Gene

Details Gene Gene

2000-09-07

2002-09-03

Chan, Christina (Department: 1644)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S252300, C435S320100, C530S350000, C536S023500

Reexamination Certificate

active

06444443

ABSTRACT:

The present invention relates to a novel inflammation-related gene designated EXP189 encoding an intercellular adhesion molecule, and to the protein molecule encoded by EXP189. The invention also relates to the use of EXP189 polynucleotide sequences for diagnostic screening of patients and the use of the protein encoded by EXP189 as a therapeutic target.
Development and maintenance of several diseases, for example many respiratory and inflammatory diseases, involve the participation of a variety of cell types that undergo a number of phenotypic changes during the development of the pathological condition. These phenotypic alterations are the result of specific changes in the expression and functioning of various genes and proteins. The detection of genes or proteins, whose expression is altered in a particular physiological or pathological condition, can therefore lead to the identification of genes or proteins of pathological and therapeutical importance.
Cells that are attracted into tissues during inflammation include various inflammatory phagocytes such as neutrophilic and eosinophilic granulocytes and monocytes. These cells have been associated with inflammation and tissue destruction in several inflammatory diseases including respiratory tract inflammation in both acute and chronic bronchitis, chronic obstructive pulmonary disease (COPD), emphysema, asthma, adult respiratory distress syndrome (ARDS), rheumatoid arthritis, inflammatory bowel disease (IBD), ulcerative colitis, primary sclerosing cholangitis and Crohn's disease.
In several inflammatory respiratory diseases, there is an increased number of neutrophils present in the inflamed tissues. Enhanced migration into the lung, as a result of the release of chemoattractants by various leukocytes and epithelial cells, and the inhibition of apoptosis of these otherwise short-lived cells contribute to the accumulation of these cells at the sites of inflammation. Increased levels of granulocyte macrophage colony stimulating factor (GM-CSF) have been shown to increase the functional life span of neutrophils and to increase both phagocytic and oxidative burst activity and the production of proinflammatory cytokines that are critical for regulating the inflammatory process. The isolation of genes and proteins whose expression is upregulated upon cytokine stimulation, for example by GM-CSF, can identify molecular targets that can be exploited to offer therapeutic benefits.
Critical steps in the action of leukocytes in inflammatory conditions include the migration of these cells into the tissues, e.g. into the airways in respiratory inflammations or to the joints in rheumatoid arthritis, cell activation and the release of a range of inflammatory mediators, leukotrienes, oxygen radicals, proteases. Leukocyte migration involves the arrest and firm adhesion of blood cells on endothelial surfaces and the migration through the endothelium into the interstitium and from there to particular microenviroments. Several of these processes are dependent on the expression of cell surface adhesion molecules, which mediate the interaction of leukocytes for example with other cells of the same or different kind as well as with extracellular matrix components. The intercellular adhesion molecules expressed on the cell surface are thus involved in cell migration, tissue localisation and modification of cell function through downstream signalling events that can follow the engagement of the adhesion molecule with its ligand.
Recently, several methods and technologies have been developed for the detection of differential gene expression and the isolation of differentially expressed genes. For example, changes can be identified at the protein level using proteomics approaches and changes in transcriptional regulation can be detected by several methods including differential display (Liang, P., and Pardee, A. B., Science 257:967-971), SAGE (serial analyses of gene expression) (Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. Science, 270:484-487 ), differerential hybridization of complex cDNA probes high density cDNA or oligonucleotide arrays bound to solid support (Chee, M., Yang, R., Hubbell, E., Berno, A. Huang, X. C., Stern, D., Winkler,J.,Lockhart, D. J., Morris, M. S. and Fodor, S. P. A. Science (1996) 274:610-614; Lockhart, D. J., Dong, H., Byrne,M. C., Follettie, M. T., Gallo, M. V., Chee, M. M., Wang, C., Kobayashi, M., Horton, H. and Brown, E. L. Nature Biotechnology (1996) 14:1675-1680; Shena, M., Shalon, D., Davis, R. W. and Brown, P. O. Science (1995) 250:467-470.) and cDNA subtraction methods such as representational difference analysis (Hubank, M., and Schatz, D. G. Nucleic Acids Res. 22: 5640-5648).
One method, which can be used to identify differentially expressed genes is Representational Difference Analysis of cDNA (cDNA-RDA). cDNA-RDA is a PCR-based subtractive enrichment procedure. Originally developed for the identification of differences between complex genomes it has been adapted to enable isolation of genes with altered expression between various tissues or cell samples (Lisitsyn, N., and Wigler, M. Science 259:946-951; Hubank, M., and Schatz, D. G. Nucleic Acids Res. 22: 5640-5648; O'Neill, M. J., and Sinclair, A. H. Nucleic Acids Res. 25: 2681-2682). This technique offers several advantages including the isolation of few false positives, the fact that unwanted difference products can be competitively eliminated and genes producing rare transcripts can also be detected and isolated.
Identification of novel genes that are expressed in neutrophils under inflammatory conditions would provide an important opportunity for the understanding of the inflammatory conditions from which a number of clinically important applications would arise. Novel genes and proteins identified may lead to the development of therapeutics (small molecule drugs, antisense molecules, antibody molecules) directly targeted to the gene or protein product of the gene, or may target the biochemical pathway at an upstream or downstream location if the development of such drugs is easier than directly targeting the gene. Polynucleotide sequences comprising the gene and sequence variants thereof may be used to develop a clinical diagnostic test for inflammatory conditions. Finally, information about the DNA sequences of the novel genes involved in inflammatory conditions and the amino acid sequences encoded by these genes facilitate large scale production of proteins by recombinant techniques and identification of the tissues and cells naturally producing the proteins. Such sequence information also permits the preparation of antibody substances or other novel binding molecules specifically reactive with the proteins encoded by the novel genes that may be used in modulating the natural ligand/antiligand binding reactions in which the proteins are involved.
Accordingly, the present invention provides, in one aspect, an isolated polynucleotide, hereinafter alternatively referred to as EXP189, comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:19 or SEQ ID NO:20 or a functionally equivalent variant of said amino acid sequence, i.e. a variant thereof which retains the biological or other functional activity thereof, e.g. a variant which is capable of raising an antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:19 or SEQ ID NO:20.
Terms used herein have the following meanings:
“Isolated” refers to material removed from its original environment.
“Hybridization” or “hybridizes” refers to any process by which a strand of a polynucleotide binds with a complementary strand through base pairing.
“Stringent conditions” refer to experimental conditions which allow up to 20% base pair mismatches, typically two 15 minute washes in 0.1 XSSC (15 mM NaCl, 1.5 mM sodium citrate, pH 7.0) at 65° C.
“Homology” or “homologous” refers to a degree of similarity between nucleotide or amino acid sequences, which may be partial or, when sequenc

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