Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-10-28
2003-07-08
Wehbé, Anne M. (Department: 1632)
Chemistry: molecular biology and microbiology
Vector, per se
C435S325000, C435S455000, C424S093200, C424S093210, C514S04400A, C536S023100
Reexamination Certificate
active
06589782
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the treatment of the cardiovascular system and its diseases through effects on anatomy, conduit function, and permeability, and more particularly to a method of treating cardiovascular disease by stimulating vascular cell proliferation using a growth factor thereby stimulating endothelial cell growth and vascular permeability.
Cardiovascular diseases are generally characterized by an impaired supply of blood to the heart or other target organs. Myocardial infarction (MI), commonly referred to as heart attacks, are a leading cause of mortality as 30% are fatal in the in the first months following the heart attack. Heart attacks result from narrowed or blocked coronary arteries in the heart which starves the heart of needed nutrients and oxygen. When the supply of blood to the heart is compromised, cells respond by generating compounds that induce the growth of new blood vessels so as to increase the supply of blood to the heart. These new blood vessels are called collateral blood vessels. The process by which new blood vessels are induced to grow out of the existing vasculature is termed angiogenesis, and the substances that are produced by cells to induce angiogenesis are the angiogenic factors.
Unfortunately, the body's natural angiogenic response is limited and often inadequate. For this reason, the discovery of angiogenic growth factors has lead to the emergence of an alternative therapeutic strategy which seeks to supplement the natural angiogenic response by supplying exogenous angiogenic substances.
Attempts have been made to stimulate angiogenesis by administering various growth factors. U.S. Pat. No. 5,318,957 to Cid et al. discloses a method of stimulating angiogenesis by administering haptoglobins (glyco-protein with two polypeptide chains linked by disulfide bonds). Intracoronary injection of a recombinant vector expressing human fibroblast growth factor-5 (FGF-5) (i.e., in vivo gene transfer) in an animal model resulted in successful amelioration of abnormalities in myocardial blood flow and function. (Giordano, F. J.,et. al.
Nature Med
2, 534-539, 1996). Recombinant adenoviruses have also been used to express angiogenic growth factors in-vivo. These included acidic fibroblast growth factor (Muhlhauser, J., et. al.
Hum. Gene Ther.
6, 1457-1465, 1995), and one of the VEGF forms, VEGF
165
(Muhlhauser, J., et. al.
Circ. Res.
77, 1077-1086, 1995).
One of the responses of heart muscle cells to impaired blood supply involves activation of the gene encoding Vascular Endothelial Growth Factor (“VEGF”) (Banai, S., et. al.
Cardiovasc. Res.
28:1176-1179, 1994). VEGFs are a family of angiogenic factors that induce the growth of new collateral blood vessels. The VEGF family of growth factors are specific angiogenic growth factors that target endothelial (blood vessel-lining) cells almost exclusively. (Reviewed in Ferrara et al., Endocr.
Rev.
13:18-32 (1992); Dvorak et al.,
Am. J. Pathol.
146:1029-39 (1995); Thomas,
J. Biol. Chem.
271:603-06 (1996)). Expression of the VEGF gene is linked in space and time to events of physiological angiogenesis, and deletion of the VEGF gene by way of targeted gene disruption in mice leads to embryonic death because the blood vessels do not develop. It is therefore the only known angiogenic growth factor that appears to function as a specific physiological regulator of angiogenesis.
In vivo, VEGFs induce angiogenesis (Leung et al.,
Science
246:1306-09, 1989) and increase vascular permeability (Senger et al.,
Science
219:983-85, 1983). VEGFs are now known as important physiological regulators of capillary blood vessel formation. They are involved in the normal formation of new capillaries during organ growth, including fetal growth (Peters et al.,
Proc. Natl. Acad. Sci.
USA 90:8915-19, 1993), tissue repair (Brown et al.,
J. Exp. Med.
176:1375-79, 1992), the menstrual cycle, and pregnancy (Jackson et al.,
Placenta
15:341-53, 1994; Cullinan & Koos,
Endocrinology
133:829-37, 1993; Kamat et al.,
Am. J. Pathol.
146:157-65, 1995). During fetal development, VEGFs appear to play an essential role in the de novo formation of blood vessels from blood islands (Risau & Flamme,
Ann. Rev. Cell. Dev. Biol.
11:73-92, 1995), as evidenced by abnormal blood vessel development and lethality in embryos lacking a single VEGF allele (Carmeliet et al.,
Nature
380:435-38, 1996). Moreover, VEGFs are implicated in the pathological blood vessel growth characteristic of many diseases, including solid tumors (Potgens et al.,
Biol. Chem. Hoppe
-
Seyler
376:57-70, 1995), retinopathies (Miller et al.,
Am. J. Pathol.
145:574-84, 1994; Aiello et al.,
N. Engl. J. Med.
331:1480-87, 1994; Adamis et al.,
Am. J. Ophthalmol.
118:445-50, 1994), psoriasis (Detmar et al.,
J. Exp. Med.
180:1141-46, 1994), and rheumatoid arthritis (Fava et al.,
J. Exp. Med.
180:341-46, 1994).
Using the rabbit chronic limb ischemia model, it has been shown that repeated intramuscular injection or a single intra-arterial bolus of VEGF can augment collateral blood vessel formation as evidenced by blood flow measurement in the ischemic hindlimb (Pu, et al.,
Circulation
88:208-15, 1993; Bauters et al.,
Am. J. Physiol.
267:H1263-71, 1994; Takeshita et al.,
Circulation
90 [part 2], II-228-34, 1994; Bauters et al.,
J. Vasc. Surg.
21:314-25, 1995; Bauters et al.,
Circulation
91:2802-09, 1995; Takeshita et al.,
J. Clin. Invest.
93:662-70, 1994). In this model, VEGF has also been shown to act synergistically with basic FGF to ameliorate ischemia (Asahara et al.,
Circulation
92:[suppl 2], II-365-71, 1995). VEGF was also reported to accelerate the repair of balloon-injured rat carotid artery endothelium while at the same time inhibiting pathological thickening of the underlying smooth muscle layers, thereby maintaining lumen diameter and blood flow (Asahara et al.,
Circulation
91:2793-2801, 1995). VEGF has also been shown to induce EDRF (Endothelin-Derived Relaxin Factor (nitric oxide))-dependent relaxation in canine coronary arteries, thus potentially contributing to increased blood flow to ischemic areas via a secondary mechanism not related to angiogenesis (Ku et al.,
Am. J. Physiol.
265:H586-H592, 1993).
Activation of the gene encoding VEGF results in the production of several different VEGF variants, or isoforms, produced by alternative splicing wherein the same chromosomal DNA yields different mRNA transcripts containing different exons thereby producing different proteins. Such variants have been disclosed, for example, in U.S. Pat. No. 5,194,596 to Tischer et al. which identifies human vascular endothelial cell growth factors having peptide sequence lengths of 121, and 165 amino acids (i.e., VEGF
121
and VEGF
165
). Additionally, VEGF
189
and VEGF
206
have also been characterized and reported (Neufeld, G., et. al.
Cancer Metastasis Rev.
15:153-158, 1996).
As depicted in
FIG. 1
, the domain encoded by exons 1-5 contains information required for the recognition of the known VEGF receptors KDR/flk-1 and flt-1 (Keyt, B. A., et. al.
J. Biol Chem
271:5638-5646, 1996), and is present in all known VEGF isoforms. The amino-acids encoded by exon 8 are also present in all known isoforms. The isoforms may be distinguished however by the presence or absence of the peptides encoded by exons 6 and 7 of the VEGF gene, and the presence or absence of the peptides encoded by these exons results in structural differences which are translated into functional differences between the VEGF forms (reviewed in: Neufeld, G., et. al.
Cancer Metastasis Rev.
15, 153-158, 1996).
Exon 6 can terminate after 72 bp at a donor splice site wherein it contributes 24 amino acids to VEGF forms that contain it such as VEGF
189
. This exon 6 form is referred to as exon 6a. However, the VEGF RNA can be spliced at the 3′ end of exon 6 using an alternative splice site located 51 bp downstream to the first resulting in a larger exon 6 product containing 41 amino-acids. The additional 17 amino-ac
Keshet Eli
Neufeld Gera
Poltorak Zoya
Vlodavsky Israel
Blank Rome LLP
Technion Research & Development Co. Ltd.
Wehbe Anne M.
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