Source: http://www.google.com/patents/US6033910?dq=5,870,513
Timestamp: 2014-03-17 10:31:40
Document Index: 335586222

Matched Legal Cases: ['art  322', 'art  324', 'art  326', 'art  328', 'art  330', 'art  332', 'art  334', 'art  336', 'art  338', 'art  340', 'art  342', 'art  322', 'art  324', 'art  326', 'art  328', 'art  332', 'art  334', 'art  336', 'art  338', 'art  340', 'art  342']

Patent US6033910 - Antisense inhibition of MAP kinase kinase 6 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 MAP kinase kinase 6. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding MAP kinase kinase 6. Methods of using these compounds for modulation...http://www.google.com/patents/US6033910?utm_source=gb-gplus-sharePatent US6033910 - Antisense inhibition of MAP kinase kinase 6 expressionAdvanced Patent SearchPublication numberUS6033910 APublication typeGrantApplication numberUS 09/357,073Publication dateMar 7, 2000Filing dateJul 19, 1999Priority dateJul 19, 1999Fee statusPaidAlso published asEP1203092A1, EP1203092A4, WO2001006018A1Publication number09357073, 357073, US 6033910 A, US 6033910A, US-A-6033910, US6033910 A, US6033910AInventorsLex M. Cowsert, Brett P. MoniaOriginal AssigneeIsis Pharmaceuticals Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (5), Non-Patent Citations (34), Referenced by (16), Classifications (30), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetAntisense inhibition of MAP kinase kinase 6 expressionUS 6033910 AAbstract Antisense compounds, compositions and methods are provided for modulating the expression of MAP kinase kinase 6. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding MAP kinase kinase 6. Methods of using these compounds for modulation of MAP kinase kinase 6 expression and for treatment of diseases associated with expression of MAP kinase kinase 6 are provided.
What is claimed is: 1. An antisense compound 8 to 30 nucleobases in length targeted to a start codon, a 3'-untranslated region, a coding region, a stop codon, or a 5'-untranslated region of human MAP kinase kinase 6, wherein said antisense compound specifically hybridizes with and inhibits the expression of human MAP kinase kinase 6.
10. A method of inhibiting the expression of human MAP kinase kinase 6 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 MAP kinase kinase 6 is inhibited.
11. An antisense compound up to 30 nucleobases in length comprising at least and 8-nucleobase portion of SEQ ID NO: 9, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 43, 44, 45, 46 or 47 which inhibits the expression of human MAP kinase kinase 6.
16. The antisense compound of claim 14 wherein the modified sugar moiety is a 2'-O-methoxyethyl sugar moiety.
20. A method of inhibiting the expression of human MAP kinase kinase 6 in human cells or tissues comprising contacting said cells or tissues in vitro with the antisense compound of claim 11 so that expression of human MAP kinase kinase 6 is inhibited.
FIELD OF THE INVENTION The present invention provides compositions and methods for modulating the expression of MAP kinase kinase 6. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human MAP kinase kinase 6. Such oligonucleotides have been shown to modulate the expression of MAP kinase kinase 6.
Nearly all cell surface receptors use one or more of the mitogen-activated protein kinase (MAP kinase) cascades during signal transduction. Three distinct subgroups of MAP kinases have been identified and each of these consists of a specific module of kinases that function downstream of an activating stimulus. One subgroup of the MAP kinases is the p38 MAP kinase cascade which has been shown to be activated by proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 as well as environmental stress (e.g., osmotic shock and ultraviolet radiation). Upon activation, the p38 cascade leads to the induction of gene expression of several factors involved in inflammation and immunity including TNF, interleukin-6, granulocyte-macrophage colony stimulating factor (GM-CSF), and HIV long terminal repeat (Paul et al., Cell Signal., 1997, 9, 403-410). Modulation of the expression of one or more of the kinases in the p38 pathway is desireable in order to interfere with inflammatory or apoptotic responses associated with disease states. Various inhibitors have been shown to have efficacy in animal models of arthritis (Badger et al., J. Pharmacol. Exp. Ther., 1996, 279, 1453-1461) and angiogenesis (Jackson et al., J. Pharmacol. Exp. Ther., 1998, 284, 687-692). MacKay, K. and Mochy-Rosen, D. demonstrate that an inhibitor of p38 MAP kinases prevents apoptosis during ischemia in cardiac myocytes, suggesting that p38 MAP kinase inhibitors can be used for treating ischemic heart disease (Mackay and Mochly-Rosen, J. Biol. Chem., 1999, 274, 6272-6279). p38 MAP kinase is also required for T-cell HIV-1 replication (Cohen et al., Molecular Medicine, 1997, 3, 339-346) and may be a useful target for AIDS therapy. Disclosed in PCT application WO 98/54203 are methods to increase cancer cell sensitivity to cancer therapy by contacting said cells with a p38 pathway inhibitor. These inhibitors being ribozymes, antisense nucleic acid molecules targeting MEKK1, or dominant negative mutants of MEKK1 (Mercola, 1998). However, within this PCT publication, the composition of these inhibitors is not disclosed.
MAP kinase kinase 6 (also known as MKK6, MEK6 and SAPKK3) is a kinase within the p38 cascade that specifically activates all three isoforms of p38 MAP kinase (Cuenda et al., Embo J., 1997, 16, 295-305; Enslen et al., J. Biol. Chem., 1998, 273, 1741-1748; Goedert et al., Embo J., 1997, 16, 3563-3571; Raingeaud et al., Mol. Cell. Biol., 1996, 16, 1247-1255). MAP kinase kinase 6 was originally cloned from skeletal muscle using a probe to MKK3, a MAP kinase that also activates p38 and is very similar to MAP kinase kinase 6 (Raingeaud et al., Mol. Cell. Biol., 1996, 16, 1247-1255). Characterization of the MAP kinase kinase 6 protein and nucleotide sequence revealed that MAP kinase kinase 6 exists in a variety of alternatively spliced forms with distinct patterns of expression (Han et al., J. Biol. Chem., 1996, 271, 2886-2891). One variant, with an mRNA of 1.7 kilobases was found exclusively in skeletal muscle while other variants were found in the heart liver and kidney. This differential expression pattern implies that MAP kinase kinase 6 may play unique roles in these tissues (Han et al., J. Biol. Chem., 1996, 271, 2886-2891).
Disclosed in the PCT publication WO 97/22704 are the nucleic acid sequence encoding MAP kinase kinase 6, compositions and methods of modulating the p38 MAP kinase pathway, including antibodies to MAP kinase kinase 6, and a kit to identify agents that modulate the pathway (Stein and Yang, 1997).
Disclosed in U.S. Pat. No. 5,736,381 are MKK isoforms including MAP kinase kinase 6, a polynucleotide encoding MAP kinase kinase 6, a vector encoding MAP kinase kinase 6 and host cells expressing said vector (Davis et al., 1998). Also disclosed are polynucleotides fully complementary to MAP kinase kinase 6 or variants thereof (Davis et al., 1996). Antisense oligonucleotides to MAP kinase kinase 6 are generally disclosed. Further disclosed in the PCT publication WO 96/36642 are antibodies to MAP kinase kinase 6 and methods to measure MAP kinase kinase 6 activity and synthesis (Davis et al., 1996).
The effect of TNF on MAP kinase kinase 6 activation and signaling has been widely investigated. Subtype specific cytokine activation of a MAP kinase pathway has been described in neutrophils by Suzuki et al. who demonstrated that the MAP kinase kinase 6 is specifically activated by the TNF-α and granulocyte-macrophage colony stimulating factor (GM-CSF) in the p38 pathway (Suzuki et al., Blood, 1999, 93, 341-349). Studies of dominant negative and dominant active mutants of MAP kinase kinase 6 in endothelial cells showed that activation of the MKK6/p38 pathway is critical for the TNF-α mediated expression of monocyte chemoattractant protein 1 (MCP-1) (Goebeler et al., Blood, 1999, 93, 857-865). MCP-1 recruits leukocytes to the site of inflammation and excesses of this protein have been detected in diseases such as atherosclerosis.
The MAP kinase kinase 6 protein has also been shown to regulate signaling events associated with apoptosis. Zechner et al. demonstrated that MAP kinase kinase 6 activates NF-kappa-B and inhibits apoptosis in rat myocardial cells (Zechner et al., J. Biol. Chem., 1998, 273, 8232-8239). Myocardial cell apoptosis is believed to contribute to several cardiac disorders including heart failure and myocardial infarction. In these studies, overexpression of a constitutively active mutant of MAP kinase kinase 6 protected cells from both anisomycin- and MEKK1-induced apoptosis (Zechner et al., J. Biol. Chem., 1998, 273, 8232-8239).
Mutant forms of MAP kinase kinase 6 have also been used to demonstrate the role of MAP kinase kinase 6 in hematopoiesis. Forced activation of the p38 cascade using a gain-of-function MAP kinase kinase 6 mutant was shown to induce erythroid differentiation in SKT6 cells (Nagata et al., Blood, 1998, 92, 1859-1869).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MAP kinase kinase 6 and to date, strategies aimed at modulating MAP kinase kinase 6 function have involved the use of antibodies, dominant negative and dominant active mutants of the protein and MAP kinase pathway inhibitors. There remains, consequently, a long felt need for additional agents capable of effectively inhibiting MAP kinase kinase 6 function.
Antisense technology is 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 MAP kinase kinase 6 expression.
The present invention provides compositions and methods for modulating MAP kinase kinase 6 expression, including modulation of the alternatively spliced form of MAP kinase kinase 6, MKK6b.
SUMMARY OF THE INVENTION The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding MAP kinase kinase 6, and which modulate the expression of MAP kinase kinase 6. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of MAP kinase kinase 6 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 MAP kinase kinase 6 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.
DETAILED DESCRIPTION OF THE INVENTION The present invention employs oligomeric antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding MAP kinase kinase 6, ultimately modulating the amount of MAP kinase kinase 6 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding MAP kinase kinase 6. As used herein, the terms "target nucleic acid" and "nucleic acid encoding MAP kinase kinase 6" encompass DNA encoding MAP kinase kinase 6, 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 functions 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 MAP kinase kinase 6. 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 MAP kinase kinase 6. 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 MAP kinase kinase 6, regardless of the sequence(s) of such codons.
While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in, accordance with this invention preferably comprise from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 25 nucleobases. As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
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 C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly preferred are O[(CH.sub.2).sub.n O].sub.m CH.sub.3, O(CH.sub.2).sub.n OCH.sub.3, O(CH.sub.2).sub.n NH.sub.2, O(CH.sub.2).sub.n CH.sub.3, O(CH.sub.2).sub.n ONH.sub.2, and O(CH.sub.2).sub.n ON[(CH.sub.2).sub.n CH.sub.3)].sub.2, where n and m are from 1 to about 10.
Other preferred oligonucleotides comprise one of the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2 CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, 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--CH.sub.2 CH.sub.2 OCH.sub.3, 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(CH.sub.2).sub.2 ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH.sub.2 --O--CH.sub.2 --N(CH.sub.2).sub.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 MAP kinase kinase 6 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 MAP kinase kinase 6, 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 MAP kinase kinase 6 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 MAP kinase kinase 6 in a sample may also be prepared.
2'-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, the protected nucleoside N6-benzoyl-2'-deoxy-21-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2'-alpha-fluoro atom is introduced by a S.sub.N 2-displacement of a 2'-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3',5'-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5'-dimethoxytrityl-(DMT) and 5'-DMT-3'-phosphoramidite intermediates.
5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60 a solid that was crushed to a light tan powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%). The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4
A first solution was prepared by dissolving 3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH.sub.3 CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH.sub.3 CN (1 L), cooled to -50 stirrer. POCl.sub.3 was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10 stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1 2 evaporated. The residue was triturated with EtOAc to give the title compound.
A solution of 3'-O-acetyl-2'-O-methoxyethyl-5'-0-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH.sub.4 OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2 residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH.sub.3 gas was added and the vessel heated to 100 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.
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH.sub.2 Cl.sub.2 (1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (TLC showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO.sub.3 (1 mL). The aqueous washes were back-extracted with CH.sub.2 Cl.sub.2 (300 mL), and the extracts were combined, dried over MgSO.sub.4 and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.
51'-O-tert-Butyldiphenylsilyl-O -2'-anhydro-5-methyluridine
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluridine 2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH.sub.2 Cl.sub.2 (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at -10 to 0 washed with ice cold CH.sub.2 Cl.sub.2 and the combined organic phase was washed with water, brine and dried over anhydrous Na.sub.2 SO.sub.4. The solution was concentrated to get 2'-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was strirred for 1 h. Solvent was removed under vacuum; residue chromatographed to get 5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%).
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6mL). Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at 10 inert atmosphere. The reaction mixture was stirred for 10 minutes at 10 and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH.sub.2 Cl.sub.2). Aqueous NaHCO.sub.3 solution (5%, 10 mL) was added and extracted with ethyl acetate (2 phase was dried over anhydrous Na.sub.2 SO.sub.4, evaporated to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10 6.13 mmol) was added and reaction mixture stirred at 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO.sub.3 (25 mL) solution was added and extracted with ethyl acetate (2 SO.sub.4 and evaporated to dryness . The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH.sub.2 Cl.sub.2 to get 5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%).
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over P.sub.2.sub.O.sub.5 under high vacuum overnight at 40 the reaction mixture was dissolved in anhydrous acetonitrile (8.4mL) and 2-cyanoethyl-N,N,N.sup.1,N.sup.1 -tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 hrs under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated, then the residue was dissolved in ethyl acetate (70 mL) and washed with 5% aqueous NaHCO.sub.3 (40 mL). Ethyl acetate layer was dried over anhydrous Na.sub.2 SO.sub.4 and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%).
2'-(Aminooxyethoxy) nucleoside amidites [also known in the art as 2'-O-(aminooxyethyl) nucleoside amidites] are prepared as described in 'he following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.
The 2'-O-aminooxyethyl guanosine analog may be obtained by selective 2'-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2'-O-(2-ethylacetyl)diaminopurine riboside along with a minor amount of the 3'-O-isomer. 2'-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to 2'-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].
2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. O.sup.2 -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 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 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.
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine To 0.5 g (1.3 mmol) of 2'-O-[2(2-N,N-dimethylaminoethoxy)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 CH.sub.2 Cl.sub.2 (2 layers are washed with saturated NaHCO.sub.3 solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate. Evaporation of the solvent followed by silica gel chromatography using MeOH:CH.sub.2 Cl.sub.2 :Et.sub.3 N (20:1, v/v, with 1% triethylamine) gives the title compound.
5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite
Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound.
Oligonucleotides of the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers".
[2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides
[2'-O-(2-methoxyethyl)]-[2'-deoxy]-[-2'-O-(methoxyethyl)] 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 Phosphorothioate]-[2'-O-(2-Methoxyethyl)Phosphodiester] Chimeric Oligonucleotides
[2'-O-(2-methoxyethyl phosphodiester]-[2'-deoxy phosphorothioate]-[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.
Oligonucleotides were cleaved from support and deprotected with concentrated NH,OH at elevated temperature (55-60 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.
Example 10 Analysis of Oligonucleotide Inhibition of M kinase kinase 6 Expression
Antisense modulation of MAP kinase kinase 6 expression can be assayed in a variety of ways known in the art. For example, MAP kinase kinase 6 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.
MAP kinase kinase 6 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 MAP kinase kinase 6 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), 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.
Example 13 Real-time Quantitative PCR Analysis of MAP kinase kinase 6 mRNA Levels
Quantitation of MAP kinase kinase 6 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 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 95 PCR protocol were carried out: 95 followed by 60 kinase kinase 6 probes and primers were designed to hybridize to the human MAP kinase kinase 6 sequence, using published sequence information (GenBank accession number U39657, incorporated herein as SEQ ID NO:1).
For MAP kinase kinase 6 the PCR primers were: forward primer: AAAAGAAGCATTTGAACAACCTCA (SEQ ID NO: 2) reverse primer: TTCACCTCAAAGTTCTGATTTCCAA (SEQ ID NO: 3) and the PCR probe was: FAM-TTCCACACCACCTAGAGATTTAGACTCCAAGGCT-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.
Example 14 Northern Blot Analysis of MAP kinase kinase 6 mRNA Levels
Membranes were probed using QUICKHYB� hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions with a MAP kinase kinase 6 specific probe prepared by PCR using the forward primer AAAAGAAGCATTTGAACAACCTCA (SEQ ID NO: 2) and the reverse primer TTCACCTCAAAGTTCTGATTTCCAA (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.
Example 15 Antisense Inhibition of MAP kinase kinase 6 Expression-phosphorothioate Oligodeoxynucleotides
In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human MAP kinase kinase 6 RNA, using published sequences (GenBank accession number U39657, 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. U39657), 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 MAP kinase kinase 6 mRNA levels by quantitative 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 MAP kinase kinase 6 mRNA levels byphosphorothioate oligodeoxynucleotides     TARGET           %    SEQ IDISIS #    REGION     SITE SEQUENCE    Inhibition                           NO.__________________________________________________________________________101427    5' UTR       1  ggtgggccgaaccagaagcc                      0    8101430    5' UTR      70  gccagggctggcagctaaaa                      20   9101433    5' UTR      204 gtttctcttgaccgatgcag                      45   10101436    5' UTR      265 tctgtagctagttttgcaac                      12   11101439    Start  322 ttttcccctttcctttgatg                      0    12    Codon101442    Start  324 cattttcccctttcctttga                      0    13    Codon101445    Start  326 gacattttcccctttccttt                      15   14    Codon101448    Start  328 gagacattttcccctttcct                      34   15    Codon101451    Start  330 ctgagacattttcccctttc                      9    16    Codon101454    Start  332 gactgagacattttcccctt                      12   17    Codon101457    Start  334 tcgactgagacattttcccc                      20   18    Codon101460    Start  336 tttcgactgagacattttcc                      0    19    Codon101463    Start  338 cctttcgactgagacatttt                      0    20    Codon101466    Start  340 tgcctttcgactgagacatt                      33   21    Codon101469    Start  342 cttgcctttcgactgagaca                      42   22    Codon101472    Coding      366 aattttaaggccagggtttc                      0    23101475    Coding      428 ttggagtctaaatctctagg                      0    24101478    Coding      484 gctccaggtcatctgccttc                      35   25101481    Coding      604 gtttctgttcctggctattt                      21   26101484    Coding      672 gccataaaaggtgacagtga                      14   27101487    Coding      738 gaatttatctagtgatgtat                      0    28101490    Coding      874 cattagaaggcttgacgtct                      33   29101494    Coding     1088 ggaaatcgaaggatggccaa                      0    30101497    Coding     1235 tatgtaggccgttctttgga                      5    31101501    Coding     1290 atctgttcctttggattcat                      27   32101504    Stop  1326 ttagtctccaagaatcagtt                      4    33    Codon101505    3' UTR     1383 cttcaccccgaaacccacca                      66   34101508    3' UTR     1518 cagttcattctatactatag                      30   35101512    3' UTR     1693 ctacaaatctacttataatt                      1    36101515    3' UTR     1871 gatgcatattaaataatttc                      17   37101518    3' UTR     1996 ccctaaaagatgtggtgatc                      0    38101520    3' UTR     2103 caagtgtcagaagaaccaaa                      30   39101524    3' UTR     2209 gtgaattgccaaatagacac                      20   40101527    3' UTR     2341 acacatggcaggagggacaa                      22   41101530    3' UTR     2403 agaagaaaacaaacaaacaa                      18   42101533    3' UTR     2478 tatcaaagaattactcacta                      15   43101536    3' UTR     2574 cgctgctgaagaaagatgcg                      15   44101539    3' UTR     2656 cgagaaagaacttttgctgt                      0    45101543    3' UTR     2740 tattttccctgtcaattact                      1    46101546    3' UTR     2889 aagagaggattcagggaagc                      0    47__________________________________________________________________________
As shown in Table 1, SEQ ID NOs 10, 15, 21, 22, 25, 29, 32, 34, 35 and 39 demonstrated at least 25% inhibition of MAP kinase kinase 6 expression in this assay and are therefore preferred.
Example 16 Antisense Inhibition of MAP kinase kinase 6 Expression-phosphorothioate 2'-MOE Gapmer Oligonucleotides
In accordance with the present invention, a second series of oligonucleotides targeted to human MAP kinase kinase 6 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. U39657), to which the oligonucleotide binds.
All compounds in Table 2 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. Cytidine residues in the 2'-MOE wings are 5-methylcytidines.
TABLE 2__________________________________________________________________________Inhibition of MAP kinase kinase 6 mRNA levels by chimericphosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap     TARGET           %    SEQ IDISIS #    REGION     SITE SEQUENCE    Inhibition                           NO.__________________________________________________________________________101548    5' UTR       1  ggtgggccgaaccagaagcc                      0    8101552    5' UTR      70  gccagggctggcagctaaaa                      57   9101554    5' UTR      204 gtttctcttgaccgatgcag                      87   10101557    5' UTR      265 tctgtagctagttttgcaac                      0    11101560    Start  322 ttttcccctttcctttgatg                      16   12    Codon101563    Start  324 cattttcccctttcctttga                      0    13    Codon101566    Start  326 gacattttcccctttccttt                      56   14    Codon101569    Start  328 gagacattttcccctttcct                      64   15    Codon101572    Start `330 ctgagacattttcccctttc                      28   16    Codon101575    Start  332 gactgagacattttcccctt                      31   17    Codon101578    Start  334 tcgactgagacattttcccc                      71   18    Codon161581    Start  336 tttcgactgagacattttcc                      56   19    Codon101584    Start  338 cctttcgactgagacatttt                      73   20    Codon101587    Start  340 tgcctttcgactgagacatt                      19   21    Codon101589    Start  342 cttgcctttcgactgagaca                      77   22    Codon101592    Coding      366 aattttaaggccagggtttc                      51   23101595    Coding      428 ttggagtctaaatctctagg                      37   24101599    Coding      484 gctccaggtcatctgccttc                      30   25101601    Coding      604 gtttctgttcctggctattt                      50   26101604    Coding      672 gccataaaaggtgacagtga                      63   27101607    Coding      738 gaatttatctagtgatgtat                      0    28101611    Coding      874 cattagaaggcttgacgtct                      50   29101612    Coding     1088 ggaaatcgaaggatggccaa                      37   30101615    Coding     1235 tatgtaggccgttctttgga                      27   31101620    Coding     1290 atctgttcctttggattcat                      55   32101621    Stop  1326 ttagtctccaagaatcagtt                      55   33    Codon101625    3' UTR     1383 cttcaccccgaaacccacca                      64   34101627    3' UTR     1518 cagttcattctatactatag                      82   35101631    3' UTR     1693 ctacaaatctacttataatt                      15   36101635    3' UTR     1871 gatgcatattaaataatttc                      0    37101636    3' UTR     1996 ccctaaaagatgtggtgatc                      62   38101639    3' UTR     2103 caagtgtcagaagaaccaaa                      29   39101641    3' UTR     2209 gtgaattgccaaatagacac                      43   40101643    3' UTR     2341 acacatggcaggagggacaa                      59   41101644    3' UTR     2403 agaagaaaacaaacaaacaa                      0    42101645    3' UTR     2478 tatcaaagaattactcacta                      25   43101646    3' UTR     2574 cgctgctgaagaaagatgcg                      64   44101647    3' UTR     2656 cgagaaagaacttttgctgt                      73   45101648    3' UTR     2740 tattttccctgtcaattact                      40   46101649    3' UTR     2889 aagagaggattcagggaagc                      38   47__________________________________________________________________________
As shown in Table 2, SEQ ID NOs 9, 10, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 43, 44, 45, 46 and 47 demonstrated at least 25% inhibition of MAP kinase kinase 6 expression in this experiment and are therefore preferred.
Example 17 Western Blot Analysis of MAP kinase kinase 6 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 MAP kinase kinase 6 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- &amp;lt;160&amp;gt; NUMBER OF SEQ ID NOS: 47- &amp;lt;210&amp;gt; SEQ ID NO 1&amp;lt;211&amp;gt; LENGTH: 2924&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Homo sapiens&amp;lt;220&amp;gt; FEATURE:&amp;lt;221&amp;gt; NAME/KEY: CDS&amp;lt;222&amp;gt; LOCATION: (341)..(1345)- &amp;lt;400&amp;gt; SEQUENCE: 1- ggcttctggt tcggcccacc tctgaaggtt ccagaatcga tagtgaattc gt - #ggttccaa  60- gtttggagct tttagctgcc agccctggcc catcatgtag ctgcagcaca gc - #cttcccta 120- acgttgcaac tgggggaaaa atcactttcc agtctgtttt gcaaggtgtg ca - #tttccatc 180- ttgattccct gaaagtccat ctgctgcatc ggtcaagaga aactccactt gc - #atgaagat 240- tgcacgcctg cagcttgcat ctttgttgca aaactagcta cagaagagaa gc - #aaggcaaa 300- gtcttttgtg ctcccctccc ccatcaaagg aaaggggaaa atg tct ca - #g tcg aaa 355#        Met Ser Gln Ser Lys#       5  1- ggc aag aag cga aac cct ggc ctt aaa att cc - #a aaa gaa gca ttt gaa 403Gly Lys Lys Arg Asn Pro Gly Leu Lys Ile Pr - #o Lys Glu Ala Phe Glu#                 20- caa cct cag acc agt tcc aca cca cct aga ga - #t tta gac tcc aag gct 451Gln Pro Gln Thr Ser Ser Thr Pro Pro Arg As - #p Leu Asp Ser Lys Ala#             35- tgc att tct att gga aat cag aac ttt gag gt - #g aag gca gat gac ctg 499Cys Ile Ser Ile Gly Asn Gln Asn Phe Glu Va - #l Lys Ala Asp Asp Leu#         50- gag cct ata atg gaa ctg gga cga ggt gcg ta - #c ggg gtg gtg gag aag 547Glu Pro Ile Met Glu Leu Gly Arg Gly Ala Ty - #r Gly Val Val Glu Lys#     65- atg cgg cac gtg ccc agc ggg cag atc atg gc - #a gtg aag cgg atc cga 595Met Arg His Val Pro Ser Gly Gln Ile Met Al - #a Val Lys Arg Ile Arg# 85- gcc aca gta aat agc cag gaa cag aaa cgg ct - #a ctg atg gat ttg gat 643Ala Thr Val Asn Ser Gln Glu Gln Lys Arg Le - #u Leu Met Asp Leu Asp#                100- att tcc atg agg acg gtg gac tgt cca ttc ac - #t gtc acc ttt tat ggc 691Ile Ser Met Arg Thr Val Asp Cys Pro Phe Th - #r Val Thr Phe Tyr Gly#           115- gca ctg ttt cgg gag ggt gat gtg tgg atc tg - #c atg gag ctc atg gat 739Ala Leu Phe Arg Glu Gly Asp Val Trp Ile Cy - #s Met Glu Leu Met Asp#       130- aca tca cta gat aaa ttc tac aaa caa gtt at - #t gat aaa ggc cag aca 787Thr Ser Leu Asp Lys Phe Tyr Lys Gln Val Il - #e Asp Lys Gly Gln Thr#   145- att cca gag gac atc tta ggg aaa ata gca gt - #t tct att gta aaa gca 835Ile Pro Glu Asp Ile Leu Gly Lys Ile Ala Va - #l Ser Ile Val Lys Ala150                 1 - #55                 1 - #60                 1 -#65- tta gaa cat tta cat agt aag ctg tct gtc at - #t cac aga gac gtc aag 883Leu Glu His Leu His Ser Lys Leu Ser Val Il - #e His Arg Asp Val Lys#               180- cct tct aat gta ctc atc aat gct ctc ggt ca - #a gtg aag atg tgc gat 931Pro Ser Asn Val Leu Ile Asn Ala Leu Gly Gl - #n Val Lys Met Cys Asp#           195- ttt gga atc agt ggc tac ttg gtg gac tct gt - #t gct aaa aca att gat 979Phe Gly Ile Ser Gly Tyr Leu Val Asp Ser Va - #l Ala Lys Thr Ile Asp#       210- gca ggt tgc aaa cca tac atg gcc cct gaa ag - #a ata aac cca gag ctc1027Ala Gly Cys Lys Pro Tyr Met Ala Pro Glu Ar - #g Ile Asn Pro Glu Leu#   225- aac cag aag gga tac agt gtg aag tct gac at - #t tgg agt ctg ggc atc1075Asn Gln Lys Gly Tyr Ser Val Lys Ser Asp Il - #e Trp Ser Leu Gly Ile230                 2 - #35                 2 - #40                 2 -#45- acg atg att gag ttg gcc atc ctt cga ttt cc - #c tat gat tca tgg gga1123Thr Met Ile Glu Leu Ala Ile Leu Arg Phe Pr - #o Tyr Asp Ser Trp Gly#               260- act cca ttt cag cag ctc aaa cag gtg gta ga - #g gag cca tcg cca caa1171Thr Pro Phe Gln Gln Leu Lys Gln Val Val Gl - #u Glu Pro Ser Pro Gln#           275- ctc cca gca gac aag ttc tct gca gag ttt gt - #t gac ttt acc tca cag1219Leu Pro Ala Asp Lys Phe Ser Ala Glu Phe Va - #l Asp Phe Thr Ser Gln#       290- tgc tta aag aag aat tcc aaa gaa cgg cct ac - #a tac cca gag cta atg1267Cys Leu Lys Lys Asn Ser Lys Glu Arg Pro Th - #r Tyr Pro Glu Leu Met#   305- caa cat cca ttt ttc acc cta cat gaa tcc aa - #a gga aca gat gtg gca1315Gln His Pro Phe Phe Thr Leu His Glu Ser Ly - #s Gly Thr Asp Val Ala310                 3 - #15                 3 - #20                 3 -#25- tct ttt gta aaa ctg att ctt gga gac taa aa - #agcagtgg acttaatcgg1365Ser Phe Val Lys Leu Ile Leu Gly Asp           330- ttgaccctac tgtggattgg tgggtttcgg ggtgaagcaa gttcactaca gc - #atcaatag1425- aaagtcatct ttgagataat ttaaccctgc ctctcagagg gttttctctc cc - #aattttct1485- ttttactccc cctcttaagg gggccttgga atctatagta tagaatgaac tg - #tctagatg1545- gatgaattat gataaaggct taggacttca aaaggtgatt aaatatttaa tg - #atgtgtca1605- tatgagtcct caagcttctc agacttctct tattctttac aaaatgaatg ca - #ttggccct1665- gacaaaaagg tgctacggta gtgatgaaat tataagtaga tttgtagttt gt - #cccattta1725- ttattttaat atttatgttt aagtgcttgg ttgaaaagat tccattttat ac - #aagaaggg1785- agattcaaaa aaaaaatata aggttgggtt agcaatattt atagggcttt ta - #ttttttaa1845- gttcaattgt gtctgtggtc cagaagaaat tatttaatat gcatctttga ga - #atattata1905- aaaatatcaa aaaggagctc ttcttgtgaa atgtctgttc cagctgttgt ga - #ctgctgcc1965- atttttggaa acatctgccc aatcctgggt gatcaccaca tcttttaggg ga - #agtgacaa2025- gatgctctgg tcatactctt tttcccaact ttggaaaaca taaaaatcac tc - #atataaca2085- gctcaaagag taaaacattt ggttcttctg acacttgtgg tatagtatta gt - #ggaaagtg2145- atttgtaata tgattttata tccacctacc tattcatcta cctgtgtgta tg - #tgtgtgtt2205- tgtgtgtcta tttggcaatt cacaagtcct gccaagtggt ttctatgagc at - #ctctgttt2265- ggtaaggagg acaattgtca gttttgaggg ggacatgtgt taaatcacag aa - #aaaaatgg2325- tgccttcttc tgcgtttgtc cctcctgcca tgtgtaagtt gtaaggattg cc - #tttgtagt2385- taatgtactc tttggctttg tttgtttgtt ttcttcttca gtgaagcagc ct - #tactattc2445- atagaagggc tagaatagga gaaaatgaaa ggtagtgagt aattctttga ta - #agatgagg2505- aaataatggg aaaggttgaa ttaattcctg ggcatggact accagatgac ca - #caagttgc2565- gttgaggccg catctttctt cagcagcgtg caatagctgg ctcctctata gg - #agatgagc2625- ttcattggga gttcctagca agttgactaa acagcaaaag ttctttctcg tg - #ggtaaata2685- tacccacagg ttctatgatt tgtagctcta ggtttcttga tgatcaagga gt - #gaagtaat2745- tgacagggaa aatatagacc tatgataaat aaccaggaag cattgctttt gg - #acaaggaa2805- gaacagaggg ttttgatttt aaaaagaaga aaaaaaaacc ttattttttc tt - #tcttggcc2865- tcaagttcaa tatggagagg attgcttccc tgaatcctct cttccttccc ct - #tttagag2924- &amp;lt;210&amp;gt; SEQ ID NO 2&amp;lt;211&amp;gt; LENGTH: 24&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: PCR Primer- &amp;lt;400&amp;gt; SEQUENCE: 2#                24caac ctca- &amp;lt;210&amp;gt; SEQ ID NO 3&amp;lt;211&amp;gt; LENGTH: 25&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: PCR Primer- &amp;lt;400&amp;gt; SEQUENCE: 3#               25 gatt tccaa- &amp;lt;210&amp;gt; SEQ ID NO 4&amp;lt;211&amp;gt; LENGTH: 34&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: PCR Probe- &amp;lt;400&amp;gt; SEQUENCE: 4#        34        gatt tagactccaa ggct- &amp;lt;210&amp;gt; SEQ ID NO 5&amp;lt;211&amp;gt; LENGTH: 19&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: PCR Primer- &amp;lt;400&amp;gt; SEQUENCE: 5# 19               gtc- &amp;lt;210&amp;gt; SEQ ID NO 6&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: PCR Primer- &amp;lt;400&amp;gt; SEQUENCE: 6# 20               tttc- &amp;lt;210&amp;gt; SEQ ID NO 7&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: PCR Probe- &amp;lt;400&amp;gt; SEQUENCE: 7# 20               agcc- &amp;lt;210&amp;gt; SEQ ID NO 8&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 8# 20               agcc- &amp;lt;210&amp;gt; SEQ ID NO 9&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 9# 20               aaaa- &amp;lt;210&amp;gt; SEQ ID NO 10&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 10# 20               gcag- &amp;lt;210&amp;gt; SEQ ID NO 11&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 11# 20               caac- &amp;lt;210&amp;gt; SEQ ID NO 12&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 12# 20               gatg- &amp;lt;210&amp;gt; SEQ ID NO 13&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 13# 20               ttga- &amp;lt;210&amp;gt; SEQ ID NO 14&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 14# 20               cttt- &amp;lt;210&amp;gt; SEQ ID NO 15&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 15# 20               tcct- &amp;lt;210&amp;gt; SEQ ID NO 16&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 16# 20               tttc- &amp;lt;210&amp;gt; SEQ ID NO 17&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 17# 20               cctt- &amp;lt;210&amp;gt; SEQ ID NO 18&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 18# 20               cccc- &amp;lt;210&amp;gt; SEQ ID NO 19&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 19# 20               ttcc- &amp;lt;210&amp;gt; SEQ ID NO 20&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 20# 20               tttt- &amp;lt;210&amp;gt; SEQ ID NO 21&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 21# 20               catt- &amp;lt;210&amp;gt; SEQ ID NO 22&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 22# 20               gaca- &amp;lt;210&amp;gt; SEQ ID NO 23&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 23# 20               tttc- &amp;lt;210&amp;gt; SEQ ID NO 24&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 24# 20               tagg- &amp;lt;210&amp;gt; SEQ ID NO 25&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 25# 20               cttc- &amp;lt;210&amp;gt; SEQ ID NO 26&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 26# 20               attt- &amp;lt;210&amp;gt; SEQ ID NO 27&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 27# 20               gtga- &amp;lt;210&amp;gt; SEQ ID NO 28&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 28# 20               gtat- &amp;lt;210&amp;gt; SEQ ID NO 29&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 29# 20               gtct- &amp;lt;210&amp;gt; SEQ ID NO 30&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 30# 20               ccaa- &amp;lt;210&amp;gt; SEQ ID NO 31&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 31# 20               tgga- &amp;lt;210&amp;gt; SEQ ID NO 32&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 32# 20               tcat- &amp;lt;210&amp;gt; SEQ ID NO 33&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 33# 20               agtt- &amp;lt;210&amp;gt; SEQ ID NO 34&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 34# 20               acca- &amp;lt;210&amp;gt; SEQ ID NO 35&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 35# 20               atag- &amp;lt;210&amp;gt; SEQ ID NO 36&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 36# 20               aatt- &amp;lt;210&amp;gt; SEQ ID NO 37&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 37# 20               tttc- &amp;lt;210&amp;gt; SEQ ID NO 38&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 38# 20               gatc- &amp;lt;210&amp;gt; SEQ ID NO 39&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 39# 20               caaa- &amp;lt;210&amp;gt; SEQ ID NO 40&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 40# 20               acac- &amp;lt;210&amp;gt; SEQ ID NO 41&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 41# 20               acaa- &amp;lt;210&amp;gt; SEQ ID NO 42&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 42# 20               acaa- &amp;lt;210&amp;gt; SEQ ID NO 43&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 43# 20               acta- &amp;lt;210&amp;gt; SEQ ID NO 44&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 44# 20               tgcg- &amp;lt;210&amp;gt; SEQ ID NO 45&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 45# 20               ctgt- &amp;lt;210&amp;gt; SEQ ID NO 46&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 46# 20               tact- &amp;lt;210&amp;gt; SEQ ID NO 47&amp;lt;211&amp;gt; LENGTH: 20&amp;lt;212&amp;gt; TYPE: DNA&amp;lt;213&amp;gt; ORGANISM: Artificial Sequence&amp;lt;220&amp;gt; FEATURE:&amp;lt;223&amp;gt; OTHER INFORMATION: Antisense Oligonucleotide- &amp;lt;400&amp;gt; SEQUENCE: 47# 20               aagc__________________________________________________________________________
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS5736381 *Sep 19, 1995Apr 7, 1998Davis; Roger J.Cytokine-, stress-, and oncoprotein-activated human protein kinase kinasesUS5801154 *Apr 8, 1997Sep 1, 1998Isis Pharmaceuticals, Inc.Antisense oligonucleotide modulation of multidrug resistance-associated proteinWO1996036642A1 *Jan 26, 1996Nov 21, 1996Roger J DavisCytokine-, stress-, and oncoprotein-activated human protein kinase kinasesWO1997022704A1 *Dec 20, 1996Jun 26, 1997Signal Pharm IncMitogen-activated protein kinase kinase mek6 and methods of use thereforWO1998054203A1 *May 28, 1998Dec 3, 1998Daniel A MercolaInhibition of stress activated protein kinase (sapk) pathway and sensitization of cells to cancer therapies* Cited by examinerNon-Patent CitationsReference1 *Badger et al., Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function, J. Pharmacol. Exp. Ther., 1996, 279:1453 1461.2Badger et al., Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function, J. Pharmacol. Exp. Ther., 1996, 279:1453-1461.3 *Cohen et al., The Critical Role of p38 MAP Kinase in T Cell HIV 1 Replication, Molecular Medicine, 1997, 3:339 346.4Cohen et al., The Critical Role of p38 MAP Kinase in T Cell HIV-1 Replication, Molecular Medicine, 1997, 3:339-346.5Crooke, Stanley T. and Lebleu, Bernard "Antisense Research & Applications" (1993) p 272-285.6 *Crooke, Stanley T. and Lebleu, Bernard Antisense Research & Applications (1993) p 272 285.7 *Cuenda et al., Activation of stress activated protein kinase 3 (SAPK3) by cytokines and cellular stresses is mediated via SAPKK3 (MKK6); comparison of the specificities of SAPK3 and SAPK2 (RK/p38), Embo J., 1997, 16:295 305.8Cuenda et al., Activation of stress-activated protein kinase-3 (SAPK3) by cytokines and cellular stresses is mediated via SAPKK3 (MKK6); comparison of the specificities of SAPK3 and SAPK2 (RK/p38), Embo J., 1997, 16:295-305.9 *Enslen et al., Selective activation of p38 mitogen activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6, J. Biol. Chem., 1998, 273:1741 1748.10Enslen et al., Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6, J. Biol. Chem., 1998, 273:1741-1748.11 *Goebeler et al., The MKK6/p38 stress kinase cascade is critical for tumor necrosis factor alpha induced expression of monocyte chemoattractant protein 1 in endothelial cells, Blood, 1999, 93:857 865.12Goebeler et al., The MKK6/p38 stress kinase cascade is critical for tumor necrosis factor-alpha-induced expression of monocyte-chemoattractant protein-1 in endothelial cells, Blood, 1999, 93:857-865.13 *Goedert et al., Activation of the novel stress activated protein kinase SAPK4 by cytokines and cellular stresses is mediated by SKK3 (MKK6); comparison of its substrate specificity with that of other SAP kinases, Embo J., 1997, 16:3563 3571.14Goedert et al., Activation of the novel stress-activated protein kinase SAPK4 by cytokines and cellular stresses is mediated by SKK3 (MKK6); comparison of its substrate specificity with that of other SAP kinases, Embo J., 1997, 16:3563-3571.15 *Han et al., Characterization of the structure and function of a novel MAP kinase kinase (MKK6), J. Biol. Chem., 1996, 271:2886 2891.16Han et al., Characterization of the structure and function of a novel MAP kinase kinase (MKK6), J. Biol. Chem., 1996, 271:2886-2891.17 *Jackson et al., Pharmacological effects of SB 220025, a selective inhibitor of P38 mitogen activated protein kinase, in angiogenesis and chronic inflammatory disease models, J. Pharmacol. Exp. Ther., 1998, 284:687 692.18Jackson et al., Pharmacological effects of SB 220025, a selective inhibitor of P38 mitogen-activated protein kinase, in angiogenesis and chronic inflammatory disease models, J. Pharmacol. Exp. Ther., 1998, 284:687-692.19James, W., "Towards Gere Inhibition Therapy: A review of progress & prospects in the field of antiviral antisense nucleic acids & ribozymes" Antiviral Chemistry & Chemotherapy (1991) 2(4), 191-214.20 *James, W., Towards Gere Inhibition Therapy: A review of progress & prospects in the field of antiviral antisense nucleic acids & ribozymes Antiviral Chemistry & Chemotherapy (1991) 2(4), 191 214.21 *Mackay et al., An inhibitor of p38 mitogen activated protein kinase protects neonatal cardiac myocytes from ischemia, J. Biol. Chem., 1999, 274:6272 6279.22Mackay et al., An inhibitor of p38 mitogen-activated protein kinase protects neonatal cardiac myocytes from ischemia, J. Biol. Chem., 1999, 274:6272-6279.23Milner, Natalic; Mir, Kalim U; Southern, Edwin "Selecting effective antisense reagents on combinatorial oligonucleotide arrays" (1997) Nature Biotechnology vol. 15, p 537-541.24 *Milner, Natalic; Mir, Kalim U; Southern, Edwin Selecting effective antisense reagents on combinatorial oligonucleotide arrays (1997) Nature Biotechnology vol. 15, p 537 541.25 *Nagata et al., Activation of p38 MAP Kinase and JNK but not ERK is Required for Erythropoietin Induced Erythroid Diferentiation, Blood, 1998, 92:1859 1869.26Nagata et al., Activation of p38 MAP Kinase and JNK but not ERK is Required for Erythropoietin-Induced Erythroid Diferentiation, Blood, 1998, 92:1859-1869.27 *Paul et al., Stress activated protein kinases: activation, regulation and function, Cell Signal., 1997, 9:403 410.28Paul et al., Stress-activated protein kinases: activation, regulation and function, Cell Signal., 1997, 9:403-410.29 *Raingeaud et al., MKK3 and MKK6 regulated gene expression is mediated by the p38 mitogen activated protein kinase signal transduction pathway, Mol. Cell. Biol., 1996, 16:1247 1255.30Raingeaud et al., MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen- activated protein kinase signal transduction pathway, Mol. Cell. Biol., 1996, 16:1247-1255.31 *Suzuki et al., Cytokine specific activation of distinct mitogen activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, and tumor necrosis factor alpha, Blood, 1999, 93:341 349.32Suzuki et al., Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha, Blood, 1999, 93:341-349.33 *Zechner et al., MKK6 activates myocardial cell NF kappaB and inhibits apoptosis in a p38 mitogen activated protein kinase dependent manner, J. Biol. Chem., 1988, 273 8232 8239.34Zechner et al., MKK6 activates myocardial cell NF-kappaB and inhibits apoptosis in a p38 mitogen-activated protein kinase-dependent manner, J. Biol. Chem., 1988, 273-8232-8239.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS7629321Oct 25, 2002Dec 8, 2009Isis Pharmaceuticals, Inc.Oligoribonucleotides and ribonucleases for cleaving RNAUS7790691Jun 3, 2004Sep 7, 2010Isis Pharmaceuticals, Inc.Double stranded compositions comprising a 3′-endo modified strand for use in gene modulationUS7888324Aug 1, 2001Feb 15, 2011Genzyme CorporationAntisense modulation of apolipoprotein B expressionUS7919612Nov 4, 2003Apr 5, 2011Isis Pharmaceuticals, Inc.2′-substituted oligomeric compounds and compositions for use in gene modulationsUS8029815Apr 27, 2005Oct 4, 2011Elford Howard LMethods for treating or preventing restenosis and other vascular proliferative disordersUS8445638Sep 2, 2009May 21, 2013Senesco Technologies, Inc.Use of a truncated eIF-5A1 polynucleotide to induce apoptosis in cancer cellsEP2116604A1Aug 5, 2003Nov 11, 2009University of RochesterProtein transducing domain/deaminase chimeric proteins, related compounds, and uses thereofEP2311852A1Feb 5, 2003Apr 20, 2011Stasys Technologies, Inc.Anti-infarction moleculesEP2363405A1Feb 5, 2003Sep 7, 2011Stasys Technologies, Inc.Anti-infarction moleculesWO2003018601A1 *Aug 21, 2002Mar 6, 2003Isis Pharmaceuticals IncUse of antisense oligonucleotide libraries for identifying gene functionWO2004037986A2 *Oct 22, 2003May 6, 2004Exelixis IncMap2k6 as modifier of branching morphogenensis and methods of useWO2008136852A2Nov 1, 2007Nov 13, 2008Univ RochesterMethods and compositions related to the structure and function of apobec3gWO2010065617A1Dec 2, 2009Jun 10, 2010University Of Utah Research FoundationPde1 as a target therapeutic in heart diseaseWO2011031974A1Sep 10, 2010Mar 17, 2011Southern Research InstituteAcridine analogs in the treatment of gliomasWO2011103224A1Feb 17, 2011Aug 25, 2011Corning IncorporatedMethods related to casein kinase ii (ck2) inhibitors and the use of purinosome-disrupting ck2 inhibitors for anti-cancer therapy agentsWO2011154453A1Jun 8, 2011Dec 15, 2011Genmab A/SAntibodies against human cd38* Cited by examinerClassifications U.S. Classification435/375, 435/325, 435/91.1, 536/24.5, 435/366, 536/23.1, 536/24.33, 536/24.31, 435/6.14International ClassificationC12N15/54, A61P29/00, A61P9/00, C12N15/09, A61K31/7125, A61K31/7088, A61P37/00, A61K31/7115, A61K31/712, A61P19/02, A61K48/00, C12N15/113Cooperative ClassificationC12N2310/315, C12Y207/12002, C12N2310/346, C12N15/1137, C12N2310/321, C12N2310/334, C12N2310/341European ClassificationC12Y207/12002, C12N15/113DLegal EventsDateCodeEventDescriptionAug 24, 2011FPAYFee paymentYear of fee payment: 12Aug 20, 2007FPAYFee paymentYear of fee payment: 8Aug 28, 2003FPAYFee paymentYear of fee payment: 4Feb 13, 2001CCCertificate of correctionJul 19, 1999ASAssignmentOwner name: ISIS PHARMACEUTICALS, INC., CALIFORNIAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MONIA, BRETT P.;COWSERT, LEX M.;REEL/FRAME:010117/0916Effective date: 19990713RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google