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Nf-kappab oligonucleotide decoy moleculesRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, , Nitrogen Containing Hetero Ring, Polynucleotide (e.g., Rna, Dna, Etc.)Nf-kappab oligonucleotide decoy molecules description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070010474, Nf-kappab oligonucleotide decoy molecules. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation application of copending U.S. application Ser. No. 11/368,060 filed on Mar. 3, 2006, which is a continuation-in-part of U.S. application Ser. No. 11/004,512 filed on Dec. 2, 2004, which claims priority under 35 U.S.C. .sctn. 119(e)(1) of U.S. provisional patent application Ser. No. 60/526,623, filed on Dec. 2, 2003, and U.S. Provisional patent application Ser. No. 60/612,029, filed on Sep. 21, 2004, the entire disclosures of which are hereby expressly incorporated by reference. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention concerns NF-.kappa.B oligonucleotide decoy molecules, and their use in the treatment of various NF-.kappa.B related diseases and pathological conditions. [0004] 2. Description of the Related Art [0005] NF-.kappa.B is a family of inducible dimeric transcription factors composed of members of the Rel family of DNA-binding proteins that recognize a common sequence motif. In its active DNA-binding form, NF-.kappa.B is a heterogeneous collection of dimers, composed of various combinations of members of the NF-.kappa.B/Rel family. At present, this family is composed of 5 members, termed p52, p50, p65, cRel and Rel B. The homology between the members of the Rel family is through the Rel homology domain, which is about 300 amino acids in size and constitutes the DNA-binding domain of these proteins. [0006] Different NF-.kappa.B dimers exhibit different binding affinities for NF-.kappa.B sites bearing the consensus sequence GGGRNNYYCC (SEQ ID NO: 1) where R is purine, Y is pyrimidine and N is any base. The Rel proteins differ in their abilities to activate transcription, such that only p65/RelA and c-Rel were found to contain potent transcriptional-activation domains among the mammalian family members. NF-.kappa.B is found in its inactive form in the cytoplasm, where it is bound to the 43-kDa protein IKB that covers the nuclear localization signal region of the p65/p50 dimer. Activation of NF-.kappa.B starts with the proteolytic destruction of IKB followed by the transport of the RelA/p50 complex into the nucleus, where it binds to its recognition site on the DNA and activates transcription of target genes. For further review of the NF.kappa.B family see, for example, Gomez et al., Frontiers in Bioscience 2:49-60 (1997). [0007] p52 and p50 do not contain transactivation domains. Dimers composed solely of p52 and/or p50 proteins that lack transcriptional activation domains are generally not activators of transcription and can mediate transcriptional repression. [0008] The transcription factors of the Rel/NF-.kappa.B family are key regulators of immune and inflammatory responses, and contribute to lymphocyte proliferation, survival and oncogenesis. Thus, NF-.kappa.B plays a key role in the expression of several genes involved in the inflammation, cell proliferation and immune responses. (D'Acquisto et al., Gene Therapy 7: 1731-1737 (2000); Griesenbach et al., Gene Therapy 7, 306-313 (2000); Morishita et al., Gene Therapy 7: 1847-1852 (2000)). Among the genes regulated by NF-.kappa.B are many which play critical roles in various diseases and conditions, such as rheumatoid arthritis, systemic lupus erythematosus, restenosis, myocardial infarction, ischemia reperfusion injury, glomerulonephritis, atopic dermatitis, saphenous vein graft, Alzheimer's disease, to name a few. See, e.g. Khaled et al. Clinical Immunology and Immunopathology 86(2): 170-179 (1998); Morishita et al., Nature Medicine 3(8): 894-899 (1997); Cho-Chung et al., Current Opinion in Molecular Therapeutics 1(3): 386-392 (1999); Nakamura et al., Gene Therapy 9: 1221-1229 (2002); Shintani et al., Ann. Thorac. Surg. 74: 1132-1138 (2002); and Li et al., J. Neurochem. 74(1): 143-150 (2000). [0009] NF-.kappa.B decoys have been proposed for the inhibition of neointimal hyperplasia after angioplasty, restenosis and myocardial infarction (Yoshimura et al., Gene Therapy 8: 1635-1642 (2001); Morishita et al., Nature Medicine 3(8): 894-899 (1997)). The greater inhibition of reperfusion injury, acute, and chronic rejection after transplantation results in a prolongation of allograft survival and decrease in graft coronary artery disease. (Feeley et al., Transplantation 70(11): 1560-1568 (2000)). In vivo transfection of an NF-.kappa.B decoy provides a novel strategy for treatment of acute myocarditis. (Yokoseki et al., supra). Ueno et al., supra reported that blocking NF.kappa.B by NF.kappa.B decoy prevented ischemia reperfusion injury in the heart. [0010] It has been shown (Ziegler-Heitbrock et al, J. Leukoc. Biol. 55(10:73-80 (1994); Kastenbauer and Ziegler-Heitbrock, Infect. Immunol. 67(4):1553-9 (1999)) that when a human monocyte cell line, Mono Mac 6, was pre-treated for two days with low doses of lipopolysaccharide (LPS), the response to subsequent LPS stimulation was strongly reduced. Upon stimulation of these LPS-tolerant cells with LPS, these cells exhibited a predominance of the p50 homodimer as shown by the gel shift assay. The authors then tested the effect of the altered NF-.kappa.B complexes on gene expression via reporter gene analysis. NF-.kappa.B-dependent HIV-1 LTR reporter gene constructs were transfected into Mono Mac 6 cells, followed by pre-culture with and without LPS, and luciferase activity was measured. When LPS-tolerant cells were tested, LPS stimulation did not increase transactivation of the NF-.kappa.B-dependent HIV-1 LTR reporter gene. This indicates that the NF-.kappa.B complexes present in LPS-tolerant cells are functionally inactive. This also was applicable to the transcription of the NF-.kappa.B-controlled TNF gene. Using a TNF promoter-controlled luciferase reporter construct, LPS-tolerant cells showed only a minimal response to LPS stimulation. Therefore, it was concluded that the p50 homodimers induced by LPS tolerance lack transactivation activity. These p50 homodimers instead occupy the cognate NF-.kappa.B-binding sites and prevent transactivation and therefore transcription by the p50/p65 complex. SUMMARY OF THE INVENTION [0011] The present invention concerns double-stranded NF-.kappa.B decoy oligodeoxynucleotide (NF-.kappa.B dsODN) molecules that contain a core sequence capable of specific binding to an NF-.kappa.B transcription factor. In a particular aspect, the invention concerns NF-.kappa.B decoy molecules that preferentially bind p50/p65 and/or cRel/p50 heterodimers over p50/p50 homodimers when p50/p50 homodimers are present. The selective decoy molecules of the invention, by not blocking p50/p50 homodimers, allow these homodimers to block the promoters of NF-.kappa.B regulated genes, which provides an additional level of negative regulation of gene transcription. As a result, the selective NF-.kappa.B decoy molecules are particularly potent inhibitors of NF-.kappa.B activity both in vitro and in vivo. [0012] If p50/p50 homodimers are not present, are present only in small amounts, or do not play a significant role in the disease state, the invention provides NF-.kappa.B decoy molecules with high binding affinity for p65. [0013] In one aspect, the invention concerns double-stranded NF-.kappa.B decoy oligodeoxynucleotide (NF-.kappa.B dsODN) molecules that preferentially bind p50/p65 and/or cRel/p50 heterodimers relative to p50/p50 homodimers. [0014] In another aspect, the invention concerns an NF-.kappa.B double-stranded decoy oligodeoxynucleotide (dsODN) molecule, comprising a sense and an antisense strand, which preferentially binds p50/p65 and/or cRel/p50 heterodimers over p50/p50 homodimers when p50/p50 homodimers are present and/or exhibits a p65 binding affinity of 45 or less, as determined by measuring the molar excess required to compete at least 50% of binding of p65/p50 in an electromobility shift assay to the non-mammalian NF-.kappa.B promoter from HIV (sequence 113/114). [0015] In yet another aspect, the invention concerns The dsODN molecule of claim 1 characterized by a specificity/affinity factor of at least about 20, where the specificity/affinity factor is determined in a competitive binding assay, and is defined as follows:Specificity/affinity factor=(S.sub.p50/p50-S.sub.p65/p50).times.S.sub.p50/p50/S.sub.p65/p50 [0016] where S.sub.p50/p50 equals the molar excess of said dsODN molecule required to compete 50% of the binding of p50/p50 to the non-mammalian NF-.kappa.B promoter from HIV (sequence 113/114) and S.sub.p65/p50 equals the molar excess of said dsODN molecule required to compete 50% of the binding of p65/p50 to the non-mammalian NF-.kappa.B promoter from HIV (sequence 113/114), and wherein the score (S) is assigned as 100 if the decoy is unable to compete at least 50% of the binding at any molar ratio tested. [0017] In another aspect, the present invention concerns NF-.kappa.B dsODN molecules that have a specificity/affinity factor of at least about 25, or at least about 30, or at least about 35, or at least about 40, or at least about 50 or at least about 60, or at least about 70, or at least about 80. In a preferred embodiment, the decoy molecules of the invention additionally show increased binding affinity to the p50/p65 heterodimers and/or have improved stability in vivo. [0018] In a further aspect, the invention concerns a dsODN molecule comprising in its first strand, in 5' to 3' direction, a sequence of the formula FLANK1-CORE-FLANK2, wherein [0019] CORE is selected from the group consisting of GGGATTTCC (SEQ ID NO: 11); GGACTTTCC (SEQ ID NO: 13); GGATTTCC (SEQ ID NO: 19); GGATTTCCC (SEQ ID NO: 21); and GGACTTTCCC (SEQ ID NO: 25); [0020] FLANK1 is selected from the group consisting of AT; TC; CTC; AGTTGA (SEQ ID NO: 79), and TTGA (SEQ ID NO: 80); [0021] FLANK2 is selected from the group consisting of GT; TC; TGT; AGGC (SEQ ID NO: 88); and AG. Continue reading about Nf-kappab oligonucleotide decoy molecules... Full patent description for Nf-kappab oligonucleotide decoy molecules Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Nf-kappab oligonucleotide decoy molecules patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. 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