Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Rodent cell – per se
Reexamination Certificate
1997-12-10
2001-04-03
Kunz, Gary L. (Department: 1646)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Rodent cell, per se
C435S252300, C435S320100, C435S325000, C435S348000, C435S356000, C435S357000, C435S365000, C435S366000, C530S350000, C536S023500, C536S024310
Reexamination Certificate
active
06210967
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application, various publications are referenced in parentheses by author and year. Full citations for these references may be found at the end of the specification immediately preceding the sequence listings and the claims. The disclosure of these publications in their entireties are hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
Biologically active phospholipids are emerging as important intracellular signaling molecules. Among these, lysophosphatidic acid (LPA; 1-acyl-glycerol-3-phosphate) is the simplest glycerophospholipid (Moolenaar, 1994). LPA produces a wide variety of biological responses such as induction of cell proliferation, stimulation of neurite retraction, platelet aggregation, smooth muscle contraction, tumor cell invasion, neurotransmitter release, chloride efflux and chemotaxis (Moolenaar, 1994; Jalink et al., 1994; Van Corven et al., 1989; Tigyi et al., 1994; Tigyi and Miledi, 1992; Jalink et al., 1993; Tokumura et al., 1994; Piazza et al., 1995). LPA is the product of the blood-clotting process and is therefore present in serum. LPA derived from platelets appears to be an important mediator of wound healing and tissue regeneration (Moolenaar, 1995).
The known effects of LPA appear to be mediated by G protein-coupled receptor(s). Specific LPA binding sites have been demonstrated in membranes from 3T3 cells and rat brain with Kd values in the low nanomolar range (Thomson et al., 1994). The actions of LPA are mediated by at least four G protein-mediated signaling pathways: stimulation of phospholipase C and phospholipase D, inhibition of adenylyl cyclase, activation of Ras and Raf/MAP kinase pathway, and tyrosine phosphorylation of focal adhesion proteins (Moolenaar, 1995).
We describe here the isolation and characterization of a novel mammalian LPA receptor, specifically a human LPA receptor. This receptor can serve as a tool for the drug design of novel therapeutic agents for various indications, including cancer (preferably tumor reduction or prevention), platelet aggregation (as an anti-coagulant), vascular restenosis, arthritis (as an anti-inflammatory), wound healing, tissue regeneration (preferably skin and nerve regeneration), blood coagulation, osteoporosis (bone regeneration), and cosmetic uses (preferably the prevention of abnormal growths or scarring or for augmentation).
Independently, the identification of a complementary DNA from Xenopus that encodes a functional high-affinity receptor for LPA has recently been reported (Guo, et al., 1996). Analysis of this receptor revealed that it is a G-protein-coupled-receptor.
SUMMARY OF THE INVENTION
This invention provides an isolated nucleic acid encoding a mammalian LPA receptor. In an embodiment, the mammalian LPA receptor is a human LPA receptor.
This invention provides a purified mammalian LPA receptor protein.
This invention provides a vector comprising a nucleic acid encoding a mammalian LPA receptor. This invention also provides for a vector comprising a nucleic acid encoding a human LPA receptor. Such a vector may be adapted for expression of the mammalian LPA receptor in mammalian or non-mammalian cells.
This invention provides a plasmid designated HL-18a (ATCC Accession No. 209448). This invention also provides a membrane preparation isolated from the cells.
This invention provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian LPA receptor, wherein the probe has a unique sequence corresponding to a sequence present within one of the two strands of the nucleic acid which encode the mammalian LPA receptor contained in plasmid HL-18a (ATCC Accession No. 209448).
This invention also provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian LPA receptor, wherein the probe has a unique sequence corresponding to a sequence present within (a) the nucleic acid sequence of shown in
FIG. 2
(Seq. I.D. No. 2) or (b) the reverse complement thereto.
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a unique fragment of the sequence of a nucleic acid molecule encoding a mammalian LPA receptor.
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to the antisense sequence of a unique fragment of the sequence of a nucleic acid molecule encoding a mammalian LPA receptor.
This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the RNA of the mammalian LPA receptor, so as to prevent translation of the RNA. This invention also provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA encoding a mammalian LPA receptor.
This invention further provides an antibody capable of binding to a mammalian LPA receptor. This invention also provides for an antibody capable of competitively inhibiting the binding of the antibody to a mammalian LPA receptor.
This invention provides a pharmaceutical composition comprising (a) an amount of the oligonucleotide capable of passing through a cell membrane and effective to reduce expression of a mammalian LPA receptor and (b) a pharmaceutically acceptable carrier capable of passing through a cell membrane.
This invention provides a transgenic, nonhuman mammal expressing DNA encoding a mammalian LPA receptor. This invention also provides a transgenic, nonhuman mammal comprising a homologous recombination knockout of the native mammalian LPA receptor. This invention further provides a transgenic, nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a mammalian LPA receptor so placed within the genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding a mammalian LPA receptor and which hybridizes to mRNA encoding a mammalian LPA receptor, thereby reducing its translation.
This invention provides for a process for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises contacting cells containing DNA encoding and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian LPA receptor.
This invention provides for a process for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises contacting a membrane fragment from a cell extract of cells containing DNA encoding and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian LPA receptor.
This invention provides for a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises separately contacting cells expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the mammalian LPA receptor, a decrease in the binding of the second chemical compound to the mammalian LPA receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian LPA receptor.
This invention provides for a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian LPA receptor
Cooper & Dunham LLP
Kunz Gary L.
O'Hara Eileen B.
Synaptic Pharmaceutical Corporation
White John P.
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