HIF oligonucleotide decoy molecules
The invention concerns double-stranded HIF decoy oligodeoxynucleotide (dsODN) molecules comprising a core sequence that is capable of specific binding to a HIF transcription factor, compositions containing such molecules, and their use in the treatment of various diseases and pathologic conditions associated with the regulation of gene transcription by a HIF transcription factor.
1 . A HIF double-stranded oligodeoxynucleotide (dsODN) molecule comprising a sense and an antisense strand, in which the sense strand comprises, in 5′ to 3′ direction, a sequence of formula FLANK1-CORE-FLANK2, wherein
CORE is the sequence ACGTG (SEQ ID NO: 126),
FLANK1, in which the nucleotide positions are designated by negative (−) numbers, is at least 6 nucleotides long, and
FLANK 2, in which the nucleotide positions are designated by positive (+) numbers, has a GC content of at least about 50%, and
wherein said dsODN molecule is capable of specific binding to HIF.
2 . The dsODN molecule of claim 1 wherein FLANK2 has a nucleotide other than G at position +1.
3 . The dsODN molecule of claim 1 wherein FLANK2 has the nucleotide A at position +1.
4 . The dsODN molecule of claim 1 wherein FLANK2 has a nucleotide A or G at position +3.
5 . The desODN molecule of claim 1 wherein FLANK2 has any nucleotide at position +2.
6 . The dsODN molecule of claim 1 wherein FLANK1 has a nucleotide other than A at position −1.
7 . The dsODN molecule of claim 1 wherein FLANK1 has a nucleotide T or C at position −1.
8 . The dsODN molecule of claim 1 wherein FLANK1 has a nucleotide other than G at position −3.
9 . The dsODN molecule of claim 1 wherein FLANK1 has the nucleotide T at position −3.
10 . The dsODN molecule of claim 1 wherein FLANK1 has the nucleotide G at position −4.
11 . The dsODN molecule of claim 1 wherein FLANK1 is at least 6 nucleotides long.
12 . The dsODN molecule of claim 1 wherein the FLANK1 is at least 7 nucleotides long.
13 . The dsODN molecule of claim 1 in which the FLANK1-CORE-FLANK2 sequence is at least 14 nucleotides long.
14 . The dsODN molecule of claim 1 in witch the FLANK1-CORE-FLANK2 sequence is at least 16 nucleotides long.
15 . The dsODN molecule of claim 1 in which the FLANK1-CORE-FLANK2 sequence is 14 to 28 nucleotides long.
16 . The dsODN molecule of claim 1 in which the FLANK1-CORE-FLANK2 sequence is 16 to 24 nucleotides long.
17 . The dsODN molecule of claim 1 , in which at least one of the sense and antisense strands has a modified backbone, comprising one or more phosphodiester linkages substituted by another linkage.
18 . The dsODN molecule of claim 14 , comprising one or more phosphodiester linkages substituted by a linkage selected from the group consisting of phosphothioate, phosphodithioate, and phosphoamidate linkages.
19 . The dsODN molecule of claim 1 , wherein FLANK1-CORE-FLANK2 is selected from the sequences listed in Tables 2A and 2B.
20 . The dsODN molecule of claim 19 wherein FLANK1-CORE-FLANK2 is selected from the group of decoy sequence Nos. 893 (SEQ ID NO: 161), 895 (SEQ ID NO: 162), 985 (SEQ ID NO: 207), 987 (SEQ ID NO: 208), 963 (SEQ ID NO: 196), 993 (SEQ ID NO: 211), and 995 (SEQ ID NO: 212).
21 . The dsODN molecule of claim 20 wherein FLANK1-CORE-FLANK2 is decoy sequence No. 895 (SEQ ID NO: 162).
22 . The dsODN molecule of claim 20 wherein FLANK1-CORE-FLANK2 is decoy sequence No. 985 (SEQ ID NO: 207).
23 . The dsODN molecule of claim 1 which is selected from the group of decoy sequence Nos. 893 (SEQ ID NO: 161), 895 (SEQ ID NO: 162), 985 (SEQ ID NO: 207), 987 (SEQ ID NO: 208), 963 (SEQ ID NO: 196), 993 (SEQ ID NO: 211), and 995 (SEQ ID NO: 212).
24 . The dsODN molecule of claim 23 which is decoy sequence No. 895 (SEQ ID NO: 162).
25 . The dsODN molecule of claim 23 which is decoy sequence No. 985 (SEQ ID NO: 207).
26 . A method for modulating the transcription of a gene that is regulated by a HIF transcription factor, comprising introducing into the nucleus of a cell containing said gene a dsODN molecule according to any one of claims 1 - 25 .
27 . The method of claim 26 wherein said HIF transcription factor is HIF-1.
28 . The method of claim 27 which is performed in vivo.
29 . The method of claim 27 which is performed ex vivo.
30 . The method of claim 27 wherein said HIF dsODN molecule is capable of episomal replication in said cell.
31 . The method of claim 27 wherein said HIF dsODN molecule is delivered as a composition.
32 . The method of claim 31 wherein said composition comprises liposomes, and said HIF dsODN is within the lumen of said liposomes.
33 . The method of claim 32 wherein said liposomes comprise lipid and a viral coat protein.
34 . The method of claim 27 wherein said HIF dsODN is introduced into the nucleus of said cell by pressure-mediated transfection.
35 . A method for the prevention or treatment in a mammalian host of a disease or condition associated with HIF-regulated gene transcription, comprising introducing into the cells of said mammal in vivo or ex vivo an effective amount of a double-stranded HIF decoy oligodeoxynucleotide (dsODN) molecule comprising a core sequence that is capable of specific binding to a HIF transcription factor.
36 . The method of claim 35 wherein said HIF transcription factor is HIF-1.
37 . The method of claim 36 wherein said dsODN molecule is any one of the dsODN molecules of claims 1 - 25 .
38 . The method of claim 37 wherein said disease or condition is cancer.
39 . The method of claim 38 wherein said cancer is selected from the group consisting of kidney, pancreatic, colon and lung cancer.
40 . The method of claim 38 further comprising the administration of an additional anti-angiogen.
41 . The method of claim 40 wherein said additional anti-angiogenic agent is selected from the group consisting of anti-EGF agents, anti-VEGF agents, matrix metalloproteinase inhibitors, vascular targeting agents, and integrin antagonists.
42 . The method of claim 40 wherein said additional anti-angiogenic agent is selected from the group consisting of Avastin™ (bevacizumab, Genentech, Inc.); angiostatin; endostatin; Panzem® (2-methoxyestradiol, EntreMed, Inc.); Iressa® (gefitinib, AstraZeneca), and thalidomide.
43 . The method of claim 37 wherein said disease or condition is an inflammatory disease.
44 . The method of claim 37 wherein said disease or condition involves hypoxia in its pathology.
45 . The method of claim 37 wherein said disease or condition is a cardiovascular disease or stroke.
46 . The method of claim 37 wherein said disease or condition is selected from the group consisting of diabetic retinopathy, Age-related Macular Degeneration, and corneal neovascularization.
47 . The method of claim 37 wherein said disease or condition is associated with pathogenic blood vessel growth.
48 . The method of claim 37 wherein said disease or condition is a musculosceletal disorder.
49 . A composition comprising a dsODN molecule according to any one of claims 1 - 25 and a carrier.
50 . The composition of claim 49 wherein said carrier facilitates delivery in the nucleus of a cell.
51 . The composition of claim 49 wherein said composition is a liposome composition.
52 . The composition of claim 51 wherein said dsODN molecule is within the lumen of the liposome.
53 . The method of claim 52 wherein said liposomes comprise lipid and a viral coat protein.