US2007078102A1PendingUtilityA1

NF-kB oligonucleotide decoy molecules

54
Individually held — no corporate assignee on recordPriority: Dec 2, 2003Filed: Mar 3, 2006Published: Apr 5, 2007
Est. expiryDec 2, 2023(expired)· nominal 20-yr term from priority
Inventors:Leslie McevoyChristi L. ParhamJie ZhangRolf Ehrhardt
A61P 43/00A61P 37/08A61P 9/00A61P 9/04A61P 7/08A61P 37/02A61P 9/10A61P 9/02A61P 3/10A61P 37/06A61P 9/12A61P 25/00A61P 27/02A61P 31/04A61P 25/28A61P 29/00A61P 35/00A61P 25/02A61P 13/12A61P 1/16C12N 2310/345C12N 15/113C12N 15/115A61P 17/02C12N 2310/315A61P 19/02A61P 21/00A61P 1/04A61P 17/00A61P 11/06A61P 1/00A61P 19/08A61P 11/00A61P 17/12A61P 19/06A61P 11/08C12N 2310/53A61P 17/04A61P 17/06C12N 2310/13
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Claims

Abstract

The present invention concerns double-stranded NF-κB decoy oligodeoxynucleotide (NF-κB dsODN) molecules that contain a core sequence capable of specific binding to an NF-κB transcription factor. In a particular aspect, the invention concerns NF-κB decoy molecules that preferentially bind p50/p65 and/or cRel/p50 heterodimers over p50/p50 homodimers. In another aspect, the invention concerns NF-κB decoy molecules with improved binding affinity to p65.

Claims

exact text as granted — not AI-modified
1 . An NF-κB 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-κB promoter from HIV (sequence 113/114; SEQ ID NO: 48).  
     
     
         2 . 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   p50/p50   −S   p65/p50 )− S   p50/p50   /S   p65/p50    where S 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-κB promoter from HIV (sequence 113/114) and S 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-κB 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.    
     
     
         3 . The dsODN molecule of  claim 2  wherein said specificity/affinity factor is at least about 25.  
     
     
         4 . The dsODN molecule of  claim 2  wherein said specificity/affinity factor is at least about 30.  
     
     
         5 . The dsODN molecule of  claim 2  wherein said specificity/affinity factor is at least about 35.  
     
     
         6 . The dsODN molecule of  claim 2  wherein said specificity/affinity factor is at least about 40.  
     
     
         7 . The dsODN molecule of  claim 2  which has a fully phosphorothioate backbone.  
     
     
         8 . The dsODN molecule of  claim 2  which has a hybrid backbone.  
     
     
         9 . The dsODN molecule of  claim 2  in which said sense and antisense strands are connected solely by Watson-Crick base pairing.  
     
     
         10 . The dsODN molecule of  claim 2  in which said sense and antisense strands are connected, completely or partially, by cross-links other than Watson-Crick base pairing.  
     
     
         11 . The dsODN molecule of  claim 10  in which the sense and antisense strands are covalently linked to each other at their 3′ and/or 5′ end.  
     
     
         12 . The dsODN molecule of  claim 2 , comprising in its sense strand, in 5′ to 3′ direction, a sequence of the formula FLANK1-CORE-FLANK2, wherein 
 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);    FLANK1 is selected from the group consisting of AT; TC; CTC; AGTTGA (SEQ ID NO:79), and TTGA (SEQ ID NO: 80);    FLANK2 is selected from the group consisting of GT; TC; TGT; AGGC (SEQ ID NO: 88); and AG.    
     
     
         13 . The dsODN molecule of  claim 12  wherein 
 CORE is selected from the group consisting of GGGATTTCC (SEQ ID NO: 11); GGACTTTCC (SEQ ID NO: 13); and GGATTTCC (SEQ ID NO: 19);    FLANK1 is AT and FLANK2 is GT; or FLANK1 is TC and FLANK2 is TC: or FLANK1 is CTC and FLANK2 is TGT; or FLANK1 is AGTTGA (SEQ ID NO:79) and FLANK 2 is AGGC (SEQ ID NO: 88); or FLANK1 is TTGA and FLANK2 is AG.    
     
     
         14 . The dsODN molecule of  claim 12  wherein CORE is GGGATTTCC (SEQ ID NO: 11); or GGACTTTCC (SEQ ID NO: 13), FLANK1 is AGTTGA (SEQ ID NO: 79) and FLANK 2 is AGGC (SEQ ID NO: 88).  
     
     
         15 . The dsODN molecule of  claim 14  wherein CORE is GGACTTTCC (SEQ ID NO: 13), FLANK1 is AGTTGA (SEQ ID NO: 79) and FLANK 2 is AGGC (SEQ ID NO: 88).  
     
     
         16 . The dsODN molecule of  claim 14 , which has a specificity/affinity factor of at least about 40.  
     
     
         17 . The dsODN molecule of  claim 14  wherein said antisense strand is at least partially complementary to said sense strand.  
     
     
         18 . The dsODN molecule of  claim 14  wherein said antisense strand fully complementary to said sense strand.  
     
     
         19 . The dsODN molecule of  claim 14  having a phosphodiesterate backbone.  
     
     
         20 . The dsODN molecule of  claim 14  having a phosphorothioate backbone.  
     
     
         21 . The dsODN molecule of  claim 14  having a mixed phosphodiesterate-phosphorothioate backbone.  
     
     
         22 . The dsODN molecule of  claim 14  in which said sense and antisense strands are connected to each other solely by Watson-Crick base pairing.  
     
     
         23 . The dsODN molecule of  claim 1  comprising a sequence, in 5′ to 3′ direction, selected from the group consisting of SEQ ID NOs 26 through 77 and 10.  
     
     
         24 . The dsODN molecule of  claim 23  comprising a strand consisting of, in 5′ to 3′ direction, a sequence selected from the group consisting of SEQ ID NOs 26 through 77 and 10.  
     
     
         25 . The dsODN molecule of  claim 1  comprising a sequence, in 5′ to 3′ direction, selected from the group consisting of SEQ ID NOs: 26 through 34.  
     
     
         26 . The dsODN molecule of  claim 25  comprising a strand consisting of, in 5′ to 3′ direction, a sequence of SEQ ID NOs: 26 through 34.  
     
     
         27 . The dsODN molecule of  claim 1  comprising a sequence, in 5′ to 3′ direction, selected from the group consisting of SEQ ID NOs: 26 through 31.  
     
     
         28 . The dsODN molecule of  claim 27  comprising a strand consisting of, in 5′ to 3′ direction, a sequence selected from the group consisting of SEQ ID NOs: 26 through 31.  
     
     
         29 . The dsODN molecule of  claim 1  comprising the sequence of SEQ ID NO: 30.  
     
     
         30 . The dsODN molecule of  claim 29  comprising a strand consisting of, in 5′ to 3′ direction, the sequence of SEQ ID NO: 30.  
     
     
         31 . The dsODN molecule of  claim 2  wherein the antisense strand comprises a sequence at least partially complementary to said FLANK1-CORE-FLANK2 sequence within the sense strand.  
     
     
         32 . The dsODN molecule of  claim 2  wherein the antisense strand comprises a sequence fully complementary to said FLANK1-CORE-FLANK2 sequence within the sense strand.  
     
     
         33 . The dsODN molecule of  claim 31  further comprising at least one single-stranded overhang.  
     
     
         34 . The dsODN molecule of  claim 31  wherein the two strands are linked at the 5′ and/or 3′ end by a covalent bond, other than a peptide bond.  
     
     
         35 . The dsODN molecule of  claim 31  which is 12 to 28 base pairs long.  
     
     
         36 . The dsODN molecule of  claim 31  which is 14 to 24 base pairs long.  
     
     
         37 . The dsODN molecule of  claim 31  which is 14 to 22 base pairs long.  
     
     
         38 . The dsODN molecule of  claim 31  comprising modified or unusual nucleotides.  
     
     
         39 . The dsODN molecule of  claim 31  having a phosphodiester backbone.  
     
     
         40 . The dsODN molecule of  claim 31  having a phosphorothioate backbone.  
     
     
         41 . The dsODN molecule of  claim 31  having a mixed phosphodiester-phosphorothioate backbone.  
     
     
         42 . The dsODN molecule of  claim 1  which exhibits a p65 binding affinity of 45 or less.  
     
     
         43 . The dsODN molecule of  claim 42  comprising in its sense strand, in 5′ to 3′ direction, a sequence of the formula FLANK1-CORE-FLANK2, wherein 
 CORE is selected from the group consisting of GGGGACTTTCCC (SEQ ID NO: 9); GGGACTTTCC (SEQ ID NO: 5); GGACTTTCCC (SEQ ID NO: 25); GGGATTTCC (SEQ ID NO: 11); and GGACTTTCC (SEQ ID NO: 13);    FLANK1 is selected from the group consisting of  AGTTGA  (SEQ ID NO: 79); CTC; TC; CT; CCTTGAA (SEQ ID NO: 6); and CT; and    FLANK 2 is selected from the group consisting of AGGC (SEQ ID NO: 88); TGT; TC; AGG; TCC; and TCA.    
     
     
         44 . The dsODN molecule of  claim 43  which has a fully phosphorothioate backbone.  
     
     
         45 . The dsODN molecule of  claim 43  which has a hybrid backbone.  
     
     
         46 . The dsODN molecule of  claim 43  in which said sense and antisense strands are connected solely by Watson-Crick base pairing.  
     
     
         47 . The dsODN molecule of  claim 43  in which said sense and antisense strands are connected, completely or partially, by cross-links other than Watson-Crick base pairing.  
     
     
         48 . The dsODN molecule of  claim 43  in which the sense and antisense strands are covalently linked to each other at their 3′ and/or 5′ end.  
     
     
         49 . The dsODN molecule of  claim 1  which comprises a sense strand of AGTTGAGGACTTTCCAGGC (SEQ ID NO: 30) and its complement.  
     
     
         50 . The dsODN molecule of  claim 1  which consists of a sense strand of AGTTGAGGACTTTCCAGGC (SEQ ID NO: 30) and its complement.  
     
     
         51 . The dsODN molecule of  claim 50  which has a fully phosphorothioate backbone.  
     
     
         52 . The dsODN molecule of  claim 50  has a hybrid backbone.  
     
     
         53 . The dsODN molecule of  claim 52  wherein in said backbone, the three most 3′ linkages are phosphorothioate bonds, and the rest of the linkages are phosphodiester bonds.  
     
     
         54 . A composition comprising an NF-κB double-stranded oligodeoxynucleotide (dsODN) molecule of  claim 1 .  
     
     
         55 . The composition of  claim 54  which is a pharmaceutical composition comprising said dsODN molecule in combination with a pharmaceutically acceptable carrier.  
     
     
         56 . A method for the treatment of an inflammatory, immune or autoimmune disease, comprising administering to a mammalian subject in need an effective amount of an NF-κB double-stranded decoy oligodeoxynucleotide (dsODN) molecule of  claim 1 .  
     
     
         57 . The method of  claim 56  wherein said mammalian subject is human.  
     
     
         58 . The method of  claim 57  wherein said inflammatory, immune or autoimmune disease is selected from the group consisting of psoriasis, eczema, atopic dermatitis; systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; surgical tissue reperfusion injury; myocardial infarction; cardiac arrest; reperfusion after cardiac surgery; constriction after percutaneous transluminal coronary angioplasty; stroke; abdominal aortic aneurysms; cerebral edema secondary to stroke; cranial trauma, hypovolemic shock; asthma; autoimmune diabetes; asphyxia; adult respiratory distress syndrome; acute-lung injury; Behcet's Disease; dermatomyositis; polymyositis; multiple sclerosis (MS); meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis; systhemic lupus erythrematosus; rheumatoid arthritis (RA), rheumatoid spondylitis; gouty arthritis; Sjorgen's syndrome, vasculitis; diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory disorder, Alzheimer's disease; multiple organ injury syndrome secondary to septicaemia or trauma; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex mediated diseases, including glomerulonephritis; sepsis; sarcoidosis; immunopathologic responses to tissue/organ transplantation; inflammations of the lung, including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), chronic pulmonary inflammatory disease; cystic fibrosis; psoriasis; pyresis; and ocular allergy.  
     
     
         59 . The method of  claim 57  wherein said inflammatory or autoimmune disease is selected from the group consisting of psoriasis, eczema, atopic dermatitis, rheumatoid arthritis (RA), rheumatoid spondylitis, gouty arthritis; autoimmune diabetes, multiple sclerosis (MS), asthma, systhemic lupus erythrematosus, adult respiratory distress syndrome, Behcet's disease, psoriasis, chronic pulmonary inflammatory disease, graft versus host reaction, Crohn's Disease, ulcerative colitis, inflammatory bowel disease (IBD), and pyresis.  
     
     
         60 . The method of  claim 57  wherein said dsODN molecule is administered by pressure mediated transfection.  
     
     
         61 . The method of  claim 57  wherein said dsODN molecule is administered by retroviral transfection.  
     
     
         62 . The method of  claim 57  wherein said dsODN molecule is administered in liposomes.  
     
     
         63 . The method of  claim 57  wherein said dsODN molecule is administered as a topical formulation.  
     
     
         64 . A method for the treatment of cancer, comprising administering to a mammalian subject in need an effective amount of an NF-κB double-stranded decoy oligodeoxynucleotide (dsODN) molecule of  claim 1 .  
     
     
         65 . The method of  claim 64  wherein said subject is human.  
     
     
         66 . A method for the treatment of reperfusion injury or restenosis, comprising administering to a mammalian subject in need an effective amount of an NF-κB double-stranded decoy oligodeoxynucleotide (dsODN) molecule of  claim 1 .  
     
     
         67 . The method of  claim 66  wherein said subject is human.

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