Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-11-07
2001-03-13
Elliott, George C. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024100
Reexamination Certificate
active
06200751
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is generally in the area of targeting and regulation of expression of recombinant gene constructs incorporating regulatory elements present in the promoter of an endothelial cell protein C/activated protein C receptor.
Atherosclerosis and most other vascular disease primarily occur in large vessels. Endothelial cells are a primary defense mechanism against cellular infiltration and thrombosis. Abnormal function of the endothelial cells contribute to myocardial infarction (MI), stroke and the development of atherosclerotic plaque. The delivery of proteins or protein expression inhibitors, directly or via gene therapy, specific to large vessel endothelial cells, is one means for addressing these clinical conditions. For example, the anti-thrombotic potential of endothelium can be increased by delivering agents that prevent thrombosis, such as thrombomodulin, heparin proteoglycans, tissue factor pathway inhibitor (TFPI, a potent inhibitor of the tissue Factor-Factor VIIa-Factor Xa complex), etc. Fibrinolytic activity can be increased by overexpression of tissue plasminogen activator (tPA) or urokinase. Expression of adhesion molecules such as P-selectin or ICAMs can be inhibited to minimize or decrease the probability of atherosclerotic plaque rupture.
Targeting endothelial cells non-specifically is often inadequate. Since more than 95% of endothelial cells are in the capillaries, any therapy directed toward endothelial cells per se runs the risk of systemic complications. One must be confident that the gene expression is limited to the desired cells when using a gene therapy approach.
It is therefore an object of the present invention to provide means and methods for selective expression of genes, especially in endothelial cells, and even more specifically in large vessel endothelial cells.
It is a further object of the present invention to provide means and methods for selective expression of genes in response to specific stimuli.
SUMMARY OF THE INVENTION
The promoter of the EPCR gene has been isolated from both murine (SEQ. ID No. 1) and human (SEQ. ID No. 2) genomic libraries. The promoter has been demonstrated to include a region which results in selective expression in endothelial cells, between −1 and −220 based on the positions relative to the ATG encoding the first amino acid of the murine EPCR protein (nucleotides 3130 to 3350 of SEQ. ID No. 1), and a region which selectively results in expression in large vessel endothelial cells, as opposed to all endothelial cells, located between −700 and −1080 (nucleotides 2270 to 2840 of SEQ. ID No. 1). A thrombin responsive element has been identified in the EPCR promoter, from −337 to −345 in the murine promoter (nucleotides 3007 to 3014 SEQ. ID No. 1) and from −360 to −368 (nucleotides 2722 to 2729 SEQ. ID No. 2) in the human promoter. The sequence is CCCACCCC (SEQ. ID No. 3). A serum response element has also been identified between −280 and −350 (nucleotides 2990 to 3061 of SEQ. ID No. 1).
The regulatory sequences present in the EPCR promoter can be used in combination with genes encoding other proteins, as well as shorter oligonucleotides, to increase expression by exposure to thrombin or serum or to effect selective expression in endothelial cells generally or preferentially in endothelial cells of the large blood vessels. The gene control elements include elements responsive to environmental stimuli (either thrombin or serum); and information to determine distribution of the desired protein expression (large vessels). Therapeutic strategies include the use of the minimal promoter (−220 to −1) for expression in all endothelial cells, for example, for any kind of gene therapy where systemic distribution is desired; the use of a promoter including an environmental stimuli response element(s), for use in delivery of agents whose expression should be increased during times of increased thrombin/platelet activation or during regional trauma; the use of the minimal promoter including an environmental stimuli response element and the element directing expression to large vessel endothelium, where a response to regional trauma is desirable but only in large vessel endothelium, and the use of the minimal promoter and element directing expression to large vessel endothelium, where expression is specifically targeted to large vessel endothelium but is not increased in response to any particular stimuli.
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Esmon Charles T.
Gu Jian-Ming
Arnall Golden & Gregory LLP
Elliott George C.
Oklahoma Medical Research Foundation
Sandals William
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