Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1997-07-21
2001-01-23
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S455000, C530S350000, C536S023100, C536S023400, C536S023500, C536S024100
Reexamination Certificate
active
06177272
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides methods for treating or preventing vascular proliferative diseases in vivo by administration of a gene which encodes p27.
2. Discussion of the Background
Vascular diseases are characterized by a fibroproliferative response to injury consisting of smooth muscle cell proliferation and migration as well as connective tissue formation. The mechanisms by which vascular smooth muscle cells (VSMC) proliferate in response to mitogenic signals are well described; however, the role of cellular gene products which cause VSMC to shift from a proliferative to a nonproliferative state during G1 phase of the cell cycle are not well understood.
It is known that transitions between phases of the cell cycle are catalyzed by a family of cyclin-dependent kinases (P. Nurs, (1990)
Nature
344, 503-508; L. Hartwell et al., (1974)
Science
183, 46-51). In many cells, transit through G1 of the cell cycle and entry into S phase requires a binding and activation of cyclin/cyclin-dependent kinase complexes (CDK), predominantly cyclin D-cdk4,6 and cyclin E-cdk2 (C. J. Sherr, (1994)
Cell
79, 551; C. J. Sherr (1996)
Science
274, 1672).
The cyclin-dependent kinase inhibitors (CKIs) are naturally-occurring gene products which inhibits cyclin-CDK activity and phosphorylation of retinoblastoma (Rb), resulting in G1/S growth arrest (D. O. Morgan,
Nature
1995, 374: 171; C. J. Sherr & J. M. Roberts,
Genes. Dev.
1995, 9: 1149). CKIs directly implicated in CDK regulation are p21
cip1/Waf1
(Y.Xiong, et al.,
Nature
1993, 366: 701; J. W. Harper et al.,
Cell
1993, 75: 805), p27
Kip1
(H. Pyoshima, T. Hunter,
Cell
1994, 78: 67; K. Polyak et al.,
Cell
1994, 78: 59; S. Coats et al.,
Science
1996, 272: 877), and p16/p15
INKN
(M. Serrano et al.,
Nature
1993, 366: 704).
Previous studies of these CKIs were focused on their potential role in malignant transformation. For example, PCT Publication No. WO95/18824 (applicant Sloan-Kettering Institute For Cancer Research) describes a method for identifying agents capable of modulating the ability of p27 to inhibit the activation of the cyclin E-Cdk2 complex. This PCT publication further provides methods for treating subjects diagnosed with a hyperproliferative disorder, such as cancer and hyperplasia, using these agents. Such agents can be both protein and non-protein moieties. Unfortunately, the involvement of CKIs in cardiovascular diseases, including atherosclerosis, angiogenesis and restenosis, has not been well studied.
There are currently a number of methods used to treat cardiovascular diseases which focus on inhibiting cell proliferation. The main problems associated with the available therapies revolve around targeting the inhibitory agent to the proliferating cells that need to be killed. Targeting has traditionally been attempted using chemo- or radiotherapeutic agents coupled with antibodies. More recently, gene therapy approaches have been used that target proliferating cells by providing gene products detrimental to specific cell types. The genes encoding these gene products are known as suicide genes. The gene products are either instilled site specifically, expressed in specific cells using vectors which target specific cells or expressed under the control of a cell type-specific promoter.
The HSV-1 thyrnidine kinase gene (TK) is the most widely used suicide gene in mammalian systems. TK efficiently phosphorylates guanosine analogs ganciclovir (GCV) and acyclovir (ACV), which are subsequently phosphorylated by cellular enzymes into their triphosphate forms. These end-products are incorporated into the growing DNA chain, leading to elongation arrest (ACV) or a drastic slow down in DNA synthesis (GCV). Death usually ensues, through a mechanism identified in some cell lines as apoptosis. The mechanism that triggers cell death is not known. In the case of GCV, another action other than at the level of the DNA polymerase inhibition might exist, since no correlation is observed between the inhibition of mutant viral DNA polymerase by GCV and growth of these mutants in the presence of GCV.
One of the peculiar features of the TK-GCV system is the bystander effect that characterized the death of untransduced cells. Two mechanisms have been proposed to explain this phenomenon. In 1993, Freeman and colleagues hypothesized that the uptake of phosphorylated GCV by bystander cells occurs via the endocytosis of apoptotic vesicles, originating from the TK-transduced cells, and containing the toxic drug. However increasing evidence suggests that the bystander effect is mediated via gap junctional intercellular communications, that allow phosphorylated ganciclovir to translocate from TK
+
to TK
−
cells. In 1995, using a flow cytometry assay to quantitate cellar coupling, Fick and colleagues found that bystander tumor cytotoxicity during GCV treatment was highly correlated with the exent of gap junction-mediated coupling. In a TK-expressing neuro-2a murine neuroblastoma cell line, which do not normally exhibit any bystander effect, adenovirus-mediated overexpression of connexin-43 was shown to confer bystander effect-mediated cell killing.
The TK-GCV system has already been successfully applied in cancer models as well as restenosis in vivo (Plautz et al.,
Circulation
1991, 83:578; Ohno et al.,
Science
1994, 265:781). However, the efficiency of gene delivery in vivo remains very low, and enhancement of TK-mediated killing at other levels must be considered. In an attempt to link TK-mediated tumor suppression and immune system, retroviral vectors were constructed carrying both the HSV-TK and interleukin-2 genes. However, using a rat 9L gliosarcoma model, no enhancement of tumor eradication was observed with this vector.
SUMMARY OF THE INVENTION
Accordingly, one object of the present invention is to provide methods for treating and preventing vascular proliferative diseases in vivo.
In one application, the present invention provides a method for treating and preventing restenosis in vivo.
In a second application, the present invention provides a method for treating and preventing atherosclerosis in vivo.
In a third application, the present invention provides a method for treating and preventing angiogenesis in vivo.
The present inventors have now found that p27 functions in arteries to control the response to acute injury and cell proliferation. The present inventors provide the first direct demonstration that p27 expression and overexpression is sufficient to produce inhibition of vascular smooth muscle cell growth in vivo. Therefore, the present invention relates to the use of the p27 gene as an anti-proliferative gene to treat vascular proliferative diseases including coronary and peripheral restenosis.
The methods described herein are aimed at increasing the bystander effect. Since the mechanism of action of TK is likely to be at the level of DNA replication, arresting proliferating cells makes them transiently insensitive to TK-GCV mediated killing, prolongs their life and the duration of TK expression, thereby increases the translocation of phosphorylated GCV to bystander cells.
REFERENCES:
patent: 5672508 (1997-09-01), Gyuris et al.
patent: 5863904 (1999-01-01), Nabel et al.
patent: WO 95/18824 (1995-07-01), None
patent: WO 9602140A1 (1996-02-01), None
patent: WO 96/25507 (1996-08-01), None
Koyama et al. Fibrillarcollagen inhibits arterial smooth muscle proliferation through regulation of cdk2 inhibitors, Dec. 13, 1996.
PCT Notification of Transmittion of The International Search Report or the Declaration (8 pages), Mailed Jan. 14, 1999.
Chen et al., “Downregulation of Cyclin-dependent Kinase 2 Activity and Cyclin A Promoter Activity in Vascular Smooth Muscle Cells by p27KIP1, an Inhibitor of Neointima Formation in the Rat Caroid Artery,” pp. 2334-2341, J. Clin. Inest. 99(10), 1997.
Toyoshima et al., “p27, a Novel Inhibitor of G1 Cyclin-Cdk Protein Kinase Activity, Is Related to p21,”Cell, vol. 78, Jul. 15, 1994, pp. 67-74.
Tanner et al., “Expression of Cyc
Nabel Elizabeth G.
Nabel Gary J.
Brinks Hofer Gilson & Lione
Sandals William
Schwartzman Robert A.
The Regents of the University of Michigan
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