Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-03-13
2003-07-29
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S455000, C435S325000, C514S04400A, C424S009100, C424S009200
Reexamination Certificate
active
06599741
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit from German application DE 299 16 160.9 and Japanese application JP 261035/99, both of which were filed Sep. 14, 1999.
A method of modulating the transcription of one or more genes in a vascular or cardiac cell, wherein the method comprises a step of contacting said cell with a composition comprising one or more double-stranded nucleic acid(s) capable of sequence-specific binding to the transcription factor AP-1 and/or C/EBP or a related transcription factor.
Coronary Heart Disease (CHD) is the leading cause of death in industrialized nations. The last decades have seen an increasing use of aortocoronary venous bypass surgery as well as percutanous transluminal coronary angioplasty (PTCA) for the treatment of Coronary Heart Disease which substantially increased survival rates as well as quality of life of heart patients.
Although these methods have been well established for decades, they still have a high probability of relapse or of complete occlusion of the treated vessel (30-50%) and often this complication is already apparent within a few months after surgery. This represents a substantial medical problem and is also an economical burden for the health systems of the industrialized world.
One of the major reasons for complications observed with PTCA as well as coronary bypass surgery seems to be the induction of migration and proliferation of cells of the vessel wall. This untimely and unwanted proliferation of vascular tissue results in remodeling processes that eventually cause relapses and obstruction of surgically treated vessels.
To prevent postoperative relapse or occlusion of a treated vessel segment, a recently employed strategy is the implantation of stents, tubular structures that are designed to maintain the postoperative shape and diameter of a vessel after PTCA. But this method is also complicated by the development of in-stent restenosis as a result of excessive proliferation and migration of smooth muscle cells due to mechanical strain on the vessel wall. Additionally, several clinical studies using a variety of adjuvant medication therapies where not able to efficiently suppress these side effects of surgical intervention.
Consequence of therapeutic failure is the (re-)occlusion of myocardial blood vessels, which leads to ischemic episodes and, in severe cases, to myocardial infarction (MI). This is accompanied by massive death of cardiomyocytes in areas of the heart muscle with insufficient oxygen supply, consequently leaving incontractile scar tissue. Thus, re-opening of vessels, induction of vessel growth in ischemic areas of the heart muscle and/or induction of proliferation of cardiomyocytes could present a possible therapeutic approach for treatment of MI.
Most cells in the body are in the G
0
-phase of the cell cycle which is called the quiescent state. Almost all quiescent body cells still have the ability to proliferate and can be induced to reenter the cell cycle by a number of stimuli the most important being growth factors and injury. Proliferation as well as remodeling processes are primarily regulated on the level of transcription. The physical stress of, for instance, coronary angioplasty and stent implantation will therefore lead to induction of a number of genes most importantly cyclins, cell cycle specific phosphatases and cell cycle specific transcription factors like for instance cyclin E, cyclin A, cyclin B, cdc25C, cdc25A, E2F-family members as well as a number of metabolically important genes or genes that are involved in the doubling of DNA like for instance PCNA, histones and dhfr. While those factors are newly synthesized upon entry into the cell cycle many transcription factors involved in the first steps of proliferation, the so called immediate-early genes, are already present in the cell and are activated upon a given stimulus, a well known member of this class is AP-1.
AP-1 is a heterodimeric transcription factor consisting of c-Jun and c-Fos protein (Curran and Franza (1988) Cell 55, 395) that interact via a leucine zipper motif with each other. Unlike c-Jun, that is able to homodimerize and bind DNA on its own, c-Fos is dependent on interaction with c-Jun for sequence specific binding to DNA. Both proteins are members of a larger family of proteins that include for instance JunB and JunD (Jun related) as well as for instance Fral and FosB (Fos related) (Curran and Vogt (1992) in Transcriptional Regulation, 797 (McKnight and Yamamoto) Cold Spring Harbor Laboratory Press). AP-1 is able to bind to a consensus sequence TGACTCA motif but many variations of this sequence can be avidly bound by various homo- and heterodimers of the AP-1 family members (Franza et al. (1988) Science 239, 1150; Rauscher et al. (1988) Genes Dev. 2, 1687; Risse et al. (1989) EMBO J. 8, 3825; Yang-Yen et al. (1990) New Biol 2, 351).
Although coexpression of Fos and Jun can lead to dramatic synergistic activation of AP-1-dependent transcription (Chiu et al. (1988) Cell 54, 541) a substantial degree of experimental variability has been encountered depending on the cell type used. Therefore, it is likely that the activities of Fos and Jun are influenced by other proteins that may be expressed in a cell type-specific manner (Baichwal and Tjian (1990) Cell 63, 815) and that in each cell type, the presence of resident or inducible transcription factors may influence both the selection of gene targets and the transcriptional effect of Fos-Jun family hetero- and homodimers. Consistent with the promoter and cell type specificity of AP-1, Fos was shown not to activate but rather to repress transcription of the c-fos promoter (Sassone-Corsi (1988) Cell 54, 553; Lucibello et al. (1989) Cell 59, 999). Consequently, it is not possible to predict what effect AP-I might have on a specific promoter in a given cell type.
A method to specifically interfere with transcriptional activation by a specific transcription factor is disclosed in WO 95/11687. It teaches that it is possible to interfere with the activating function of a transcription factor by treating a cell with a double-stranded DNA molecule, termed cis-element decoy, carrying a binding site for that specific transcription factor. Exogenous supply of a high number of transcription factor binding sites to a cell, preferentially in much higher numbers than present in endogenous promoters in the genome, creates a situation where the majority of a given transcription factor will bind specifically to the respective cis-element decoy rather than to its endogenous target genes. This approach for inhibiting the binding of a transcription factor to its endogenous binding site is also referred to as squelching. Squelching of transcription using DNA decoys has been successfully employed to inhibit proliferation of cells using DNA fragments that specifically target the transcription factor E2F (Morishita et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5855).
It is speculated by extrapolating the current results of the Human Genome Project that about 50% of all human genes are transcription factors. The complexity and diversity of organisms is known to be in large part caused by the restriction of expression of certain transcription factors to specific cell types and/or to specific stages of development. A transcription factor that has a positive effect on proliferation in one cell type might have an opposite effect in another cell type or in another species. Thus, the effect of treatment with a given transcription factor decoy may vary not only between species, but also between various tissues, for example veins and arteries, and between cell types in a given tissue, for example endothelial cells and smooth muscle cells of a vessel wall, within one organism.
Therefore, one of the objectives of the present invention is the targeting of transcription factors that modulate transcription in vascular cells, including endothelial and smooth muscle cells, or cardiac cells and which thus would be appropriate targets for squelching by double-stranded nucleic aci
Hecker Markus
Lauth Manfred
Wagner Andreas H.
Avontec GmbH
Clark & Elbing LLP
Crouch Deborah
Ton Thai-An N.
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