucleotides have been shown to modulate the expression of Beta catenin.
Critical to the development and maintenance of multicellular organisms is the regulation of intercellular communication. The primary contacts made between cells and the surrounding environment, including contacts with other cells, are mediated by surface and transmembrane proteins that can transduce signals to the interior of the cell modulating biochemical pathways. Cadherins, one class of transmembrane glycoproteins, are characterized by a long extracellular domain which interacts with cadherins on the opposite membrane of adhering cells and a cytoplasmic domain anchored to the actin cytoskeleton through another class of proteins, the catenins. Because cell adhesiveness is reduced during metastasis, much attention has been focused on the cell adhesion system and, in particular, the role of the cadherins and catenins in this process.
Beta catenin (also known as cadherin-associated protein and β-Catenin), a member of the catenin family of cytosolic proteins and key mediator of the Wnt signaling pathway, was originally isolated because of its association with the cytoplasmic domain of E-cadherin, an epithelial-specific cell-adhesion molecule (Willert and Nusse, Curr. Opin. Genet. Dev., 1998, 8, 95-102). It was demonstrated that Beta catenin undergoes phosphorylation upon growth factor stimulation resulting in reduced cell adhesion, thereby functioning as a component of adherin junctions which are multiprotein complexes that mediate cell adhesion, cell--cell communication and cytoskeletal anchoring. Since then it has been demonstrated that Beta catenin performs a dual role in the cell, in that free pools of Beta catenin mediate signal transduction pathways involving T-cell factor/lymphoid-enhancer factor (TCF/LEF) transcription factor complexes (Korinek et al., Science, 1997, 275, 1784-1787; Peifer, Science, 1993, 262, 1667-1668).
The role of Beta catenin in the development of several types of cancer, more specifically colorectal cancer and melanomas, has been investigated and shown to be regulated by the expression product of the APC (adenomatous polyposis of the colon) gene (Korinek et al., Science, 1997, 275, 1784-1787; Morin et al., Science, 1997, 275, 1787-1790). The APC protein normally binds Beta catenin in conjunction with TCF/LEF forming a transcription factor complex. It is believed that APC regulates the concentration of Beta catenin in the cytosol by controlling its degradation. In studies of the relationship between Beta catenin and APC, Ilyas et al. found four different mutations in Beta catenin including deletions and missense mutations in 21 colorectal cancer cell lines. The full-length Beta catenin protein was detected in all cell lines studied, but failed to show complex formation with APC in some cell lines (Ilyas and Tomlinson, J. Pathol., 1997, 182, 128-137). In other studies, Rubinfeld et al. detected abnormally high amounts of Beta catenin in 7 of 26 human melanoma cell lines and, in 6 of these 7, unusual splicing (deletions of exons 2 and 3 or 2,3 and 4) or missense mutations (TCT to TTT mutations causing a serine 45 to phenylalanine 37 mutation in 3 of 4 cases and a TCT to TAT mutation causing a serine 45 to tyrosine 45 mutation in another case) in Beta catenin; with APC being missing or altered in two of the six (Rubinfeld et al., Science, 1997, 275, 1790-1792). Finally Morin et al. demonstrated that mutations of Beta catenin that altered phosphorylation sites rendered the cells insensitive to APC-mediated downregulation of Beta catenin and that this disrupted mechanism was critical to colorectal tumorigenesis (Morin et al., Science, 1997, 275, 1787-1790).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Beta catenin and strategies aimed at inhibiting Beta catenin function have involved the use of antibodies, antisense oligonucleotides, chemical inhibitors and gene knock-outs in mice. Furthermore, most of these studies have been focused on elucidating the role of Beta catenin in the process of embryogenesis.
Although Beta catenin is expressed ubiquitously, it is specifically required in ectodermal cell layer development. In knock-out mice lacking Beta catenin, cells detached from the ectodermal cell layer and were dispersed into the proamniotic cavity and no mesoderm formation was observed (Haegel et al., Development, 1995, 121, 3529-3537).
Studies involving an antisense Beta catenin 18-mer phosphorothioate/phosphodiester oligodeoxynucleotide injected into Xenopus embryos demonstrated that either overexpression of cadherins or underexpression of Beta catenin, through treatment with antisense, was sufficient to inhibit dorsal mesoderm induction indicating an important role for Beta catenin in embryogenesis (Heasman et al., Cell, 1994, 79, 791-803).
Antisense mediated inhibition of Beta catenin was also used as a tool to investigate the effect of Beta catenin in the process of invasive motility in normal and uveal melanomas. In these studies, treatment of cells with a single 20-mer phosphorothioate antisense oligodeoxynucleotide targeting both human Beta catenin and human plakoglobin reduced the invasive motility of both normal and neoplastic mesenchymal cells (Kim et al., Exp. Cell. Res., 1998, 245, 79-90). Additional oligonucleotides targeted to either Beta catenin or plakoglobin (targeted to unique sequences) did not have a significant effect.
These antisense strategies are untested as therapeutic protocols and consequently there remains a long felt need for additional agents capable of effectively inhibiting Beta catenin function.
Antisense technology is, however, emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of Beta catenin expression.
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding Beta catenin, and which modulate the expression of Beta catenin. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of Beta catenin in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of Beta catenin by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.
The present invention employs oligomeric antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Beta catenin, ultimately modulating the amount of Beta catenin produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Beta catenin. As used herein, the terms "target nucleic acid" and "nucleic acid encoding Beta catenin" encompass DNA encoding Beta catenin, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as "antisense". The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of Beta catenin. In the context of the present invention, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.
It is preferred to target specific nucleic acids for antisense. "Targeting" an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding Beta catenin. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding Beta catenin, regardless of the sequence(s) of such codons.
Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; O--, S--, or N-alkyl; O--, S--, or N-alkenyl; O--, S-- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)n O]m CH3, O(CH2)n OCH3, O(CH2)n NH2, O(CH2)n CH3, O(CH2)n ONH2, and O(CH2)n ON[(CH2)n CH3)]2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-O--CH2 CH2 OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2 ON(CH3)2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylamino-ethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2 --O--CH2 --N(CH2)2, also described in examples hereinbelow.
The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Beta catenin is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.
The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Beta catenin, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding Beta catenin can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of Beta catenin in a sample may also be prepared.
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham, Mass. or Glen Research, Inc. Sterling, Va.). Other 2'-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2'-alkoxy amidites, the standard cycle for unmodified oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.
A solution of 3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH3 OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2�200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH3 gas was added and the vessel heated to 100� C. for 2 hours (TLC showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.
5'-O-tert-Butyldiphenylsilyl-O2 -2-anhydro-5-methyluridine
O2 -2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, aItaly, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between dichloromethane (1 L) and saturated sodium bicarbonate (2�1 L) and brine (1 L). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) and the solution was cooled to -10� C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3�200 mL) and dried (40� C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMR were consistent with pure product.
In a 2 L stainless steel, unstirred pressure reactor was added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided. 5'-O-tert-Butyldiphenylsilyl-O2 -2'-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160� C. was reached and then maintained for 16 h (pressure<100 psig). The reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T side product, ethyl acetate) indicated about 70% conversion to the product. In order to avoid additional side product formation, the reaction was stopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100� C.) with the more extreme conditions used to remove the ethylene glycol. [Alternatively, once the low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water. The product will be in the organic phase.] The residue was purified by column chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield based on starting material less pure recovered starting material was 58%. TLC and NMR were consistent with 99% pure product.
2[2-(Dimethylaminoethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. O2 -,2'-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oil bath and heated to 155� C. for 26 hours. The bomb is cooled to room temperature and opened. The crude solution is concentrated and the residue partitioned between water (200 mL) and hexanes (200 mL). The excess phenol is extracted into the hexane layer. The aqueous layer is extracted with ethyl acetate (3�200 mL) and the combined organic layers are washed once with water, dried over anhydrous sodium sulfate and concentrated. The residue is columned on silica gel using methanol/methylene chloride 1:20 (which has 2% triethylamine) as the eluent. As the column fractions are concentrated a colorless solid forms which is collected to give the title compound as a white solid.
To 0.5 g (1.3 mmol) of 2'-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reaction mixture is poured into water (200 mL) and extracted with CH2 Cl2 (2�200 mL). The combined CH2 Cl2 layers are washed with saturated NaHCO3 solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate. Evaporation of the solvent followed by silica gel chromatography using MeOH:CH2 Cl2 :Et3 N (20:1, v/v, with 1% triethylamine) gives the title compound.
[2'-O-(2-methoxyethyl)]--[2'-deoxy]--[-2'-O-(methoxy-ethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2'-O-methyl chimeric oligonucleotide, with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites.
[2'-O-(2-methoxyethyl phosphodiester]--[2'-deoxy phoshorothioate]--[2'-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2'-O-methyl chimeric oligonucleotide with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites, oxidization with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.
Analysis of oligonucleotide inhibition of Beta catenin expression
Antisense modulation of Beta catenin expression can be assayed in a variety of ways known in the art. For example, Beta catenin mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions. Other methods of PCR are also known in the art.
Beta catenin protein levels can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to Beta catenin can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mi.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.
Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine, Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70� C. was added to each well, the plate was incubated on a 90� C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.
Total mRNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.
Real-time Quantitative PCR Analysis of Beta catenin mRNA Levels
Quantitation of Beta catenin mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE or FAM, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1� TAQMAN™ buffer A, 5.5 mM MgCl2, 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL poly(A) mRNA solution. The RT reaction was carried out by incubation for 30 minutes at 48� C. Following a 10 minute incubation at 95� C. to activate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carried out: 95� C. for 15 seconds (denaturation) followed by 60� C. for 1.5 minutes (annealing/extension). Beta catenin probes and primers were designed to hybridize to the human Beta catenin sequence, using published sequence information (GenBank accession number X87838, incorporated herein as SEQ ID NO:1).
For Beta catenin the PCR primers were: forward primer: AGTGGGAACAGGGATTTTCTCA (SEQ ID NO: 2) reverse primer: CCTCATCTAATGTCTCAGGGAACA (SEQ ID NO: 3) and the PCR probe was: FAM-TGATGGACAGTATGCAATGACTCGAGCTCA-TAMRA (SEQ ID NO: 4) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.
Northern blot analysis of Beta catenin mRNA levels
Membranes were probed using QUICKHYB™ T hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions with a Beta catenin specific probe prepared by PCR using the forward primer AGTGGGAACAGGGATTTTCTCA (SEQ ID NO: 2) and the reverse primer CCTCATCTAATGTCTCAGGGAACA (SEQ ID NO: 3). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.). Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.
Antisense inhibition of Beta catenin expression-phosphorothioate oligodeoxynucleotides
In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human Beta catenin RNA, using published sequences (GenBank accession number X87838, incorporated herein as SEQ ID NO: 1). The oligonucleotides are shown in Table 1. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X87838), to which the oligonucleotide binds. All compounds in Table 1 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. The compounds were analyzed for effect on Beta catenin mRNA levels by quantitive real-time PCR as described in other examples herein. Data are averages from two experiments. If present, "N.D." indicates "no data".
TABLE 1__________________________________________________________________________Inhibition of Beta catenin mRNA levels by phosphorothioate oligodeoxynucleotides   TARGET           %                            ISIS                           # REGION SITE SEQUENCE Inhibition SEQ ID                           NO.__________________________________________________________________________102453    5' UTR   39  cccctcgctctccgctcccg                    93     8   - 102456 5' UTR 103 cgacctgcggtggcggctcg 64 9   - 102460 5' UTR 171 gttgtatggtatacttcaaa 31 10   - 102465 Start 196 ttgtccacgctggattttca 48 11   Codon   - 102468 Start 198 cattgtccacgctggatttt 28 12   Codon   - 102473 Start 200 gccattgtccacgctggatt 0 13   Codon   - 102477 Start 202 tagccattgtccacgctgga 77 14   Codon   - 102481 Start 204 agtagccattgtccacgctg 63 15   Codon   - 102484 Start 206 tgagtagccattgtccacgc 65 16   Codon   - 102488 Start 208 cttgagtagccattgtccac 0 17   Codon   - 102493 Start 210 agcttgagtagccattgtcc 66 18   Codon   - 102497 Start 212 tcagcttgagtagccattgt 69 19   Codon   - 102501 Start 214 aatcagcttgagtagccatt 69 20   Codon   - 102505 Start 216 caaatcagcttgagtagcca 51 21   Codon   - 102509 Coding 276 ttgctgccagtgactaacag 54 22   - 102513 Coding 408 agaaaatccctgttcccact 68 23   - 102517 Coding 537 aaactgtgtagatgggatct 43 24   - 102520 Coding 675 tagcagttttgtcagttcag 0 25   - 102524 Coding 786 agcagacaccatctgaggag 70 26   - 102528 Coding 847 aggtcccagcggtacaacga 79 27   - 102531 Coding 930 accaagcattttcaccaggg 60 28   - 102536 Coding 1031 agcccaccagctaaacgcac 60 29   - 102539 Coding 1487 ttattgcaagtgaggttaga 49 30   - 102544 Coding 1544 gtacgcacaagagcctctat 71 31   - 102550 Coding 1773 aggtgcatgatttgcgggac 38 32   - 102556 Coding 1823 gcacgaacaagcaactgaac 63 33   - 102561 Coding 1970 cctctgataacaattcggtt 48 34   - 102566 Coding 2023 tgttttcaatgggagaataa 37 35   - 102571 Coding 2067 gtcctgagcaagttcacaga 63 36   - 102576 Coding 2119 ctgtcagaggagctgtggct 36 37   - 102581 Coding 2152 tcgccacaccttcattccta 63 38   - 102586 Coding 2234 ctggtcagctcaactgaaag 59 39   - 102591 Coding 2430 accagggtggtggccaccca 52 40   - 102596 Stop 2553 ctaaaggatgatttacaggt 46 41   Codon   - 102602 3' UTR 2603 cctctcagcaactctacagg 76 42   - 102607 3' UTR 2669 cccataggaaactcagcttg 71 43   - 102612 3' UTR 2804 aggctagggtttgctaaatt 53 44   - 102617 3' UTR 3028 tagagtgattatgaattaat 30 45   - 102622 3' UTR 3230 caaaacaaggttccaaataa 44 46   - 102627 3' UTR 3298 ttctcttgaagcatcgtatc 80 47__________________________________________________________________________
As shown in Table 1, SEQ ID NOs 8, 9, 11, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 33, 34, 36, 38, 39, 40, 41, 42, 43, 44, 46 and 47 demonstrated at least 40% inhibition of Beta catenin expression in this assay and are therefore preferred.
Antisense inhibition of Beta catenin expression-phosphorothioate 2'-MOE gapmer oligonucleotides
In accordance with the present invention, a second series of oligonucleotides targeted to human Beta catenin were synthesized. The oligonucleotide sequences are shown in Table 2. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. X87838), to which the oligonucleotide binds.
TABLE 2__________________________________________________________________________Inhibition of Beta catenin mRNA levels by chimeric  phosphorothioate oligonucleotides having 2'-MOE wings  and a deoxy gap   TARGET           %  ISIS# REGION SITE SEQUENCE Inhibition SEQ ID NO.__________________________________________________________________________102630    5' UTR   39  cccctcgctctccgctcccg                    58     8   - 102635 5' UTR 103 cgacctgcggtggcggctcg 78 9   - 102638 5' UTR 171 gttgtatggtatacttcaaa 67 10   - 102642 Start 196 ttgtccacgctggattttca 42 11   Codon   - 102646 Start 198 cattgtccacgctggatttt 55 12   Codon   - 102651 Start 200 gccattgtccacgctggatt 65 13   Codon   - 102655 Start 202 tagccattgtccacgctgga 54 14   Codon   - 102659 Start 204 agtagccattgtccacgctg 76 15   Codon   - 102663 Start 206 tgagtagccattgtccacgc 81 16   Codon   - 102667 Start 208 cttgagtagccattgtccac 67 17   Codon   - 102671 Start 210 agcttgagtagccattgtcc 73 18   Codon   - 102675 Start 212 tcagcttgagtagccattgt 79 19   Codon   - 102679 Start 214 aatcagcttgagtagccatt 82 20   Codon   - 102683 Start 216 caaatcagcttgagtagcca 53 21   Codon   - 102687 Coding 276 ttgctgccagtgactaacag 67 22   - 102691 Coding 408 agaaaatccctgttcccact 73 23   - 102695 Coding 537 aaactgtgtagatgggatct 76 24   - 102699 Coding 675 tagcagttttgtcagttcag 58 25   - 102704 Coding 786 agcagacaccatctgaggag 77 26   - 102708 Coding 847 aggtcccagcggtacaacga 86 27   - 102712 Coding 930 accaagcattttcaccaggg 64 28   - 102715 Coding 1031 agcccaccagctaaacgcac 0 29   - 102719 Coding 1487 ttattgcaagtgaggttaga 58 30   - 102723 Coding 1544 gtacgcacaagagcctctat 84 31   - 102728 Coding 1773 aggtgcatgatttgcgggac 23 32   - 102732 Coding 1823 gcacgaacaagcaactgaac 66 33   - 102735 Coding 1970 cctctgataacaattcggtt 86 34   - 102739 Coding 2023 tgttttcaatgggagaataa 47 35   - 102742 Coding 2067 gtcctgagcaagttcacaga 80 36   - 102745 Coding 2119 ctgtcagaggagctgtggct 73 37   - 102748 Coding 2152 tcgccacaccttcattccta 62 38   - 102751 Coding 2234 ctggtcagctcaactgaaag 71 39   - 102753 Coding 2430 accagggtggtggccaccca 55 40   - 102755 Stop 2553 ctaaaggatgatttacaggt 76 41   Codon   - 102757 3' UTR 2603 cctctcagcaactctacagg 79 42   - 102759 3' UTR 2669 cccataggaaactcagcttg 79 43   - 102761 3' UTR 2804 aggctagggtttgctaaatt 0 44   - 102763 3' UTR 3028 tagagtgattatgaattaat 62 45   - 102764 3' UTR 3230 caaaacaaggttccaaataa 63 46   - 102766 3' UTR 3298 ttctcttgaagcatcgtatc 88 47__________________________________________________________________________
As shown in Table 2, SEQ ID NOs 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46 and 47 demonstrated at least 40% inhibition of Beta catenin expression in this experiment and are therefore preferred.
Western blot analysis of Beta catenin protein levels
Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to Beta catenin is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale, Calif.).
__________________________________________________________________________#             SEQUENCE LISTING   - -  - - &lt;160&gt; NUMBER OF SEQ ID NOS: 47   - - &lt;210&gt; SEQ ID NO 1  &lt;211&gt; LENGTH: 3362  &lt;212&gt; TYPE: DNA  &lt;213&gt; ORGANISM: Homo sapiens  &lt;220&gt; FEATURE:  &lt;221&gt; NAME/KEY: CDS  &lt;222&gt; LOCATION: (215)..(2560)   - - &lt;400&gt; SEQUENCE: 1   - - aagcctctcg gtctgtggca gcagcgttgg cccggccccg ggagcggaga gc -#gaggggag     60   - - gcggagacgg aggaaggtct gaggagcagc ttcagtcccc gccgagccgc ca -#ccgcaggt    120   - - cgaggacggt cggactcccg cggcgggagg agcctgttcc cctgagggta tt -#tgaagtat    180   - - accatacaac tgttttgaaa atccagcgtg gaca atg gct act c - #aa gct gat 232              - #                  - #  Met Ala Thr Gln Ala Asp              - #                  - #    1              - # 5  - - ttg atg gag ttg gac atg gcc atg gaa cca ga - #c aga aaa gcg gct gtt280 Leu Met Glu Leu Asp Met Ala Met Glu Pro As - #p Arg Lys Ala Ala Val         10     - #             15     - #             20  - - agt cac tgg cag caa cag tct tac ctg gac tc - #t gga atc cat tct ggt328 Ser His Trp Gln Gln Gln Ser Tyr Leu Asp Se - #r Gly Ile His Ser Gly     25         - #         30         - #         35  - - gcc act acc aca gct cct tct ctg agt ggt aa - #a ggc aat cct gag gaa376 Ala Thr Thr Thr Ala Pro Ser Leu Ser Gly Ly - #s Gly Asn Pro Glu Glu 40             - #     45             - #     50  - - gag gat gtg gat acc tcc caa gtc ctg tat ga - #g tgg gaa cag gga ttt424 Glu Asp Val Asp Thr Ser Gln Val Leu Tyr Gl - #u Trp Glu Gln Gly Phe  55                 - # 60                 - # 65                 - #70  - - tct cag tcc ttc act caa gaa caa gta gct ga - #t att gat gga cag tat472 Ser Gln Ser Phe Thr Gln Glu Gln Val Ala As - #p Ile Asp Gly Gln Tyr             75 - #                 80 - #                 85  - - gca atg act cga gct cag agg gta cga gct gc - #t atg ttc cct gag aca520 Ala Met Thr Arg Ala Gln Arg Val Arg Ala Al - #a Met Phe Pro Glu Thr         90     - #             95     - #            100  - - tta gat gag ggc atg cag atc cca tct aca ca - #g ttt gat gct gct cat568 Leu Asp Glu Gly Met Gln Ile Pro Ser Thr Gl - #n Phe Asp Ala Ala His    105          - #       110          - #       115  - - ccc act aat gtc cag cgt ttg gct gaa cca tc - #a cag atg ctg aaa cat616 Pro Thr Asn Val Gln Arg Leu Ala Glu Pro Se - #r Gln Met Leu Lys His120              - #   125              - #   130  - - gca gtt gta aac ttg att aac tat caa gat ga - #t gca gaa ctt gcc aca664 Ala Val Val Asn Leu Ile Asn Tyr Gln Asp As - #p Ala Glu Leu Ala Thr 135                 1 - #40                 1 - #45                 1 -#50   - - cgt gca atc cct gaa ctg aca aaa ctg cta aa - #t gac gag gac caggtg      712  Arg Ala Ile Pro Glu Leu Thr Lys Leu Leu As - #n Asp Glu Asp Gln Val            155  - #               160  - #               165  - - gtg gtt aat aag gct gca gtt atg gtc cat ca - #g ctt tct aaa aag gaa760 Val Val Asn Lys Ala Ala Val Met Val His Gl - #n Leu Ser Lys Lys Glu        170      - #           175      - #           180  - - gct tcc aga cac gct atc atg cgt tct cct ca - #g atg gtg tct gct att808 Ala Ser Arg His Ala Ile Met Arg Ser Pro Gl - #n Met Val Ser Ala Ile    185          - #       190          - #       195  - - gta cgt acc atg cag aat aca aat gat gta ga - #a aca gct cgt tgt acc856 Val Arg Thr Met Gln Asn Thr Asn Asp Val Gl - #u Thr Ala Arg Cys Thr200              - #   205              - #   210  - - gct ggg acc ttg cat aac ctt tcc cat cat cg - #t gag ggc tta ctg gcc904 Ala Gly Thr Leu His Asn Leu Ser His His Ar - #g Glu Gly Leu Leu Ala 215                 2 - #20                 2 - #25                 2 -#30   - - atc ttt aag tct gga ggc att cct gcc ctg gt - #g aaa atg ctt ggttca      952  Ile Phe Lys Ser Gly Gly Ile Pro Ala Leu Va - #l Lys Met Leu Gly Ser            235  - #               240  - #               245  - - cca gtg gat tct gtg ttg ttt tat gcc att ac - #a act ctc cac aac ctt    1000 Pro Val Asp Ser Val Leu Phe Tyr Ala Ile Th - #r Thr Leu His Asn Leu        250      - #           255      - #           260  - - tta tta cat caa gaa gga gct aaa atg gca gt - #g cgt tta gct ggt ggg    1048 Leu Leu His Gln Glu Gly Ala Lys Met Ala Va - #l Arg Leu Ala Gly Gly    265          - #       270          - #       275  - - ctg cag aaa atg gtt gcc ttg ctc aac aaa ac - #a aat gtt aaa ttc ttg    1096 Leu Gln Lys Met Val Ala Leu Leu Asn Lys Th - #r Asn Val Lys Phe Leu280              - #   285              - #   290  - - gct att acg aca gac tgc ctt caa att tta gc - #t tat ggc aac caa gaa    1144 Ala Ile Thr Thr Asp Cys Leu Gln Ile Leu Al - #a Tyr Gly Asn Gln Glu 295                 3 - #00                 3 - #05                 3 -#10   - - agc aag ctc atc ata ctg gct agt ggt gga cc - #c caa gct tta gtaaat     1192  Ser Lys Leu Ile Ile Leu Ala Ser Gly Gly Pr - #o Gln Ala Leu Val Asn            315  - #               320  - #               325  - - ata atg agg acc tat act tac gaa aaa cta ct - #g tgg acc aca agc aga    1240 Ile Met Arg Thr Tyr Thr Tyr Glu Lys Leu Le - #u Trp Thr Thr Ser Arg        330      - #           335      - #           340  - - gtg ctg aag gtg cta tct gtc tgc tct agt aa - #t aag ccg gct att gta    1288 Val Leu Lys Val Leu Ser Val Cys Ser Ser As - #n Lys Pro Ala Ile Val    345          - #       350          - #       355  - - gaa gct ggt gga atg caa gct tta gga ctt ca - #c ctg aca gat cca agt    1336 Glu Ala Gly Gly Met Gln Ala Leu Gly Leu Hi - #s Leu Thr Asp Pro Ser360              - #   365              - #   370  - - caa cgt ctt gtt cag aac tgt ctt tgg act ct - #c agg aat ctt tca gat    1384 Gln Arg Leu Val Gln Asn Cys Leu Trp Thr Le - #u Arg Asn Leu Ser Asp 375                 3 - #80                 3 - #85                 3 -#90   - - gct gca act aaa cag gaa ggg atg gaa ggt ct - #c ctt ggg act cttgtt     1432  Ala Ala Thr Lys Gln Glu Gly Met Glu Gly Le - #u Leu Gly Thr Leu Val            395  - #               400  - #               405  - - cag ctt ctg ggt tca gat gat ata aat gtg gt - #c acc tgt gca gct gga    1480 Gln Leu Leu Gly Ser Asp Asp Ile Asn Val Va - #l Thr Cys Ala Ala Gly        410      - #           415      - #           420  - - att ctt tct aac ctc act tgc aat aat tat aa - #g aac aag atg atg gtc    1528 Ile Leu Ser Asn Leu Thr Cys Asn Asn Tyr Ly - #s Asn Lys Met Met Val    425          - #       430          - #       435  - - tgc caa gtg ggt ggt ata gag gct ctt gtg cg - #t act gtc ctt cgg gct    1576 Cys Gln Val Gly Gly Ile Glu Ala Leu Val Ar - #g Thr Val Leu Arg Ala440              - #   445              - #   450  - - ggt gac agg gaa gac atc act gag cct gcc at - #c tgt gct ctt cgt cat    1624 Gly Asp Arg Glu Asp Ile Thr Glu Pro Ala Il - #e Cys Ala Leu Arg His 455                 4 - #60                 4 - #65                 4 -#70   - - ctg acc agc cga cac caa gaa gca gag atg gc - #c cag aat gca gttcgc     1672  Leu Thr Ser Arg His Gln Glu Ala Glu Met Al - #a Gln Asn Ala Val Arg            475  - #               480  - #               485  - - ctt cac tat gga cta cca gtt gtg gtt aag ct - #c tta cac cca cca tcc    1720 Leu His Tyr Gly Leu Pro Val Val Val Lys Le - #u Leu His Pro Pro Ser        490      - #           495      - #           500  - - cac tgg cct ctg ata aag gct act gtt gga tt - #g att cga aat ctt gcc    1768 His Trp Pro Leu Ile Lys Ala Thr Val Gly Le - #u Ile Arg Asn Leu Ala    505          - #       510          - #       515  - - ctt tgt ccc gca aat cat gca cct ttg cgt ga - #g cag ggt gcc att cca    1816 Leu Cys Pro Ala Asn His Ala Pro Leu Arg Gl - #u Gln Gly Ala Ile Pro520              - #   525              - #   530  - - cga cta gtt cag ttg ctt gtt cgt gca cat ca - #g gat acc cag cgc cgt    1864 Arg Leu Val Gln Leu Leu Val Arg Ala His Gl - #n Asp Thr Gln Arg Arg 535                 5 - #40                 5 - #45                 5 -#50   - - acg tcc atg ggt ggg aca cag cag caa ttt gt - #g gag ggg gtc cgcatg     1912  Thr Ser Met Gly Gly Thr Gln Gln Gln Phe Va - #l Glu Gly Val Arg Met            555  - #               560  - #               565  - - gaa gaa ata gtt gaa ggt tgt acc gga gcc ct - #t cac atc cta gct cgg    1960 Glu Glu Ile Val Glu Gly Cys Thr Gly Ala Le - #u His Ile Leu Ala Arg        570      - #           575      - #           580  - - gat gtt cac aac cga att gtt atc aga gga ct - #a aat acc att cca ttg    2008 Asp Val His Asn Arg Ile Val Ile Arg Gly Le - #u Asn Thr Ile Pro Leu    585          - #       590          - #       595  - - ttt gtg cag ctg ctt tat tct ccc att gaa aa - #c atc caa aga gta gct    2056 Phe Val Gln Leu Leu Tyr Ser Pro Ile Glu As - #n Ile Gln Arg Val Ala600              - #   605              - #   610  - - gca ggg gtc ctc tgt gaa ctt gct cag gac aa - #g gaa gct gca gaa gct    2104 Ala Gly Val Leu Cys Glu Leu Ala Gln Asp Ly - #s Glu Ala Ala Glu Ala 615                 6 - #20                 6 - #25                 6 -#30   - - att gaa gct gag gga gcc aca gct cct ctg ac - #a gag tta ctt cactct     2152  Ile Glu Ala Glu Gly Ala Thr Ala Pro Leu Th - #r Glu Leu Leu His Ser            635  - #               640  - #               645  - - agg aat gaa ggt gtg gcg aca tat gca gct gc - #t gtt ttg ttc cga atg    2200 Arg Asn Glu Gly Val Ala Thr Tyr Ala Ala Al - #a Val Leu Phe Arg Met        650      - #           655      - #           660  - - tct gag gac aag cca caa gat tac aag aaa cg - #g ctt tca gtt gag ctg    2248 Ser Glu Asp Lys Pro Gln Asp Tyr Lys Lys Ar - #g Leu Ser Val Glu Leu    665          - #       670          - #       675  - - acc agc tct ctc ttc aga aca gag cca atg gc - #t tgg aat gag act gct    2296 Thr Ser Ser Leu Phe Arg Thr Glu Pro Met Al - #a Trp Asn Glu Thr Ala680              - #   685              - #   690  - - gat ctt gga ctt gat att ggt gcc cag gga ga - #a ccc ctt gga tat cgc    2344 Asp Leu Gly Leu Asp Ile Gly Ala Gln Gly Gl - #u Pro Leu Gly Tyr Arg 695                 7 - #00                 7 - #05                 7 -#10   - - cag gat gat cct agc tat cgt tct ttt cac tc - #t ggt gga tat ggccag     2392  Gln Asp Asp Pro Ser Tyr Arg Ser Phe His Se - #r Gly Gly Tyr Gly Gln            715  - #               720  - #               725  - - gat gcc ttg ggt atg gac ccc atg atg gaa ca - #t gag atg ggt ggc cac    2440 Asp Ala Leu Gly Met Asp Pro Met Met Glu Hi - #s Glu Met Gly Gly His        730      - #           735      - #           740  - - cac cct ggt gct gac tat cca gtt gat ggg ct - #g cca gat ctg ggg cat    2488 His Pro Gly Ala Asp Tyr Pro Val Asp Gly Le - #u Pro Asp Leu Gly His    745          - #       750          - #       755  - - gcc cag gac ctc atg gat ggg ctg cct cca gg - #t gac agc aat cag ctg    2536 Ala Gln Asp Leu Met Asp Gly Leu Pro Pro Gl - #y Asp Ser Asn Gln Leu760              - #   765              - #   770  - - gcc tgg ttt gat act gac ctg taa atcatccttt ag - #ctgtattg tctgaacttg    2590 Ala Trp Phe Asp Thr Asp Leu 775                 7 - #80  - - cattgtgatt ggcctgtaga gttgctgaga gggctcgagg ggtgggctgg ta -#tctcagaa   2650   - - agtgcctgac acactaacca agctgagttt cctatgggaa caattgaagt aa -#actttttg   2710   - - ttctggtcct ttttggtcga ggagtaacaa tacaaatgga ttttgggagt ga -#ctcaagaa   2770   - - gtgaagaatg cacaagaatg gatcacaaga tggaatttag caaaccctag cc -#ttgcttgt   2830   - - taaaattttt tttttttttt ttttaagaat atctgtaatg gtactgactt tg -#cttgcttt   2890   - - gaagtagctc tttttttttt tttttttttt tttttttgca gtaactgttt tt -#taagtctc   2950   - - tcgtagtgtt aagttatagt gaatactgct acagcaattt ctaattttta ag -#aattgagt   3010   - - aatggtgtag aacactaatt aattcataat cactctaatt aattgtaatc tg -#aataaagt   3070   - - gtaacaattg tgtagccttt ttgtataaaa tagacaaata gaaaatggtc ca -#attagttt   3130   - - cctttttaat atgcttaaaa taagcaggtg gatctatttc atgtttttga tc -#aaaaacta   3190   - - tttgggatat gtatgggtag ggtaaatcag taagaggtgt tatttggaac ct -#tgttttgg   3250   - - acagtttacc agttgccttt tatcccaaag ttgttgtaac ctgctgtgat ac -#gatgcttc   3310   - - aagagaaaat gcggttataa aaaatggttc agaattaaac ttttaattca tt - #3362  - - &lt;210&gt; SEQ ID NO 2 &lt;211&gt; LENGTH: 22 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: PCR Primer  - - &lt;400&gt; SEQUENCE: 2  - - agtgggaaca gggattttct ca           - #                  - #   22  - -  - - &lt;210&gt; SEQ ID NO 3 &lt;211&gt; LENGTH: 24 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: PCR Primer  - - &lt;400&gt; SEQUENCE: 3  - - cctcatctaa tgtctcaggg aaca          - #                  - #   24  - -  - - &lt;210&gt; SEQ ID NO 4 &lt;211&gt; LENGTH: 30 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: PCR Probe  - - &lt;400&gt; SEQUENCE: 4  - - tgatggacag tatgcaatga ctcgagctca         - #                  - #   30  - -  - - &lt;210&gt; SEQ ID NO 5 &lt;211&gt; LENGTH: 19 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: PCR Primer  - - &lt;400&gt; SEQUENCE: 5  - - gaaggtgaag gtcggagtc             - #                  - #  - # 19  - -  - - &lt;210&gt; SEQ ID NO 6 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: PCR Primer  - - &lt;400&gt; SEQUENCE: 6  - - gaagatggtg atgggatttc            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 7 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: PCR Probe  - - &lt;400&gt; SEQUENCE: 7  - - caagcttccc gttctcagcc            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 8 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 8  - - cccctcgctc tccgctcccg            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 9 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 9  - - cgacctgcgg tggcggctcg            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 10 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 10  - - gttgtatggt atacttcaaa            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 11 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 11  - - ttgtccacgc tggattttca            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 12 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 12  - - cattgtccac gctggatttt            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 13 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 13  - - gccattgtcc acgctggatt            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 14 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 14  - - tagccattgt ccacgctgga            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 15 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 15  - - agtagccatt gtccacgctg            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 16 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 16  - - tgagtagcca ttgtccacgc            - #                  - #  - #  20  - -  - - &lt;210&gt; SEQ ID NO 17 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 17  - - cttgagtagc cattgtccac            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 18 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 18  - - agcttgagta gccattgtcc            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 19 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 19  - - tcagcttgag tagccattgt            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 20 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 20  - - aatcagcttg agtagccatt            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 21 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 21  - - caaatcagct tgagtagcca            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 22 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 22  - - ttgctgccag tgactaacag            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 23 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 23  - - agaaaatccc tgttcccact            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 24 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 24  - - aaactgtgta gatgggatct            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 25 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 25  - - tagcagtttt gtcagttcag            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 26 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 26  - - agcagacacc atctgaggag            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 27 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 27  - - aggtcccagc ggtacaacga            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 28 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 28  - - accaagcatt ttcaccaggg            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 29 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 29  - - agcccaccag ctaaacgcac            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 30 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 30  - - ttattgcaag tgaggttaga            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 31 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 31  - - gtacgcacaa gagcctctat            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 32 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 32  - - aggtgcatga tttgcgggac            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 33 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 33  - - gcacgaacaa gcaactgaac            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 34 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 34  - - cctctgataa caattcggtt            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 35 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 35  - - tgttttcaat gggagaataa            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 36 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 36  - - gtcctgagca agttcacaga            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 37 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 37  - - ctgtcagagg agctgtggct            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 38 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 38  - - tcgccacacc ttcattccta            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 39 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 39  - - ctggtcagct caactgaaag            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 40 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 40  - - accagggtgg tggccaccca            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 41 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 41  - - ctaaaggatg atttacaggt            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 42 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 42  - - cctctcagca actctacagg            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 43 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 43  - - cccataggaa actcagcttg            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 44 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 44  - - aggctagggt ttgctaaatt            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 45 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 45  - - tagagtgatt atgaattaat            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 46 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 46  - - caaaacaagg ttccaaataa            - #                  - #  - # 20  - -  - - &lt;210&gt; SEQ ID NO 47 &lt;211&gt; LENGTH: 20 &lt;212&gt; TYPE: DNA &lt;213&gt; ORGANISM: Artificial Sequence &lt;220&gt; FEATURE: &lt;223&gt; OTHER INFORMATION: Antisense Oligonucleotide  - - &lt;400&gt; SEQUENCE: 47  - - ttctcttgaa gcatcgtatc            - #                  - #  - # 20__________________________________________________________________________
Non-Patent CitationsReference1 *Branch, TIBS 23 pp. 45 50, Feb. 1998.2Branch, TIBS 23 pp. 45-50, Feb. 1998.3 *Candidus et al, Cancer Res. 56, pp. 49 52, Jan. 1996.4Candidus et al, Cancer Res. 56, pp. 49-52, Jan. 1996.5 *Crooke, Basic Principles of Antisense Therapeutics, Antisense Research and Application, pp. 1 50, 1998.6Crooke, Basic Principles of Antisense Therapeutics, Antisense Research and Application, pp. 1-50, 1998.7 *Flanagan et al, Nature Biotech 17:48 52, Jan. 1999.8Flanagan et al, Nature Biotech 17:48-52, Jan. 1999.9 *Haegel et al., Lack of beta catenin affects mouse development at gastrulation, Development, 1995, 121:3529 3537.10Haegel et al., Lack of beta-catenin affects mouse development at gastrulation, Development, 1995, 121:3529-3537.11 *Heasman et al., Overexpression of cadherins and underexpression of beta catenin inhibit dorsal mesoderm induction in early Xenopus embryos, Cell, 1994, 79:791 803.12Heasman et al., Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos, Cell, 1994, 79:791-803.13 *Ilyas et al., The interactions of APC, E cadherin and beta catenin in tumor development and progression, J. Pathol., 1997, 182:128 137.14Ilyas et al., The interactions of APC, E-cadherin and beta-catenin in tumor development and progression, J. Pathol., 1997, 182:128-137.15 *James, W. Antiviral Chem & Chemo. 2(4) 191 214, 1991.16James, W. Antiviral Chem & Chemo. 2(4) 191-214, 1991.17 *Kim et al., Tissue specific expression of beta catenin in normal mesenchyme and uveal melanomas and its effect on invasiveness, Exp. Cell. Res., 1998, 245:79 90.18Kim et al., Tissue-specific expression of beta-catenin in normal mesenchyme and uveal melanomas and its effect on invasiveness, Exp. Cell. Res., 1998, 245:79-90.19 *Korinek et al., Constitutive transcriptional activation by a beta catenin Tcf complex in APC / colon carcinoma, Science, 1997, 275:1784 1787020Korinek et al., Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/-colon carcinoma, Science, 1997, 275:1784-1787021 *Morin et al., Activation of beta catenin Tcf signaling in colon cancer by mutations in beta catenin or APC, Science, 1997, 275:1787 1790.22Morin et al., Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC, Science, 1997, 275:1787-1790.23 *Nollet et al, Genomics 32, pp. 413 424, 1996.24Nollet et al, Genomics 32, pp. 413-424, 1996.25 *Peifer, Cancer, catenins, and cuticle pattern: a complex connection, Science, 1993, 262:1667 1668.26Peifer, Cancer, catenins, and cuticle pattern: a complex connection, Science, 1993, 262:1667-1668.27 *Robbins et al, J. Exp. Med, vol. 183, pp. 1185 1192, Mar. 1996.28Robbins et al, J. Exp. Med, vol. 183, pp. 1185-1192, Mar. 1996.29 *Rubinfeld et al., Stabilization of beta catenin by genetic defects in melanoma cell lines, Science, 1997, 275:1790 1792.30Rubinfeld et al., Stabilization of beta-catenin by genetic defects in melanoma cell lines, Science, 1997, 275:1790-1792.31 *Uhlman et al, Chem Rev, vol. 90, No. 4, pp. 543 584, 1990.32Uhlman et al, Chem Rev, vol. 90, No. 4, pp. 543-584, 1990.33 *Willert et al., Beta catenin: a key mediator of Wnt signaling, Curr. Opin. Genet. Dev., 1998, 8:95 102.34Willert et al., Beta-catenin: a key mediator of Wnt signaling, Curr. Opin. Genet. Dev., 1998, 8:95-102.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS7662791 *Aug 1, 2001Feb 16, 2010University Of Southern CaliforniaGene silencing using mRNA-cDNA hybridsUS7915401 *Jan 21, 2009Mar 29, 2011Enzon Pharmaceuticals, Inc.Compounds for the modulation of beta-catenin expressionUS8039446Sep 2, 2010Oct 18, 2011Enzon Pharmaceuticals, Inc.Compounds for the modulation of beta-catenin expressionUS8080652Dec 2, 2009Dec 20, 2011University Of Southern CaliforniaGene silencing using mRNA-cDNA hybridsUS8110674Mar 8, 2004Feb 7, 2012Alnylam Pharmaceuticals, Inc.Therapeutic compositionsUS8420799Jul 16, 2010Apr 16, 2013Alnylam Pharmaceuticals, Inc.Therapeutic compositionsUS8445665Apr 6, 2010May 21, 2013Alnylam Pharmaceuticals, Inc.Therapeutic compositionsUS8754201Mar 10, 2010Jun 17, 2014Alnylam Pharmaceuticals, Inc.Therapeutic compositionsUS8809516Sep 25, 2012Aug 19, 2014Alnylam Pharmaceuticals, Inc.Therapeutic compositionsEP1297186A1 *Jul 2, 2001Apr 2, 2003The University of Utah Research FoundationMethod of screening for chemotherapeutic treatments for cancerEP1605978A2 *Mar 8, 2004Dec 21, 2005Alnylam Pharmaceuticals Inc.Therapeutic compositionsEP1651674A2 *Aug 12, 2004May 3, 2006Exelixis, Inc.Ups as modifiers of the beta catenin pathway and methods of useWO2008132234A2Apr 30, 2008Nov 6, 2008Santaris Pharma AsRna antagonist compounds for the modulation of beta-cateninWO2014065849A1 *Feb 19, 2013May 1, 2014Phaserx, Inc.Rna targeted to beta catenin* Cited by examinerClassifications U.S. Classification435/375, 435/91.1, 536/24.5, 536/23.1, 536/24.33, 536/24.31, 435/366, 435/6.14International ClassificationA61K38/00, C12N15/113Cooperative ClassificationC12N2310/346, C12N15/113, C12N2310/315, C12N2310/3341, A61K38/00, C12N2310/321, C12N2310/341European ClassificationC12N15/113Legal EventsDateCodeEventDescriptionJun 25, 1999ASAssignmentOwner name: ISIS PHARMACEUTICALS, INC., CALIFORNIAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BENNETT, C. FRANK;COWSERT, LEX M.;REEL/FRAME:010067/0245Effective date: 19990623Sep 26, 2003FPAYFee paymentYear of fee payment: 4Sep 14, 2007FPAYFee paymentYear of fee payment: 8Sep 23, 2011FPAYFee paymentYear of fee payment: 12RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services