Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
1993-07-27
2002-06-25
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C536S024500
Reexamination Certificate
active
06410322
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the field of antisense oligonucleotides for use in the inhibition of vascular endothelial cell growth factor (VEGF) expression.
2. Description of Related Art
Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor (VPF), is a 34-43 kDa (with the predominant species at about 45 kDa) dimeric, disulphide-linked glycoprotein synthesized and secreted by a variety of tumor and normal cells. Leung et al.,
Science
246, 1306 (1989), observed three VEGF transcripts (121, 165, and 189 amino acids long, respectively), suggesting that an alternative splicing mechanism is involved. More recently, Houck et al. discovered a fourth VEGF transcript having a length of 206 amino acids. Tischer et al.,
J. Biol. Chem.
266, 11947 (1991), have determined that the human VEGF coding region is comprised of eight exons. Furthermore, this group proved that three mRNA transcripts (encoding for the 121, 165, and 189 amino acid long peptides) were the result of alternative splicing. Transcripts analogous to the 121 and 165 amino acid polypeptides have been identified in the bovine system. Leung et al.,
supra
. The transcript corresponding to the 165 amino acid transcript have also been identified in the rodent system—rat (Conn et al.,
Proc. Natl. Acad. Sci. U.S.A.
87, 2628 (1989)), guinea pig (Sanger et al.,
Cancer Res.
50, 1774 (1990)), and mouse (Claffey et al.,
J. Biol. Chem.
257, 16317 (1992)).
Tischer et al.,
supra
, reported the nucleic acid sequence for three forms of human VEGF coding region. Claffey et al.,
supra
, published the sequence for murine VEGF. Comparisons have revealed greater than 85% interspecies conservation of the VEGF molecule. All the alternatively spliced VEGF molecules have not been identified yet, but based on the conservation between species, they should be in the near future.
The following discussion presents several pathological states in which VEGF is involved and emphasizes the importance of VEGF as a potential target for therapeutic treatment.
Diabetic Retinopathy
Diabetic retinopathy is the leading cause of blindness among working age adults (20-64) in the United States. During the course of Diabetes Mellitus, one complication that can arise is an occlusion of the retinal veins. This venous occlusion results in the formation of microaneurysms due to the expansion of the vessel wall, hemorrhaging (leaking of blood into surrounding areas), “cotton wool” spots representing cellular exudates (i.e., cellular damage) and neovascularization of the retina extending into the vitreous, resulting in bleeding. Classic treatments for diabetic retinopathy are 1) the control of blood glucose and blood pressure and 2) pan retinal laser photocoagulation (PRP). Treatment #1 can prolong the onset of the disease depending on the diligence of the affected individual. Treatment #2 is quite effective, but can lead to additional hemorrhaging as well as damage to critical areas needed for visions (i.e., foveal fibers). Additional treatments for this disease which have less side effects would prove extremely valuable.
Recent observations have shown an increase in VEGF protein levels in retinal membranes from patients with diabetes, suggesting that this cytokine/growth factor may play an important role in the disease. The following characteristics of VEGF provide evidence that it may be an important regulator of diabetic retinopathy: (1) The action of VEGF is specific for endothelial cells; (2) VEGF has been shown to be angiogenic as well as mitogenic; (3) VEGF is a secreted molecule; (4) VEGF induces vascular permeability; and 5) VEGF is induced under hypoxic conditions (i.e., during retinal vein occlusion).
Atherosclerotic Plaque Formation
VEGF may play a role in the development of an atherosclerotic plaque. Atherosclerosis describes a state where the formation of lipid-containing lesions occurs in medium and large arteries. It is the primary cause of myocardial and cerebral infarctions in the United States. Lesions form within the intima, the innermost layer of the arterial wall, and are separated into two forms: the fatty streak (early), and the fibrous plaque (advanced). Both of these forms are characterized by lipid-filled macrophages (derived from blood-borne monocytes) and smooth muscle cells. The fibrous plaque is further characterized by the deposition of connective tissue and cholesterol crystals. These lesions occlude the lumen of the blood vessel diminishing the blood flow, leading to ischemia and necrosis. Research has shown that neovascularization can also occur in the atherosclerotic lesion. Levels of VEGF protein in these affected areas have not been determined, but it has been shown that both monocytes and macrophages express VEGF.
Wound Healing
VEGF may also be important in maintaining normal states of wound healing. See Brown et al.,
J. Exp. Med.
176, 1375 (1992). Wound healing is usually a regulated response to injury or trauma. Focal hemorrhaging is followed by the extravasation (leaking) of fibrinogen from the plasma to form a fibrin gel or clot. This initial matrix is replaced by granulation tissue (fibronectin, collagen, proteoglycan) and finally by scar tissue. In addition, keratinocytes migrate and form a covering to protect against fluid loss and bacterial infection. One major characteristic of wound healing is that vessel hyperpermeability occurs for some time after bleeding has stopped. In addition, angiogenic activity is detectable during this time period. Recent work has shown that keratinocytes, located at the border of the wound as well as in the wound covering, produce VEGF. Brown et al.,
supra
. This result suggests that VEGF may be responsible for hyperpermeable and angiogenic activity associated with wound healing.
Aberrant would healing associated with surgery can result in complications such as hypertrophic scarring (excessive collagen deposition), keloid formation (scar tissue invading normal surrounding tissue), and adhesions in the peritoneal cavity. Other problems related with unregulated wound healing occur during the formation of lung fibrosis and in diabetes mellitus (wounds do not heal). It is believed that VEGF plays a role in these processes as well.
Tumor Angiogenesis
VEGF may be a tumor angiogenesis factor. Plate et al.,
Nature
359, 845 (1992). Angiogenesis is the tightly regulated processes by which new blood vessels develop. The development of a vascular system is necessary for the flow of nutrients and waste to and from tissues and organs. Smaller solid tumors (<1-2 mm) do not require an extensive vascular system to survive, but instead derive their nourishment through the diffusion of needed nutrients. However, in order for these cell masses to grow beyond several millimeters in size, additional vascularization is needed. See, e.g., Folkman,
J. Natl. Cancer Inst.
82, 4 (1990). It has been suggested that inhibition of tumor angiogenesis might be an effective strategy to combat tumor growth and circumvent acquired resistance to traditional anti-cancer therapeutic agents. Kerbel,
BioEssays
13, 31(1991). Kim et al.,
Nature
362, 841 (1993) reported that monoclonal antibodies specific for VEGF inhibited the growth of tumors in vivo.
The tumor stroma, which contains both connective tissue and the vascular system, is essentially the “lifeline” of the tumor. Whereas normal tissue vasculature is organized and can respond to changes in metabolism, the tumor stroma is poorly organized and closely resembles scar tissue found during wound healing. The tumor stroma may represent only a small portion of the total tumor (e.g., medullary carcinoma of the breast) or may exist as 80-90% of the total cell mass (e.g., desmoplastic carcinoma). Tumor blood vessels also differ from those found in normal tissue in that they are hyperpermeable to plasma and plasma proteins. Whereas this porosity is seen in normal tissue only during wound healing, solid tumors maintain this porous characteristic indefinitely.
While a necess
LandOfFree
Antisense oligonucleotide inhibition of vascular endothelial... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Antisense oligonucleotide inhibition of vascular endothelial..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Antisense oligonucleotide inhibition of vascular endothelial... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2969070