Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2002-08-09
2004-04-06
Lacourciere, Karen A. (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S091100, C435S325000, C435S375000, C435S366000, C536S023100, C536S024300, C536S024330, C514S04400A
Reexamination Certificate
active
06716975
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of EDG1. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding EDG1. Such compounds have been shown to modulate the expression of EDG1.
BACKGROUND OF THE INVENTION
A vast majority of biologically active molecules including growth factors, cytokines, neurotransmitters, phospholipids and hormones transduce signals via specific cell-surface receptors. Some of these receptors are then coupled to heterotrimeric GTP-binding proteins (G-proteins) which, upon activation, relay signals to a variety of cellular effectors.
One family of G-protein coupled receptors is the endothelial differentiation genes or EDGs. Five members of this family have been identified and these have been further divided into two subfamilies or clusters. EDG1, EDG3 and EDG5/H218/AGR16 recognize and respond to the phospholipid mediators sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) while EDG2 and EDG4 recognize and respond only to lysophosphatidic acid (An et al.,
J. Cell Biochem. Suppl
., 1998, 31, 147—157). These phospholipids have diverse biological effects, acting both as intracellular second messengers as well as extracellular mediators (Van Brocklyn et al.,
J. Cell Biol
., 1998, 142, 229—240). They have been shown to play roles in development, wound healing, tissue regeneration, induction of cellular proliferation, alterations in cellular differentiation and suppression of apoptosis. Other effects include cytoskeletal responses such as contraction, adhesion, secretion and chemotaxis (Goetzl and An,
Faseb J
., 1998, 12, 1589—1598; Hla et al.,
Biochem. Pharmacol
., 1999, 58, 201—207).
EDG1 (also known endothelial differentiation gene 1, ECGF1, G-protein coupled receptor 1, GPCR1 and sphingolipid G-protein coupled receptor 1) was the first member of the EDG family to be isolated and characterized. Hla and Maciag isolated an immediate early gene which is rapidly induced when cells are treated with the tumor promoter phorbol 12-myristate 13-acetate (PMA) and superinduced upon treatment with cycloheximide. The protein is was found to be abundantly expressed in endothelial cells but was also detected at lower levels in vascular smooth muscle cells, fibroblasts, melanocytes, and cells of epithelial origin (Hla and Maciag,
J. Biol. Chem
., 1990, 265, 9308—9313).
EDG1 encodes a protein that contains seven transmembrane domains making it structurally similar to other G-protein coupled receptors (Hla and Maciag,
J. Biol. Chem
., 1990, 265, 9308—9313). Since then both LPA and S1P have been confirmed to be the natural ligands of this orphan receptor (Lee et al.,
J. Biol. Chem
., 1998, 273, 22105—22112; Lee et al.,
Science
, 1998, 279, 1552—1555).
EDG1 is linked via the G1 family of C-proteins to multiple signaling cascades including phospholipase C activation, calcium mobilization, MAPK/ERK activation, and adenylate cyclase inhibition (Okamoto et al.,
J. Biol. Chem
., 1998, 273, 27104—27110).
EDG1 also plays a role in angiogenesis, the process of new blood vessel formation which, if uncontrolled, can lead to numerous pathologic conditions including diabetic retinopathy, rheumatoid arthritis, and solid tumor growth. Overexpression of EDG1 in human embryonic kidney 293 cells and NIH3T3 cells transfected with a clone expressing the human EDG1 transcript results in the sustained activation of the MAP kinase pathway (Lee et al.,
J. Biol. Chem
., 1996, 271, 11272—11279). This pathway is important in the control of cell growth and differentiation. Other studies have directly indicated that S1P activates EDG1 on endothelial cells to regulate the process of angiogenesis (Lee et al.,
Cell
, 1999, 99, 301—312).
Further support for a role in endothelial regulation comes from studies of cells exposed to fluid shear stress. These cells showed a marked increase in the levels of the EDG1 transcript under physiologic blood flow conditions (Takada et al.,
Biochem. Biophys. Res. Commun
., 1997, 240, 737—741).
EDG1 is also involved in the inflammation process. Rizza et al. showed that in human aortic endothelial cells (HAEC), treatment with LPA activated these cells and increased their interaction with monocytes, neutrophils and HL60 cells. This interaction was the result of the upregulation of VCAM-1 and E-selectin, cell adhesion molecules, which in turn was mediated through EDG1 (Rizza et al.,
Lab. Invest
., 1999, 79, 1227—1235).
The pharmacological modulation of EDG1 activity and/or expression is therefore believed to be an appropriate point of therapeutic intervention in pathological conditions involving excessive cellular proliferation such as cancer and angiogenesis as well as the process of inflammation.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of EDG1. To date, strategies aimed at investigating EDG1 function have involved the use of antibodies, protein labeling for use in localization of the protein within the cell and molecules that block cellular pathways such as MAPK inhibitors. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting EDG1 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of EDG1 expression.
The present invention provides compositions and methods for modulating EDG1 expression.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding EDG1, and which modulate the expression of EDG1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of EDG1 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of EDG1 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding EDG1, ultimately modulating the amount of EDG1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding EDG1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding EDG1” encompass DNA encoding EDG1, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of EDG1. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In
Gibbs Terra C.
ISIS Pharmaceuticals Inc.
Lacourciere Karen A.
Licata & Tyrrell P.C.
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