Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-28
2002-04-16
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S325000, C435S375000, C536S024500
Reexamination Certificate
active
06372433
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Inhibitor of DNA binding-1. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Inhibitor of DNA binding-1. Such oligonucleotides have been shown to modulate the expression of Inhibitor of DNA binding-1.
BACKGROUND OF THE INVENTION
Transcription factors represent a group of molecules within the cell that function to connect the pathways from extracellular signals to intracellular responses.
Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to specific DNA sequences in the promoter elements of target genes and activate the transcription of these target genes.
One family of transcription factors are the basic Helix-Loop-Helix (bHLH) proteins. These proteins contain a motif that consists of two segments capable of forming amphipathic alpha helices connected by a nonconserved loop. A basic region is found just to the amino terminal side of this motif and consists of two to three clusters of basic amino acid residues. Various proteins containing the bHLH motif can form homodimeric and heterodimeric complexes with other bHLH proteins and it is through the basic region that these complexes bind to the target DNA (Murre et al.,
Cell
, 1989, 56, 777-783; Murre et al.,
Cell
, 1989, 58, 537-544).
A distinct subfamily of proteins containing the helix-loop-helix domain yet lacking the basic region are still able to form heterodimers with other bHLH transcription factors affecting transcription. These proteins lack DNA-binding ability and are therefore negative regulators of the bHLH transcription factors. Consequently, they have been termed “Id” (inhibitor of DNA binding) proteins.
Four members of the Id family have been identified in mammals and the first, Inhibitor of DNA binding-1 (Id-1), originally isolated in the mouse, has been shown to exist in two forms in the human as a result of alternative splicing (Benezra et al.,
Cell
, 1990, 61, 49-59; Deed et al.,
Biochim. Biophys. Acta
., 1994, 1219, 160-162; Hara et al.,
J. Biol. Chem
., 1994, 269, 2139-2145; Nehlin et al.,
Biochem. Biophys. Res. Commun
., 1997, 231, 628-634; Zhu et al.,
Brain Res. Mol. Brain Res
., 1995, 30, 312-326).
In fibroblasts, Inhibitor of DNA binding-1 is essential for proliferation, repressed by senescence and is regulated during the cell cycle (Desprez et al.,
Mol. Cell. Biol
., 1995, 15, 3398-3404; Hara et al.,
Dev. Genet
., 1996, 18, 161-172; Hara et al.,
J. Biol. Chem
., 1994, 269, 2139-2145). In these studies, one antisense oligonucleotide targeting each of the splice variants (complementary to 18 nucleotides overlapping the translation initiation codon) was used to define the role of Inhibitor of DNA binding-1 in the cell cycle. Treatment of early passage fibroblasts with the oligonucleotides prevented the cells from entering the S-phase of the cell cycle (Hara et al.,
J. Biol. Chem
., 1994, 269, 2139-2145).
Sun et al. established lines of transgenic mice that constitutively expressed the mouse Inhibitor of DNA binding-1 gene in lymphoid cells and detected severe defects in B-cell development as well as the necessity for the downregulation of Inhibitor of DNA binding-1 for B-cell differentiation to proceed (Sun,
Cell
, 1994, 79, 893-900).
In muscle tissue that has been paralyzed or denervated, the expression of Inhibitor of DNA binding-1 is elevated. To test the role of Inhibitor of DNA binding-1 in active muscle, mice overexpressing Inhibitor of DNA binding-1, under the control of regulatory elements which control tissue and fiber-type specific expression in differentiated muscle cells, were created. These mice showed increased muscle atrophy compared to the wild-type littermates. These studies suggest that Inhibitor of DNA binding-1 is involved in muscle fiber size during disuse and/or myogenesis (Gundersen and Merlie,
Proc. Natl. Acad. Sci. U. S. A
., 1994, 91, 3647-3651).
More recently, Lyden et al. have created double knockout mice lacking both Inhibitor of DNA binding-1 and Inhibitor of DNA binding-3 for use in tumor xenograft studies. These mice displayed premature withdrawal of neuroblasts from the cell cycle and expression of neural-specific differentiation markers as well as vascular malformations in the forebrain and an absence of branching and sprouting of blood vessels into the neuroectoderm. When examined for tumor growth in these mice, the tumors showed poor vascularization and extensive necrosis indicating a prominent role for the Id proteins in neurogenesis, angiogenesis and vascularization (Lyden et al.,
Nature
, 1999, 401, 670-677).
Ectopic expression of the Inhibitor of DNA binding-1 protein results in immortalization and activation of telomerase activity in keratinocytes (Alani et al.,
Proc. Natl. Acad. Sci. U. S. A
., 1999, 96, 9637-9641). Further disclosed in the PCT publication, WO 99/35244, are methods for the immortalization of cells including keratinocytes by modulating the expression of Inhibitor of DNA binding-1 (Alani et al., 1999).
Inhibitor of DNA binding-1 is also overexpressed in pancreatic cancers and in dysplastic lesions in chronic pancreatitis (Maruyama et al.,
Am. J. Pathol
., 1999, 155, 815-822).
The pharmacological modulation of Inhibitor of DNA binding-1 activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions such as cancer and disorders involving the control of angiogenesis or vascularization.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Inhibitor of DNA binding-1 and to date, investigative strategies aimed at modulating Inhibitor of DNA binding-1 function have involved the use of antibodies, antisense expression vectors, antisense oligonucleotides, and transgenic mice.
These strategies are untested as therapeutic protocols, and consequently, there remains a long felt need for additional agents capable of effectively inhibiting Inhibitor of DNA binding-1 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 Inhibitor of DNA binding-1 expression.
The present invention provides compositions and methods for modulating Inhibitor of DNA binding-1 expression, including modulation of expression of the alternative splice variant of Inhibitor of DNA binding-1.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding Inhibitor of DNA binding-1, and which modulate the expression of Inhibitor of DNA binding-1. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of Inhibitor of DNA binding-1 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 Inhibitor of DNA binding-1 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 Inhibitor of DNA binding-1, ultimately modulating the amount of Inhibitor of DNA binding-1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Inhibitor of DNA binding-1. As used herein, the terms “target
Baker Brenda F.
Bennett C. Frank
Wyatt Jacqueline
ISIS Pharmaceuticals Inc.
Licata & Tyrrell P.C.
Wang Andrew
LandOfFree
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