Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2001-09-28
2004-05-11
Lacourciere, Karen A. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C435S091100, C435S325000, C536S024500, C536S024300, C536S024310
Reexamination Certificate
active
06734017
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of vascular endothelial growth factor receptor-2. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding vascular endothelial growth factor receptor-2. Such compounds have been shown to modulate the expression of vascular endothelial growth factor receptor-2.
BACKGROUND OF THE INVENTION
As a mitogen that acts primarily on endothelial cells, vascular endothelial growth factor (VEGF, or VEGF-A) is essential for endothelial cell differentiation (vasculogenesis) and for the sprouting of new capillaries from pre-existing vessels (angiogenesis) during embryonic development and wound repair. Signaling by VEGF affects a number of biological functions, including endothelial cell survival via inhibition of apoptosis, cell proliferation, vascular permeability, monocyte activation, chemotaxis, and cell migration. Thus, VEGF is believed to play a key role in wound healing, postnatal angiogenesis during pregnancy, and in human pathophysiological conditions such as cancer, rheumatoid arthritis, ocular neovascular disorders, and cardiovascular disease (Zachary and Gliki,
Cardiovasc. Res.,
2001, 49, 568-581).
For transmission of the VEGF signal, VEGF binds to three receptor protein tyrosine kinases, vascular endothelial growth factor receptors-1, -2, and -3, that are structurally related to the PDGF family of class III receptors, characterized by cytoplasmic regions with an insert sequence within the catalytic domain, a single transmembrane domain, and seven immunoglobulin-like extracellular domains. Monomeric vascular endothelial growth factor receptors have 100-fold less affinity for VEGF, and thus, ligands preferentially bind to predimerized receptors. Upon ligand binding, the receptors auto- or trans-phosphorylate specific cytoplasmic tyrosine residues to initiate an intracellular cascade of signaling that ultimately reaches nuclear transcription factor effectors (Zachary and Gliki,
Cardiovasc. Res.,
2001, 49, 568-581).
Most biological functions of VEGF are mediated through vascular endothelial growth factor receptor-2, and the role of vascular endothelial growth factor receptor-1 is currently less well understood (Zachary and Gliki,
Cardiovasc. Res.,
2001, 49, 568-581).
Human vascular endothelial growth factor receptor-2 (also known as VEGF receptor-2, VEGFR2, kinase insert domain receptor, KDR, tyrosine kinase growth factor receptor, and FLK1) binds VEGF, albeit with weaker affinity than does vascular endothelial growth factor receptor-1, and specifically binds VEGF-E, VEGF-C and VEGF-D (alternative splice forms of VEGF), but does not bind the closely related placenta growth factor (PlGF) (Shibuya,
Int. J. Biochem. Cell Biol.,
2001, 33, 409-420; Zachary and Gliki,
Cardiovasc. Res.,
2001, 49, 568-581).
The vascular endothelial growth factor receptor-2 gene was cloned using a PCR approach in which degenerate primers were designed based on the conserved kinase domains flanking the insert domain (characteristic of type III receptor tyrosine kinases). A PCR product was then amplified from a human endothelial cell cDNA library and the product was used as a probe to isolate two overlapping clones (together comprising the entire gene) from that cDNA library (Terman et al.,
Oncogene,
1991, 6, 1677-1683). The vascular endothelial growth factor receptor-2 gene was mapped to the 4q12 human chromosomal locus in a cluster of protein tyrosine kinases including two other type III receptor genes, KIT (mast/stem cell growth factor receptor) and PDGFRA (platelet derived growth factor receptor, A-type) (Spritz et al.,
Genomics,
1994, 22, 431-436).
Disclosed and claimed in U.S. Pat. No. 6,204,011 is an isolated nucleic acid molecule encoding human vascular endothelial growth factor receptor-2 and purified forms of the encoded protein, as well as recombinant expression vectors, a process for the expression of the vascular endothelial growth factor receptor-2 protein, and host cells which express the protein (Kendall et al., 2001).
A degenerate PCR approach was also used to amplify sequences from the kinase domains of the mouse vascular endothelial growth factor receptor-2 gene, using cDNAs synthesized from total RNA from mouse cell populations enriched for hematopoietic stem and progenitor cells. These PCR products were then used to clone the cDNA encoding vascular endothelial growth factor receptor-2 from a library constructed from day 12.5 whole mouse embryo, and the gene was mapped to mouse chromosome 5 (Matthews et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1991, 88, 9026-9030).
Vascular endothelial growth factor receptor-1 may act as a negative regulator of vascular endothelial growth factor receptor-2. Differential splicing of the vascular endothelial growth factor receptor-1 transcript results in a full-length receptor and a naturally occurring, soluble form of the extracellular domain of vascular endothelial growth factor receptor-1 (sVEGFR-1 or sFLT-1). This sFLT-1 isoform can form heterodimers with vascular endothelial growth factor receptor-2 (Kendall et al.,
Biochem. Biophys. Res. Commun.,
1996, 226, 324-328), and when overexpressed, sFLT-1 but not an artificial, soluble vascular endothelial growth factor receptor-2, can act as a receptor antagonist and inhibit VEGF-induced cell proliferation and migration of human microvascular endothelial cells and human umbilical vein endothelial cells (HUVECs) by forming and inactive complex with VEGF and with full length vascular endothelial growth factor receptor-2 (Roeckl et al.,
Exp. Cell Res.,
1998, 241, 161-170; Zachary and Gliki,
Cardiovasc. Res.,
2001, 49, 568-581).
Like other receptor protein kinases, vascular endothelial growth factor receptor-2 associates with a number of src homology 2 (SH2) domain-containing proteins, including the adapter proteins Grb2, Nck, and Shc, as well as the protein tyrosine phosphatases, SHP-1 and SHP-2, involved in a multitude of signal transduction pathways (Zachary and Gliki,
Cardiovasc. Res.,
2001, 49, 568-581). It has also been suggested that vascular endothelial growth factor receptor-2 may be unique among receptor tyrosine kinases in potentially activating the MAPK/ERK kinase cascade via a Ras-independent mechanism mediated by protein kinase C (Wu et al.,
J. Biol. Chem.,
2000, 275, 5096-5103).
Expression of VEGF receptor-2 was once believed to be restricted to proliferating endothelial cells (Millauer et al.,
Nature,
1994, 367, 576-579; Millauer et al.,
Cell,
1993, 72, 835-846; Shalaby et al.,
Nature,
1995, 376, 62-66), but expression of both VEGF receptor-1 and VEGF receptor-2 has been demonstrated more recently in atherosclerotic lesions and in several non-endothelial tumor cell types (Epstein et al.,
Cardiovasc. Res.,
2001, 49, 532-542). For example, co-expression of both receptors with VEGF is found in melanoma cells derived from primary and metastatic lesions (Graeven et al.,
J. Cancer Res. Clin. Oncol.,
1999, 125, 621-629). Vascular endothelial growth factor receptor-2 is also believed to be important in development and maintenance of glomerular capillaries (Robert et al.,
Am. J. Physiol. Renal Physiol.,
2000, 279, F275-282).
The majority of severe visual loss in the United States results from complications associated with retinal neovascularization, in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion and retinopathy of prematurity. VEGF and its receptors have been shown to have a causal role in retinal angiogenesis (Aiello et al.,
Proc. Natl. Acad. Sci. U.S.A.,
1995, 92, 10457-10461). Furthermore, in experimentally induced choroidal neovascularization (CNV), a devastating complication of macular diseases such as age-related macular degeneration, expression levels of both VEGF and vascular endothelial growth factor receptor-2 were shown to increase, and thus, vascular endothelial growth factor receptor-2 may play a role in the pathologic process
Bennett C. Frank
Watt Andrew T.
ISIS Pharmaceuticals Inc.
Lacourciere Karen A.
Licata & Tyrrell P.C.
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