WO2000006206A1 - Compositions and methods for the treatment and diagnosis of cardiovascular disease - Google Patents

Abstract

The present invention relates to methods and compositions for the treatment and diagnosis of cardiovascular disease, including, but not limited to, atherosclerosis, ischemia/reperfusion, hypertension, restenosis, and arterial inflammation. Specifically, the present invention identifies and describes genes which are differentially expressed in cardiovascular disease states, relative to their expression in normal, or non-cardiovascular disease states, and/or in response to manipulations relevant to cardiovascular disease. Further, the present invention identifies and describes genes via the ability of their gene products to interact with gene products involved in cardiovascular disease. Still further, the present invention provides methods for the identification and therapeutic use of compounds as treatments of cardiovascular disease. Moreover, the present invention provides methods for the diagnostic monitoring of patients undergoing clinical evaluation for the treatment of cardiovascular disease, and for monitoring the efficicacy of compounds in clinical trials. Additionally, the present invention describes methods for the diagnostic evaluation and prognosis of various cardiovascular diseases, and for the identification of subjects exhibiting a predisposition to such conditions.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT AND DIAGNOSIS OF CARDIOVASCULAR DISEASE

This application is a continuation-in-part of application Serial No. 08/870,434 filed June 6, 1997, which is a continuation-in-part of application Serial No.

08/799,910, filed February 13, 1997, which claims the benefit under 35 U.S.C. §119 (e) of co-pending provisional Application

No. 60/011,787 filed February 16, 1996, each of which is incorporated herein by reference in its entirty.

INTRODUCTION

The present invention relates to methods and compositions for the treatment and diagnosis of cardiovascular disease, including, but not limited to, atherosclerosis, ischemia/reperfusion, hypertension, restenosis, and arterial inflammation. The present invention further relates to methods and compositions for the treatment and diagnosis of fibroprpliferative and oncogenic disorders, especially TGF-/?-related disorders, including diabetic retinopathy, artherosclerosis, angiogenesis, inflammation, fibrosis, tumor growth and vascularization. The present invention still further relates to screening methods to identify compositions for such therapeutic and diagnostic uses. Genes which are differentially expressed in cardiovascular or oncogenic disease states, relative to their expression in normal, or non-disease states are identified. Genes are also identified via the ability of their gene products to interact with other gene products involved in cardiovascular or oncogenic disease. The genes identified may be used diagnostically or as targets for therapeutic intervention. Additionally, methods are provided for the diagnostic monitoring of patients undergoing clinical evaluation for the treatment of cardiovascular, fibroproliferative or oncogenic related disorders, for monitoring the efficacy of compounds in clinical trials, and for identifying subjects who may be predisposed to such disorders. 2 . BACKGROUND OF THE INVENTION

Cardiovascular disease is a major health risk throughout the industrialized world. Atherosclerosis, the most prevalent of cardiovascular diseases, is the principal cause of heart attack, stroke, and gangrene of the extremities, and thereby the principal cause of death in the United States. Atherosclerosis is a complex disease involving many cell types and molecular factors (for a detailed review, .see Ross, 1993, Nature 362: 801-809). The process, In normal circumstances a protective response to insults to the endothelium and smooth muscle cells (SMCs) of the wall of the artery, consists of the formation of fibrofatty and fibrous lesions or plaques, preceded and accompanied by inflammation. The advanced lesions of atherosclerosis may occlude the artery concerned, and result from an excessive inflammatory- fibroproliferative response to numerous different forms of insult. For example, shear stresses are thought to be responsible for the frequent occurrence of atherosclerotic plaques in regions of the circulatory system where turbulent blood flow occurs, such as branch points and irregular structures.

The first observable event in the formation of an atherosclerotic plaque occurs when blood-borne monocytes adhere to the vascular endothelial layer and transmigrate through to the sub-endothelial space. Adjacent endothelial cells at the same time produce oxidized low density lipoprotein (LDL) . These oxidized LDL's are then taken up in large amounts by the monocytes through scavenger receptors expressed on their surfaces. In contrast to the regulated pathway by which native LDL (nLDL) is taken up by nLDL specific receptors, the scavenger pathway of uptake is not regulated by the monocytes.

These lipid-filled monocytes are called foam cells, and are the major constituent of the fatty streak. Interactions between foam cells and the endothelial and SMCs which surround them lead to a state of chronic local inflammation which can eventually lead to smooth muscle cell proliferation and migration, and the formation of a fibrous plaque. Such plaques occlude the blood vessel concerned- and thus restrict the flow of blood, resulting in ischemia.

Ischemia is a condition characterized by a lack of oxygen supply in tissues of organs due to inadequate perfusion. Such inadequate perfusion cεn have number of natural causes, including atherosclerotic or restenotic lesions, anemia, or stroke, to name a few. Many medical interventions, such as the interruption o the flow of blood during bypass surgery, for example, also lead to ischemia. In addition to sometimes being caused by diseased cardiovascular tissue, ischemia may sometimes affect cardiovascular tissue, such as in ischemic heart disease. Ischemia may occur in any organ, however, that is suffering a lack of oxygen supply.

The most common cause of ischemia in the heart is atherosclerotic disease of epicardial coronary arteries. By reducing the lumen of these vessels, atherosclerosis causes an absolute decrease in myocardial perfusion in the basal state or limits appropriate increases in perfusion when the demand for flow is augmented. Coronary blood flow can also be limited by arterial thrombi, spasm, and, rarely, coronary emboli, as well as by ostial narrowing due to luetic aortitis. Congenital abnormalities, such as anomalous origin of the left anterior descending coronary artery from the pulmonary artery, may cause myocardial ischemia and infarction in infancy, but this cause is very rare in adults. Myocardial ischemia can also occur if myocardial oxygen demands are abnormally increased, as in severe ventricular hypertrophy due to hypertension or aortic stenosis. The latter can be present with angina that is indistinguishable from that caused by coronary atherosclerosis. A reduction in the oxygen-carrying capacity of the blood, as in extremely severe anemia or in the presence of carboxy-hemoglobin, is a rare cause of myocardial ischemia. Not infrequently, two or more causes of ischemia will coexist, such as an increase in oxygen demand due to left ventricular hypertrophy and a reduction in oxygen supply secondary to coronary atherosclerosis .

The principal surgical approaches to the treatment of ischemic atherosclerosis are bypass grafting, endarterectomy, and percutaneous translumenal angioplasty (PCTA) . The failure rate after these approaches due to restenosis, in which the occlusions recur and often become even worse, is extraordinarily high (30-50%) . It appears that much of the restenosis is due to further inflammation, smooth muscle accumulation, and thrombosir.

A moαified balloon angioplasty approach was used to treat arterial restenosis in pigs by gene therapy (Ohno et al., 1994, Science 265: 781-784). A specialized catheter was used to introduce a recombinant adenovirus carrying the gene encoding thymidine kinase (tk) into the cells at the site of arterial blockage. Subsequently, the pigs were treated with ganciclovir, a nucleoside analog which is converted by tk into a toxic form which kills cells when incorporated into DNA. Treated animals had a 50% to 90% reduction in arterial wall thickening without any observed local or systemic toxicities.

Because of the presumed role of the excessive inflammatory-fibroproliferative response in atherosclerosis and ischemia, a number of researchers have investigated, in the context of arterial injury, the expression of certain factors involved in inflammation, cell recruitment and proliferation. These factors include growth factors, cytokines, and other chemicals, including lipids involved in cell recruitment and migration, cell proliferation and the control of lipid and protein synthesis.

For example, the expression of PDGF (platelet derived growth factor) or its receptor was studied: in rats during repair of arterial injury (Majesky et al., 1990, J. Cell Biol. Ill: 2149) ; in adherent cultures of human monocyte- derived macrophages treated with oxidized LDL (Maiden et al., 1991, J. Biol. Chem. 266: 13901) ; and in bovine aortic endothelial cells subjected to fluid shear stress (Resnick et al., 1993, Proc. Natl. Acad. Sci. USA 90: 4591-4595). Expression of IGF-I (insulin-like growth factor-I) was studied after balloon deendothelialization of rat aorta (Cercek et al., 1990, Circulation Research 66: 1755-1760). Other studies have focused on the expression of adhesion-molecules on the surface of activated endothelial ceiτ«s which mediate monocyte adhesion. These adhesic. molecules include intracellular adhesion molecule-1, ICAM-1 (Simmons et al., 1988, Nature, 331: 624-627), ELAM (Bevilacqua et al., 1989, Science 243: 1160-1165; Bevilacqua et al., 1991, Cell 67: 233), and vascular cell adhesion molecule, VCAM-1 (Osborn et al., 1989, Cell 59: 1203-1211); all of these surface molecules are induced transcriptionally in the presence of IL-1. Histological studies reveal that ICAM-1, EL'VM and VCAM-1 are expressed on endothelial cells in areas of lesion formation in vivo (Cybulsky et al., 1991, Science 251: 788-791; 1991, Arterioscler. Thromb. 11: 1397a; Poston et al., 1992, Am. J. Pathol. 140: 665-673). VCAM-1 and ICAM-1 were shown to be induced in cultured rabbit arterial endothelium, as well as in cultured human iliac artery endothelial cells by lysophophatidylcholine, a major phospholipid component of atherogenic lipoproteins. (Ku e et al., 1992, J. Clin. Invest. 90: 1138-1144). VCAM-I, ICAM-1, and class II major histocompatibility antigens were reported to be induced in response to injury to rabbit aorta (Tanaka, et al., 1993, Circulation 88: 1788-1803).

Cytomegalovirus (CMV) has been implicated in restenosis as well as atherosclerosis in general (Speir, et al., 1994, Science 265: 391-394). It was observed that the CMV protein IE84 apparently predisposes smooth muscle cells to increased growth at the site of restenosis by combining with and inactivating p53 protein, which is known to suppress tumors in its active form.

The foregoing studies are aimed at defining the role of particular gene products presumed to be involved in the excessive inflammatory-fibroproliferative response leading to atherosclerotic plaque formation. However, such approaches cannot identify the full panoply of gene products that are involved in the disease process, much less id intify those which may serve as therapeutic targets for the diagnosis and treatment of various forms of cardiovascular disease.

3. SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for the treatment and diagnosis of cardiovascular disease, including but not limited to, atherosclerosis, ischemia/reperfusion, hypertension, restenosis, and arterial inflammation. Specifically, genes are identified and described which are differentially expressed in cardiovascular disease states, relative to their expression in normal, or non-cardiovascular disease states. The present invention further relates to screening methods to identify compositions which can be utilized as part of such diagnostic therapeutic uses.

The present invention further relates to methods and compositions for the treatment and diagnosis of fibroproliferative and oncogenic disorders, especially TGF-/S- related disorders, including diabetic retinopathy, artherosclerosiε, angiogenesis, inflammation, fibrosis, tumor growth and vascularization. Specifically, genes are identified and described which are differentially expressed in oncogenic disease states, relative to their expression in normal, or non-oncogenic disease states. The present invention still further relates to screening methods to identify compositions involved in activation or enhancement of the TGF-β signaling pathway and their diagnostic and therapeutic uses for such disorders.

The term "activation" as used herein shall represent any alteration of a signaling pathway or biological response including, for example, increases above basal levels, restoration to basal levels from an inhibited state, and stimulation of the pathway above basal levels.

"Differential expression", as used herein, refers to both quantitative as well as qualitative differences in the genes' temporal and/or tissue expression patterns. Differentially expressed genes may represent "fingerprint genes," and/or "target genes." "Fingerprint gene," as used herein, refers to a differentially expressed gene whose expression pattern may be utilized as part of a prognostic or diagnostic cardiovascular disease evaluation, or which, alternatively, may be used in methods for identifying compounds useful for the treatment of cardiovascular disease. "Target gene", as used herein, refers to a differentially expressed gene involved in cardiovascular disease such that modulation of the level of target gene expression or of target gene product activity may act to ameliorate a cardiovascular disease condition. Compounds that modulate target gene expression or activity of the target gene product can be used in the treatment of cardiovascular disease.

Further, "pathway genes" are defined via the ability of their products to interact with other gene products involved in cardiovascular disease. Pathway genes may also exhibit target gene and/or fingerprint gene characteristics. Although the genes described herein may be differentially expressed with respect to cardiovascular disease, and/or their products may interact with gene products important to cardiovascular disease, the genes may also be involved in mechanisms important to additional cardiovascular processes. The invention includes the products of such fingerprint, target, and pathway genes, as well as antibodies to such gene products. Furthermore, the engineering and use of cell- and animal-based models of cardiovascular disease to which such gene products may contribute are also described. The present invention encompasses methods for prognostic and diagnostic evaluation of cardiovascular, fibroproliferative and oncogenic related disease conditions, and for the identification of subjects exhibiting a predisposition to such conditions. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of cardiovascular, fibroproliferative and oncogenic related diseases.

The invention also provides methods for the identification of compounds that modulate the expression of genes or the activity of gene products involved in cardiovascular disease, as well as methods for the treatment of cardiovascular disease which may involve the administration of such compounds to individuals exhibiting cardiovascular disease symptoms or tendencies. The invention also provides methods for the identification of compounds that modulate the expression of genes or the activity of gene products involved in fibroproliferative or oncogenic disorders, including tumorigenesis and the vascularization of tumors. The invention is based, in part, on systematic search strategies involving in vivo and in vitro cardiovascular disease paradigms coupled with sensitive and high throughput gene expression assays. In contrast to approaches that merely evaluate the expression of a given gene product presumed to play a role in a disease process, the search strategies and assays used herein permit the identification of all genes, whether known or novel, that are expressed or repressed in the disease condition, as well as the evaluation of their temporal regulation and function during disease progression. This comprehensive approach and evaluation permits the discovery of novel genes and gene products, as well as the identification of an array of genes and gene products (whether novel or known) involved in novel pathways that play a major role in the disease pathology. Thus, the invention allows one to define targets useful for diagnosis, monitoring, rational drug screening and design, and/or other therapeutic intervention.

In the working examples described herein, five novel human genes are identified that are demonstrated to be differentially expressed in different cardiovascular disease states. The identification of these genes and the characterization of their expression in particular disease states provide newly identified roles in cardiovascular disease for these genes.

Specifically, fchd531, fchd540, and fchd545 are novel genes that are each differentially regulated in endothelial cells subjected to shear stress. fchd531 and fchd545 are each down-regulated, whereas fchd540 is up-regulated by shear stress. fchd602 and fchd605 are novel genes that are each up-regulated in monocytes treated with oxidized LDL. Accordingly, methods are provided for the .diagnosis, monitoring in clinical trials, screening for therapeutically effective compounds, and treatment of cardiovascular disease based upon the discoveries herein regarding the expression patterns of fchd531, fchd540, fchd545, fchd602, and fchd605. Both fchd540 and rchd534 are up-regulated in response to laminar shear stress and are specifically expressed in vascular tissue. fchd540 is also shown herein to be upregulated in oncogenic related disorders, such as pancreatic cancers. Further, overexpression of fchd540 expression constructs in pancreatic cells results in complete loss of the TGF-β response in such cells. These findings combined with the observations that both fchd540 and rchd534 specifically inhibit TGF-3 signalling and that these genes are located in an area of the human genome implicated in the pathogenesis of several human malignancies indicates that they are excellent and specific targets for therapeutic intervention in the treatment of fibroproliferative and oncogenic disorders including tumorigenesis and vascularization.

The characteristic up-regulation of genes fchd540, fchd602, and fchd605 can be used to design cardiovascular disease treatment strategies. For those up-regulated genes that have a causative effect on the disease conditions, treatment methods can be designed to reduce or eliminate their expression, particularly in endothelial cells or monocytes. Alternatively, treatment methods include inhibiting the activity of the protein products of these genes. For those up-regulated genes that have a protective effect, treatment nethods can be designed for enhancing the activity of the products of such genes.

In either situation, detecting expression of these genes in excess of normal expression provides for the diagnosis of cardiovascular disease. Furthermore, in testing the efficacy of compounds during clinical trials, a decrease in the level of the expression of these genes corresponds to a return from a disease condition to a normal state, and thereby indicates a positive effect of the compound. The cardiovascular diseases that may be so diaσnosed, monitored in clinical trials, arid treated include but are not limited to atherosclerosis, ischemia/reperfusion, hypertension, restenosis, and arterial inflammation.

The characteristic down-regulation of fchd531 and fchd545 can also be used to design cardiovascular disease treatment strategies. For those genes whose down-regulation has a pathogenic effect, treatment methods can be designed to restore or increase their expression, particularly in endothelial cells. Alternatively, treatment methods include increasing the activity of the protein products of these genes. For those genes whose down-regulation has a protective effect, treatment methods can be designed for decreasing the amount or activity of the products of such genes. In either situation, detecting expression of these genes in below normal expression provides for the diagnosis of cardiovascular disease. Furthermore, in testing the efficacy of compounds during clinical trials, an increase in the level of the expression of these genes corresponds to a return from a disease condition to a normal state, and thereby indicates a positive effect of the compound. The cardiovascular diseases that may be so diagnosed, monitored in clinical trials, and treated include but are not limited to atherosclerosis, ischemia/reperfusion, hypertension, restenosis, and arterial inflammation.

The invention encompasses methods for screening compounds and other substances for treating cardiovascular disease by assaying their ability to modulate the expression of the target genes disclosed herein or activity of the protein products of the target genes. The invention further encompasses methods for screening compounds and other substances for treating fibroproliferative disorders and oncogenic disorders by assaying their ability to modulate the expression of the target genes disclosed herein or a>. ity of the protein products of the target genes. Such screening methods include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the target gene protein products.

In addition, the invention encompasses methods for treating cardiovascular, fibroproliferative and oncogenic related diseases by administering compounds and other substances .that modulate the overall activity of the target gene products. Compounds and other substances can effect such modulation either on the level of target gene expression or target protein activity.

The invention is based in part on the identification of novel protein-protein interactions of the rchd534 protein with itself and with the fchd540 protein, as well as interactions of the rchd534 protein or the fchd540 protein with other protein members of the TGF-/3 signalling pathway. The rchd534 gene was described in Applicant's co-pending Application No. 08/485,573, filed June 7, 1995, which is hereby incorporated by reference in its entirety. Screening methods are provided for identifying compounds and other substances for treating cardiovascular disease by assaying their ability to inhibit these interactions. Furthermore, methods are provided for identifying compounds and other substances that enhance the TGF- response by modulating the activity or the expression of the rchd534 or fchd540 genes or the activity of their gene products, for the treatment of fibroproliferative and oncogenic disease. For example, an increase in activity or expression of fchd540 can contribute to proliferative and oncogenic disorders by inhibiting _the TGF-3 response. Therapeutic targets that activate or enhance the TGF-S response as mediated through, e.g., fchd540, rchd534 or other TGF-S signaling protein are ->f significant use. In addition, methods are provided for treating cardiovascular disease by administering compounds and other substances that inhibit these protein interactions.

The invention is based in part on the identification of the endothelial cell specific expression pattern of two genes, rchd534 and fchd540, whose protein products inhibit the TGF-/3 response. The_^fchd540 gene has been mapped to regions of the human genome that have been implicated in the pathogenesis of several human malignancies. The invention is further based on the finding that these genes and mutants thereof may be used to modulate TGF-/S induced signalling in endothelial cells. Accordingly, the rchd534 and rchd540 genes may be targets for intervention in a. variety of inflammatory and fibroproliferative disorders that involve endothelial cells, including, but not limited to, oncology related disorders, disorders related to vascularization, such as cancer angiogenesis, inflammation, and fibrosis. Membrane bound target gene products containing extracellular domains can be a particularly useful target for treatment methods as well as diagnostic and clinical monitoring methods. The fchd602 gene, for example, encodes a transmembrane protein, which contains multiple transmembrane domains and, therefore, can be readily contacted by other compounds on the cell surface. Accordingly, natural ligands, derivatives of natural ligands, and antibodies that bind to the fchd602 gene product can be utilized to inhibit its activity, or alternatively, to target the specific destruction of cells that express the gene. Furthermore, the extracellular domains of the fchd602 gene product provide targets which allow for the design of especially efficient screening systems for identifying compounds that bind to the fchd602 gene product. Such an assay system can also be used to screen and identify antagonists of the interaction between the fchd602 gene product and ligands that bind to the fchd602 gene product. For example, the compounds can act as decoys by binding to the endogenous (i.e., natural) ligand for the fchd602 gene product. The resulting reduction in the amount of ligand-bound fchd602 gene transmembrane protein will modulate the activity of disease state cells, such as monocytes. Soluble proteins or peptides, such as peptides comprising one or more of the extracellular domains, or portions and/or analogs thereof of the fchd602 gene product, including, for example, soluble fusion proteins such as Ig- tailed fusion proteins, can be particularly useful for this purpose.

Similarly, antibodies that are specific to one or more of the extracellular domains of the fchd602 product provide for the ready detection of this target gene product in diagnostic tests or in clinical test monitoring.

Accordingly, endothelial cells can be treated, either in vivo or in vitro, with such a labeled antibody to determine the disease state of endothelial cells. Because the fchd602 gene product is up-regulated in monocytes in the disease state, its detection positively corresponds with cardiovascular disease.

Such methods for treatment, diagnosis, and clinical test monitoring which use the fchd602 gene product as described above can also be applied to other target genes that encode transmembrane gene products, including but not limited to the fchd545 gene, which encodes multiple transmembrane domains and extracellular domains.

The examples presented in Sections 6 and 7, below, demonstrate the use of the cardiovascular disease paradigms of the invention to identify cardiovascular disease target genes.

The example presented in Section 8, below, demonstrates the use of fingerprint genes in diagnostics and as surrogate markers for testing the efficacy of candidate drugs in basic research and in clinical trials. The example presented in Section^, below, demonstrates the use of fingerprint genes, particularly fchd545, in the imaging of a diseased cardiovascular tissue.

The example presented in Section 11, below, demonstrates the interaction of two target gene products, the rchd534 and fchd540 proteins, and the further characterization of their roles in oncology, angiogenesiε, cardiovascular disease and the TGF-/3 signalling pathway.

The example presented in Section 13, below, demonstrates that fchd540 can be overexpressed in oncorgenic related disorders such as pancreatic cancers, and shows that overexpression of fchd540 constructs in pancreatic cell lines results in completely loss of the TGF-3 response.

4. DESCRIPTION OF THE FIGURES

FIG.1A-1D. Nucleotide sequence and encoded amino acid sequence of the fchd531 gene.

FIG.2A-2E. Nucleotide sequence and encoded amino acid sequence of the fchd540 gene. FIG.3A-3C. Nucleotide sequence and encoded amino acid sequence of the fchd545 gene.

FIG.4A-4B. Nucleotide sequence and encoded amino acid sequence from the fchd602 gene.

FIG.5A-5B. Nucleotide sequence and encoded amino acid sequence from the fchd605 gene.

FIG.6A-6D. Nucleotide sequence and encoded amino acid sequence of the rchd534 gene.

FIG.7A-7B. Effects of fchd540 on TGF-31 mediated growth inhibition. FIG.8A-8C. Anchorage independent growth of fchd540 transfected cell lines.

5. DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for the diagnosis and treatment of cardiovascular disease, including but not limited to atherosclerosis, ischemia/reperfusion, hypertension, restenosiε, and arterial inflammation, are described. Methods and compositions for the treatment of oncogenic related disorders, including tumorigenesis-and the vascularization of tumors, are also described. The invention is based, in part, on the evaluation of the expression and role of all genes that are differentially expressed in paradigms that are physiologically relevant to the disease condition. This permits the definition of disease pathways and the identification of targets in the pathway that are useful both diagnostically and therapeutically. Genes, termed "target genes" and/or "fingerprint genes" which are differentially expressed in cardiovascular disease conditions, relative to their expression in normal, or non- cardiovascular disease conditions, are described in Section 5.4. Additionally, genes, termed "pathway geneε" whose gene products exhibit an ability to interact with gene products involved in cardiovascular disease are also described in Section 5.4. Pathway genes may additionally have fingerprint and/or target gene characteristics. Methods for the identification of such fingerprint, target, and pathway genes are described in Sections 5.1, 5.2, and 5.3.

Further, the gene products of such fingerprint, target, and pathway genes are described in Section 5.4.2, antibodies to such gene products are described in Section 5.4.3, as are cell- and animal-based models of cardiovascular disease and oncogenic related disorders to which such gene products may contribute, in Section 5.4.4.

Methods for the identification of compounds which modulate the expression of genes or the activity of gene products involved in cardiovascular disease and fibroproliferative and oncogenic related disorders including tumorigenesis are described in Section 5.5. Methods for monitoring the efficacy of compounds during clinical trials are described in Section 5.5.4. Additionally described below, in Section 5.6, are methods for the treatment of cardiovascular disease and oncogenic related disorders.

Also discussed below, in Section 5.8, are methods for prognostic and diagnostic evaluation of cardiovascular disease, including the identification of subjects exhibiting a predisposition to this disease, and the imaging of cardiovascular disease conditions.

5.1. IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES

This section describes methods for the identification of genes which are involved in cardiovascular disease, including but not limited to atherosclerosis, ischemia/reperfusion, hypertension, restenosis, and arterial inflammation. Such genes may represent genes which are differentially expressed in cardiovascular disease conditions relative to their expression in normal, or non-cardiovascular disease conditions. Such differentially expressed genes may represent "target" and/or "fingerprint" genes. Methods for the identification of such differentially expressed genes are described, below, in this section. Methods for the further characterization of such differentially expressed genes, and for their identification as target and/or fingerprint genes, are presented, below, in Section 5.3. "Differential expression" as used herein refers to both quantitative as well as qualitative differences in the genes' temporal and/or tissue expression patterns. Thus, a differentially expressed gene may have its expression activated or completely inactivated in normal versus cardiovascular disease conditions (e.g. , treated with oxidized LDL versus untreated) , or under control versus experimental conditions. Such a qualitatively regulated gene will exhibit an expression pattern within a given tissue or cell type which is detectable in either control or cardiovascular disease subjects, but is not detectable in both. Alternatively, such a qualitatively regulated gene will exhibit an expression pattern within a given tissue or cell type which is detectable in either control or experimental subjects, but is not detectable in both. "Detectable", as used herein, refers to an RNA expression pattern which is detectable via the standard techniques of differential display, reverse transcriptase- (RT-) PCR and/or Northern analyses, which are well known to those of skill in the art.

Alternatively, a differentially expressed gene may have its expression modulated, i.e. , quantitatively increased or decreased, in normal versus cardiovascular disease states, or under control versus experimental conditions. The deπ-ee to whih expression differs in normal versus cardiovascular disease or control versus experimental states need only be large enough to be visualized via standard characterization techniques, such as, for example, the differential display technique described below. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to quantitative RT-PCR and Northern analyses. Differentially expressed genes may be further described as target genes and/or fingerprint genes. "Fingerprint gene," as used herein, refers to a differentially expressed gene whose expression pattern may be utilized as part of a prognostic or diagnostic cardiovascular disease evaluation, or which, alternatively, may be used in methods for identifying compoundε useful for the treatment of cardiovascular disease. A fingerprint gene may also have the characteristics of a target gene.

"Target gene", as used herein, refers to a differentially expressed gene involved in cardiovascular disease in a manner by which modulation of the level of target gene expression or of target gene product activity may act to ameliorate symptoms of cardiovascular disease. A target gene may also have the characteristics of a fingerprint gene.

A variety of methods may be utilized for the identification of genes which are involved in cardiovascular disease. These methods include but are not limited to the experimental paradigms described, below, in Section 5.1.1. Material from the paradigms may be characterized for the presence of differentially expressed gene sequences as discussed, below, in Section 5.1.2. 5.1.1. PARADIGMS FOR THE IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES

One strategy for identifying genes that are involved in cardiovarmilar disease is to detect genes that are expressed

₅ differentially under conditions associated with the disease versus non-disease conditions. The sub-sections below describe a number of experimental systemε, called paradigms, which may be used to detect such differentially expressed genes. In general, the paradigms include at least one experimental condition in which subjects or sa pleε are treated in a manner associated with cardiovascular disease, in addition to at least one experimental control condition lacking such disease aεsociated treatment. Differentially expressed genes are detected, as described herein, below, by

. comparing the pattern of gene expression between the experimental and control conditions.

Once a particular gene has been identified through the use of one such paradigm, its expression pattern may be further -characterized by studying its expression in a

_ different paradigm. A gene may, for example, be regulated one way in a given paradigm (e.g., up-regulation) , but may be regulated differently in some other paradigm (e.g., down- regulation) . Furthermore, while different genes may have similar expression patterns in one paradigm, their respective

₂_ expression patterns may differ from one another under a different paradigm. Such use of multiple paradigms may be useful in distinguishing the roles and relative importance of particular genes in cardiovascular disease.

30 5.1.1.1. FOAM CELL PARADIGM - 1 Among the paradigms which may be utilized for the identification of differentially expressed genes involved in atherosclerosis, for example, are paradigms designed to analyze those genes which may be involved in foam cell ₃₅ formation. Such paradigms may serve to identify genes involved in the differentiation of this cell type, or their uptake of oxidized LDL. One embodiment of such a paradigm, hereinafter referred to as Paradigm A, is carried out as follows: First, human blood is drawn and peripheral onocyteε are isolated by methods routinely practiced in the art. These human monocyteε can then be used immediately or cultured in vitro, using methods routinely practiced in the art, for 5 to 9 days where they develop more macrophage-like characteristics such as the up-regulation of scavenger receptors. These cellε are then treated for variouε lengths of time with agents thought to be involved in foam cell formation. Theεe agentε include but are not limited to oxidized LDL, acetylated LDL, lysophosphatidylcholine, and homocysteine. Control monocytes that are untreated or treated with native LDL are grown in parallel. At a certain time after addition of the test agents, the cellε are harveεted and analyzed for differential expreεεion as described in detail in Section 5.1.2., below. The Example presented in Section 6, below, demonstrates in detail the use of such a ^"foam cell paradigm to identify genes which are differentially expresεed in treated versus control cells.

5.1.1.2. FOAM CELL PARADIGM - 2 Alternative paradigms involving monocytes for detecting differentially expresεed geneε aεεociated with atheroεclerosis involve the simulation of the phenomenon of transmigration. When monocytes encounter arterial injury, they adhere to the vascular endothelial layer, transmigrate acrosε thiε layer, and locate between the endothelium and the layer of smooth muscle cellε that ring the artery. This phenomenon can be mimicked in vitro by culturing a layer of endothelial cells isolated, for example, from human umbilical cord. Once the endothelial monolayer forms, monocytes drawn from peripheral blood are cultured on top of the endothelium in the presence and absence of LDL. After several hours, the monocytes transmigrate through the endothelium and develop into foam cells after 3 to 5 days when exposed to LDL. In this εystem, as in vivo, the endothelial cells carry out the oxidation of LDL which is then taken up by the monocytes. As described in sub-section 5.1.2. below, the pattern of gene expresεion can then be compared between theεe foam cellε and untreated monocytes.

5.1.1.3. FOAM CELL PARADIGM - 3

Yet another εyεtem includes the third cell type, smooth muscle cell, that plays a critical role in atherogeaesis (Navab et al., 1988, J. Clin. Invest., 82: 1853). In this εyεtem, a multilayer of human aortic εmooth muεcle cellε waε grown on a micropore filter covered with a gel layer of native collagen, and a monolayer of human aortic endothelial cellε waε grown on top of the collagen layer. Expoεure of thiε coculture to human monocyteε in the preεence of chemotactic factor rFMLP reεulted in monocyte attachment to the endothelial cellε followed by migration across the endothelial monolayer into the collagen layer of the subendothelial εpace. Thiε type of culture can also be treated with LDL to generate foam cells. The foam cells can then be harveεted and their pattern of gene expreεεion compared to that of untreated cells as explained below in sub-section 5.1.2.

5.1.1.4. IN VIVO MONOCYTE PARADIGM An alternative embodiment of such paradigms for the study of monocytes, hereinafter referred to as Paradigm B, involves differential treatment of human subjects through the dietary control of lipid consumption. Such human subjects are held on a low fat/low cholesterol diet for three weeks, at which time blood is drawn, monocyteε are iεolated according to the methods routinely practiced in the art, and RNA is purified, as deεcribed below, in sub-section 5.1.2. These same patients are εubsequently switched to a high fat /high cholesterol diet and monocyte RNA is purified again. The patients may also be fed a third, combination diet containing high fat/low choleεterol and monocyte RNA may be purified once again. The order in which patientε receive the d.'.ets may be varied. The RNA derived from patients maintained on two of the dietε, or on all three dietε, may then be compared and analyzed for differential gene expreεεion as, explained below in εub-εection 5.1.2.

5.1.1.5. ENDOTHELIAL CELL - IL-1 PARADIGM In addition to the detection of differential gene expreεεion in monocyteε, paradigmε focuεing on endothelial cellε may be uεed to detect genes involved in cardiovaεcular disease. In one such paradigm, hereinafter referred to as Paradigm C, human umbilical vein endothelial cells (HUVECε) are grown in vitro. Experimental cultures are treated with human "ΪL-lS, a factor known to be involved in the inflammatory responεe, in order to mimic the phyεiologic conditions involved in the atherosclerotic state.

Alternatively experimental HUVEC cultures may be treated with lysophosphatidylcholine, a major phospholipid component of atherogenic lipoproteinε or oxidized human LDL. Control cultures are grown in the absence of these compounds. After a certain period of treatment, experimental and control cells are harvested and analyzed for differential gene expreεεion as described in εub-section 5.1.2, below.

5.1.1.6. ENDOTHELIAL CELL - SHEAR STRESS PARADIGM

In another paradigm involving endothelial cells, hereinafter referred to as Paradigm D, cultures are exposed to fluid εhear stresε which iε thought to be responsible for the prevalence of atherosclerotic leεionε in areas of unuεual circulatory flow. Unusual blood flow also plays a role in the harmful effects of ischemia/reperfuεion, wherein an organ receiving inadequate blood supply is suddenly reperfused with an overabundance of blood when the obstruction iε overcome. Cultured HUVEC monolayers are expoεed to laminar shear stresε by rotating the culture in a specialized apparatus containing liquid culture medium (Nagel et al., 1994, J. Clin. Invest. 94: 885-891). Static cultures grown in the same medium serve as controls. After a certain period of exposure to shear streεε, experimental and control cellε are harveεted and analyzed for differential gene expreεεion aε deεcribed in εub-εection 5.1.2, below. The Example preεented in Section 7, below, demonεtrates the use of εuch a shear streεεed endothelial cell paradigm to identify sequences which are differentially expresεed in expoεed versus control cells. In all such paradigms caεigned to identify geneε which are involved in cardiovaεcular disease, including but not limited to those deεcribed above in Sections 5.1.1.1 through 5.1.1.6, compoundε εuch as drugs known to have an ameliorative effect on the disease symptomε may be incorporated into the experimental syεtem. Such compounds may include known therapeutics, as well as compounds that are not useful as therapeuticε due to their harmful εide effectε. Teεt cells that are cultured aε explained in the paradigmε deεcribed in Sectionε 5.1.1.1 through 5.1.1.6, for example, may be expoεed to one of these compounds and analyzed for differential gene expression with respect to untreated cells, according to the methods described below in Section 5.1.2. In principle, according to the particular paradigm, any cell type involved in the disease may be treated at any stage of the disease procesε by these compounds.

Test cells may also be compared to unrelated cells (e.g., fibroblaεtε) that are also treated with the compound, in order to screen out generic effects on gene expression that might not be related to the disease. Such generic effects might be manifest by changes in gene expression that are common to the test cells and the unrelated cells upon treatment with the compound.

By these methodε, the geneε and gene productε upon which theεe compounds act can be identified and used in the asεayε described below to identify novel therapeutic compounds for the treatment of cardiovascular diεease. 5.1.2. ANALYSIS OF PARADIGM MATERIAL In order to identify differentially expreεεed geneε, RNA, either total or mRNA, may be isolated from one or more tisεueε of the εubjects utilized in paradigms such as those described earlier in thiε Section. RNA samples are obtained from tissues of experimental εubjects and from corresponding tissues of control εubjectε. Any RNA iεolation techi αe which doeε not εelect against the isolation of mRNA may be utilized for the purification of εuch RNA εampleε. See, for example, Sambrook et al. , 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Preεε, N.Y.; and Auεubel, F.M. et al. , edε., 1987-1993, Current Protocols in Molecular Biology, John Wiley & Sonε, Inc. New York, both of which are incorporated herein by reference in their entirety. Additionally, large numberε of tiεεue εamples may readily be procesεed uεing techniqueε well known to thoεe of skill in the art, such aε, for example, the single-step RNA isolation process of Chomczynski, P. (1989, U.S. Patent No. 4,843,155), which is incorporated herein by reference in itε entirety. Tranεcriptε within the collected RNA εampleε which repreεent RNA produced by differentially expreεεed geneε may be identified by utilizing a variety of methodε which are well known to thoεe of εkill in the art. For example, differential εcreening (Tedder, T.F. et al., 1988, Proc. Natl. Acad. Sci. USA 85:208-212), subtractive hybridization (Hedrick, S.M. et al. , 1984, Nature 308:149-153; Lee, S.W. et al., 1984, Proc. Natl. Acad. Sci. USA 88:2825), and, preferably, differential display (Liang, P., and Pardee, A.B., 1993, U.S. Patent No. 5,262,311, which is incorporated herein by reference in its entirety) , may be utilized to identify nucleic acid sequences derived from genes that are differentially expreεsed.

Differential εcreening involves the duplicate screening of a cDNA library in which one copy of the library is screened with a total cell cDNA probe corresponding to the mRNA population of one cell type while a duplicate copy of the cDNA library iε εcreened with a total cDNA probe correεponding to the mRNA population of a εecond cell type. For example, one cDNA probe may correεpond to a total cell cDNA probe of a cell type derived from a control εubject, while the second cDNA probe may correspond to a total cell cDNA probe of the same cell type derived from an experimental subject. Those clones which hybridize to one probe but not to the other potentially repreεent clones derived from geneε differentially expreεεed in the cell type of interest in control verεuε experimental εubjects. Subtractive hybridization techniques generally involve the iεolation of mRNA taken from two different εourceε, e.g. _f control and experimental tiεsue, the hybridization of the mRNA or εingle-εtranded cDNA reverse-transcribed from the isolated mRNA, and the removal of all hybridized, and therefore double-stranded, εequenceε. The_^remaining non- hybridized, εingle-εtranded cDNAε, potentially repreεent cloneε derived from geneε that are differentially expreεεed in the two mRNA εources. Such single-stranded cDNAs are then uεed aε the starting material for the construction of a library compriεing cloneε derived from differentially expressed genes.

The differential display technique describeε a procedure, utilizing the well known polymeraεe chain reaction (PCR; the experimental embodiment set forth in Mullis, K.B., 1987, U.S. Patent No. 4,683,202) which allows for the identification of sequences derived from genes which are differentially expressed. First, isolated RNA is reverse- transcribed into single-stranded cDNA, utilizing standard techniques which are well known to those of skill in the art. Primers for the reverse transcriptase reaction may include, but are not limited to, oligo dT-containing primers, preferably of the reverse primer type of oligonucleotide described below. Next, this technique uses pairs of PCR primers, as described below, which allow for the amplification of clones representing a random subset of the RNA transcriptε present within any given cell. Utilizing different pairs of primers allowε each of the mRNA transcripts present in a cell to be amplified. Among such amplified transcriptε may be identified thoεe which have been produced from differentially expreεεed geneε.

The reverεe oligonucleotide primer of the primer pairs may contain an oligo dT stretch of nucleotideε, preferably eleven nucleotides long, at its 5' end, which hybridizes to the poly (A) tail of mRNA or to the complement of a cDNA reverse transcribed from an mRNA poly(A) tail. Second, in order to increase the specificity of the reverse primer, the primer may contain one or more, preferably two, additional nucleotideε at itε 3' end. Becauεe, statiεtically, only a εubεet of the mRNA derived εequences present in the sample of interest will hybridize to such primerε, the additional nucleotides allow the primers to amplify only a subset of the mRNA derived sequenceε present in the sample of interest. This is preferred in that it allows more accurate and complete visualization and characterization of each of the bands representing amplified sequences.

The forward primer may contain a nucleotide sequence expected, statistically, to have the ability to hybridize to cDNA sequenceε derived from the tiεsueε of interest. The nucleotide sequence may be an arbitrary one, and the length of the forward oligonucleotide primer may range from about 9 to about 13 nucleotides, with about 10 nucleotides being preferred. Arbitrary primer sequences cause the lengths of the amplified partial cDNAs produced to be variable, thus allowing different clones to be separated by using standard denaturing sequencing gel electrophoresis. PCR reaction conditions should be chosen which optimize amplified product yield and specificity, and, additionally, produce amplified products of lengthε which may be reεolved utilizing standard gel electrophoresis techniques. Such reaction conditions are well known to those of skill in the art, and important reaction parameters include, for example, length and nucleotide sequence of oligonucleotide primers as discuεsed above, and annealing and elongation step temperatures and reaction times. The pattern of clones resulting from the reverse transcription and amplification of the mRNA of two different cell types is displayed via sequencing gel electrophoresis and compared. Differences in the two banding patterns 5 indicate potentially differentially expressed genes.

Once potentially differentially expreεεed gene εequences have been identified via bulk techniques εuch aε, for example, thoεe deεcribed above, the differential expreεεion of εuch putatively differentially expresεed genes should be 0 corroborated. Corroboration may be accomplished via, for example, such well known techniques as Northern analysis and/or RT-PCR.

Upon corroboration, the differentially expresεed geneε may be further characterized, and may be identified aε target § and/or fingerprint genes, as discusεed, below, in Section 5.3.

Alεo, amplified εequenceε of differentially expreεεed geneε obtained through, for example, differential display may be used to iεolate full length cloneε of the correεponding 0 gene. The full length coding portion of the gene may readily be iεolated, without undue experimentation, by molecular biological techniqueε well known in the art. For example, the iεolated differentially expreεεed amplified fragment may be labeled and uεed to screen a cDNA library. Alternatively, 5 the labeled fragment may be used to screen a genomic library. PCR technology may also be utilized to isolate full length cDNA εequences. As described, above, in this Section, the isolated, amplified gene fragments obtained through differential display have 5' terminal ends at some random 0 point within the gene and have 3' terminal ends at a position preferably corresponding to the 3 ' end of the transcribed portion of the gene. Once nucleotide sequence information from an amplified fragment is obtained, the remainder of the gene (i.e.. the 5' end of the gene, when utilizing 5 differential diεplay) may be obtained uεing, for example, RT- PCR. In one embodiment of such a procedure for the identification and cloning of full length gene sequenceε, RNA may be iεolated, following εtandard procedureε, from an appropriate tiεεue or cellular source. A reverse tranεcription reaction may then be performed on the RNA using an oligonucleotide primer complimentary to the mRNA that corresponds to the amplified fragment, for the priming of first strand synthesis. Because the primer is anti-parallel to the mRNA, extension will proceed toward the 5' end of the mRNA. The resulting RNA/DNA hybrid may then be "tailed" with guanineε uεing a standard terminal tranεferaεe reaction, the hybrid may be digeεted with RNAaεe H, and εecond strand synthesis may then be primed with a poly-C primer. Using the two primerε, the 5' portion of the gene iε amplified uεing PCR. Sequences obtained may then be isolated and recombined with previously isolated sequenceε to generate a full-length cDNA of the differentially expressed genes of the invention. For a review of cloning strategies and recombinant DNA techniques, see e.g. , Sambrook et al., 1989, supra; and Auεubel et al. , 1989, supra .

5.2. IDENTIFICATION OF PATHWAY GENES

Thiε εection deεcribeε methodε for the identification of geneε, termed "pathway geneε", involved in cardiovaεcular diεease. "Pathway gene", aε uεed herein, referε to a gene whoεe gene product exhibitε the ability to interact with gene productε involved in cardiovaεcular diεease. A pathway gene may be differentially expressed and, therefore, may additionally have the characteristicε of a target and/or fingerprint gene.

Any method εuitable for detecting protein-protein interactionε may be employed for identifying pathway gene productε by identifying interactionε between gene products and gene products known to be involved in cardiovascular disease. Such known gene products may be cellular or extracellular proteins. Those gene products which interact with εuch known gene products repreεent pathway gene products and the genes which encode them represent pathway genes.

Among the traditional methodε which may be employed are co-immunoprecipitation, croεslinking and co-purification through gradients or chromatographic columnε. Utilizing procedureε εuch aε theεe allows for the identification of pathway gene products. Once identified, a pathway gene

_* product may be used, in conjunction with standard techniques, to identify itε correεponding pathway gene. For example, at leaεt a portion of the amine acid εequence of the pathway gene product may be aεcertained using techniques well known to those of εkill in the art, such as via the Edman degradation technique (see, e.g. , Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., pp.34-49). The amino acid εequence obtained may be uεed as a guide for the generation of oligonucleotide mixtures that can be used to screen for pathway gene sequences. Screening may be accomplished, for example by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and screening are well-known. (See, e.g. , Ausubel, supra . , and PCR Protocols: A Guide to Methods and Applications, 1990, Innis, M. et al., eds . Academic Press, Inc. , New York) .

Additionally, methods may be employed which reεult in the εimultaneouε identification of pathway geneε which encode the protein interacting with a protein involved in cardiovaεcular disease. These methods include, for example, probing expression libraries with labeled protein known or suggeεted to be involved in cardiovascular diεease, using this protein in a manner similar to the well known technique of antibody probing of λgtll librarieε.

One such method which detects protein interactions in vivo , the two-hybrid εystem, is described in detail for illustration only and not by way of limitation. One version of thiε εyεtem haε been deεcribed (Chien et al. , 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and iε commercially available from Clontech (Palo Alto, CA) . Briefly, utilizing such a syεtem, plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to a known protein, and the other consistε of the activator protein'ε activation domain fuεed to an unknown protein that iε encoded by a cDNA which has been recombined into thiε plaεmid aε part of a cDNA library. The pla._^..idε are tranεformed into a εtrain of the yeaεt Saccharomyceε cereviεiae that contains a reporter gene (e.g., lacZ) whose regulatory region contains the activator's binding siteε. Either hybrid protein alone cannot activate transcription of the reporter gene; the DNA-binding domain hybrid, because it does not provide activation function and the activation domain hybrid, becauεe it cannot localize to the activator'ε binding εi"ieε. Interaction of the two proteinε reconεtituteε the functional activator protein and resultε in expreεεion of the reporter gene, which iε detected by an aεεay for the reporter gene product.

The two-hybrid εyεtem or related methodology may be used to screen activation domain libraries for proteins that interact with a known "bait" gene protein. Total genomic or cDNA sequenceε may be fuεed to the DNA encoding an activation domain. Such a library and a plasmid encoding a hybrid of the bait gene protein fused to the DNA-binding domain may be cotransformed into a yeast reporter strain, and the resulting transformantε may be screened for thoεe that expreεε the reporter gene. These colonieε may be purified and the library plasmids responsible for reporter gene expression may be isolated. DNA sequencing may then be used to identify the proteins encoded by the library plasmids.

For example, and not by way of limitation, the bait gene may be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. Also by way of example, for the isolation of geneε involved in cardiovaεcular diεease, previously isolated genes known or suggested to play a part in cardiovascular disease may be used as the bait genes. These include but are not limited to the geneε for bFGF, IGF-I, VEGF, IL-1, M-CSF, TGF/3,.TGF , TNFα, HB-EGF, PDGF, IFN-γ, and GM-CSF, to name a few.

A cLiNA library of the cell line from which proteinε that interact with bait gene are to be detected can be made uεing methodε routinely practiced in the art. According to the particular εyεtem deεcribed herein, for example, the cDNA fragments may be inεerted into a vector εuch that they are tranεlationally fuεed to_^the activation domain of GAL4. Thiε library may be co-tranεformed along with the bait gene-GAL4 fusion plaεmid into a yeaεt εtrain which contains a lacZ gene driven by a promoter which containε the GAL4 activation εequence. A cDNA encoded protein, fuεed to the GAL4 activation domain, that interactε with bait gene will reconεtitute an active GAL4 protein and thereby drive expreεεion of the lacZ gene. Colonieε which expreεε lacZ may be detected by their blue color in the presence of X-gal. The cDNA may then be purified from these strainε, and uεed to produce and iεolate the bait gene-interacting protein uεing techniqueε routinely practiced in the art.

Once a pathway gene haε been identified and isolated, it may be further characterized as, for example, discusεed below, in Section 5.3.

A preferred embodiment of the use of the yeaεt two- hybrid syεtem iε deεcribed in detail in the example in Section 12, below. Aε described in Section 12, the yeast two-hybrid system was used to detect the interaction between the protein products of two target genes, rchd53 and fchd540.

5.3. CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED AND PATHWAY GENES

Differentially expreεεed geneε, such aε thoεe identified via the methodε discuεεed, above, in Section 5.1.1, pathway geneε, εuch as thoεe identified via the methods diεcussed, above, in Section 5.2, aε well aε geneε identified by alternative means, may be further characterized by utilizing, for example, methods εuch as thoεe diεcuεsed herein. Such genes will be referred to herein as "identified geneε". Analyεeε εuch as thoεe deεcribed herein will yield information regarding the biological function of the identified geneε. An aεεessment of the biological function of the differentially expreεsed genes, in addition, will allow for their designation as target and/or fingerprint genes. Specifically, any of the differentially expreεεed genes whose further characterization indicates that a modulation of the gene's expresεion or a modulation of the gene product'ε activity may ameliorate cardiovaεcular disease will be designated "target genes", as defined, above, in Section 5.1. Such target genes and target gene products, along with thoεe diεcuεεed below, will constitute the focus of the compound discovery strategieε diεcussed, below, in Section 5,5.

Any of the differentially expresεed genes whoεe further characterization indicateε that such modulations may not positively affect cardiovascular diεeaεe, but whoεe expreεεion pattern contributeε to a gene expreεεion "fingerprint pattern" correlative of, for example, a cardiovascular disease condition will be designated a "fingerprint gene". "Fingerprint patterns" will be more fully discuεεed, below, in Section 5.8. It εhould be noted that each of the target geneε may also function aε fingerprint geneε, aε may all or a subset of the pathway genes.

It should further be noted that the pathway genes may alεo be characterized according to techniqueε such as those described herein. Those pathway genes which yield information indicating that they are differentially expreεsed and that modulation of the gene'ε expreεεion or a modulation of the gene product's activity may ameliorate cardiovascular disease will be also be deεignated "target geneε". Such target geneε and target gene products, along with those discussed above, will constitute the focus of the compound diεcovery εtrategies diεcuεεed, below, in Section 5.5. It should be additionally noted that the characterization of one or more of the pathway genes may reveal a lack of differential expression, but evidence that modulation of the gene's activity or expression may, 5 nonetheless, ameliorate cardiovascular diseaεe εymptoms. In such cases, these geneε and gene products would alεo be conεidered a focuε of the compound discovery strategies of Section 5.5, below.

In instances wherein a pathway gene's characterization

10 indicateε that modulation of gene expreεεion or gene product activity may not poεitively affect cardiovaεcular diεease, but whose expresεion iε differentially expreεεed and which contributeε to a gene expreεεion fingerprint pattern correlative of, for example, a cardiovaεcular diεease state, l's such pathway genes may additionally be designated as fingerprint genes.

Among the techniques whereby the identified genes may be further characterized, the nucleotide sequence of the identified genes, which may be obtained by utilizing standard

20 techniques well known to those of skill in the art, may be used to further characterize εuch geneε. For example, the εequence of the identified genes may reveal homologies to one or more known εequence motifε which may yield information regarding the biological function of the identified gene

25 product.

Second, an analysis of the tissue distribution of the mRNA produced by the identified genes may be conducted, utilizing standard techniques well known to those of skill in the art. Such techniques may include, for example, Northern

30 analyses and RT-PCR. Such analyseε provide information aε to whether the identified geneε are expreεεed in tissueε expected to contribute to cardiovascular disease. Such analyses may also provide quantitative information regarding steady state mRNA regulation, yielding data concerning which

35 of the identified genes exhibitε a high level of regulation in, preferably, tiεεueε which may be expected to contribute to cardiovascular diεease. Such analyεeε may alεo be performed on an iεolated cell population of a particular cell type derived from a given tissue. Additionally, standard in situ hybridization techniques may be utilized to provide information regarding which cells within a given tissue express the identified gene. Such analyses may provide in^formation regarding the biological function of an identified gene relative to cardiovascular diεease in instanceε wherein only a εubεet of the cellε within the tiεεue iε thought to ^'be relevant to cardiovascular diseaεe. ~

Third, the sequences of the identified genes may be used, utilizing standard techniques, to place the geneε onto genetic mapε, e.g. , mouse (Copeland & Jenkins, 1991, Trends in Genetics 7: 113-118) and human genetic mapε (Cohen, et al., 1993, Nature 366: 698-701). Such mapping information may yield information regarding the geneε' importance to human diεeaεe by, for example, identifying geneε which map near genetic regionε to which known genetic cardiovascular diεease tendencies map. Fourth, the biological function of the identified genes may be more directly assessed by utilizing relevant in vivo and in vitro syεtems. In vivo εyεtems may include, but are not limited to, animal systemε which naturally exhibit cardiovaεcular diseaεe prediεpoεition, or ones which have been engineered to exhibit εuch εymptomε, including but not limited to the apoE-deficient atherosclerosis mouse model (Plump et al. , 1992, Cell 71: 343-353). Such systems are discusεed in Section 5.4.4.1, below.

In vitro systems may include, but are not limited to, cell-based syεtemε compriεing cell typeε known or suεpected of involvement in cardiovaεcular diεease. Such systems are discussed in detail, below, in Section 5.4.4.2.

In further characterizing the biological function of the identified genes, the expression of these genes may be modulated within the in vivo and/or in vitro systems, i.e. , either over- or underexpresεed, and the εubsequent effect on the system then assayed. Alternatively, the activity of the product of the identified gene may be modulated by either increasing or decreasing the level of activity in the in vivo and/or in vitro εyεtem of intereεt, and itε εubsequent effect then asεayed. The information obtained through εuch characterizationε may suggest relevant methods for the treatment of cardiovascular diεeaεe involving the gene of intereεt. For example, treatment may include a modulation of gene expreεεion and/or gene product activity. Characterization procedures εuch as those described herein may indicate where εuch modulation εhould involve an increaεe or a decreaεe in the expreεεion or activity of the gene or gene product of intereεt.

For example, geneε which are up-regulated under diεease conditionε may be involved in causing or exacerbating the diseaεe condition. Treatmentε directed at down-regulating the activity of εuch harmfully expreεεed geneε will ameliorate the diεeaεe condition. On the other hand, the up- regulation of genes under disease conditions may be part of a protective reεponse by affected cells. Treatments directed at increasing or enhancing the activity of εuch up-regulated gene productε, eεpecially in individuals lacking normal up- regulation, will similarly ameliorate diεeaεe conditionε. Such methods of treatment are discussed, below, in Section 5.6.

5.4. DIFFERENTIALLY EXPRESSED AND PATHWAY GENES Identified genes, which include but are not limited to differentially expressed geneε such as those identified in Section 5.1.1, above, and pathway genes, εuch aε thoεe identified in Section 5.2, above, are deεcribed herein.

Specifically, the nucleic acid sequences and gene products of such identified genes are described herein. Further, antibodies directed against the identified geneε' productε, and cell- and animal-based models by which the identified geneε may be further characterized and utilized are also discusεed in this Section. 5.4.1. DIFFERENTIALLY EXPRESSED AND PATHWAY GENE SEQUENCES

The differentially expreεεed and pathway genes of the invention c.re listed below, in Table 1. Differentially expressed and pathway gene nucleotide sequences are shown in FIGS. 8, 12, 15, 18, 22, 28, 31, and 35.

Table 1 liεtε differentially expreεsed genes id Lfied through, for example, the paradigms discuεεed, above, in Section 5.1.1, and below, in the exampleε preεented in Sections 6 through 9. Table 1 also εummarizes information regarding the further characterization of such geneε.

Firεt, the paradigm uεed initially to detect the differentially expreεεed gene is described under the column headed "Paradigm of Original Detection" . The expresεion patternε of thoεe geneε which have been εhown to be differentially expreεεed, for example, under one or more of the paradigm conditionε deεcribed in Section 5.1.1 are εummarized under the column headed "Paradigm Expreεsion Pattern". For each of the tested genes, the paradigm which was used and the difference in the expression of the gene among the εampleε generated is shown. "It" indicateε that gene expreεεion is up-regulated (i.e. , there is an increase in the amount of detectable mRNA) among the εampleε generated, while "11" indicates that gene expreεsion iε downregulated (i.e.. there is a decrease in the amount of detectable mRNA) among the samples generated. "Detectable" aε uεed herein, referε to levelε of mRNA which are detectable via, for example, standard Northern and/or RT-PCR techniques which are well known to those of skill in the art.

Cell types in which differential expression was detected are also summarized in Table 1 under the column headed "Cell Type Detected in" . The column headed "Chromosomal Location" provides the human chromosome number on which the gene is located. Additionally, in instances wherein the geneε contain nucleotide sequenceε similar or homologous to sequenceε found in nucleic acid databases, references to such similaritieε are listed. The genes listed in Table 1 may be obtained using cloning methods well known to those skilled i.i the art, including but not limited to the use of appropriate probes to detect the genes within an appropriate cDNA or gDNA (genomic DNA) library. (See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, which iε incorporated by reference herein in its entirety) . Probes for the novel sequenceε reported herein may be obtained directly from the iεolated clones deposited with the ATCC, as indicated in Table 2, below.

Alternatively, oligonucleotide probes for the novel geneε may be syntheεized baεed on the DNA sequences disclosed herein in FIGS 1-5.

The sequence obtained from clones containing partial coding sequences or non-coding sequences can be used to obtain the entire coding region by using the RACE method (Chenchik, et al., 1995, CLONTECHniques (X) 1: 5-8; Barneε, 1994, Proc. Natl. Acad. Sci. USA 91: 2216-2220; and Cheng et al., Proc. Natl. Acad. Sci. USA 91: 5695-5699). Oligonucleotideε can be deεigned baεed on the εequence obtained from the partial clone that can amplify a reverse transcribed mRNA encoding the entire coding εequence. Alternatively, probeε can be uεed to screen cDNA libraries prepared from an appropriate cell or cell line in which the gene is transcribed. For example, the genes described herein that were detected in monocytes may be cloned from a cDNA library prepared from monocytes isolated as described in Section 6.1.1, below.

The geneε described herein that were detected in endothelial cells may also be cloned from a cDNA library conεtructed from endothelial cells isolated as described in Progreεε in Hemoεtaεiε and Thrombosis, Vol. 3, P. Spaet, editor, Grune & Stratton Inc., New York, 1-28. Alternatively, the genes may be retrieved from a human placenta cDNA library (Clontech Laboratorieε, Palo Alto, CA) , according to Takahaεhi et al., 1990, εupra; a HUVEC cDNA library aε deεcribed in Joneε et al. 1993, εupra; or an acute lymphoblastic leukemia (SUP-B2) cDNA library as described in Cleary et al., 1986, εupra, for example. Genomic DNA libraries can be prepared from any source.

TABLE 1 Differentially Expressed and Pathway Genes

1. GenBank accession num er U05 4 .

Drosohlla Mothers against dpp (Kadi , Sekelβky et al. 1995, Genetics 139: 1347-1358. 3. Human Voltage-dependent Anion Channel, Blachly-Dyaon, E., et al., 1993, J. Biol. Chem. 268: 1835-1841; and EST T24012 4. Rat Cl-6, Diamond, R.H., et al., 1993, J. Biol. Chem. 2S8: 1S18S-15192.

Mouse gly96, Charles, C.H., et al., 1993, Oncogene 8: 797-801; and EST T49532.

Table 2, below, lists the εtrainε of E. coli deposited with the ATCC that contain plasmids bearing the novel geneε liεted in Table 1.

TABLE 2

Aε uεed herein, "differentially expressed gene" (i.e. target and fingerprint gene) or "pathway gene" refers to (a) a gene containing at least one of the DNA sequences discloεed herein (aε εhown in FIGS. 1-5) , or contained in the cloneε liεted in Table 2, aε deposited with the ATCC; (b) any DNA sequence that encodes the amino acid sequence encoded by the DNA sequences disclosed herein (as shown in FIGS.1-5), contained in the clones, liεted in Table 2, aε depoεited with the ATCC or contained within the coding region of the gene to which the DNA sequences disclosed herein (as εhown in FIGS.l- 5) or contained in the cloneε liεted in Table 2, as deposited with the ATCC, belong; (c) any DNA sequence that hybridizeε to the complement of the coding εequences disclosed herein, contained in the clones listed in Table 2, as deposited with the ATCC, or contained within the coding region of the gene to which the DNA sequences disclosed herein (as shown in FIGS.1-5) or contained in the clones listed in Table 2, as depoεited with the ATCC, belong, under highly stringent conditionε, e.g. , hybridization to filter-bound DNA in 0.5 M NaHPO„, 7% εodium dodecyl εulfate (SDS), 1 mM EDTA at 65°C, and washing in O.lxSSC/0.1% SDS at 68°C (Ausubel F.M. et al., edε., 1989, Current Protocolε in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sonε, Inc., New York, at p. 2.10.3) and encodes a gene product functionally equivalent to a gene product encoded by sequenceε contained within the cloneε liεted in Table 2; and/or (d) any DNA sequence that hybridizes to the complement of the coding sequences discloεed herein, (as εhown in FIGS.1-5) contained in the cloneε liεted in Table 2, aε depoεited with the ATCC or contained within the coding region of the gene to which DNA εequenceε diεcloεed herein (aε εhown in FIGS.1-5) or contained in the cloneε, liεted in Table 2, aε deposited with the ATCC, belong, under less stringent conditions, such as moderately stringent conditions, e.g. , washing in 0.2xSSC/0.1% SDS at 42 °C (Ausubel et al. , 1989, εupra) , yet which εtill encodes a functionally equivalent gene product.

The invention also includes nucleic acid moleculeε, preferably DNA molecules, that hybridize to, and are therefore the complementε of, the DNA sequences (a) through (c) , in the preceding paragraph. Such hybridization conditions may be highly stringent or lesε highly εtringent, aε deεcribed above. In inεtanceε wherein the nucleic acid moleculeε are deoxyoligonucleotideε ("oligoε") , highly stringent conditions may refer, e.g. , to washing in 6xSSC/0.05% sodium pyrophoεphate at 37°C (for 14-baεe oligoε) , 48°C (for 17-baεe oligoε) , 55°C (for 20-baεe oligoε) , and 60°C (for 23-baεe oligoε) . Theεe nucleic acid molecules may act as target gene antisense molecules, useful, for example, in target gene regulation and/or as antisense primers in amplification reactions of target gene nucleic acid sequenceε. Further, such sequences may be used as part of ribozyme and/or triple helix sequences, also uεeful for target gene regulation. Still further, εuch molecules may be used aε componentε of diagnoεtic methodε whereby the preεence of a cardiovascular diεeaεe-cauεing allele, may be detected.

The invention alεo encompasseε (a) DNA vectorε that contain any of the foregoing coding εequences and/or their complementε (i.e.. antiεenεe) ; (b) DNA expreεεion vectorε that contain any of the foregoing coding εequences operatively associated with a regulatory element that directs the expression of the coding sequences-; and (c) genetically engineered host cells that contain any of the foregoing coding sequences operatively associated with a regulatory element that directs the expresεion of the coding εequenceε in the hoεt cell. Aε used herein, regulatory elementε include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to thoεe εkilled in the art that drive and regulate expreεεion. The invention includeε fragmentε of any of the DNA εequences disclosed herein.

In addition to the gene sequenceε described above, homologues of such sequences, as may, for example be preεent in other εpecieε, may be identified and may be readily iεolated, without undue experimentation, by molecular biological techniques well known in the art. Further, there may exist genes at other genetic loci within the genome that encode proteins which have extensive homology to one or more domains of such gene products. These genes may alεo be identified via εimilar techniqueε. For example, the iεolated differentially expreεεed gene sequence may be labeled and used to screen a cDNA library constructed from mRNA obtained from the organism of intereεt. Hybridization conditions will be of a lower stringency when the cDNA library was derived from an organism different from the type of organism from which the labeled sequence was derived. Alternatively, the labeled fragment may be used to εcreen a genomic library derived from the organism of interest, again, using appropriately stringent conditions. Such low εtringency conditionε will be well known to thoεe of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions εee, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springε Harbor Press, N.Y.; and Ausubel et al. , 1989, Current Protocolε in

Molecular Biology, Green Publiεhing Aεεociateε and Wiley Interscience, N.Y. Further, a previously unknown differentially expreεεed or pathway gene-type εequence may be iεolated by performing PCR using two degenerate oligonucleotide primer poolε designed on the basiε of amino acid sequenceε within the gene of intereεt. The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from human or non-human cell lines or tisεue known or suεpected to expreεε a differentially expreεsed or pathway gene allele.

The PCR product may be εubcloned and "sequenced to insure that the amplified εequenees repreεent the εequenceε of a differentially expressed or pathway gene-like nucleic acid sequence. The PCR fragment may then be used to iεolate a full length cDNA clone by a variety of methodε. For example, the amplified fragment may be labeled and uεed to εcreen a bacteriophage cDNA library. Alternatively, the labeled fragment may be uεed to screen a genomic library. PCR technology may also be utilized to isolate full length cDNA sequences. For example, RNA may be isolated, following standard procedures, from an appropriate cellular or tissue εource. A reverεe tranεcription reaction may be performed on the RNA uεing an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand syntheεis. The reεulting RNA/DNA hybrid may then be "tailed" with guanineε uεing a standard terminal transferaεe reaction, the hybrid may be digested with RNAase H, and second strand εynthesiε may then be primed with a poly-C primer. Thuε, cDNA εequences upstream of the amplified fragment may eaεily be isolated. For a review of cloning strategies which may be used, see e.g. , Sambrook et al., 1989, εupra .

In caseε where the differentially expreεεed or pathway gene identified iε the normal, or wild type, gene, thiε gene may be uεed to isolate mutant alleles of the gene. Such an iεolation is preferable in processeε and disorders which are known or suεpected to have a genetic basis. Mutant alleles may be isolated from individuals either known or suεpected to have a genotype which contributes to cardiovascular diεeaεe symptoms. Mutant alleles and mutant allele products may then be utilized in the therapeutic and diagnostic asεay εyεtems described below.

A cDNA of the mutant gene may be iεolated, for example, by using PCR, a technique which is well known to those of skill in the art. In this case, the first cDNA strand may be syntheεized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from tisεue known or suspected to be expressed in an individual putatively carrying the mutant allele, and by extending the new strand tfith reverse transcriptaεe. The εecond εtrand of the cDNA iε then εyntheεized uεing an oligonucleotide that hybridizeε εpecifically to the 5' end of the normal gene. Using theεe two primers, the product is then amplified via PCR, cloned into a suitable vector, and εubjected to DNA εequence analyεiε through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant gene to that of the normal gene, the mutation(ε) reεponεible for the loεs or alteration of function of the mutant gene product can be aεcertained. Alternatively, a genomic or cDNA library can be constructed and screened using DNA or RNA, respectively, from a tisεue known to or εuεpected of expreεsing the gene of interest in an individual suεpected of or known to carry the mutant allele. The normal gene or any εuitable fragment thereof may then be labeled and used aε a probed to identify the correεponding mutant allele in the library. The clone containing thiε gene may then be purified through methodε routinely practiced in the art, and εubjected to εequence analysiε aε deεcribed, above, in thiε Section. Additionally, an expreεεion library can be conεtructed utilizing DNA iεolated from or cDNA εyntheεized from a tiεεue known to or suspected of expressing the gene of interest in an individual suspected of or known to carry the mutant allele. In thiε manner, gene products made by the putatively mutant tissue may be expresεed and εcreened using standard antibody screening techniques in conjunction with antibodieε raiεed againεt the normal gene product, aε described, below, in Section 5.4.3. (For screening techniques, see, for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor.) in cases where the mutation results in an expreεsed 5 gene product with altered function (e.g. ,^' as a result of a missense mutation) , a polyclonal set of antibodies are likely to sross-react with the mutant gene product. Library loneε detected via their reaction with εuch labeled antibodies can be purified and subjected to sequence analyεiε aε deεcribed 10 in thiε Section, above.

5.4.2. DIFFERENTIALLY EXPRESSED AND PATHWAY GENE PRODUCTS

Differentially expressed and pathway gene products

. include those proteins encoded by the differentially expresεed and pathway gene sequences described in Section

5.4.1, above. Specifically, differentially expresεed and pathway gene productε may include differentially expreεεed and pathway gene polypeptideε encoded by the differentially expressed and pathway gene sequences contained in the clones listed in Table 2, above, as deposited with the ATCC, or contained in the coding regions of the genes to which DNA sequences disclosed herein (in FIGS. 1-5) or contained in the clones, liεted in Table 2, aε deposited with the ATCC,

_₅ belong, for example.

In addition, differentially expreεεed and pathway gene productε may include proteinε that represent functionally equivalent gene productε. Such an equivalent differentially expreεεed or pathway gene product may contain deletionε, additionε or εubεtitutions of amino acid residueε within the amino acid εequence encoded by the differentially expreεεed or pathway gene εequenceε deεcribed, above, in Section 5.4.1, but which result in a silent change, thuε producing a functionally equivalent differentially expreεεed on pathway

₃₅ gene product. Amino acid εubstitutions may be made on the basis of similarity in polarity, charge, εolubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residueε involved.

For example, nonpolar (hydrophobic) amino acidε include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, .nd methionine; polar neutral amino acids include glycine, εerine, threonine, cyεteine, tyroεine, aεparagine, and glutamine; poεitively charged (baεic) amino acidε include arginine, lyεine, and hiεtidine; and negatively charged (acidic) amino acidε include aspartic acid and glutamic acid. "Functionally equivalent", as utilized herein, refers to a protein capable of exhibiting a εubεtantially εimilar in vivo activity aε the endogenouε differentially expreεεed or pathway gene productε encoded by the differentially expreεεed or pathway gene sequences described in Section 5.4.1, above. Alternatively, when utilized aε part of aεsays such aε thoεe described, below, in Section 5.5, "functionally equivalent" may refer to peptideε capable of interacting with other cellular or extracellular moleculeε in a manner substantially similar to the way in which the corresponding portion of the endogenous differentially expreεεed or pathway gene product would.

The differentially expreεεed or pathway gene productε may be produced by recombinant DNA technology uεing techniqueε well known in the art. Thuε, methodε for preparing the differentially expressed or pathway gene polypeptides and peptideε of the invention by expreεsing nucleic acid encoding differentially expresεed or pathway gene sequences are described herein. Methods which are well known to those skilled in the art can be used to construct expreεsion vectors containing differentially expreεεed or pathway gene protein coding εequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, εynthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniqueε deεcribed in Sambrook et al., 1989, εupra, and Auεubel et al. , 1989, εupra . Alternatively, RNA capable of encoding differentially expreεεed or pathway gene protein εequenceε may be chemically εyntheεized uεing, for example, εyntheεizerε. See, for example, the techniqueε described in "Oligonucleotide Synthesiε", 1984, Gait, M.J. ed. , IRL Preεε, Oxford, which iε incorporated by reference herein in itε entirety.

A variety of host-expresεion vector systemε may be utilized to expreεε the differentially expressed or pathway gene coding sequenceε of the invention. Such hoεt-expreεεion εystemε represent vehicleε by which the coding sequences of interest^"may be produced and subsequently purified, but also represent cells which may, when tranεformed or transfected with the appropriate nucleotide coding sequences, exhibit the differentially expressed or pathway gene _r,protein of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli , B . εubtiliε) transformed with recombinant bacteriophage DNA, plaεmid DNA or coεmid DNA expreεsion vectors containing differentially expresεed or pathway gene protein coding sequences; yeast (e.g. Saccharomyceε, Pichia) tranεformed with recombinant yeaεt expreεεion vectorε containing the differentially expreεεed or pathway gene protein coding sequences; insect cell syεtemε infected with recombinant viruε expression vectors (e.g., baculovirus) containing the differentially expresεed or pathway gene protein coding εequenceε; plant cell εyεtemε infected with recombinant viruε expreεεion vectorε (e.g., cauliflower moεaic viruε, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing differentially expressed or pathway gene protein coding sequences; or mammalian cell syεtemε (e.g. COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cellε (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) .

In bacterial systems, a number of expresεion vectors may be advantageously selected depending upon the use intended for the differentially expressed or pathway gene protein being expresεed. For example, when a large quantity of such a protein iε to be produced, for the generation of antibodieε or to εcreen peptide librarieε, for example, vectorε which direct the expreεεion of high levelε of fuεion protein productε that are readily purified may be deεirable. Such vectors include, but are not limited, to the E. coli expresεion vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the differentially expresεed or pathway gene protein coding εequence may be ligated individually into the vector in frame with the lac Z coding region εo that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids-Reε. 13:3101-3109; Van Heeke & Schuεter, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectorε may also be used to expreεε foreign polypeptideε aε fuεion proteins with glutathione S-transferase (GST) . In general, such fusion proteins are soluble and can easily be purified from lyεed cellε by adεorption to glutathione- agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage εiteε εo that the cloned target gene protein can be releaεed from the GST moiety.

In a preferred embodiment, full length cDNA sequences are appended with in-frame Bam HI sites at the amino terminus and Eco RI sites at the carboxyl terminus using -standard PCR methodologies (Innis et al., 1990, supra) and ligated into the pGEX-2TK vector (Pharmacia, Uppεala, Sweden) . The reεulting cDNA construct contains a kinase recognition site at the amino terminus for radioactive labelling and glutathione S-transferaεe sequenceε at the carboxyl terminuε for affinity purification (Nilsson, et al., 1985, EMBO J. 4: 1075; Zabeau and Stanley, 1982, EMBO J. 1: 1217.

In an insect syεtem, Autographa californica nuclear polyhedrosis virus (AcNPV) iε used as a vector to expresε foreign geneε. The virus grows in Spodoptera frugiperda cellε. The differentially expressed or pathway gene coding ε-ϋquence may be cloned individually into non-eεεential regionε (for example the polyhedrin gene) of the viruε and placed under control of an AcNPV promoter (for example the polyhedrin promoter) . Succeεsful insertion of differentially expressed or pathway gene coding sequence will result in inactivation of the polyhedrin gene and production of non- occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene) . Theεe recombinant viruses are then used to infect Spodoptera frugiperda cellε in which the inεerted gene iε expressed.

(E.g., see Smith et al. , 1983, J. Virol. 46: 584; Smith, U.S. Patent No. 4,215,051).

In mammalian host cellε, a number of viral-baεed expression syεtemε may be utilized. In cases where an adenovirus is uεed aε an expression vector, the differentially expresεed or pathway gene coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader εequence. Thiε chimeric gene may then be inεerted in the adenoviruε genome by in vitro or in vivo recombination. Insertion in a non-esεential region of the viral genome (e.g., region El or E3) will reεult in a recombinant viruε that iε viable and capable of expreεsing differentially expressed or pathway gene protein in infected hosts. (E.g., See Logan & Shenk, 1984, Proc. Natl. Acad.

Sci. USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted differentially expressed or pathway gene coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire differentially expresεed or pathway gene, including itε own initiation codon and adjacent εequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the differentially expressed or pathway gene coding sequence is inserted, exogenouε tranεlational control εignals, including, perhapε, the ATG initiation codon, muεt be provided. Furthermore, the initiation codon must be in phaεe with the reading frame of the desired coding εequence to ensure tranεlation of the-entire inεert. These exogenous tranεlational control εignalε and initiation codonε can be of 5 a variety of originε, both natural and synthetic. The efficiency of expreεεion may be enhanced by the inclusion of appropriate transcription enhancer elementε, tranεcription terminatorε, etc. (εee Bittner et al., 1987, Methodε in Enzymol. 153:516-544).

10 In a preferred embodiment, cDNA εequenceε encoding the full-length open reading frameε are ligated into pCMVS replacing the 3-galactoεidaεe gene εuch that cDNA expreεεion iε driven by the CMV promoter (Alam, 1990, Anal. Biochem. 188: 245-254; MacGregor & Caεkey, 1989, Nucl. Acidε Reε. 17:

15 2365; Norton & Corrin, 1985, Mol. Cell. Biol. 5: 281).

In addition, a hoεt cell εtrain may be chosen which modulates the expresεion of the inserted sequenceε, or modifieε and proceεεeε the gene product in the εpecific fashion desired. Such modificationε (e.g., glycosylation)

20 and proceεεing (e.g., cleavage) of protein productε may be important for the function of the protein. Different host cellε have characteriεtic and εpecific mechaniεms for the poεt-tranεlational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to

25 ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cellε include

30 but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

For long-term, high-yield production of recombinant proteinε, stable expression is preferred. For example, cell lineε which stably express the differentially expressed or

35 pathway gene protein may be engineered. Rather than using expresεion vectorε which contain viral originε of replication, hoεt cellε can be tranε ormed with DNA controlled by appropriate expresεion control elements (e.g., promoter, enhancer, sequenceε, tranεcription terminatorε, polyadenylation εites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cellε may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. Th^ selectable marker in the recombinant plaεmid confers resistance to the selection and allows cellε to εtably integrate the plaεmid into their chromosomes and gro to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lineε which expreεε the differentially expreεεed or pathway gene protein. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the differentially expresεed or pathway gene protein.

A number of εelection εyεtems may be used, including but not limited to the herpes simplex viruε thymidine kinase (Wigler, et al. , 1977, Cell 11:223), hypoxanthine-guanine phosphoriboεyltranεferaεe (Szybalεka & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoriboεyltranεferaεe (Lowy, et al., 1980, Cell 22:817) geneε can be employed in tk^", hgprt^" or aprt^" cellε, reεpectively. Also, antimetabolite resistance can be used as the basis of εelection for dhfr, which conferε reεiεtance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al. , 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which conferε reεiεtance to -mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which conferε reεiεtance to the aminoglycoεide G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, which conferε reεistance to hygromycin (Santerre, et al., 1984, Gene 30:147) genes.

An alternative fusion protein system allows for the ready purification of non-denatured fusion proteins expresεed in human cell lineε (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-8976). In thiε εyεtem, the gene of intereεt iε εubcloned into a vaccinia recombination plaεmid εuch that the gene'ε open reading frame iε tranεlationally fuεed to an amino-terminal tag conεisting of εix hiεtidine reεidueε. Extractε from cellε infected with recombinant vaccinia virus are loaded onto Ni²⁺- nitriloacetic acid-agaroεe columnε and hiεtidine-tagged proteins are εelectively eluted with imidazole-containing bufferε.

When used aε a component in asεay εystems εuch^* aε thoεe deεcribed, below, in Section 5.5, the differentially expreεεed or pathway gene protein may be labeled, either directly or indirectly, to facilitate detection of a complex formed between the differentially expreεεed or pathway gene protein and a teεt subεtance. Any of a variety of εuitable labeling εyεtemε may be uεed including but not limited to radioisotopes εuch as ^12SI; enzyme labelling syεtemε that generate a detectable colorimetric εignal or light when exposed to subεtrate; and fluoreεcent labelε.

Where recombinant DNA technology iε uεed to produce the differentially expreεεed or pathway gene protein for εuch aεεay εyεtems, it may be advantageous to engineer fusion proteins that can facilitate labeling, immobilization and/or detection.

Indirect labeling involveε the uεe of a protein, such as a labeled antibody, which εpecifically bindε to either a differentially expreεεed or pathway gene product. Such antibodieε include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by an Fab expresεion library.

5.4.3. DIFFERENTIALLY EXPRESSED OR

PATHWAY GENE PRODUCT ANTIBODIES

Described herein are methods for the production of antibodies capable of εpecifically recognizing one or more differentially expressed or pathway gene epitopes. Such antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs) , humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, fragmentε produced by a Fab expreεεion library, anti-idiotypic (anti-Id) antibodieε, and epitope- binding fragments of any of the above. Such antibodies may be used, for example, in the detection of a fingerprint, target, or pathway gene in a biological sample, or, alternatively, as a method for the inhibition of abnormal= target gene activity. Thuε, εuch antibodieε may be utilized as part of cardiovascular diseaεe treatment methods, and/or - may be used as part of diagnostic techniques whereby patients may be teεted for abnormal levelε of fingerprint, target, or pathway gene proteins, or for the presence of abnormal forms of the such proteins.

For the production of antibodies to f.a differentially expreεεed or pathway gene, various hoεt animalε may be immunized by injection with a differentially expressed or pathway gene protein, or a portion thereof. Such host animalε may include but are not limited to rabbitε, mice, and ratε, to name but a few. Variouε adjuvantε may be uεed to increaεe the immunological reεponεe, depending on the hoεt εpecies, including but not limited to Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active subεtances εuch aε lyεolecithin, pluronic polyolε, polyanions, peptideε, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjυvantε εuch aε BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

In a preferred embodiment, peptide sequences corresponding to amino εequenceε of target gene productε were εelected and εubmitted to Reεearch Geneticε (Huntεville, AL) for εyntheεiε and antibody production. Peptides were modified aε deεcribed (Tam, J.P., 1988, Proc. Natl. Acad. Sci. USA 85: 5409-5413; Tam, J.P., and Zavala, F., 1989, J. Immunol. Methodε 124: 53-61; Tam, J.P., and Lu, Y.A. , 1989, Proc. Natl. Acad. Sci. USA 86: 9084-9088), emulεified in an equal volume of Freund's adjuvant and injected into rabbits at 3 to 4 subcutaneouε dorsal siteε for a total volume of 1.0 ml (0.5 mg peptide) per immunization. The animalε were booεted after 2 and 6 weekε and bled at weekε 4 , 8 , and 10. The blood was allowed to clot and serum waε collected by centrifugation. The generation of polyclonal antibodies against the fchd545 gene product is described in detail in the example in Section 10, below.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animalέ immunized with an antigen, εuch aε target gene product, or a antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as those described above, may be immunized by injection with differentially expreεεed or pathway gene product εupplemented with adjuvantε aε alεo described above.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV- hybridoma technique (Cole et al., 1985, Monoclonal Antibodieε And Cancer Therapy, Alan R. Liεε, Inc., pp. 77-96). Such antibodieε may be of any immunoglobulin claεε including IgG, IgM, IgE, IgA, IgD and any εubclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titerε of mAbε in vivo makeε this the presently preferred method of production. In addition, techniques developed for the production of "chimeric antibodieε" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al. , 1985, Nature, 314:452-454) by εplicing the geneε from a mouse antibody molecule of appropriate antigen specificity together with geneε from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portionε are derived from different animal species, such aε thoεe having a variable region derived from a murine mAb and a human immunoglobulin conεtant region.

Alternatively, techniques described for the production of single chain antibodies (U.S. Patent 4,946,778; Bird, 1988, Science 242:423-426; Huston et al. , 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adapted to produce differentially expreεεed or pathway gene-εingle chain an ibodieε. Single chain antibodieε are formed by linking the heavy and light chain fragmentε of the Fv region via an amino acid bridge, reεulting in a single chain polypeptide.

Antibody fragments which recognize εpecific epitopes may be generated by known techniques. For example, such fragmentε include but are not limited to: the F(ab')₂ fragmentε which can be produced by pepεin digeεtion of the antibody molecule and the Fab .fragmentε which can be generated by reducing the diεulfide bridgeε of the F(ab')₂ fragmentε. Alternatively, Fab expreεεion librarieε may be conεtructed (Huεe et al., 1989, Science, 246:1275-1281) to allow rapid and eaεy identification of monoclonal Fab fragmentε with the desired εpecificity.

5.4.4. CELL- AND ANIMAL-BASED MODEL SYSTEMS Deεcribed herein are cell- and animal-baεed εyεtemε which act aε modelε for cardiovaεcular diεeaεe. Theεe εyεtemε may be uεed in a variety of applicationε. For example, the cell- and animal-baεed model εyεtemε may be uεed to further characterize differentially expreεεed and pathway geneε, aε deεcribed, above, in Section 5.3. Such further characterization may, for example, indicate that a differentially expreεεed gene is a target gene. Second, such assays may be utilized as part of εcreening εtrategieε designed to identify compounds which are capable of ameliorating cardiovascular diεeaεe εymptomε, as deεcribed, below, in Section 5.5.4. Thuε, the animal- and cell-baεed modelε may be uεed to identify drugs, pharmaceuticalε, therapies and interventions which may be effective in treating cardiovascular disease. In addition, as described in detail, below, in Section*5.7.1, such animal modelε may be uεed to determine the LD_S0 and the ED_S0 in animal εubjects, and such data can be used to determine the in vivo efficacy of potential cardiovascular diseaεe treatments.

5.4.4.1. ANIMAL-BASED SYSTEMS Animal-based model syεtemε of cardiovascular disease may include, but are not limited to, non-recombinant and engineered transgenic animals.

Non-recombinant animal modelε for cardiovaεcular diεeaεe may include, for example, genetic models. Such genetic cardiovaεcular diεease modelε may include, for example, apoB or apoR deficient pigs (Rapacz, et al., 1986, Science

234:1573-1577) and Watanabe heritable hyperlipidemic (WHHL) rabbitε (Kita et al., 1987, Proc. Natl. Acad. Sci USA 84: 5928-5931) .

Non-recombinant, non-genetic animal modelε of atheroεclerosis may include, for example, pig, rabbit, or rat models in which the animal haε been expoεed to either chemical wounding through dietary supplementation of LDL, or mechanical wounding through balloon catheter angioplasty, for example. Additionally, animal modelε exhibiting cardiovaεcular disease symptoms may be engineered by utilizing, for example, target gene sequences such as those described, above, in Section 5.4.1, in conjunction with techniques for producing transgenic animals that are well known to those of skill in the art. For example, target gene sequenceε may be introduced into, and overexpreεεed in, the genome of the animal of intereεt, or, if endogenouε target gene sequences are present, they may either be overexpresεed or, alternatively, be disrupted in order to underexpress or inactivate target gene expreεεion, such as described for the diεruption of apoE in mice (Plump et al. , 1992, Cell 71: 343- 353) . In order to overexpreεs a target gene sequence, the coding portion of the target gene sequence may be ligated to a regulatory εequence which is capable of driving gene expresεion in the animal and cell type of intereεt. Such regulatory regionε will be well known to thoεe of εkill in the art, and may be utilized in the absence of undue exp-≥rimentation.

For underexpreεεion of an endogenous target gene sequence, such a sequence may be iεolated and engineered εuch that when reintroduced into the genome of the animal of intereεt, the endogenouε target gene alleleε will be inactivated. Preferably, the engineered target gene εequence is introduced via gene targeting εuch that the endogenouε target εequence is disrupted upon integration of the engineered .,target gene sequence into the animal'ε genome. Gene targeting is discuεεed, Taelow, in thiε Section.

Animalε of any εpecieε, including, but not limited to, mice, ratε, rabbitε, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g. , baboonε, monkeyε, and chimpanzeeε may be uεed to generate cardiovaεcular diεeaεe animal modelε.

Any technique known in the art may be uεed to introduce a target gene tranεgene into animalε to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene tranεfer into germ lineε (Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic εtem cells (Thompson et al., 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723); etc. For a review of such techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which iε incorporated by reference herein in itε entirety.

The preεent invention provides for transgenic animals that carry the tranεgene in all their cells, as well aε animals which carry the tranεgene in εome, but not all their cells; i.e. , oεaic animalε. The transgene may be integrated aε a εingle tranεgene or in concatamers, e.g. _f head-to-head tandemε or head-to-tail tandemε. The tranεgene may alεo be εelectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236) . The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the target gene transgene be integrated into the chromosomal site of the endogenouε target gene, gene targeting iε preferred. Briefly, when εuch a technique is to be utilized, vectors containing εome nucleotide εequences homologouε to the endogenouε target gene of interest are deεigned for the purpose of integrating, via homologous recombination with chromosomal εequences, into and disrupting the function of the nucleotide sequence of the endogenous target gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene of interest in only that cell type, by following, for example, the teaching of Gu et al. (Gu, et al., 1994, Science 265: 103-106). The regulatory sequenceε required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to thoεe of εkill in the art. Recombinant methodε for expreεεing target geneε are deεcribed in Section 5.4.2, above. Once transgenic animals have been generated, the expresεion of the recombinant target gene and protein may be aεεayed utilizing εtandard techniqueε. Initial screening may be accompliεhed by Southern blot analyεiε or PCR techniqueε to analyze animal tiεεues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tiεεues of the transgenic animals may also be asεeεεed uεing techniques which include but are not limited to Northern blot analysiε of tiεεue εampleε obtained from the animal, in situ hybridization analysiε, and RT-PCR. Sa pleε of target gene-expreεεing tiεεue, may also be evaluated immunocytochemically using antibodies εpecific for the target gene transgene gene product of interest.

The target gene transgenic animals that express target gene mRNA or target gene transgene peptide (detected immunocytochemically, using antibodieε directed against the - target gene product's epitopes) at easily detectable levelε εhould then be further evaluated to identify thoεe animalε which diεplay characteriεtic cardiovaεcular diεeaεe εymptoms. Such symptoms may include, for example, increaεed prevalence and εize of fatty εtreakε and/or cardiovaεcular disease plaques. Additionally, specific cell types within the transgenic animals may be analyzed and asεayed for cellular phenotypes characteristic of cardiovaεcular disease. In the case of monocytes, such phenotypeε may include but are not limited to increaεeε in rateε of LDL uptake, adhesion to endothelial cellε, transmigration, foam cell formation, fatty streak formation, and production of foam cell εpecific productε. Cellular phenotype aεεayε are diεcuεεed in detail in Section 5.4.4.2, below. Further, εuch cellular phenotypeε may include a particular cell type'ε fingerprint pattern of expreεεion aε compared to known fingerprint expreεεion profileε of the particular cell type in animalε exhibiting cardiovascular diεeaεe sy ptomε. Fingerprint profileε are deεcribed in detail in Section 5.8.1, below. Such tranεgenic animalε serve aε εuitable model εystemε for cardiovaεcular diεease.

Once target gene transgenic founder animals are produced, they may be bred, inbred, outbred, or croεεbred to produce colonieε of the particular animal. Exampleε of εuch breeding εtrategies include but are not limited to: outbreeding of founder animalε with more than one integration εite in order to establish separate lines; inbreeding of separate lines in order to produce compound target gene transgenics that express the target gene transgene of interest at higher levels because of the effects of additive expresεion of each target gene tranεgene; crossing of heterozygous transgenic animalε to produce animalε homozygous for a given integration site in order both to augment expresεion and eliminate the poεεible need for εcreening of animalε by DNA analyεiε; croεsing of separate homozygouε lineε to produce compound heterozygouε or homozygouό lineε; breeding animalε to different inbred genetic backgroundε εo aε to examine effects of modifying alleles on expresεion of the target gene transgene and the development of cardiovaεcular diεeaεe εymptomε. One such approach iε to croεs the target gene tranεgenic founder animalε with a wild type εtrain to produce an FI generation that exhibitε cardiovascular diεeaεe εymptoms . The FI generation may then be inbred in order to develop a homozygouε line, if it is found that homozygous target gene transgenic animals are viable.

5.4.4.2. CELL-BASED ASSAYS

Cells that contain and expresε target gene εequenceε which encode target gene protein, and, further, exhibit cellular phenotypes asεociated with cardiovascular diseaεe, may be utilized to identify compoundε that exhibit anti- cardiovascular diεease activity.

Such cells may include non-recombinant monocyte cell lines, such as U937 (ATCC# CRL-1593) , THP-1 (ATCC# TIB-202) , and P388D1 (ATCC# TIB-63) ; endothelial cellε εuch aε HUVEC'ε and bovine aortic endothelial cellε (BAEC'ε); aε well aε generic mammalian cell lines εuch as HeLa cells and COS cells, e.g., COS-7 (ATCC# CRL-1651) . Further, such cells may include recombinant, transgenic cell lineε. For example, the cardiovaεcular diεeaεe animal models of the invention, discusεed, above, in Section 5.4.4.1, may be uεed to generate cell lines, containing one or more cell typeε involved in cardiovascular disease, that can be used as cell culture modelε for thiε diεorder. While primary cultureε derived from the cardiovascular disease transgenic animals of the invention may be utilized, the generation of continuouε cell lineε is preferred. For exampleε of techniques which may be uεed to derive a continuous cell line from the transgenic animalε, see Small et al., 1985, Mol. Cell Biol. 5:642-648.

Alternatively, cells of a cell type known to be involved in cardiovaεcular diεeaεe may be transfected with sequenceε capable of increaεing or decreaεing the amount of target gene expreεεion within the cell. For example, ^target gene sequences may be introduced into, and overexpresεed in, the genome of the cell of intereεt, or, if endogenous target gene εequenceε are preεent, they may be either overexpreεεed or, alternatively diεrupted in order to underexpress or inactivate target gene expresεion. In order to overexpress a target gene sequence, the coding portion of the target gene sequence may be ligated to a regulatory sequence which is capable of driving gene expresεion in the cell type of intereεt. Such regulatory regions will be well known to those of skill in the art, and may be utilized in the abεence of undue experimentation.

Recombinant methods for expreεεing target geneε are deεcribed in Section 5.4.2, above.

For underexpreεεion of an endogenous target gene εequence, εuch a sequence may be isolated and engineered such that when reintroduced into the genome of the cell type of interest, the endogenous target gene alleles will be inactivated. Preferably, the engineered target gene sequence is introduced via gene targeting such that the endogenous target sequence is disrupted upon integration of the engineered target gene sequence into the cell's genome.

Tranεfection of host cellε with target geneε iε diεcuεεed, above, in Section 5.4.4.1.

Cellε treated with compoundε or tranεfected with target geneε can be examined for phenotypeε associated with cardiovascular diseaεe. In the case of monocytes, such phenotypes include but are not limited to increases in rates of LDL uptake, adheεion to endothelial cells, tranεmigration, foam cell formation, fatty streak formation, and production by foam cells of growth factors such as bFGF, IGF-I, VEGF, IL-1, M-CSF, TGF3, TGFα, TNFσ, HB-EGF, PDGF, IFN-γ, and GM- CSF. Transmigration rates, for example, may be eaεured uεing the in vitro εyεtem of Navab et al., deεcribed in Section 5.1.1.3, above, by quantifying the number of monocytes that migrate acroεs the endothelial monolayer and into the collagen layer of the subendothelial space.

Similarly, HUVEC's can be treated with test compounds or transfected with genetically engineered target genes deεcribed in Section 5.4.2, above. The HUVEC's can then be examined for phenotypeε aεεociated with cardiovaεcular diεeaεe, including, but not limited to changeε in cellular morphology, cell proliferation, cell migration, and mononuclear cell adheεion; or for the effectε on production of other proteinε involved in cardiovaεcular diεeaεe εuch aε ICAM, VCAM, PDGF-S, and E-εelectin.

Tranεfection of target gene εequence nucleic acid may be accompliεhed by utilizing εtandard techniqueε. See, for example, Ausubel, 1989, εupra . Tranεfected cellε εhould be evaluated for the preεence of the recombinant target gene εequenceε, for expreεεion and accumulation of target gene mRNA, and for the preεence of recombinant target gene protein production. In inεtanceε wherein a decrease in target gene expresεion iε deεired, εtandard techniques may be used to demonstrate whether a decrease in endogenous target gene expression and/or in target gene product production is achieve .

5.5. SCREENING ASSAYS FOR COMPOUNDS THAT INTERACT WITH THE TARGET GENE PRODUCT AND/OR MODULATE TARGET GENE EXPRESSION

The following assayε are designed to identify compoundε that bind to target gene productε, bind to other cellular or extracellular proteins that interact with a target gene product, and interfere with the interaction of the target gene product with other cellular or extracellular proteins. Such compounds can act as the basis for amelioration of such cardiovascular diseaεes as atherosclerosiε, ischemia/reperfuεion, hypertenεion, restenoεiε, and arterial inflammation by modulating the activity of the protein products of target geneε. Such compounds may alεo act, for example, as activators or enhancerε of the TGF-b εigna¹ling response, e.g. , of the TGF- cell growth inhibitory i <—ponse, for the baεiε for the amelioration of fibroproliferative and oncogenic related diεorders, including tumorigenesiε and the vaεcularization of tumorε. Such compoundε may include, but are not limited to peptides, antibodieε, or εmall organic or inorganic compounds. Methods for the identification of such compounds are described in Section 5.5.1, below. Such compounds may also include other cellular proteins. Methods for the identification of such cellular proteins are described, below, in Section 5.5.2.

Compoundε identified via assays such as those described herein may be uεeful, for example, in elaborating the biological function of the target gene product, and for ameliorating cardiovaεcular, fibroproliferative and oncogenic related disease. In instanceε whereby a cardiovaεcular diεeaεe condition reεultε from an overall lower level of target gene expreεsion and/or target gene product in a cell or tiεsue, compounds that interact with the target gene product may include compounds which accentuate or amplify the activity of the bound target gene protein. Such compounds would bring about an effective increase in the level of target gene product activity, thus ameliorating symptomε.

In εome caseε, a target gene obεerved to be up-regulated under disease conditions may be exerting a protective effect. Compounds that enhance the expresεion of such up-regulated geneε, or the activity of their gene productε, would also ameliorate diεease symptoms, eεpecially in individualε whoεe target gene iε not normally up-regulated. In other inεtanceε mutationε within the target gene may cauεe aberrant typeε or exceεεive amountε of target gene proteinε to be made which have a deleterious effect that leads to cardiovascular, fibroproliferative and oncogenic related disease. Similarly, physiological conditions may cause an excessive increase in target gene expression leading to cardiovascular, fibroproliferative and oncogenic related diseaεe. In εuch caεeε, compounds that bind target gene protein may be identified that inhibit the activity of the bound target gene protein. Asεayε for teεting the effectiveneεε of compoundε, identified by, for example, techniqueε εuch aε those_^deεcribed in thiε Section are diεcuεsed, below, in Section 5.5.4.

5.5.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO THE TARGET GENE PRODUCT

In vitro εyεterns may be deεigned to identify compoundε capable of binding the target gene of the invention. Such compoundε may include, but are not limited to, peptideε made of D-and/or L-configuration amino acidε (in, for example, the form of random peptide librarieε; see e.g. , Lam, K.S. et al. , 1991, Nature 354:82-84), phosphopeptideε (in, for example, the form of random or partially degenerate, directed phoεphopeptide libraries; see, e.g. , Songyang, Z. et al., 1993, Cell 72:767-778), antibodieε, and small organic or inorganic molecules. Compounds identified may be uεeful, for example, in modulating the activity of target gene proteinε, preferably mutant target gene proteinε, may be useful in elaborating the biological function of the target gene protein, may be utilized in screens for identifying compounds that disrupt normal target gene interactions, or may in themεelveε diεrupt εuch interactionε. For inεtance, the example in Section 12, below, deεcribeε the interaction between the rchd534 protein and the fchd540 protein. Compoundε that diεrupt the interaction between theεe two proteinε may be uεeful in the treatment of cardiovaεcular diεease. The principle of the asεays used to identify compoundε that bind to the target gene protein involveε preparing a reaction mixture of the target gene protein and the teεt compound under conditions and for a time εufficient to_^ allow the two componentε to interact and bind, thuε forming a complex which can be removed and/or detected in the reaction mixture. Theεe aεεayε can be conducted in a variety of wayε. For example, one method to conduct εuch an assay would involve anchoring the target gene or the test εubεtance onto a solid phase and detecting target gene/test εubεtance complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the target gene protein may be anchored onto a εolid εurface, and the teεt compound, which iε not anchored, may be labeled, either directly or indirectly.

In practice, microtitre plateε are conveniently utilized. The anchored component may be immobilized by non- covalent or covalent attachmentε. Non-covalent attachment may be accompliεhed εimply by coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, εpecific for the protein may be uεed to anchor the protein to the εolid εurface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction iε complete, unreacted componentε are removed (e.g. , by waεhing) under conditionε such that any complexes formed vill remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of wayε. Where the previouεly nonimmobilized component iε pre-labeled, the detection of label immobilized on the surface indicateε that complexes were formed. Where the previously nonimmobilized component iε not pre-labeled, an indirect label can be uεed to detect complexeε anchored on the εurface; e.g. , uεing a labeled antibody εpecific for the previouεly nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody) . Alternatively, a reaction can be ^conducted in a liquid phaεe, the reaction productε εeparated from unreacted componentε, and complexes detected; e.g. , using an immobilized antibody specific for target gene product or the teεt compound to anchor any complexeε formed in εolution, and a labeled antibody εpecific for the other component of the poεεible complex to detect anchored complexeε.

Compoundε that are εhown to bind to a_particular target gene product through one of the methodε deεcribed above can be further teεted for their ability to elicit a biochemical reεponεe from the target gene protein. A particular embodiment iε deεcribed herein for receptor proteinε involved in εignal tranεduction. Compoundε that interact with a target gene product receptor domain, can be εcreened for their ability to function aε ligandε, i.e., to bind to the receptor protein in a manner that triggers the signal tranεduction pathway. Uεeful receptor fragmentε or analogε in the invention are thoεe which interact with ligand. The receptor component can be aεεayed functionally, i.e., for its ability to bind ligand and mobilize Ca⁺⁺ (see below) . These asεayε include, as components, ligand and a recombinant target gene product (or a suitable fragment or analog) configured to permit detection of binding.

For example, and not by way of limitation, a recombinant receptor may be used to screen for ligands by its ability to mediate ligand-dependent mobilization of calcium. Cellε, preferably myeloma cellε or Xenopuε oocytes, transfected with a target gene expresεion vector (conεtructed according to the methodε described in Section 5.4.2, above) are loaded with FURA-2 or INDO-1 by standard techniqueε. Mobilization of Ca²⁺ induced by ligand iε meaεured by fluoreεcence εpectroεcopy aε previouεly deεcribed (Grynkiewicz et al., 1985, J. Biol . Chem . 260:3440). Ligandε that react with the target gene product receptor domain, therefore, can be identified by their ability to produce a fluoreεcent εignal. Their receptor binding activitieε can be quantified and compared by meaεuring the level of fluoreεcence produced over background. Identification of ligand, and measuring the activity of the ligand-receptor complex, leads to the identification of antagonists of this interaction, as described in Section 5.5.3, below. Such antagonistε are uεeful in the treatment of cardiovaεcular diεeaεe.

5.5.2. ASSAYS FOR CELLULAR OR EXTRACELLULAR PROTEINS THAT INTERACT WITH THE TARGET GENE PRODUCT

Any method suitable for detecting protein-protein interactions may be employed for identifying novel target protein-cellular or extracellular protein interactions. These methods are outlined in Section 5.2., εupra, for the identification of pathway genes, and may be utilized herein with respect to the identification of proteins which interact with identified target proteins. In such a case, the target gene serves as the known "bait" gene.

The example presented in Section 12, below, demonεtrateε the use of this method to detect the interaction between the rchd534 protein and the fchd540 protein, which both had been identified as target proteins.

5.5.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH INTERACTION BETWEEN TARGET GENE PRODUCT AND OTHER COMPOUNDS The target gene proteins of the invention may, in vivo, interact with one or more cellular or extracellular proteinε. Such proteinε may include, but are not limited to, thoεe proteinε identified via methodε such aε thoεe deεcribed, above, in Section 5.5.2. For the purpoεeε of this discuεεion, target gene productε and εuch cellular and extracellular proteinε are referred to herein as "binding partners". Compoundε that diεrupt εuch interactionε may be uεeful in regulating the activity of the target gene proteins, especially mutant target gene proteins. Such compounds may include, but are not limited to molecules such as antibodies, peptides, and the like deεcribed in Section 5.5.1. above. The baεic principle of the aεsay systems used to identify compounds that interfere with the interaction between the target gene protein, and its cellular or extracellular protein binding partner or partners involveε preparing a reaction mixture containing the target gene protein and the binding partner under conditionε and for a time εufficient to allow the two proteinε to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture iε prepared in the presence and abεence of the teεt compound. The teεt compound may be initially included in the reaction mixture or may be added at a time εubεequent to the addition of target gene and itε cellular or extracellular binding partner. Control reaction mixtureε are incubated without the teεt compound or with a placebo. The formation of any complexes between the target gene protein and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the target gene protein and the interactive binding partner protein. Additionally, complex formation within reaction mixtures containing the test compound and a normal target gene protein may alεo be compared to complex formation within reaction mixtureε containing the teεt compound and mutant target gene protein. Thiε comparison may be important in thoεe cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene proteins. The assay for compounds that interfere with the interaction of the binding partners can be conducted in a heterogeneouε or homogeneouε format. Heterogeneous aεεayε involve anchoring one of the binding partnerε onto a εolid phaεe and detecting complexeε anchored on the εolid phase at the end of the reaction. In homogeneous assayε, the entire reaction iε carried out in a liquid phaεe. In either approach, the order of addition of reactantε can be varied to obtain different information about the compounds being tested. For example, teεt compoundε that interfere with the interaction betwee the binding partnerε, e.g. , by competition, can be identified by conducting the reaction in the preεence of the teεt εubεtance; i.e. , by adding the teεt substance to the reaction mixture prior to or simultan^ouεly witl; the target gene protein and interactive cellular _^r extracellular protein. Alternatively, teεt compoundε that diεrupt preformed complexeε, e.g. compoundε with higher binding conεtantε that displace one of the binding partners from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are deεcribed briefly below.

In a heterogeneouε aεεay εyεtem, either the target gene protein or --the interactive cellular or extracellular binding partner protein, iε anchored onto a εolid surface, and its binding partner, which is not anchored, is labeled, either directly or indirectly. In practice, microtitre plates are conveniently utilized. The anchored species may be immobilized by non-covalent or covalent attachments. Non- covalent attachment may be accomplished simply by coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody specific for the protein may be used to anchor the protein to the εolid surface. The surfaceε may be prepared in advance and stored. In order to conduct the assay, the binding partner of the immobilized species is exposed to the coated εurface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g.. by washing) and any complexes formed will remain immobilized on the εolid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the binding partner was pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the binding partner iε not pre-labeled, an indirect label can be used to detect complexes anchored-on the εurface; e.g. , uεing a labeled antibody εpecific for the binding partner (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody) . Depending upon the order of addition of reaction componentε, test compounds which inhibit complex formation or which disrupt, preformed complexeε can be detected.

Alternatively, the reaction can be conducted in a liquid phaεe in the preεence or abεence of the teεt compound, the reaction productε εeparated from unreacted componehtε, and complexeε detected; e.g.7 using an immobilized antibody specific for one binding partner to anchor any complexeε formed in solution, and a labeled antibody specific for the other binding partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compoundε which inhibit complex or which diεrupt preformed complexeε can be identified.

In an alternate embodiment of the invention, a homogeneouε aεεay can be uεed. In thiε approach, a preformed complex of the target gene protein and the interactive cellular or extracellular protein is prepared in which one of the binding partners iε labeled, but the εignal generated by the label iε quenched due to complex formation (see, e.g. , U.S. Patent No. 4,109,496 by Rubenstein which utilizes this approach for immunoaεεayε) . The addition of a teεt εubεtance that competeε with and diεplaces one of the binding partners from the preformed complex will result in the generation of a εignal above background. In thiε way, test subεtanceε which diεrupt target gene protein-cellular or extracellular protein interaction can be identified.

In a particular embodiment, the target gene protein can be prepared for immobilization uεing recombinant DNA techniqueε deεcribed in Section 5.4.2, εupra . For example, the target gene coding region can be fuεed to a glutathione- S-tranεferaεe (GST) gene, uεing a fusion vector such aε pGEX- 5X-1, in εuch a manner that itε binding activity iε maintained in the reεulting fuεion protein. The interactive cellular or extracellular protein can be purified and used to raiεe a monoclonal antibody, uεing methodε routinely practiced in the art and deεcribed above, in Section 5.4.3. Thiε antibody can be labeled with the radioactive iεotope ^12SI, for example, by methodε routinely practiced in the art. In a heterogeneous asεay, e.g. , the GST-target gene fuεion protein can be anchored to glutathione-agarose beads. The interactive cellular or extracellular binding partner protein can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to - occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the syεtem and allowed to bind to the complexed binding partners. The interaction between the target gene protein and the interactive cellular or extracellular binding partner protein can be detected by measuring the amount of radioactivity that remains associated with the glutathione- agarose beads. A succeεεful inhibition of the interaction by the teεt compound will result in a decrease in meaεured radioactivity.

Alternatively, the GST-target gene fusion protein and the interactive cellular or extracellular binding partner protein can be mixed together in liquid in the absence of the solid glutathione-agarose beadε. The teεt compound can be added either during or after the binding partnerε are allowed to interact. This mixture can then be added to the glutathione-agarose beadε and unbound material iε washed away. Again the extent of inhibition of the binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity asεociated with the beads. In another embodiment of the invention, theεe same techniques can be employed using peptide fragments that correspond to the binding domains of the target gene protein and the interactive cellular or extracellular protein, respectively, in place of one or both of the full length proteinε. Any number of methods routinely practiced in the art can be used to identify and iεolate the protein'ε binding εite. Theεe methods include, but are not limited to, mutagenesiε of one of the genes encoding the proteins and εcreening for disruption of binding in a co- immunoprecipitation asεay. Compensating mutations in the target gene can be selected. Sequence analyεiε of the geneε encoding the reεpective proteinε will reveal the mutationε that correspond to the region of the protein involved in interactive binding. Alternatively, one protein can be anchored to a solid εurface using methods described in thiε Section above, and allowed to interact with and bind to itε labeled binding partner, which has been treated with a proteolytic enzyme, such as trypεin. After waεhing, a εhort, labeled peptide compriεing the binding domain may remain associated with the solid material, which can be iεolated and identified by amino" acid εequencing. Alεo, once the gene coding for the for the cellular or extracellular protein iε obtained, short gene εegments can be engineered to expresε peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.

For example, and not by way of limitation, target gene can be anchored to a solid material as deεcribed above in thiε Section by making a GST-target gene fusion protein and allowing it to bind to glutathione agarose beads. The interactive cellular or extracellular binding partner protein can be labeled with a radioactive iεotope, εuch as ³⁵S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-target gene fusion protein and allowed to bind. After waεhing away unbound peptides, labeled bound material, representing the cellular or extracellular binding partner protein binding domain, can be eluted, purified, and analyzed for amino acid sequence by techniques well known in the art; e.g. , uεing the Edman degradation procedure (see e.g. , Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., pp. 34-49). Peptides so identified can be produced, using techniques well known in the art, either synthetically (see e.g. , Creighton, 1983, εupra at pp. 50-60) or, if the gene has already been isolated, by using recombinant DNA technology, aε deεcribed in Section 5.4.2, εupra .

A particular embodiment of the invention featureε a method of εcreening candidate compounds for their ability to antagonize the interaction between ligand and the receptor domain of a target gene product. The method involveε: a) mixing a candidate antagoniεt compound with a firεt compound which includeε a recombinant target gene product compriεing a receptor domain (or ligand-binding fragment or analog) on the one hand and with a second compound which includes ligand on the other hand; b) determining whether the firεt and second compoundε bind; and c) identifying antagoniεtic compoundε aε thoεe which interfere with the binding of the firεt compound to the second compound and/or which reduce the ligand- mediated release of intracellular Ca⁺⁺.

By an "antagonist" iε meant a molecule which inhibitε a particular activity, in this case, the ability of ligand to interact with a target gene product receptor domain and/or to trigger the biological events resulting from such an interaction (e.g., release of intracellular Ca⁺⁺) . Preferred therapeutics include antagonistε, e.g., peptide fragmentε (particularly, fragments derived from the N-terminal extracellular domain) , antibodies (particularly, antibodies which recognize and bind the N-terminal extracellular domain) , or drugs, which block ligand or target gene product function by interfering with the ligand-receptor interaction.

Becauεe the receptor component of the target gene product can be produced by recombinant techniqueε and because candidate antagonists may be screened in vitro, the instant invention provides a simple and rapid approach to the identification of useful therapeutics.

Specific receptor fragments of interest include any portionε of the target gene products that are capable of interaction with ligand, for example, all or part of the N- terminal extracellular domain. Such portions include the transmembrane segments and portions of the receptor deduced to be extracellular. Such fragments may be useful as antagoniεtε (aε described above) , and are also useful as immunogens for producing antibodies which neutralize the activity of the target gene product in vivo (e.g., by interfering with the interaction between the receptor and ligand; see below) . Extracellular regionε may be identified by compariεon with related proteinε of εimilar εtructure, uεeful regions are thoεe exhibiting homology to the extracellular domainε of well-characterized memberε of the family. Alternatively, from the primary amino acid εequence, the εecondary protein εtructure and, therefore, the extracellular domain regionε may be deduced εemi-empirically using a hydrophobicity/hydrophilicity calculation such aε the Chou- Faεman method (see, e.g., Chou and Fasman, Ann. Rev. Biochem . 47:251, 1978). Hydrophilic domains, particularly oneε εurrounded by hydrophobic stretches (e.g., transmembrane domains) present themselveε aε strong candidates for extracellular domains. Finally, extracellular domainε may be identified experimentally uεing εtandard enzymatic digeεt analyεis, e.g., tryptic digest analysiε.

Candidate fragmentε (e.g., all or part of the tranεmembrane εegmentε or any extracellular fragment) are teεted for interaction with ligand by the aεεayε deεcribed herein (e.g., the aεεay deεcribed above). Such fragmentε are also tested for their ability to antagonize the interaction between ligand and its endogenouε receptor using the asεays described herein. Analogs of useful receptor fragments (as deεcribed above) may alεo be produced and tested for efficacy as εcreening components or antagonistε (uεing the aεεayε described herein) ; such analogs are also considered to be uεeful in the invention.

Of particular intereεt are receptor fragmentε encompassing the extracellular main-terminal domain (or a ligand binding fragment thereof) . Also of interest are the target gene product extracellular loops. Peptide fragments derived from these extracellular loops may also be used aε antagoniεts, particularly if the loopε cooperate with the amino-terminal domain to facilitate ligand binding. Alternatively, εuch loops and extracellular N-terminal domain (as well as the full length target gene product) provide immunogenε for producing anti-target gene product antibodies. Binding of ligand to its receptor may be asεayed by any of the methodε deεcribed above in Section 5.5.1. Pref^erably, cell_'s expressing recombinant target gene product (or a suitable target gene product fragment or analog) are immobilized on a εolid substrate (e.g. , the wall of a microtitre plate or a column) and reacted with detectably- labelled ligand (as described above) . Binding is assayed by the detection label in asεociation with the receptor component (and, therefore, in aεεociation with the εolid εubstrate) . Binding of labelled ligand to receptor-bearing cells is ed as a "control" against which antagoniεt aεεayε are meaεured. The antagonist assays involve incubation of the target gene product-bearing cells with an appropriate amount of candidate antagonist. To this mix, an equivalent amount to labelled ligand is added. An antagoniεt uεeful in the invention specifically interferes with labelled ligand binding to the immobilized receptor-expresεing cellε. An antagonist is then tested for its ability to interfere with ligand function, i.e., to specifically interfere with labelled ligand binding without resulting in signal tranεduction normally mediated by the receptor. To teεt thiε uεing a functional assay, stably transfected cell lineε containing the target gene product can be produced aε deεcribed herein and reporter compoundε such as the calcium binding agent, FURA-2, loaded into the cytoplaεm by standard techniqueε. Stimulation of the heterologouε target gene product with ligand or another agonist leadε to intracellular calcium release and the concomitant fluorescence of the calcium-FURA-2 complex. This provides a convenient means for measuring agonist activity. Incluεion of potential antagoniεtε along with ligand allowε for the εcreening and identification of authentic receptor antagoniεtε aε those which effectively block ligand binding without producing fluorescence (i.e., without cauεing the mobilization of intracellular Ca**) . Such an antagoniεt may ta expected to be a useful therapeutic agent for cardiovascular disorderε.

Appropriate candidate antagoniεtε include target gene product fragments, particularly fragments containing a ligand-binding portion adjacent to or including one or more transmembrane εegmentε or an extracellular domain of the receptor (deεcribed above) ; εuch fragmentε would preferably including five or more amino acidε. Other candidate antagoniεtε include analogε of ligand and other peptideε aε well aε non-peptide compoundε and anti-target gene product antibodieε designed or derived from analysiε of the receptor.

In one embodiment, candidate compoundε can be εcreened for their ability to antagonize the interaction between a target gene product which iε a member of the TGF-b εignaling pathway and a εecond member of εaid pathway. The method involveε: a) contacting a candidate antagoniεt compound with a firεt compound comprising at least that portion of a target gene product involved in binding to a second compound, and at least that portion of the second compound which is involved in binding to the target gene product; b) determining whether the first and εecond compounds bind in the presence of the test compound; and c) identifying antagonistic compounds aε those which interfere with the binding of the first compound to the εecond compound relative to the binding observed in the absence of test compound and/or which activate or enchance the TGF-β reεponse.

One such target gene product which can be utilized as part of εuch an embodiment iε the fchd540 target gene product. Alternatively, for example, the rchd534 target gene product can be utilized. Second TGF- εignalling related compounds can include, for example, fchd540, rchd534 and activated TjSlR.

Binding of ligand to its receptor may be aεεayed by any of the methods deεcribed above in Section 5.5.1. Preferably, downεtream signaling events may be monitored as a readout for asεay reεults. For example, many signaling proteins phoεphorylate εubstrate proteins that are "downstream", in a εignaling pathway. The phoεphorylation εtate of a εubεtrate protein could therefore be examined in an assay to determine the activity of a signaling pathway, such as the TGF-/3 pathway. Becauεe the receptor component of the target gene product can be produced by recombinant techniqueε and becauεe candidate antagoniεtε may be εcreened, for example, in vitro, the inεtant invention provideε a εimple and rapid approach to the identification of useful therapeutics.

5.5.3. ASSAYS FOR AMELIORATION OF CARDIOVASCULAR AND CELL PROLIFERATIVE DISEASE SYMPTOMS

Any of the binding compoundε, including but not limited to compoundε εuch as those identified in the foregoing aεεay εyεtems, may be tested for the ability to ameliorate cardiovaεcular diεeaεe εymptoms. Cell-based and animal model-based asεayε for the identification of compounds exhibiting such an ability to ameliorate cardiovascular diεease symptoms are described below. Firεt, cell-baεed εystems εuch aε those described, above, in Section 5.4.4.2., may be used to identify compoundε which may act to ameliorate cardiovaεcular diεeaεe εymptomε. For example, εuch cell εyεtemε may be expoεed to a compound, suspected of exhibiting an ability to ameliorate cardiovascular diεease symptomε, at a εufficient concentration and for a time εufficient to elicit εuch an amelioration of cardiovaεcular diεeaεe symptomε in the expoεed cellε. After expoεure, the cells are examined to determine whether one or more of the cardiovascular diεeaεe cellular phenotypeε haε been altered to reεemble a more normal or more wild type, non-cardiovaεcular diεeaεe phenotype. For example, and not by way of limitation, in the case of monocytes, such more normal phenotypes may include but are not limited to decreaεed rates of LDL uptake, adhesion to endothelial cells, transmigration, foam cell formation, fatty streak formation, and production by foam cells of growth factors such as bFGF, IGF-I, VEGF, IL-1, M- CSF, TGF/3, TGFα, TNFα, HB-EGF, PDGF, JFN-γ, and GM-CSF. Tranεmigration rates, for example, may be measured using the in vitro system of Navab et al., described in Section 5.1.1.3, above, by quantifying the number of monocytes that migrate acrosε the endothelial monolayer and into the collagen layer of the εubendothelial space.

In addition, animal-based cardiovascular diseaεe εyεtems, such as those described, above, in Section 5.4.4.1, may be uεed to identify compoundε capable of ameliorating cardiovaεcular diεeaεe εymptoms. Such animal models may be used aε teεt εubεtrateε for the identification of drugs, pharmaceuticals, therapies, and interventions which may be effective in treating cardiovascular diseaεe. For example, animal modelε may be expoεed to a compound, suspected of exhibiting an ability to ameliorate cardiovascular diseaεe symptoms, at a sufficient concentration and for a time εufficient to elicit such an amelioration of cardiovascular diseaεe εymptoms in the exposed animals. The responεe of the animalε to the exposure may be monitored by asεeεsing the reversal of diεorderε aεεociated with cardiovaεcular diεease, for example, by counting the number of atherosclerotic plaques and/or measuring their size before and after treatment.

Further, both cell-based syεtems and animal-based syεtemε aε deεcribed herein may be used to identify compounds which act to ameliorate symptoms of fibroproliferative and oncogenic related disorderε, including tumorigenesiε and the vaεcularization of tumors. Such cell-based and animal-based systems may be exposed to a compound, suspected of exhibiting an ability to ameliorate TGF-β asεociated fibroproliferative or oncogenic disease symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration of fibroproliferative diseaεe εymptomε in the expoεed εyεtem. The reεponεe may be monitored by assesεing the reverεal of diεorderε aεεociated with fibroproliferative diεeaεe, for example by meaεuring the εize and growth of tumors or vascularization of tumors before and after treatment. With regard to intervention, any treatments which reverse any aspect of cardiovascular, fibroproliferative and oncogenic related diεeaεe εymptoms should be considered as candidates for human cardiovascular, fibroproliferative and oncogenic related diseaεe therapeutic intervention. Doεages of test agents may be determined by deriving doεe-reεponεe curves, as diεcuεεed in Section 5.7.1, below.

Additionally, gene expreεεion patternε may be utilized to assess the ability of a compound to ameliorate cardiovascular diεeaεe εymptoms. For example, the expression pattern of one or more fingerprint genes may form part of a "fingerprint profile" which may be then be uεed in εuch an aεεessment. "Fingerprint profile", aε uεed herein, referε to the pattern of mRNA expreεsion obtained for a given tisεue or cell type under a given set of conditions. Such conditionε may include, but are not limited to, atheroεcleroεiε, ischemia/reperfusion, hypertension, restenosis, and arterial inflammation, including any of the control or experimental conditions described in the paradigms of Section 5.1.1, above. Fingerprint profiles may be generated, for example, by utilizing a differential display procedure, as diεcuεεed, above, in Section 5.1.2, Northern analyεiε and/or RT-PCR. Any of the gene εequences described, above, in Section 5.4.1. may be used as probeε and/or PCR primerε for the generation and corroboration of such fingerprint profileε.

Fingerprint profileε may be characterized for known εtateε, either cardiovaεcular diεease or normal, within the cell- and/or animal-based model systemε. Subεequently, these known fingerprint profiles may be compared to aεcertain the effect a teεt compound haε to modify εuch fingerprint profileε, and to cause the profile to more closely reεemble that of a more deεirable fingerprint.

For example, adminiεtration of a compound may cauεe the fingerprint profile of a cardiovaεcular diεeaεe model system to more closely reεemble the control εyεtem. Adminiεtration of a compound may, alternatively, cauεe the fingerprint profile of a control εyεtem to begin to mimic a cardiovaεcular diεeaεe εtate. Such a compound may, for example, be uεed in _^further characterizing the compound of interest, or may be used in the generation of additional animal models.

5.5.4. MONITORING OF EFFECTS DURING CLINICAL TRIALS

Monitoring the influence of compounds on cardiovascular, fibroproliferative and oncogenic related diεeaεe εtateε may be. applied not only in baεio drug εcreening, but alεo in clinical trialε. In εuch clinical trialε, the expression of a panel of genes that have been diεcovered in one of the paradigraε deεcribed in Section 5.1.1.1 through 5.1.1.6 may be uεed aε a "read out" of a particular drug'ε effect on a cardiovascular, fibroproliferative oncogenic related diseaεe εtate.

For example, and not by way of limitation, Paradigm A provideε for the identification of fingerprint geneε that are up-regulated in monocytes treated with oxidized LDL. Thuε, to study the effect of anti-oxidant drugs, for example, in a clinical trial, blood may be drawn from patients before and at different stageε during treatment with εuch a drug. Their monocyteε may then be iεolated and RNA prepared and analyzed by differential diεplay aε deεcribed in Sectionε 6.1.1 and 6.1.2. The levelε of expreεsion of these fingerprint genes may be quantified by Northern blot analysis or RT-PCR, as described in Section 6.1.2, or by one of the methods described in Section 5.8.1, or alternatively by measuring the amount of protein produced, by one of the methodε deεcribed in Section 5.8.2. In thiε way, the fingerprint profileε may εerve aε εurrogate markerε indicative of the phyεiological reεponse of monocytes that have taken up oxidized LDL. Accordingly, this response state may be determined before, and at various points during, drug treatment. This method is deεcribed in further detail in the example in Section 8, below. Specifically, the up-regulation of fchd602 and fchd605 under treatment with oxidized LDL provides a fingerprint profile for monocytes under oxidative εtreεε. The fchd602 and fchd605 geneε can εerve, therefore, aε εurrogate markerε _during clinical treatment of cardiovaεcular diεeaεe. Accordingly, the influence of anti-oxidant drugε on 5 oxidative potential iε meaεured by recording the differential diεplay of fchd602 and fchd605 in the monocyteε of pa ^'entε undergoing clinical treatment.

Likewise, the expreεεion of fchd540 and/or rchd534 can be utilized as surrogate markerε during clinical treatment of 10 fibroproliferative and oncogenic related diεorderε. In thiε inεtance, a lowering of expreεsion from theεe geneε would be εought.

5.5.5. ASSAYS FOR COMPOUNDS THAT MODULATE

15 EXPRESSION OF TARGET GENES

Compoundε and other εubstances that modulate expresεion of target geneε can be εcreened using in vitro cellular syεtemε. In a manner analogouε to the monitoring of compoundε clinical εampleε deεcribed in Section 5.5.5, above,

20 a εample of cellε, εuch as a tisεue culture iε expoεed to a teεt εubεtance. Appropriate tissue culture cells include, but are not limited to, human umbilical vein endothelial cells (HUVECs) , bovine aortic endothelial cells (BAECs) , and 293 cellε (embryonic human kidney cells) . The RNA is then

__ extracted from the cells. The level of transcription of a specific target gene can be detected using, for example, standard RT-PCR amplification techniqueε and/or Northern analyεiε (aε described in the example in Section 6.1.2, below) . Alternatively, the level of target protein

₃₀ production can be assayed by using antibodies that detect the target gene protein, aε deεcribed in Section 5.5.1, above. The level of expreεεion iε compared to a control cell sample which was not expoεed to the teεt εubstance.

Compounds that can be screened for modulation of

₃₅ expresεion of the target gene include, but are not limited to, εmall inorganic or organic moleculeε, peptideε, εuch aε peptide hormones analogs, εteroid hormones, analogs of such hormones, and other proteins. Compounds that down-regulate expression include, but are not limited to, oligonucleotides that are complementary to the 5 '-end of the mRNA of the target gene and inhibit transcription by forming triple helix εtructures, and ribozymes or antisense molecules which inhibit translation of the target gene mRNA. Techniqueε and εtrategieε for deεigning εuch down-regulating test compounds are described in detail in Section 5.6, below.

5.6. COMPOUNDS AND METHODS FOR ACTIVATION OF TGF-B INHIBITORY RESPONSE AND TREATMENT OF CARDIOVASCULAR AND FIBROPROLIFERATIVE DISEASE

Deεcribed below are methodε and compoεitions whereby TGF-β modulated fibroproliferative and oncogenic disease symptoms may be ameliorated by compounds tliat activate or enchance the TGF-β responεe, and whereby cardiovaεcular disease symptoms may be ameliorated. In certain instanceε, fibroproliferative and oncogenic diεeaεe aεεociated with aberrant TGF-/3 εignaling, or inεtanceε of cardiovascular disease, are brought about, at least in part, by an excesεive level of gene product, or by the preεence of a gene product exhibiting an abnormal or exceεεive activity. Aε εuch, the reduction in the level and/or activity of εuch gene productε would bring about the activation or enhancement of the TFG-b reεponεe and, therefore, amelioration of diεeaεe symptoms. Techniques for the reduction of target gene expreεεion levelε or target gene product activity levelε are diεcuεεed in Section 5.6.1, below.

Alternatively, certain other fibroproliferative and oncogenic diεeaεe associated with abarrent TGF-b signaling, or inεtanceε of cardiovaεcular diεeaεe, are brought about, at leaεt in part, by the abεence or reduction of the level of gene expreεεion, or a reduction in the level of a gene product'ε activity. As such, an increase in the level of gene expresεion and/or the activity of such gene products would bring about the amelioration of cardiovaεcular diεeaεe εymptomε. In εome caεes, the up-regulation of a gene in a diεeaεe εtate reflectε a protective role for that gene product in reεponding to the diεeaεe condition. Enhancement of εuch a target gene'ε expreεεion, or the activity of the target gene product, will reinforce the protective effect it exertε. Fibroproliferative and oncogenic diεeaεe aεεociated with aberrant TGF-β εignaling, or inεtances of cardiovascular diseaεe, εtates may result from an abnormally low level of activity of εuch a protective gene. In theεe cases also, an increase in the level of gene expreεεion and/or the activity of εuch gene productε would bring about the amelioration of cardiovaεcular diεeaεe εymptomε. Techniques for increaεing target gene expreεεion levelε or target gene product activity levelε are diεcuεεed in Section 5.6.2, below. With reεpect to εpecific fibroproliferative and oncogenic disease asεociated with aberrant TGF-β εignaling, the diεeaεeε that can be treated or prevented by the methodε of the present invention include but are not limited to: human sarcomaε and carcinomaε, e .g. , fibroεarcoma, myxoεarcoma, lipoεarcoma, chondroεarcoma, oεteogenic εarcoma, chordoma, angioεarcoma, endothelioεarcoma, lymphangioεarcoma, lymphangioendothelioεarcoma, εynovioma, meεothelioma, Ewing'ε tumor, leiomyoεarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamouε cell carcinoma, baεal cell carcinoma, adenocarcinoma, εweat gland carcinoma, εebaceouε gland carcinoma, papillary carcinoma, papillary adenocarcinomaε, cyεtadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, εeminoma, embryonal carcinoma, Wilmε' tumor, cervical cancer, teεticular tumor, lung carcinoma, εmall cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, aεtrocytoma, medulloblaεtoma, craniopharyngio a, ependymoma, pinealoma, hemangioblaεtoma, acouεtic neuroma, oligodendroglioma, meningioma, melanoma, neuroblaεtoma, retinoblastoma; leukemias, e .g. , acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia) ; chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia) ; and polycythemia vera, lymphoma (Hodgkin's diseaεe and non- Hodgkin'ε disease), multiple myeloma, Waldenstrδm'ε macroglobulinemia, and heavy chain diεeaεe.

5.6.1. COMPOUNDS THAT INHIBIT EXPRESSION, SYNTHESIS OR ACTIVITY OF MUTANT TARGET GENE ACTIVITY

Aε diεcusεed above, target genes involved in cardiovascular diseaεe diεorderε can cauεe εuch diεorderε via an increaεed level of target gene activity. Aε εummarized in Table 1, above, and detailed in the exampleε in Sections 6 and 7, below, a number of genes have been demonstrated to be up-regulated in monocytes and endothelial cells under diseaεe conditionε. Specifically, fchd602 and fchd605 are each up- regulated in monocyteε treated with oxidized LDL. Furthermore, fchd540 iε up-regulated in endothelial cellε εubjected to εhear εtreεε and in εome cancer cellε. In εome cases, such up-regulation may have a cauεative or exacerbating effect on the diεease state. A variety of techniques may be utilized to inhibit the expreεεion, εyntheεiε, or activity of εuch target genes and/or proteins.

For example, compounds such as thoεe identified through assays described, above, in Section 5.5, which exhibit inhibitory activity, may be uεed in accordance with the invention to ameliorate cardiovascular diseaεe symptomε. Aε diεcuεsed in Section 5.5, above, such molecules may include, but are not limited to small organic molecules, peptides, antibodieε, and the like. Inhibitory antibody techniqueε are deεcribed, below, in Section 5.6.1.2.

For example, compoundε can be adminiεtered that compete with endogenouε ligand for a transmembrane target gene product. The resulting reduction in the amount of ligand- bound target gene transmembrane protein will modulate cell physiology. Compounds that can be particularly useful for thiε purpoεe include, for example, soluble proteins or peptides, εuch aε peptides comprising one 5r more of the extracellular domains, or portions and/or analogs thereof, of the target gene product, including, for example, soluble fusion proteins εuch as Ig-tailed fusion proteinε. (For a discusεion of the production of Ig-^ailed fuεion proteinε, εee, for example, U.S. Patent No. 5,116,964.). Alternatively, compoundε, εuch aε ligand analogε or antibodies, that bind to the target gene product receptor site, but do not activaterthe protein, (e.g., receptor-ligand antagonistε) can be effective in inhibiting target gene product activity.

Further, antiεenεe and ribozyme moleculeε which inhibit expreεεion of the target gene may alεo be uεed in accordance with the invention to inhibit the aberrant target gene activity. Such techniques are described, below, in Section 5.6.1.1. Still further, alεo aε deεcribed, below, in Section 5.6.1.1, triple helix moleculeε may be utilized in inhibiting the aberrant target gene activity.

5.6.1.1. INHIBITORY ANTISENSE, RIBOZYME,

TRIPLE HELIX, AND GENE INACTIVATION APPROACHES

Among the compoundε which may exhibit the ability to ameliorate cardiovaεcular diεeaεe εymptomε are antiεenεe, ribozyme, and triple helix moleculeε. Such moleculeε may be designed to reduce or inhibit mutant target gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.

Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation.

Antisenεe approacheε involve the deεign of oligonucleotideε (either DNA or RNA) that are complementary to target gene mRNA. The antiεenεe oligonucleotideε will bind to the complementary target gene mRNA transcripts and prevent translation. Abεolute complementarity, although preferred, is not -required. A sequence "complementary" to a portion of an RNA, as referred to herein, means a εequence having εufficient complementarity to be able to hybridize with the RNA, forming a εtable duplex; in the caεe of double- εtranded antiεenεe nucleic acidε, a εingle strand of the duplex DNA may thus be tested, or triplex formation may be aεεayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antiε^'ense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and εtill form a εtable duplex (or triplex, as the case may be) . One skilled in the art can ascertain a tolerable degree of miεmatch by use of standard procedures to determine the melting point of the hybridized complex. Oligonucleotides that are complementary to the 5' end of the meεεage, e.g. , the 5' untranεlated sequence up to and including the AUG initiation codon, εhould work moεt efficiently at inhibiting translation. However, sequenceε complementary to the 3 ' untranεlated εequenceε of mRNAε have recently shown to be effective at inhibiting tranεlation of mRNAs as well. See generally, Wagner, R. , 1994, Nature 372:333-335. Thuε, oligonucleotideε complementary to either the 5'- or 3'- non- translated, non-coding regions of the target gene could be used in an antisense approach to inhibit translation of endogenous target gene mRNA. Oligonucleotideε complementary to the 5' untranεlated region of the mRNA εhould include the complement of the AUG start codon. Antiεenεe oligonucleotideε complementary to mRNA coding regionε are leεs efficient inhibitors of translation but could be used in accordance with the invention. Whether deεigned to hybridize to the 5'-, 3'- or coding region of target gene mRNA, antiεenεe nucleic acidε εhould be at leaεt εix nucleotideε in length, and are preferably oligonucleotideε ranging from 6 to about 50 nucleotides in length. In specific aεpects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotideε or at leaεt 50 nucleotides. Regardleεε of the choice of target εequence, it iε preferred that in vitro εtudieε are first performed to quantitate the ability of the antisenεe oligonucleotide to inhibit gene expreεεion. It iε preferred that these studies utilize controls that distinguish between antisenεe gene inhibition and nonεpecific biological effects of oliςOnucleotides. It iε alεo preferred that theεe st dies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisenεe oligonucleotide are compared with thoεe obtained uεing a control oligonucleotide. It iε preferred that the control oligonucleotide iε of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differε from the antiεenεe εequence no more than iε neceεεary to prevent εpecific hybridization to the target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures or derivativeε or modified verεionε thereof, single- stranded or double-stranded. The oligonucleotide can be modified at the base moiety, εugar moiety, or phoεphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groupε such aε peptideε (e.g. , for targeting host cell receptorε in vivo) , or agents facilitating transport across the cell membrane (see, e .g. , Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810, publiεhed December 15, 1988) or the blood-brain barrier (see, e .g. , PCT Publication No. WO89/10134, publiεhed April 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988, BioTechniqueε 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered crosε-linking agent, transport _agent, hybridization-triggered cleavage agent, etc. The antisenεe oligonucleotide may compriεe at least one modified base moiety which iε εelected from tue group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactoεylqueoεine, inoεine, N6-iεopentenyladenine, ^* 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytoεine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, b.ta-D-mannosylqueoεine, 5 ' -methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-iεopentenyladenine, uracil-5-oxyacetic acid (v) , wybutoxosine, -pεeudouracil, queoεine, 2-thiocytoεine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil- 5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v) , 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

The antisenεe oligonucleotide may alεo compriεe at leaεt one modified εugar moiety εelected from the group including but not limited to arabinoεe, 2-fluoroarabinoεe, xyluloεe, and hexoεe. In yet another embodiment, the antiεenεe oligonucleotide compriεeε at leaεt one modified phoεphate backbone selected from the group conεiεting of a phosphorothioate, a phoεphorodithioate, a phoεphoramidothioate, a phoεphora idate, a phosphordiamidate, a methyIphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisenεe oligonucleotide is an α-anomeric oligonucleotide. An o-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual 3-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2'- O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

Oligonucleotideε of the invention may be εyntheεized by εtandard methodε known in the art, e .g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Bioεyεtemε, etc.). Aε exampleε, phoεphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotideε can be prepared by use of controlled pore glasε polymer εupportε (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.

While antiεenεe nucleotideε complementary to the target gene coding region εequence could be uεed, thoεe complementary to the tranεcribed untranεlated region are moεt preferred.

Specific antiεenεe oligonucleotideε for the rchd534 gene and fchd540 gene are deεcribed in the Example in Section 13, below.

The antisense molecules should be delivered to cellε which expreεε the target gene in vivo, e.g. , endothelial cells. A number of methods have been developed for delivering antisense DNA or RNA to cells; e.g. , antiεenεe moleculeε can be injected directly into the tiεεue εite, or modified antisenεe moleculeε, deεigned to target the desired cells (e.g.. antisenεe linked to peptides or antibodies that εpecifically bind receptors or antigens expressed on the target cell εurface) can be adminiεtered εyεtemically.

However, it iε often difficult to achieve intracellular concentrations of the antisenεe sufficient to suppress translation of endogenouε mRNAε. Therefore a preferred approach utilizeε a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a εtrong pol III or pol II promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of εufficient amountε of εingle εtranded RNAε that will form complementary baεe pairε with the endogenouε target gene transcriptε and thereby prevent translation of the target gene mRNA. ^rFor example, a vector can be introduced in vivo εuch that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain epiεomal or become chromoεomally integrated, aε long aε it can be tranεcribed to produce the deεired antiεenεe RNA. Such vectorε can be constructed by recombinant DNA technology methodε εtandard in the art. Vectorε can be plaεmid, viral, or otherε known in -the art, uεed for replication and expreεεion in mammalian cells. Expresεion of the εequence encoding the antiεense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such^"- promoterε include but are not limited to: the SV40 early promoter region (Bernoiεt and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous εarcoma viruε (Yamamoto et al., 1980, Cell 22:787-797), the herpeε thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441- 1445) , the regulatory sequenceε of the metallothionein gene (Brinεter et al. , 1982, Nature 296:39-42), etc. Any type of plasmid, cosmid, YAC or viral vector can be uεed to prepare the recombinant DNA construct which can be introduced directly into the tisεue site; e.g. , atherosclerotic vascular tissue. Alternatively, viral vectors can be uεed which selectively infect the deεired tiεεue, in which case administration may be accomplished by another route (e.g.. syεtemically) .

Ribozymeε are enzymatic RNA moleculeε capable of catalyzing the εpecific cleavage of RNA. The mechanism of ribozyme action involveε εequence εpecific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. Ribozyme moleculeε deεigned to catalytically cleave target gene mRNA transcripts can also be used to prevent translation of target gene mRNA and expression of target gene. (See, e.g. , PCT International Publication WO90/11364, published October 4, 1990; Sarver et al., 1990, Science 247:1222-1225). While ribozymes that cleave mRNA at site εpecific recognition εequenceε can be used to destroy target gene mRNAs, the uεe'of hammerhead ribozymeε iε preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The εole requirement iε that the target mRNA have the following εequence of two baseε: 5'-UG-3'. The conεtruction and production of hammerhead ribozymes iε well known in the art and iε deεcribed more fully in Haseloff arid Gerlach, 1988, Nature, 334:585-591. For example, there are hundreds of potential hammerhead ribozyme cleavage εiteε within the nucleotide εequence of rchd534 and fchd540 cDNA. Preferably the ribozyme iε engineered εo that the cleavage recognition εite iε located near the 5' end of the target mRNA; i.e. , to increaεe efficiency and minimize the intracellular accumulation of non-functional mRNA tranεcriptε.

Specific hammerhead ribozymeε moleculeε for the rchd534 and fchd540 geneε are deεcribed in the Example in Section 13, below. The ribozymeε of the preεent invention alεo include RNA endoribonucleaεes (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena Thermophila (known aε the IVS, or L-19 IVS RNA) and which haε been extenεively deεcribed by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al. , 1986, Nature, 324:429- 433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). The Cech-type ribozymes have an eight baεe pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The invention encompaεses thoεe Cech-type ribozymeε which target eight baεe-pair active site sequenceε that are present in target gene. As in the antiεenεe approach, the ribozymeε can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which expreεs the ^"target gene in vivo, e.g. , endothelial cells. A preferred method of delivery involveε uεing a DNA construct "encoding" the ribozyme under the control of a strong conεtitutive pol III or pol II promoter, εo that tranεfected cellε will produce εufficient quantitieε of the ribozyme to deεtroy endogenouε target gene meεεages and inhibit translation. Because ribozymeε, unlike antiεense molecules, are catalytic, a lower intracellular concentration is required for efficiency. Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription should be εingle εtranded and compoεed of deoxyribonucleotideε. The baεe compoεition of theεe oligonucleotideε muεt be deεigned to promote triple helix formation via Hoogεteen baεe pairing ruleε, which generally require εizeable εtretcheε of either purineε or pyrimidineε to be preεent on one εtrand of a duplex. Nucleotide εequenceε may be pyrimidine-based, which will result in TAT and CGC* triplets across the three asεociated εtrandε of the reεulting triple helix. The pyrimidine-rich moleculeε provide baεe complementarity to a purine-rich region of a εingle εtrand of the duplex in a parallel orientation to that εtrand. In addition, nucleic acid moleculeε may be choεen that are purine-rich, for example, containing a εtretch of G residues. These moleculeε will form a triple helix with a DNA duplex that iε rich in GC pariε, in which the majority of the purine reεidueε are located on a εingle εtrand of the targeted duplex, reεulting in GGC tripletε across the three strandε in the triplex. Alternatively, the potential εequenceε that can be targeted for triple helix formation may be increaεed by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3 '-5' manner, such that they base pair with firεt one εtrand of a duplex and then the other, eliminating the neceεεity for a εizeable εtretch of either purineε or pyrimidineε to be preεent on one εtrand of a duplex. It is possible that the antisenεe, ribozyme, and/or triple helix molecules described herein may reduce or inhibit the transcription _(triple helix) and/or tranεlation (antiεenεe, ribozyme) of mRNA produced by both normal and 5 mutant target gene alleleε. In order to enεure that εubstantially normal levelε of target gene activity ar° maintained, nucleic acid moleculeε that encode and e cess target gene polypeptides exhibiting normal activity may be introduced into cells via gene therapy methods such as those

10 described, below, in Section 5.7. that do not contain sequenceε εuεceptible to whatever antiεense, ribozyme, or triple helix treatments are being utilized. Alternatively, it may be preferable to coadminister normal target gene protein into the cell or tisεue in order to maintain the

15 requiεite level of cellular or tiεεue target gene activity.

Endogenouε target gene expreεεion can also be reduced by inactivating or "knocking out" the target gene or itε promoter using targeted homologous recombination. (E.g. , see Smithies et al., 1985, Nature 317:230-234; Thomas & Capecchi,

20 1987, Cell 51:503-512; Thompson et al., 1989 Cell 5:313-321; each of which is incorporated by reference herein in its entirety) . For example, a mutant, non-functional target (or a completely unrelated DNA sequence) flanked by DNA homologouε to the endogenouε target gene (either the coding

25 regionε or regulatory regionε of .the target gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cellε that expreεε target in vivo. Inεertion of the DNA conεtruct, via targeted homologouε recombination, resultε in inactivation of the

30 target gene. Such approacheε can be adapted for uεe in humans provided the recombinant DNA conεtructε are directly adminiεtered or targeted to the required site in vivo using appropriate viral vectors, e.g. , vectors for delivery vascular tisεue.

35 Alternatively, endogenous target gene expresεion can be reduced by targeting deoxyribonucleotide εequences complementary to the regulatory region of the target gene (i.e. , the target promoter and/or enhancerε) to form triple helical εtructureε that prevent tranεcription of the target gene in target cellε in the body. (See generally, Helene, C. 1991, Anticancer Drug Deε., 6(6): 569-84; Helene, C. , et al. , 5 1992, Ann, N.Y. Acad. Sci., 660:27-36; and Maher, L.J. , 1992, Bioaεεays 14 (12) : 807-15) .

In yet another embodiment of the invention, the activity of a target can be reduced using a "dominant negative" approach to effectuate reduction in cardiovascular disease 0 symptomε. For example, if two gene productε interact, εuch aε the rchd534 and fchd540 proteinε, then the presence of a mutant version of one or both of these proteins in the cell can reduce the overall pool of complexes consiεting of entirely wild-type proteinε. In this manner, the overall 5 level of activity resulting from the rchd5€4/fchd540 protein interaction can be reduced.

5.6.1.2. ANTIBODIES FOR TARGET GENE PRODUCTS Antibodieε that are both εpecific for target gene 0 protein and interfere with itε activity may be uεed to inhibit target gene function. Such antibodieε may be generated uεing εtandard techniqueε described in Section 5.4.3., εupra , against the proteins themselveε or againεt peptideε correεponding to portionε of the proteinε. Such 5 antibodieε include but are not limited to polyclonal, monoclonal, Fab fragmentε, εingle chain antibodieε, chimeric antibodieε, etc.

In inεtanceε where the target gene protein iε intracellular and whole antibodieε are uεed, internalizing 0 antibodies may be preferred. However, lipofectin liposomeε may be uεed to deliver the antibody or a fragment of the Fab region which bindε to the target gene epitope into cellε. Where fragmentε of the antibody are uεed, the smallest inhibitory fragment which binds to the target protein's 5 binding domain is preferred. For example, peptideε having an amino acid εequence corresponding to the domain of the variable region of the antibody that binds to the target gene protein may be used. Such peptides may be syntheεized chemically or produced via recombinant DNA technology uεing methodε well known in the art (e.g. , εee Creighton, 1983, εupra ; and Sambrook et al., 1989, εupra) . Alternatively, εingle chain neutralizing antibodieε which bind to intracellular target gene epitopeε may alεo be ad iniεtered. Such εingle chain antibodieε may be adminiεtered, for example, by expreεεing nucleotide εequenceε encoding εingle- - chain antibodieε within the target cell population by utilizing, for example, techniqueε εuch as those described in Maraεco et al. (Maraεco, W. et al., 1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).

In εome inεtances, the target gene protein is extracellular, or is a tranεmembrane protein, εuch aε the fchd545 and fchd602 gene productε. Antibodieε that are εpecific for one or more extracellular domainε of these gene products, for example, and that interfere with itε activity, are particularly uεeful in treating cardiovaεcular diεeaεe. Such antibodieε are eεpecially efficient becauεe they can access the target domains directly from the bloodstream. Any of the administration techniques deεcribed, below in Section 5.7 which are appropriate for peptide adminiεtration may be utilized to effectively administer inhibitory target gene antibodies to their site of action.

5.6.2. METHODS FOR RESTORING OR ENHANCING TARGET GENE ACTIVITY

Target geneε that cauεe cardiovaεcular diεease may be underexpresεed within cardiovaεcular diεeaεe εituationε. Aε εummarized in Table 1, above, and detailed in the example in Section 7, below, εeveral geneε are now known to be downregulated in endothelial cellε under diεeaεe conditionε. Specifically, fchd531 and fchd545 are down-regulated in endothelial cellε εubjected to εhear εtreεε. Alternatively, the activity of target gene productε may be decreaεed, leading to the development of cardiovaεcular diεeaεe εymptomε. Such down-regulation of target gene expreεεion or decreaεe of target gene product activity might have a cauεative or exacerbating effect on the diseaεe εtate.

In εome caεeε, target geneε that are up-regulated in the diεeaεe εtate might be exerting a protective effect. Aε εummarized in Table 1, above, and detailed in the examples in Sections 6 and 7, below, a number of genes are now known to be up-regulated in monocyteε and endothelial cellε ^"under diεeaεe conditionε. Specifically, fchd602 and fchdό05 are each up-regulated in monocyteε treated with oxidized LDL. Furthermore, fchd540 iε up-regulated in endothelial cellε εubjected to εhear streεε. A variety of techniqueε may be utilized to increaεe the expreεεion, εyntheεiε, or activity of εuch target genes and/or proteinε, for thoεe geneε that exert a protective effect in reεponεe to disease conditions. Described in this Section are methods whereby the level of target gene activity may be increased to levelε wherein cardiovaεcular diεeaεe εymptomε are ameliorated. The level of gene activity may be increased, for example, by either increasing the level of target gene product present or by increasing the level of active target gene product which is present.

For example, a target gene protein, at a level sufficient to ameliorate cardiovascular disease symptoms maybe administered to a patient exhibiting such symptoms. Any of the techniques discuεεed, below, in Section 5.7, may be utilized for εuch adminiεtration. One of skill in the art will readily know how to determine the concentration of effective, non-toxic doεeε of the normal target gene protein, utilizing techniqueε such aε thoεe deεcribed, below, in Section 5.7.1.

Additionally, RNA εequenceε encoding target gene protein may be directly adminiεtered to a patient exhibiting cardiovaεcular diεeaεe εymptomε, at a concentration sufficient to produce a level of target gene protein such that cardiovaεcular diεeaεe symptoms are ameliorated. Any of the techniques discussed, below, in Section 5.7, which achieve intracellular administration of compounds, such as, f Dr example, liposome adminiεtration, may be utilized for the adminiεtration of such RNA molecules. The- RNA moleculeε may be produced, for example, by recombinant techniques εuch aε those deεcribed, above, in Section 5.4.2. Further, patientε may be treated by gene replacement therapy. One or more copieε of a normal target gene, or a portion of the gene that directε the production of a normal target gene protein with target gene function, may be inεerted into cellε uεing vectorε which include, but are not limited to adenoviruε, adeno-aεεociated viruε, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such aε lipoεomes. Additionally, techniques such as those described above may be utilized for the introduction of normal target gene sequenceε into human cellε. Cellε, preferably, autologouε cellε, containing normal target gene expreεεing gene εequences may then be introduced or reintroduced into the patient at poεitionε which allow for the amelioration of cardiovascular disease symptomε. Such cell replacement techniqueε may be preferred, for example, when the target gene product iε a εecreted, extracellular gene product.

5.7. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION The identified compoundε that inhibit target gene expreεεion, synthesiε and/or activity can be adminiεtered to a patient at therapeutically effective doεeε to treat or ameliorate cardiovaεcular diεeaεe. A therapeutically effective doεe referε to that amount of the compound εufficient to reεult in amelioration of εymptomε of cardiovascular diseaεe. 5.7.1. SFFECTIVE DOSE Toxicity and therapeutic efficacy of εuch compounds can be determined by εtandard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expresεed as the ratio LD_S0/ED_5O. Compounds which exhibit large therapeutic indices are preferred. Whi¹e compounds that exhibit toxic side effects may be uεed, care εhould be taken to deεign a delivery εyεtem that targetε such compounds to the site of affected tiεεue in order to minimize potential damage to uninfected cellε and, thereby, reduce εide effectε. The data obtained from the cell culture aεεayε and animal εtudies can be uεed in formulating a range of doεage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within thiε range depending upon the doεage form employed and the route of adminiεtration utilized. For any compound uεed in the method of the invention, the therapeutically effective doεe can be eεtimated initially from cell culture aεεayε. A doεe may be formulated in animal modelε to achieve a circulating plaεma concentration range that includes the IC_S0 (i.e. , the concentration of the teεt compound which achieveε a half-maximal inhibition of εymptomε) aε determined in cell culture. Such information can be uεed to more accurately determine uεeful doεeε in humanε. Levelε in plaεma may be measured, for example, by high performance liquid chromatography. 5.7.2. FORMULATIONS AND USE Pharmaceutical compoεitionε for uεe in accordance with the preεent invention may be formulated in conventional manner uεing one or more phyεiologically acceptable carriers or excipients.

Thus, the compoundε and their phyεiologically acc^eptable εalϊε and εolvateε may be formulated for adminiεtrati^.. by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal adminiεtration. For oral adminiεtration, the pharmaceutical compoεitionε may take the form of, for example, tabletε or capεuleε prepared by conventional meanε with pharmaceutically acceptable excipientε εuch as binding agents (e.g. , pregelatiniεed maize εtarch, polyvinylpyrrolidone or hydroxypropyl methylcelluloεe) ; fillerε (e.g. , lactoεe, microcryεtalline celluloεe or calcium hydrogen phoεphate) ; lubricantε (e.g. , magneεium stearate, talc or silica) ; disintegrants (e.g. , potato starch or sodium starch glycolate) ; or wetting agentε (e.g. , εodium lauryl εulphate) . The tablets may be coated by methodε well known in the art. Liquid preparationε for oral adminiεtration may take the form of, for example, εolutionε, εyrupε or εuεpenεionε, or they may be preεented aε a dry product for conεtitution with water or other εuitable vehicle before uεe. Such liquid preparationε may be prepared by conventional meanε with pharmaceutically acceptable additives such as suεpending agentε (e.g. , εorbitol εyrup, celluloεe derivativeε or hydrogenated edible fatε) ; emulεifying agentε (e.g. , lecithin or acacia); non-aqueouε vehicleε (e.g.. almond oil, oily eεterε, ethyl alcohol or fractionated vegetable oilε) ; and preservatives (e.g. , methyl or propyl-p-hydroxybenzoates or εorbic acid) . The preparationε may alεo contain buffer εalts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal adminiεtration the compoεitions may take the form of tablets or lozenges formulated in con/entional manner.

For administration by inhalation, the compounds for use according to the preεent invention are conveniently delivered in the form of an aerosol spray presentation from preεεurized packε or a nebuliεer, with the uεe of a εuitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other εuitable gaε. In the caεe of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsuleε and cartridgeε of e.g. gelatin for uεe in an inhaler or inεufflator may be formulated containing a powder mix of the compound and a εuitable powder baεe εuch aε lactoεe or εtarch.

The compoundε may be formulated for parenteral adminiεtration by injection, e.g. , by boluε injection or continuouε infuεion. Formulationε for injection may be preεented in unit doεage form, e.g. , in ampouleε or in multi- dose containers, with an added preservative. The compositionε may take εuch formε aε εuεpenεionε, εolutionε or emulεionε in oily or aqueouε vehicleε, and may contain formulatory agentε such aε εuεpending, εtabilizing and/or diεperεing agentε. Alternatively, the active ingredient may be in powder form for constitution with a εuitable vehicle, e.g.. εterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositionε εuch as suppositories or retention enemas, e.g.. containing conventional suppoεitory bases such aε cocoa butter or other glycerideε.

In addition to the formulationε deεcribed previouεly, the compoundε may alεo be formulated aε a depot preparation. Such long acting formulations may be adminiεtered by implantation (for example εubcutaneouεly or intramuεcularly) or by intramuεcular injection. Thuε, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulεion in an acceptable oil) or ion exchange reεins, or aε εparingly εoluble derivativeε, for example, aε a εparingly εoluble εalt.

The compoεitionε may, if deεired, be preεented in a pack or diεpenεer device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example compriεe metal or plaεtic foil, εuch aε a bliεter pack. The pack or diεpenser device may be accompanied by instructions for adminiεtration.

5.8. DIAGNOSIS OF CARDIOVASCULAR AND FIBROPROLIFERATIVE DISEASE ABNORMALITIES

A variety of methodε may be employed, utilizing reagentε εuch aε fingerprint gene nucleotide εequenceε deεcribed in Section 5.4.1, and antibodieε directed againεt differentially expreεεed and pathway gene peptides, as deεcribed, above, in Sectionε 5.4.2. (peptideε) and 5.4.3. (antibodieε). Specifically, εuch reagentε may be uεed, for example, for the detection of the preεence of target gene mutationε, or the detection of either over or under expreεεion of target gene mRNA.

The methods described herein may be performed, for example, by utilizing pre-packaged diagnoεtic kitε compriεing at leaεt one εpecific fingerprint gene nucleic acid or anti- ₅ fingerprint gene antibody reagent deεcribed herein, which may be conveniently uεed, e.g. , in clinical εettingε, to diagnoεe patientε exhibiting cardiovaεcular diεeaεe εymptomε or at riεk for developing cardiovaεcular diεease.

Any cell type or tiεεue, preferably monocyteε, _Q endothelial cellε, or εmooth muscle cells, in which the fingerprint gene is expresεed may be utilized in the diagnoεticε deεcribed below.

5.8.1. DETECTION OF FINGERPRINT GENE NUCLEIC

ACIDS 5

DNA or RNA from the cell type or tiεεue to be analyzed may easily be isolated using procedures which are well known to thoεe in the art. Diagnoεtic procedures may also be performed "in εitu" directly upon tiεεue εections (fixed and/or frozen) of patient tiεεue obtained from biopsies or resections, εuch that no nucleic acid purification iε necesεary. Nucleic acid reagents such aε thoεe deεcribed in Section 5.1. may be uεed as probeε and/or primers for εuch in εitu procedureε (εee, for example, Nuovo, G.J. , 1992, PCR in εitu hybridization: protocolε and applications, Raven Preεε, NY) . Fingerprint gene nucleotide εequenceε, either RNA or DNA, may, for example, be uεed in hybridization or amplification assayε of biological εampleε to detect cardiovaεcular diεeaεe-related gene εtructureε and expreεεion. Such aεεayε may include, but are not limited to, Southern or Northern analyεeε, εingle εtranded conformational polymorphiεm analyεeε, in εitu hybridization aεεayε, and polymeraεe chain reaction analyεeε. Such analyεeε may reveal both quantitative aεpectε of the expreεεion pattern of the fingerprint gene, and qualitative aεpectε of the fingerprint gene expression and/or gene composition. That iε, εuch aεpects may include, for example, point mutations, insertionε, deletionε, chromoεomal rearrangements, and/or activation or inactivation of gene expresεion.

Preferred diagnoεtic methodε for the detection of fingerprint gene-εpecific nucleic acid moleculeε may involve for example, contacting and incubating nucleic acids, derived from the cell type or tiεεue being analyzed, with one or more labeled nucleic acid reagents as are described in Section 5.1, under conditions favorable for the specific annealing of theεe reagentε to their complementary εequenceε within the nucleic acid molecule of intereεt. Preferably, the lengths of these nucleic acid reagentε are at leaεt 9 to 30 nucleotideε. After incubation, all non-annealed nucleic acidε are removed from the nucleic acid: fingerprint molecule hybrid. The preεence of nucleic acidε from the fingerprint tiεsue which have hybridized, if any such molecules exist, iε then detected. Uεing εuch a detection scheme, the nucleic acid from the tiεεue or cell type of intereεt may be immobilized, for example, to a solid support such aε a membrane, or a plaεtic εurface εuch aε that on a microtitre plate or polyεtyrene beadε. In thiε caεe, after incubation, non-annealed, labeled fingerprint nucleic acid reagentε of the type deεcribed m Section 5.1. are easily removed. Detection of the remaining, annealed, labeled nucleic acid reagents is accomplished using εtandard techniqueε well-known to thoεe in the art. Alternative diagnoεtic methodε for the detection of fingerprint gene εpecific nucleic acid moleculeε may involve their amplification, e.g., by PCR (the experimental embodiment εet forth in Mulliε, K.B., 1987, U.S. Patent No. 4,683,202), ligaεe chain reaction (Barany, F. , 1991, Proc. Natl. Acad. Sci. USA 88:189-193), εelf εuεtained εequence replication (Guatelli, J.C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), tranεcriptional amplification εyεtem (Kwoh, D.Y et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicaεe (Lizardi, P.M. et al., 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified moleculeε uεing techniqueε well known to thoεe of εkill in the art. Theεe detection εchemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

In one embodiment of εuch a detection εcheme, a cDNA molecule iε obtained from an RNA molecule of inter »est (e.g—.. by reverse transcription of the RNA molecule into cDNA) . Cell types or tisεueε from which εuch RNA may be iεolated include any tiεεue in which wild type fingerprint gene is known to be expresεed, including, but not limited, to monocytes, endothelium, and/or smooth muεcle. A fingerprint εequence within the cDNA iε then uεed as the template for a nucleic acid amplification reaction, such aε a PCR amplification reaction, or the like. The nucleic acid reagentε uεed aε εyntheεiε initiation reagentε (e.g.. primerε) in the reverεe tranεcription and nucleic acid amplification εteps of this method are chosen from among the fingerprint gene nucleic acid reagents described in Section 5.1. The preferred lengths of such nucleic acid reagents are at leaεt 15-30 nucleotideε. For detection of the amplified product, the nucleic acid amplification may be performed uεing radioactively or non-radioactively labeled nucleotideε. Alternatively, enough amplified product may be made εuch that the product may be visualized by standard ethidium bromide εtaining or by utilizing any other εuitable nucleic acid εtaining method.

With reεpect to mutationε or polymorphiεmε in gene εtructure, the methodε deεcribed below can be uεed in addition to, or in cunjuction with those thoεe diεcussed above. In thiε context, mutationε or polymorphiεmε within any of target geneε of the invention, e.g. , fchd540 or a related gene, can be detected by utilizing a number of techniqueε. Nucleic acid from any nucleated cell can be uεed aε the εtarting point for εuch aεεay techniqueε, and may be iεolated according to εtandard nucleic acid preparation procedureε which are well known to thoεe of εkill in the art. Genomic DNA may be uεed in hybridization or amplification assays of biological sampleε to detect abnormalitieε involving a target gene structure including point mutations, insertions, deletions and chromosomal rearrangements. Such asεayε may include, but are not limited to, Southern analyses, single stranded conformation polymorphism analyseε (SSCP) , and PCR analyses.

Further, well-known genotyping techniques can be performed to type polymorphisms that are in close proximity to mutations in the target gene itself, including mutationε aεεociated with fibroproliferative, oncogenic or cardiovaεcular disorderε. Such polymorphisms can be used to identify individualε of a population likely to carry mutations in the target gene e.g. _f fchd540 or a related gene. If a polymorphism exhibits linkage disequilibrium with mutations in the target gene e.g. _f fchd540, the polymorphism can alεo be used to identify individuals in the general population who are likely to carry such mutations. Polymorphiεmε that can be uεed in thiε way include reεtriction fragment length polymorphiεmε (RFLPs) , which involve sequence variations in restriction enzyme target εequenceε, εingle-baεe polymorphiεmε, and εimple εequence length polymorphiεmε (SSLPs) .

.-- For example, Weber (U.S. Pat. No. 5,075,217) de^_^-ibes a DNA marker baεed on length polymorphiεmε in blockε of (dc- dA)n-(dG-dT)n εhort tandem repeatε. The average εeparation of (dC-dA)n-(dG-dT)n blockε iε eεtimated to be 30,000-60,000 bp. Markerε that are εo cloεely εpaced exhibit a high frequency co-inheritance, and are extremely uεeful in the identification of genetic mutationε, such as, for example, mutations within the fchd540 or a related gene, and the diagnosis of diseaεeε and diεorderε related to mutationε in the target gene.

Also, Caskey et al . (U.S. Pat.No. 5,364,759) deεcribe a DNA profiling aεεay for detecting εhort tri and tetra nucleotide repeat sequences. The process includes extracting the DNA of intereεt, εuch as the target gene, e.g. , fchd540 or a related gene, amplifying the extracted DNA, and labelling the repeat sequenceε to form a genotypic map of the individual'ε DNA.

A target gene, e.g. , fchd540 or a related probe could additionally be uεed to directly identify RFLPε. Further, a target gene or a related probe or primerε .derived from the target gene sequence could be used to iεolate genomic cloneε εuch aε YACε, BACε, PACε, coεmidε, phage, or plaεmids. The DNA contained in these clones can be screened for single-base polymorphisms or SSLPs using standard hybridization or sequencing procedureε.

Further, tranεgenic animalε developed from of the invention include animalε that expreεε a mutant variant or polymorphism of a target gene, e.g. , fchd540 or a related gene, particularly a mutant variant or polymorphism of a fchd540 or a related gene that is associated with fibroproliferative, oncogenic or cardiovaεcular diεorders is of use in diagnosis and treament of such diseiεes.

In addition to methods which focus primarily on the detection of one nucleic acid sequence, fingerprint profileε, aε diεcuεεed in Section 5.5.4, may alεo be aεεeεεed in εuch detection εchemeε. Fingerprint profiles may be generated, for example, by utilizing a differential display procedure, aε diεcussed, above, in Section 5.1.2, Northern analyεiε and/or RT-PCR. Any of the gene εequenceε deεcribed, above, in Section 5.4.1. may be uεed as probes and/or PCR primers for the generation and corroboration of such fingerprint profiles.

5.8.2. DETECTION OF FINGERPRINT.; GENE PEPTIDES

Antibodies directed against wild type or mutant fingerprint gene peptides, which are diεcuεεed, above, in Section 5.4.3, may alεo be uεed aε cardiovaεcular disease diagnostics and prognosticε, as described, for example, herein. Such diagnoεtic methodε, may be uεed to detect abnormalitieε in the level of fingerprint gene protein expreεεion, or abnormalitieε in the εtructure and/or tisεue, cellular, or εubcellular location of fingerprint gene protein. Structural differences may include, for example, differenceε in the size, electronegativity, or antigenicity of the mutant fingerprint gene protein relative to the normal fingerprint gene protein.

Protein from the tiεεue or cell type to be analyzed may eaεily be detected or isolated using techniqueε which are well known to thoεe of εkill in the art, including but not limited to western blot analysis. For a detailed explanation of methods for carrying out western blot analysis, see Sambrook et al, 1989, supra, at Chapter 18. The protein detection and isolation methods employed herein may also be such aε thoεe deεcribed in Harlow and Lane, for example, (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Presε, Cold Spring Harbor, New York) , which iε incorporated herein by reference in itε entirety.

Preferred diagnoεtic methodε for the detection of wild type or mutant fingerprint gene peptide moleculeε may involve, for example, immunoaεεays wherein fingerprint gene peptideε are detected by their interaction with an anti- fingerprint gene εpecific peptide antibody.

For example, antibodieε, or fragmentε of antibodieε, εuch aε thoεe deεcribed, above, in Section 5.4.3, uεeful in the preεent invention may be uεed to quantitatively or qualitatively detect the preεence of wild type or mutant fingerprint gene peptideε. Thiε can be accompliεhed, for example, by immunofluoreεcence techniqueε employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorimetric detection. Such techniques are especially preferred if the fingerprint gene peptides are expreεεed on the cell εurface.

The antibodieε (or fragmentε thereof) useful in the present invention may, additionally, be employed histologically, aε in immunofluorescence or immunoelectron microscopy, for in situ detection of fingerprint gene peptides. In situ detection may be accomplished by removing a histological εpecimen from a patient, and applying thereto a labeled antibody of the present invention. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological εa ple. Through the uεe of such a procedure, it iε possible to determine not only the presence of the fingerprint gene peptides, but alεo their diεtribution in the examined tiεsue. Uεing the preεent invention, thoεe of ordinary skill will readily perceive that any of a wide variety of histological methodε (εuch aε εtaining procedureε) can be modified in order to achieve such in εitu detection.

Immunoassayε for wild type or mutant fingerprint gene peptideε typically compriεe incubating a biological εample, εuch aε a biological fluid, a tiεεue extract, freεhly harveεted cellε, or cells which have been incubated in tisεue culture, in the presence of a detectably labeled antibody capable of identifying fingerprint gene peptides, and detecting the bound antibody by any of a number of techniques well known in the art. The biological sample may be brought in contact with and immobilized onto a solid phase εupport or carrier εuch as nitrocellulose, or other solid εupport which is capa÷._ of immobilizing cells, cell particles or soluble proteinε. The εupport may then be washed with suitable ufferε followed by treatment with the detectably labeled fingerprint gene specific antibody. The solid phaεe εupport may then be waεhed with the buffer a εecond time to remove unbound antibody. The amount of bound label on εolid εupport may then be detected by conventional means. By "solid phaεe εupport or carrier" iε intended any εupport capable of binding an antigen or an antibody. Well- known εupports or carrierε include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloεeε, polyacrylamide , gabbroε, and magnetite. The nature of the carrier can be either εoluble to εome extent or insoluble for the purposeε of the preεent invention. The εupport material may have virtually any poεεible εtructural configuration εo long aε the coupled molecule iε capable of binding to an antigen or antibody. Thus, the support configuration may be εpherical, aε in a bead, or cylindrical, as in the inside surface of a test tube, or the external εurface of a rod. Alternatively, the εurface may be flat εuch as a sheet, test εtrip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-wild type or mutant fingerprint gene peptide antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal asεay conditions for each determination by employing routine experimentation.

One of the ways in which the fingerprint gene peptide- εpecific antibody can be detectably labeled iε by linking the εame to an enzyme and uεe in an enzyme immunoassay (EIA) (Voller, "The Enzyme Linked Immunosorbent Asεay (ELISA)", Diagnostic Horizonε 2:1-7, 1978, Microbiological Aεεociateε Quarterly Publication, Walkerεville, MD; Voller, etal., J. Clin. Pathol. 31:507-520- (1978) ; Butler, Meth. Enzymol. 73:482-523 (1981); Maggio, ed.) Enzyme Immunoaεεay, CRC Preεε, Boca Raton, FL, 1980; Iεhikawa, et al., (edε.) Enzyme Immunoassay , Kgaku Shoin, Tokyo, 1981) . The enzyme which iε bound to the antibody will react with an appropriate εubstrate, preferably a chromogenic εubεtrate, in εuch a manner aε to produce a chemical moiety which can be detected, for example, by εpectrophoto etric, fluorimetric or by viεual meanε. Enzymeε which can be uεed to detectably label the antibody include, but are not limited to, malate dehydrogenaεe, εtaphylococcal nuclease, delta-5-steroid iεomeraεe, yeaεt alcohol dehydrogenaεe, alpha- glycerophoεphate, dehydrogenaεe, trioεe phosphate isomeraεe, horεeradiεh peroxidase, alkaline phoεphatase, asparaginase, glucose oxidaεe, beta-galactoεidase, ribonuclease, urease, catalaεe, glucose-6-phoεphate dehydrogenaεe, glucoamylaεe and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may alεo be accompliεhed by viεual compariεon of the extent of enzymatic reaction of a εubεtrate in compariεon with similarly prepared standards. Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragmentε, it iε poεεible to detect fingerprint gene wild type or mutant peptideε through the uεe of a radioimmunoaεεay (RIA) (εee, for example, Weintraub, B., Principles of Radio immunoaεε ay ε ,

Seventh Training Courεe on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein) . The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

It iε alεo posεible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody iε expoεed to light of the proper wave length, itε presence can then be detected due to fluorescence. ng the moεt commonly uεed fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocy&ήin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can alεo be detectably labeled uεing fluorescence emitting metalε εuch aε ¹⁵²Eu, or others of the lanthanide εerieε. Theεe metalε can be attached to the antibody using εuch metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA) .

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemilumineεcent-tagged antibody iε then determined by detecting the preεence of lumineεcence that ariεes during the courεe of a chemical reaction. Exampleε of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium eεter, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody of the preεent invention. Bioluminescence is a type of chemiluminescence found in biological εyεtemε in, which a catalytic protein increaεeε the efficiency of the chemiluminescent reaction. The preεence of a biolumineεcent protein iε determined by detecting the preεence of lumineεcence. Important biolumineεcent compoundε for purposes of labeling are luciferin, luciferase and aequorin.

5.8.3. IMAGING CARDIOVASCULAR DISEASE CONDITIONS

In εome cases, differentially expresεed gene productε identified herein may be up-regulated under cardiovascular diεease conditions and expresεed on the εurface of the affected tiεεue. Such target gene products allow for the non-invaεive imaging of damaged or diεeaεed cardiovaεcular tiεεue for the purpoεed of diagnoεiε and directing of 5 treatment of the diεeaεe. For example, such differentially expreεεed gene productε may include but are not limited to atherosclerosis εpecific adhesion moleculeε responsible for atherogeneεiε, or monocyte scavenger receptors that'* are up- regulated in response to -oxidized LDL, which are discuεεed in

10 Section 2, above. Alternatively, other such surface proteins may be specifically up-regulated in tisεueε εuffering from iεchemia/reperfusion or other tiεεueε with atherosclerotic or restenotic leεionε.

Aε deεcribed in the example in Section 6, below,

15 fchd602 iε a gene that iε up-regulated in monocytes under diεeaεe conditions. Furthermore, the fchd602 gene encodes a novel protein containing multiple transmembrane domains. Not only is the fchd602 gene expresεed in monocytes, which play a role in the initiation and progresεion of atherosclerotic

2_.0 leεionε, it iε alεo upregulated in monocytes under εuch diεeaεe conditions. The fchd602 gene product, therefore, provideε and excellent tool for imaging cardiovaεcular diεeaεe conditionε.

Thiε method can be applied in a εimilar manner to other

25 tranεmembrane target gene productε, εuch aε the fchd545 gene product. As described in the example in Section 7, below, the fchd545 gene encodes a novel anion channel, containing multiple transmembrane domains. Because the fchd545 gene product might be more readily detected in normal tissue, as

30 oppoεed to tiεεue in the diεeaεe εtate, it alεo provideε an excellent tool for imaging cardiovaεcular diεeaεe conditionε.

An example illustrating the use of this method in accordance with the invention is provided in Section 9, below. Monoclonal and polyclonal antibodieε, aε deεcribed in

35 Section 5.6.1.2, above, which εpecifically bind to εuch εurface proteins, εuch aε the fchd602 and fchd545 gene productε, can be uεed for the diagnoεis of cardiovascular disease by in vivo tissue imaging techniques. Such antibodies raised against the fchd5 5 gene^"product are deεcribed in detail in the example in Section 10, below. An antibody εpecific for a target gene product, or preferably an antigen binding fragment thereof, is conjugated to a label (e.g. , a gamma emitting radioisotop ) which generateε a detectable εignal and administered to a subject (human or animal) εuεpected of having cardiovaεcular diεeaεe. After εufficient time to allow the detectably-labeled antibody to localize^{^}at the diεeaεed or damaged tiεεue site (or sites) , the signal generated by the label is detected by a photoscanning device. The detected εignal iε then converted to an image of the tiεεue. Thiε image makeε it possible to localize the tisεue in vivo. Thiε data can then be uεed to develop an appropriate therapeutic εtrategy.

Antibody fragmentε, rather than whole antibody molecules, are generally preferred for use in tisεue imaging. Antibody fragmentε accumulate at the tiεsue(ε) more rapidly becauεe they are diεtributed more readily than are entire antibody moleculeε. Thus an image can be obtained in less time than is poεεible uεing whole antibody. Theεe fragmentε are also cleared more rapidly from tisεueε, reεulting in a lower background signal. See, e.g. _f Haber et al., U.S. Patent No. 4,036,945; Goldenberg et al., U.S. Patent No. 4,331,647. The divalent antigen binding fragment (Fab')₂ and the monovalent Fab are especially preferred. Such fragments can be prepared by digestion of the whole immunoglobulin molecule with the enzymes pepsin or papain according to any of several well known protocols. The types of labels that are εuitable for conjugation to a monoclonal antibody for diseased or damaged tisεue localization include, but are not limited to radiolabels (i.e. , radioisotopes) , fluorescent labels and biotin labels.

Among the radioisotopeε that can be uεed to label antibodies or antibody fragments, gamma-emitterε, poεitron- emitters, X-ray-emitters and fluorescence-emitterε are εuitable for localization. Suitable radioisotopes for

- Ill - labeling antibodies include Iodine-131,. Iodine-123, Iodine- 125, Iodine-126, Iod.ine-133, Bromine-77, Indium-Ill, Indium- 113m, Gallium-67, Gallium-eδ" Ruthenium-95, Ruthenium-97 , Ruthenium-103 , Ruthenium-105, Mercury-107 , Mercury-203, Rhenium-99m, Rhenium-105, Rhenium-101, Tellurium-121m, Tellurium-122m, Tellurium-125m, Thulium-165, Thulium-167, Thulium-168, Technetium-99m and Fluorine-18. The halogens can be used more or leεε interchangeably aε labelε since halogen-labeled antibodieε and/or normal immunoglobulinε would have εubεtantially the εame kineticε and diεtribution and εimilar metaboliεm.

The gamma-emitters Indium-Ill and Technetium-99m are preferred because these radiometals are detectable with a gamma camera and have favorable half liveε for imaging in vivo. Antibody can be labelled with Indium-Ill or

Technetium-99m via a conjugated metal chelator, εuch aε DTPA (diethlenetriaminepentaacetic acid). See Krejcarek et al., 1977, Biochem. Biophys. Res. Comm. 77:581; Khaw et al. , 1980, Science 209:295; Ganεow et al., U.S. Patent No. 4,472,509; Hnatowich, U.S. Patent No. 4,479,930, the teachings of which are incorporated herein by reference.

Fluoreεcent compoundε that are εuitable for conjugation to a monoclonal antibody include fluoreεcein εodium, fluorescein isothiocyanate, and Texas Red sulfonyl chloride. See, DeBelder & Wik, 1975, Carbohydrate Research 44:254-257. Those skilled in the art will know, or will be able to aεcertain with no more than routine experimentation, other fluorescent compounds that are εuitable for labeling monoclonal antibodieε.

6. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN RESPONSE TO PARADIGM A: IN VITRO FOAM CELL PARADIGM

According to the invention, differential diεplay may be uεed to detect genes that are differentially expresεed in monocytes that were treated so aε to εimulate the conditions under which foam cells develop during atherogeneεiε. By uεe cf Paradigm A, the novel genes fchd602 and fchd605 were identified. Both fchd602 and fchd605 are -up-regulated under the diεease condition of treatment with oxidized LDL.

The fchd602 gene product contains multiple transmembrane domains, and has sequence similarity to the rat Cl-6 gene, ^"which is induced in regenerating rat liver, is insulir indicible, and also contains multiple transmembrane uu.„ains (Diamond, R.H. , et al., 1993, J. Biol. Chem. 268: 15185- 15192) . The fchd605 gene product has seqaence similarity to the mouse gly96 gene (Charles, CH. , et al., 1993, Oncogene 8: 797-801), and to EST T49532.

The discovery of the up-regulation of these two genes provides a fingerprint profile, e.g., markerε, for monocyteε in the proceεs of foam cell formation. Thiε profile can be uεed in tha treatment and diagnoεiε of cardiovaεcular disease, including but not limited to atheroscleroεiε, iεchemia/reperfuεion, hypertenεion, reεtenoεiε, and arterial inflammation.

Furthermore, aε a tranεmembrane protein, the fchd602 gene product can be. readily acceεεed or detected on the monocyte cell εurface by other compounds. It provides, therefore, an excellent target for detection of cardiovascular diseaεe εtateε in diagnoεtic εyεtemε, aε well aε in the monitoring of the efficacy of compoundε in clinical trialε. Furthermore, the extracellular domains of this gene product provide targets which allow for the design of eεpecially efficient εcreening εyεtemε for identifying compoundε that bind to them. Such compounds can be useful in treating cardiovaεcular diεeaεe by modulating the activity of the tranεmembrane gene product.

6.1. MATERIALS AND METHODS

6.1.1. CELL ISOLATION AND CULTURING Blood (~200 ml) waε drawn into chilled 20 ml vacutainer tubeε to which 3 ml of citrate phoεphate dextroεe (Sigma) waε added. Blood waε then pooled into 50 ml tubes and spun in the Beckman GS-6R at 1250 RPM for 15 minutes at 4°C. The upper clear layer f~25 ^ml) ^was then removed with a pipette and d->-εcarded and replaced with the εame voluι..e of 4°C PBS. The blood waε then mixed, an εpun again at 2680 RPM for 15 inuteε at 4°C. The upper layer waε then removed and diεcarded, ar.d the buffy coat at the interface was removed in ~5 ml and placed in a εeparate 50 ml tube, and the pipette waε washed with 20 ml PBS. Cells were added to a T flaεk and εtored at 4°C for 16 hourε. A εmall aliquot of the"cellε were then removed and counted uεing a hemacytometer. The final red blood cell concentration in the buffy coat population waε then adjusted to 1.5 X 10⁹/D_1 with PBS, the cellε were added to Leucoprep tubeε (Becton Dickinson) after being allowed to come to room temperature, and spun at 2300 RPM for 25 minutes at 25°C. The upper clear layer was removed and discarded and the turbid layer -over the gel waε removed and pooled in 50 ml tubeε. Samples were then diluted to 50 ml with PBS (25°C) and εpun at 1000 RPM for 10 minuteε. The εupernatant waε then removed, and the pellet waε reεuεpended in 50 ml PBS. This procedure was repeated 3 more times. After the last spin, the cellε were reεuspended in a small volume of PBS and counted.

Tisεue culture diεheε were coated with bovine collagen before monocytes were plated out. 1/6 volume of 7X RPMI (JRH Bioεcienceε) waε added to Vitrogen 100 collagen (Celtrix) which waε then diluted 1:10 with RPMI to a final concentration of 0.35 mg/ml. Collagen mixture waε then added to plateε (2.5 ml/100 mm diεh) and placed at 37°C for at leaεt one hour to allow for gel formation. After gel formation haε taken place, the RPMI waε removed and cellε were added in RPMI/10% plaεma derived εerum (PDS) . PDS was prepared by drawing blood into chilled evacuated tubes containing 1/lOth volume 3.8% εodium citrate. Blood waε then tranεferred into new Sorvall tubeε and εpun at 14,000- 16,000 RPM for 20 minuteε at 4°C. Plaεma layer waε removed and pooled in new tubes to which l/50th volume IM CaCl₂ waε added. Plaεma waε mixed and aliquoted into new Sorvall tubes and incubated at 37% for 2 hourε to allow for fibrin clot formation. The clot was then disturbed with a pipette to allow it to contract and tubes were spun at 14,500 RPM for 20 minutes at 25°C. Supernatant was collected, pooled, and heat inactivateu at 56°C prior to sterile filtration and freezing. Purified human monocytes were cultured in 10% PDS/RPMI containing 5 units/ml of Genzyme recombinant human MCSF for 5 days before being treated with LDL, oxidized LDL, ac lated LDL (all LDL at 50 μg/ml) , lyεophoεphatidylcholine (Sigma, 37.5 μM) , or homocyεteine (Sigma, ImM) . After incubation with these reagentε for periods ranging from 2 hours up to 3 dayε, the media waε withdrawn and the cellε were diεsolved in RNA lyεiε buffer and RNA waε prepared aε deεcribed, above, in Section 6.1.

Lipoproteinε For oxidation, human LDL (Sigma) was firεt diluted to 1 mg/ml with PBS and then dialyzed againεt PBS at 4°C overnight. LDL waε then diluted to 0.3 mg/ml with PBS. CuSO₄-5H₂0 waε then added to 5uM final concentration, and the εolution waε incubated in a T flaεk in a 37°C incubator for 24 hr. LDL εolution was then dialyzed at 4°C against 0.15M NaCl/0.3mM EDTA for 2 days with several changeε, before being removed and concentrated uεing an A icon spin column by spinning for 1 hr. 4000 RPM at 4°C.

For acetylation, 1 ml of 5 mg/ml LDL was added to 1 ml of a saturated solution of NaOAc in a 15 ml tube on ice"on a shaker at 4°C. 8 μl of acetic anhydride waε added 2 μl at a time over 1 hr. LDL was then dialyzed for 48' hr. against 0.15M NaCI/0.3 mM EDTA at 4°C for 48 hr. with several changes. Final concentrations of derivatized LDL's were determined by comparing to a dilution curve of native LDL analyzed at OD₂₈₀, with 0.15M NaCl/0.3mM EDTA used as diluent in all caεes. 6.1.2. ANALYSIS OF PARADIGM- MATERIAL Differential Diεplay: Removal of DNA: The RNA pellet was resuspended in H₂0 and quantified by spectrophotometry at OD_2€0. Approximately half of the sample waε then treated with DNAεe I to remove contaminating chromoεomal DNA. RNA waε amplified by PCR uεing the following procedure. 50 ul RNA εample (10-20 μg) , 5.7 μl lOx PCR buffer (Perkin-Elmer/Cetus) ,_ 1 μl RN se inhibitor (40 units/μl) (Boehringer Mannheim, Germany) were mixed together, vortexed, and briefly spun. 2 μl DNAse I (10 unitε/μl) (Boehringer Mannheim) waε added to the reaction which waε incubated for 30 min. at 37°C. The total volume waε brought to 200 μl with DEPC H₂0, extracted once with phenol/chloroform, once with chloroform, and precipitated by adding 20 μl 3M NaOAc, pH 4.8, (DEPC-treated) , 500 μl abεolute ETOH and incubating for 1 hour on dry ice or -20°C overnight. The precipitated εample waε centrifuged for 15 min., and the pellet waε waεhed with 70% ETOH. The sample waε re-centrifuged, the remaining liquid waε aεpirated, and the pellet waε reεuεpended in 100 μl H₂0. The concentration of RNA waε meaεured by reading the OD₂₆₀.

Firεt εtrand cDNA εyntheεiε: For each RNA εample duplicate reactionε were carried out in parallel. 400 ng RNA pluε DEPC H₂0 in a total volume of 10 μl were added to 4 μl T_X1XX reverse primer (10 μM) (Operon) . The specific primers uεed in each experiment are provided in the Deεcription of the Figureε in Section 4, above. The mixture waε incubated at 70°C for 5 min. to denature the RNA and then placed at r.t. 26 μl of reaction mix containing the following componentε waε added to each denatured RNA/primer εample: 8 μl 5x Firεt Strand Buffer (Gibco/BRL, Gaitherεburg, MD) , 4 μl 0.1M DTT (Gibco/BRL), 2 μl RNAεe inhibitor (40 unitε/μl) (Boehringer Mannheim), 4 μl 200 μM dNTP mix, 6 μl H₂0, 2 μl Superεcript reverεe tranεcriptaεe (200 unitε/μl) (Gibco/BRL) . The reactions were mixed gently and incubated for 30 min. at 42 °C. 60 μl of H₂0 (final volume = 100 μl) were then added aid the εamples were denatured for 5 min. at 85°C and εtored at -20°C.

PCR reactions: 13 μl of reaction mix was added to each tube of a 96 well plate on ice. The reaction mix contained 6.4 μl H₂0, 2 μl lOx PCR Buffer (Perkin-Zlmer) , 2 μl 20 μM dNTP'ε, 0.4 μl ^3SS dATP (12.5 μCi/μl; 50 μCi total) (Dupont/NEN) , 2 μl forward (for-) primer (10 μM) (Operon) , and 0.2 μl AmpliTaq Polymerase (5 units/μx) (Perkin-Elmer) . Next, 2 £1 of reverse (rev-) primer (T_X1XX, 10 μM) were added to the εide of each tube followed by 5 μl of cDNA alεo to the εideε of the tubeε, which were εtill on ice. The εpecific primers used in each experiment were as followε: fchd602: rev-T^XC and for-GTGAGGCGTC fchd605: rev-T_xlXC and for-TGGACCGGTG

Tubeε were capped and mixed, and brought up to 1000 RPM in a centrifuge then returned immediately to ice. The PCR machine (Perkin-Elmer 9600) was programmed for differential display as follows: 94°c 2 min.

*94°C 15 sec. *40°C 2 min. * = X40*ramp 72°C 1 min. *72°C 30 sec. 72°C 5 min.

4°C hold

When the PCR machine reached 94 °C, the plate was removed from ice and placed directly into the Perkin-Elmer 9600 PCR machine . Following PCR, 15 μl of loading dye, containing 80% formamide, 10 mM EDTA, 1 mg/ml xylene cyanol, 1 mg/ml bromphenol blue were added. The loading dye and reaction were mixed, incubated at 85°C for 5 min. , cooled on ice, centrifuged, and placed on ice. Approximately 4 μl from each tube were loaded onto a prerun (60V) 6% acrylamide gel. The gel was run at approximately 80V until top dye front was about 1 inch from bottom. The gel was transferred to 3MM paper (Whatman Paper, England) and dried under vacuum. Bands were viεualized by aμtoradiography.

Band iεolation and amplification: Differentially expreεεed bandε were exciεed from the dried gel with a razor blade and placed into a microfuge tube with 100 μl H₂0 and heated at 100°C for 5 min., vortexed, heated again to 100°C for _^5 min., and vortex again. After cooling, 100 μl H₂0, 20 μl 3.1 NaOAc, 1 μl glycogen (20 mg/ml) , and 500 μl ethanol were added and chilled. .After centrifugation, the pellet waε waεhed and reεuεpended in 10 μl H₂0.

The iεolated differentially expressed bands were then amplified by PCR uεi-ng the following reaction conditionε:

58 μl H₂0 10 μl lOx PCR Buffer

10 μl 200 μm dNTP'ε 10 μl 10 μM reverεe primer 10 μl 10 μM forward primer 1.5 μl amplified band 0.5 μl AmpliTaq polymeraεe (5 unitε/μl)

(Perkin Elmer) PCR waε performed uεing the program deεcribed in thiε Section, above, for differential diεplay. After PCR, glycerol loading dyeε were added and εampleε were loaded onto a 2% preparative TAE/Biogel (BiolOl, La Jolla, CA) agaroεe gel and eluted. Bandε were then exciεed from the gel with a razor blade and vortexed for 15 min. at r.t., and purified uεing the Mermaid kit from BiolOl by adding 3 volumes of Mermaid high salt binding solution and 8 μl of resuεpended glassfog in a microfuge tube. Glassfog waε then pelleted, waεhed 3 timeε with ethanol waεh εolution, and then DNA waε eluted twice in 10 μl at 50°C.

Subcloning: The TA cloning kit (Invitrogen, San Diego, CA) waε uεed to εubclone the amplified bandε. The ligation reaction typically conεiεted of 4 μl εterile H₂0, 1 μl ligation buffer, 2 μl TA cloning vector, 2 μl PCR product, and 1 μl T4 DNA ligase. The volume of PCR product can vary, but the total volume of PCR product pluε H₂-0 waε always 6 μl. Ligations (including vector alone) were incubated overnight at 12 °C before bacterial transformation. TA cloning kit competent bacteria (INVαF': endal , recAl, hsdR17 (r- , m+k) , supE44 , λ-, thi-1 , gyrA, relAl , φ80? acZαAMlδΔ (lacZYA-a-gF) , deoI+, F') were thawed on ice and 2 μl of 0.5 H β- mercaptoethanol were added to each tube. 2 μl from each ligation were added to each tube of competent cells (50 μl) , mixed without vortexing, and incubated on ice for 30 min. Tubes were then placed in 42°C bath for exactly 30 sec, before being returned to ice for 2 min. 450 μl of SOC media (Sambrook et al., 1989, εupra) were then added to each tube which were then shaken at 37°C for 1 hr. Bacteria were then pelleted, resuspended in -200 μl SOC and plated on Luria broth agar plates containing X-gal and 60 μg/ml ampicillin and incubated overnight at 37^CC. White colonies were then picked and screened for inεertε uεing PCR.

A maεter mix containing 2 μl lOx PCR buffer, 1.6 μl 2.5 mM dNTP'ε, 0.1 μl 25 mM MgCl₂, 0.2 μl M13 reverεe primer (100 ng/μl) , 0.2 μl M13 forward primer (100 ng/μl) , 0.1 μl AmpliTaq (Perkin-Elmer), and 15.8 μl H₂0 waε made. 40 μl of the maεter mix were aliquoted into tubes of a 96 well plate, and whole bacteria were added with a pipette tip prior to PCR. The PCR machine (Perkin-Elmer 9600) was programmed for insert screening as follows:

94°C 2 min. *94°C 15 sec. *47°C 2 min. * = X35 *ramp 72°C 30 sec.

*72°C 30 sec. 72°C 10 min. 4°C hold Reaction products were eluted on a 2% agarose gel and compared to vector control. Colonieε with vectorε containing inserts were purified by streaking onto LB/Amp plateε. Vectorε were iεolated from εuch εtrainε and εubjected to εequence analyεiε, uεing an Applied Biosystems Automated Seque cer (Applied Biosyεtemε, Inc. Seattle, ,JA) .

Northern analysis: Northern analysis was performed to confirm the differential expression of the genes corresponding to the amplified bands. The probes used to detect mRNA were syntheεized as follows: typically 2 μl amplified band (-30 ng) , 7 μl H₂0, and 2 μl lOx Hexanucleotide mix (Boehringer-Mannheim)- were mixed and heated to 95°C for 5 min., and then allowed to crol on ice. The volume of the amplified band can vary, but the total volume of the band plus H₂0 waε alwayε 9 μl. 3 μl dATP/dGTP/dTTP mix (1:1:1 of 0.5 mM each), 5 μl α³²P dCTP 3000 Ci/mM (50 μCi total) (Amersham, Arlington Heights. IL) , and 1 μl Klenow (2 units) (Boehringer-Mannheim) were mixed and incubated at 37°C.

After 1 hr., 30 μl TE were added and the reaction was loaded onto a Biospin-6™ column (Biorad, Hercules, CA) , and centrifuged. A 1 μl aliquot of eluate was used to meaεure incorporation in a εcintillation counter with εcintillant to enεure that 10⁶cpm/μl of incorporation was achieved.

The sampleε were loaded onto a denaturing agaroεe gel. A 300 ml 1% gel waε made by adding 3 g of agaroεe (SeaKem^w LE, FMC BioProductε, Rockland, ME) and 60 ml of 5x MOPS buffer to 210 ml εterile H20. 5x MOPS buffer (0.1M MOPS (pH 7.0), 40 mM NaOAc, 5mM EDTA (pH 8.0)) waε made by adding 20.6 g of MOPS to 800 ml of 50mM NaOAc (13.3 ml of 3M NaOAc pH 4.8 in 800 ml sterile H₂0) ; then adjusting the pH to 7.0 with 10M NaOH; adding 10 ml of 0.5M EDTA (pHδ.O); and adding H₂0 to a final volume of IL. The mixture was heated until melted, then cooled to 50°C, at which time 5 μl ethidium bromide (5mg/ml) and 30 ml of 37% formaldehyde of gel were added. The gel waε swirled quickly to mix, and then poured immediately.

2μg RNA εample, lx final 1.5x RNA loading dyeε (60% formamide, 9% formaldehyde, 1.5X MOPS, .075% XC/BPB dyes) and H₂0 were mixed to a final volume of 40 μl. The tubeε were heated at 65°C for 5 min. and then cooled on ice. 10 μg of RNA MW standards (New England Biolabs, Beverly, MA) were also denatured with dye and loaded onto the gel. The gel waε run overnight at 32V in MOPS running buffer.

The gel waε then soaked in 0.5 μg/ml Ethidium Bromide for 45 min., 50 mM NaOH/0.1 M NaCI for 30 min., 0.1 M Tris pH 8.0 for 30 min., and 2Ox SSC for 20 min. Each soaking steo waε done at r.t. with εhaking. The gel waε then photogrt_^ 3d along with a fluorescent ruler before blotting with Hybond-N- membrane (Amerεham) , according to the methodε of Sambrook et al., 1989, εupra , in 20x SSC overnight.

Northern blot hybridizationε were carried out aε followε: for pre-hybridization, the blot waε placed into roller bottle containing 10 ml of rapid-hyb εolution (Amerεham) , and placed into 65°C incubator for at leaεt 1 hr For hybridization, lxlO⁷ cpm αf the probe was then heated to 95°C, chilled on ice, and added to 10 ml of rapid-hyb εolution. The prehybridization εolution was then replaced with probe solution and incubated for 3 hr at 65°C. The following day, the blot was waεhed once for 20 min. at r.t. in 2x SSC/0.1% SDS and twice for 15 min. at 65°C in O.lx SSC/0.1% SDS before being covered in plaεtic wrap and put down for expoεure.

RT-PCR Analyεiε: RT-PCR waε performed to detect differentially expressed levels of mRNA from the geneε correεponding to amplified bands. First εtrand εyntheεiε waε conducted by mixing 20 μl DNaεed RNA (~2 μg) , 1 μl oligo dT (Operon) (1 μg) , and 9.75 μl H₂0. The εampleε were heated at 70°C for 10 min., and then allowed to cool on ice. 10 μl firεt strand buffer (Gibco/BRL), 5 μl 0.1M DTT, 1.25 μl 20 mM dNTP's (500 μM final), 1 μl RNAεin (40 units/μl) (Boehringer Mannheim) , and 2 μl Superscript Reverse Transcriptase (200 units/μl) (Gibco/BRL) were added to the reaction, incubated at 42°C for 1 hr., and then placed at 85°C for 5 min., and stored at -20°C.

PCR waε performed on the reverεe tranεcribed εamples. Each reaction contained 2 μl lOx PCR buffer, 14.5 μl H₂0, 0.2 μl 20 mM dNTP's (200 μM final), 0.5 μl 20 μM forward primer (0.4 μM final), 0.5 μl 20 μM reverεe primer (0.4 μM final), 0.3 μl AmpliTaq polymerase (Perkin-Elmer/Cetus) , 2 μl cDNA dilution or positive control (-40 pg) . The specific piimers used in each experiment are provided in the Description of the Figures in Section 4, above. Samples were placed in the PCR 5 9600 machine at 94°C (hot start), which was programmed as follows:

94 °C 2 min. (samples loaded) *94°C 45 sec. * = 35x *55°C 45" sec. 10 *72°C 2 min.

72°C 5 min. 4°C hold _..Reactions were carried out on cDNA dilution εerieε and tubeε were removed at variouε cycles from the machine during 15 72 °C εtep. Reaction productε were eluted on a 1.8% agaroεe gel and viεualized with ethidiura bromide.

Gene Retrieval: Amplified εequences, which contained portionε of the genes, were subcloned and then uεed

20 individually to retrieve a cDNA encoding the corresponding gene. Probes were prepared by isolating the εubcloned inεert DNA from vector DNA, and labeling with ³²P aε deεcribed above in Section 6.1.2. Labeled inεert DNA containing fchd602 εequenceε waε uεed to probe a cDNA library prepared from

25 human macrophage cell line U937. Labeled inεert DNA containing fchd605 sequences was used to probe a cDNA library prepared from human primary blood monocytes. The cDNA libraries were prepared and screened according to methods routinely practiced in the art (εee Sambrook et al . , 1989,

30 εupra) . Plaqueε from the libraries that were detected by the probes were isolated and the cDNA insert within the phage vector was εequenced.

The RACE procedure kit waε uεed either aε an alternative to cDNA library εcreening, or, when the cDNA library did not

35 yield a clone encoding the full-length gene, to obtain adjacent εequences of the gene. The procedure waε carried out according to the manufacturer's instructions (Clontech, Palo Alto, CA; εee alεo: Chenchik, et al. , 1995, CLONTECHniqueε (X) 1: 5-8; Barneε, 1994, Proc. Natl. Acad. Sci. USA 91: 2216-2220; and Cheng et al., Proc. Natl. Acad. Sci. USA 91: 5695-5699). Primers were deεigned baεed either on amplified εequenceε, or on εequenceε obtained from iεolates from the cDNA libraries. ^memplate mRNA for fchd605 was iεolated from human primary blood monocyteε.

6.1.3. CHROMOSOMAL LOCALIZATION O' TARGET GENES Once the nucleotide εequence has been determined, the presence of the gene on a particular chromoεome iε detected. Oligonucleotide primers based on the nucleotide sequence of the target gene are uεed in PCR reactions uεing individual human chromoεomeε as templates. Individual sampleε of each the twenty-three human chromoεomeε are commercially available (Coriel Inεtitute for Medical Reεearch, Camden, NJ) . The chromosomal DNA is amplified according to the following conditionε: lOng chromosomal DNA, 2μl lOx PCR buffer, 1.6μl 2.5mM dNTP'ε, O.lμl 25mM MgCl₂, 0.2μl reverεe primer (lOOng/μl) , 0.2μl forward primer (lOOng/μl) , 0.1 μl Taq polymeraεe, and 15.8μl H₂0. Samples are placed in the PCR 9600 machine at 94 °C (hot εtart) , which iε programmed as follows:

94°C 2 min. (sampleε loaded)

*94°C 20 εec.

* = 35x *55°C 30 sec.

*72°C 30 εec.

72°C 5 min.

4°C hold

6.2. RESULTS

Differential diεplay waε performed on monocytes treated with oxidized LDL and untreated monocytes. Bands correεponding to fchd602 and fchd605 were detected as up- regulated by oxidized LDL, aε compared with the untreated monocytes. The up-regulation was confirmed by northern blot analysis.

The fchd602 gene produced a 2.5kb mRNA that was up- regulated after 5 hours of treatment with oxidized LDL, minimally oxidized LDL, and lysophoεphatidylcholine. No message was detected in untreated or native LDL treated control monocytes. The amplified DNA sequence waε 'uεed to recover a cDNA of approximately 875 bp comprising ar. open reading frame encoding approximately 182 amino acidε. The DNA εequence and encoded am_^no acid εequence of this cDNA from the fchd602 gene is shown in FIG. 4A-4B. The open reading frame haε 88% εequence εimilarity to the rat Cl-6 gene, which iε induced in regenerating rat liver, iε inεulin inducible, and alεo containε multiple tranεmembrane domainε (Diamond, R.H., et al., 1993, J. Biol. Chem. 268: 15185- 15192) .

The fchd605 gene produced a 1.5kb mRNA that iε up-regulated after 5 hourε treatment with oxidized LDL, and to a lesser degree with native LDL, as compared to untreated monocytes. The amplified DNA was sequenced and used to recover a cDNA of approximately 2.2kb, which was εequenced to reveal a partial open reading frame of approximately 258 bp, encoding approximately 86 amino acidε. The DNA εequence and encoded amino acid εequence from the fchd605 gene iε εhown in FIG. 5A-5B. The sequence haε εimilarity to the mouse gly96 gene, which encodes a cytokine inducible glycosylated protein expreεεed in mouse lung, testeε, and uteruε.

EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED

IN RESPONSE TO PARADIGM D: ENDOTHELIAL CELL

SHEAR STRESS

According to the invention, differential diεplay was used to detect geneε that are differentially expreεεed in endothelial cellε that were εubjected to fluid εhear εtreεε in vitro. Shear εtress is thought to be responsible for the prevalence of atherosclerotic lesionε in areas of unusual circulatory flow. Uεing the method of Paradigm D, three novel DNA εequenceε were identified.

The fchd531 gene iε down-regulated in endothelial cells under both turbulent and laminar εhear εtreεs, as compared to the static control. The fchd531 gene encodes a novel 570 amino acid polypeptide, and has 94% sequence similarit^y to the τnouse penta zinc finger gene (Pzf) , which haε not „een publiεhed, but is contained in the GenBank sequence data base under accession no U05343. The fchd540 gene is up-regulated in endothelial cellε under laminar εhear stresε, but is not up-regulated by IL-l treatment. The fchd540 gene encodes a novel intracellular protein which has εequence similarity to the Drosophila Mad protein (Sekelsky et al., 1995, Genetics 139: 1347-1358). The fchά545 gene is down-regulated in endothelial cells under laminar shear streεε aε compared to endothelial cellε under turbulent εhear εtreεε and εtatic control endothelial cellε. The fchd545 gene encodes an 848 amino acid polypeptide which has 73% sequence similarity to the human Voltage-dependent Anion Channel protein (Blachly-Dyεon, E., et al., 1993, J. Biol. Chem. 268: 1835-1841.). The fchd545 gene iε also expressed in' the human heart, smooth muscles, and testes.

The up-regulation of the fchd540 gene and down-regulation of the fchd531 and fchd545 geneε in shear streεsed endothelial cells provides a fingerprint for the study of cardiovascular diseaεeε, including but not limited to atherosclerosiε, ischemia/reperfusion, hypertension, and restenosiε. The fact that one of theεe geneε, fchd540, is not up-regulated under Paradigm C (IL-l induction) provides an extremely uεeful meanε of diεtinguiεhing and targeting physiological phenomena specific to shear stress.

Furthermore, as a transmembrane protein, the fchd545 gene product can be readily accesεed or detected on the endothelial cell surface by other compounds. It provides, therefore, an excellent target for detection of cardiovascular diseaεe εtateε in diagnoεtic εyste s, aε well aε in the monitoring of the efficacy of compounds in clinical trialε. Furthermore, the extracellular domaii s of this gene product provide targets which-allow for deεigning especially efficient εcreening εyεtems for identifying compoundε that bind to them. Such compounds can be uεeful in treating cardiovaεcular diεeaεe by modulating the activity of the transmembrane gene product.

7.1. MATERIALS AND METHODS Primary cultureε of HUVEC -> were established from normal term umbilical cords as described (In Progresε in Hemostaεiε and Thrombosis, Vol. 3, P. Spaet, editor, Grune & Stratton Inc., New York, 1-28). Cells were grown in 20% fetal calf serum complete media (1989, J. Immunol. 142: 2257-2263) and paεεaged 1-3 timeε before εhear stress induction.

.For induction, εecond paεεage HUVEC'ε were plated on tiεεue culture-treated polyεtyrene and εubjected to 10 dyn/cm² laminar flow for 1 and 6 hr. aε described (1994, J. Clin. Invest. 94: 885-891) or 3-10 dyn/cm² turbulent flow as previously described (1986 Proc. Natl. Acad. Sci. U.S.A. 83: 2114-2117) . RNA was isolated as described, above, in Section 6.1. Differential diεplay, Northern analyεiε, RT-PCR, εubcloning, and DNA εequencing were carried out aε deεcribed, above, in Section 6.1.2. Specific primerε uεed in differential diεplay were aε followε: fchd531: for-TnXA and rev-AGACGTCCAC fchd540: for-T_xlXA and rev-ACTTCGCCAC fchd545: for-T_ι:ιXC and rev-TCGGACGTGA

Amplified εequences, which contained portionε of the geneε, were εubcloned and then used individually to retrieve a cDNA encoding the corresponding gene. Probes were prepared by iεolating the εubcloned inεert DNA from vector DNA, and labeling with ³P aε deεcribed above in Section 6.1.2. Labeled inεert DNA waε uεed to probe cDNA library prepared from shear εtreεε induced endothelial cellε. The library was prepared and probed using methodε routinely practiced in the art (see Sambrook et al . , 1989, εupra) . Plaqueε from the librarieε that were detected by the probes were isolated and the cDNA inεert within the phage vector was εequenced.

The RACE procedure kit was used either as an alternative to cDNA library screening, or, when the cDNA' library did not yield a clone encoding the full-length gene, to obtain adjacent εequenceε of the gene. The procedure waε ca ed out according to the manufacturer'ε inεtructionε (Clontech, Palo Alto, CA; see alεo: Chenchik, et al., 1995, CLONTECHiϊiqueε (X) 1: 5-8; Barneε, 1994, Proc. Natl. Acad. Sci. USA 91: 2216-2220; and Cheng et al., Proc. Natl. Acad. Sci. USA 91: 5695-5699) . Primerε were deεigned baεed either on amplified sequences, or on sequenceε obtained from iεolateε from the cDNA librarieε. Teuplate mRNA waε iεolated from εhear .εtreεsed HUVEC's.

Northern blot analysiε of RNA extracted from variouε human organs and tissues was performed using commercially available pre-blotted filters (Clontech, Palo Alto, CA) .

7.2. RESULTS

An amplified fchd531 fragment obtained from differential display was subcloned and εequenced, and used to obtain a 1.9 kb cDNA containing the entire fchd53l coding region. The DNA sequence and encoded amino acid sequence of the novel fchd531 gene is shown in FIG. 1A-1D. The fchd531 gene encodeε a 570 amino acid polypeptide, and haε 94% εequence εimilarity to the mouse penta zinc finger gene (Pzf) (GenBank accession number U05343) . Northern analysis of HUVEC's which were subjected turbulent and laminar shear streεε demonεtrated that the fchd531 gene produceε an approximately 5 kb meεεage which iε down-regulated under laminar εhear stress, but not turbulent shear streεε, compared with the static control.

The fchd540 gene was detected as an up-regulated message under shear streεε. The amplified fragment waε uεed to probe a Northern blot containing εampleε from HUVECs treated with laminar shear streεε. A 4.4 kb fchd540 mRNA iε up-regulated after 6 hourε treatment with laminar εhear εtreεε. The fchd540 gene iε not induced by IL-l by the method of Paradigm C, (Section 5.1.1.5, above). The amplified f-agment waε εequenced and uεed to obtain a 2.7 kb cDNA containing the entire fchd540 coding region. The DNA εequence and encoded 5 amino acid ε uence from the fchd540 gene iε εhown in FIG. 2A-2E. The fchd540 gene encodes a 426 amino acid polypeptide and has sequence similarity to the Drosophila Mad gene (Sekelεky et al. , 1995, Geneticε 139: 1347-1358).

The fchd545 gene waε detected aε a down-regulated meεεage 10 under εhear εtreεε. Northern analyεiε revealed that the fchd545 gene produceε a l.4kb meεεage which iε down regulated by turbulent εhear εtreεs, but not by laminar shear streεε, aε compared with εtatic control. The amplified fragment waε εequenced and uεed to iεolate a 1.4kb cDNA containing the

15 complete fchd545 coding εequence. The DNA- εequence and encoded amino acid εequence of the fchd545 gene iε εhown in FIG. 3A-3C. The fchd545 gene encodeε a 283 amino acid polypeptide which haε 73% εequence εimilarity to the human Voltage-dependent Anion Channel (Blachly-Dyεon, E., et al. ,

20 1993, J. Biol. Chem. 268: 1835-1841). Northern analyεiε of a commercially available (Clontech, Palo Alto, California) northern blot revealed that the fchd545 gene iε expreεsed in human heart, smooth muscle, and testeε.

25 8. EXAMPLE: USE OF GENES UNDER PARADIGM A AS SURROGATE MARKERS IN CLINICAL TRIALS

According to the invention, the fingerprint profile derived from any of the paradigms described in Sections 5.1.1.1 through 5.1.1.6 may be uεed to monitor clinical trialε of

30 drugε in human patientε. The fingerprint profile, deεcribed generally in Section 5.5.4, above, indicateε the characteriεtic pattern of differential gene regulation correεponding to a particular disease state. Paradigm A, described in Section 5.1.1.1, and illustrated in the example

35 in Section 6, above, for example, provideε the fingerprint profile of monocyteε under oxidative εtreεε. The target genes, therefore, serve as εurrogate markerε by giving an indicative reading of the physiological response of monocytes to the uptake of oxidized LDL. Accordingly, the influence of anti-oxidant drugs on the oxidative potential may be measured by performing differential display on the monocyteε of patientε undergoing clinical teεtε.

8.1. TREATMENT OF PATIENTS AND CELL ISOLATION

Teεt patients may be adminiεtered compounds suεpected of having anti-oxidant activity. Control patients may be given a placebo.

Blood may be drawn from each patient after a 12 hour period of fasting and monocytes may be purified aε described, above, in Section 7.1.1. RNA may be iεolated as deεcribed in Section 6.1.1, above. Primers may then be deεigned for amplification based on the DNA εequence of target geneε identified aε up-regulated, such as fchd602 and fchd605, or down-regulated under Paradigm A.

8.2. ANALYSIS OF SAMPLES RNA may be subjected to differential diεplay analyεiε aε deεcribed in Section 6.1.2, above. A decreaεe in the phyεiological reεponεe state of the monocyteε iε indicated by a decreaεed intenεity of thoεe bandε corresponding to fchd602 and fchd605, which were up-regulated by oxidized LDL under Paradigm A, as described in Section 6.2, above.

9. EXAMPLE: IMAGING OF A CARDIOVASCULAR DISEASE CONDITION

According to the invention, differentially expressed gene products which are localized on the εurface of affected tiεεue may be uεed aε markerε for imaging the diεeaεed or damaged tiεεue. Conjugated antibodieε that are specific to the differentially expresεed gene product may be administered to a patient or a teεt animal intravenouεly. Thiε method provideε the advantage of allowing the diεeaεed or damaged tiεsue to be visualized non-invasively.

For the purposes of illustration, this method iε described in detail for the fchd602 gene product. The principles and techniques can be applied to any tranεmembrane target gene product, including, for example, the fchd545 gene product.

9.1. MONOCLONAL CONJUGATED ANTIBODIES

5 The differentially expreεεed surface gene product, εuch as the fchd602 gene product, is expresεed in a recombinant hoεt and purified uεing methodε deεcribed in Section 5.4.2, above. Preferably, a protein fragment compriεing one or more of the extracellular domainε of the fchd602 product iε produced. 0 Once purified, it iε be use to produce F(ab')₂ or Fab fragmentε, aε deεcribed in Section 5.4.3, above. Theεe fragmentε are then labelled with technetium-99m (^99mTc) uεing a conjugated metal ehelator, εuch as DTPA aε deεcribed in εection 5.8.3, above. 5

9.2. ADMINISTRATION AND DETECTION OF IMAGING AGENTS Labeled MAb may be adminiεtered intravenously to a patient being diagnosed for atheroscleroεiε, reεtenosis, or iεchemia/reperfuεion. Sufficient time iε allowed for the 0 detectably-labeled antibody to localize at the diεeaεed or damaged tiεεue εite (or εiteε) , and bind to the fchd6C2 gene product. The εignal generated by the label is detected by a photoscanning device. The detected εignal iε then converted to an image of the tiεεue, revealing cells, such as 5 monocyteε, in which fchd602 gene expression is up-regulated.

10. POLYCLONAL ANTIBODIES TO TARGET GENE PEPTIDE SEQUENCES

Peptide εequenceε corresponding to the indicated amino sequenceε of cDNAs were selected and submitted to Research

30 Genetics (Huntsville, AL) for εyntheεiε and antibody production. Peptideε were modified aε described (Tam, J.P., 1988, Proc. Natl. Acad. Sci. USA 85: 5409-5413; Tam, J.P. , and Zavala, F., 1989, J. Immunol. Methods 124: 53-61; Tam, J.P. , and Lu, Y.A. , 1989, Proc. Natl. Acad. Sci. USA 86:

35 9084-9088) , emulsified in an equal volume of Freund'ε adjuvant and injected into rabbitε at 3 to 4 εubcutaneouε dorεal εites for a total volume of 1.0 ml (0.5 mg peptide) per immunization. The animals were boosted after 2 and 6 weekε and bled at weekε 4, 8, and 10. The-blood waε allowed to clot and εeru was collected by centrifugation. The peptideε uεed are εummarized below:

fchd545 Peptide Antigens

Name Position Sequence fchd545.1 48-63 YTDTϋJKASGNLETKYK fchd545.2 107-121 TGKKSGKLKASYKRD

11. EXAMPLE: THE RCHD534 AND FCHD540 GENE PRODUCTS INTERACT

The ^"novel fchd540 gene and its nucleotide sequence is described in Section 7, above. The fchd540 gene shares homology with the Droεophila Mad gene. The rchd534 gene (described in Applicant's co-pending Application No. 08/485,573, filed June 7, 1995, which is incorporated by reference in its entirety herein) iε another gene that iε up- regulated in endothelial cellε by εhear εtreεε. The DNA and encoded amino acid εequence of the rchd534 gene iε εhown in FIG. 6A-6D. The rchd534 gene waε deposited in the Agricultural Reεearch Service Culture Collection (NRRL) in microorganiεm FCHD534 on June 6, 1995 and aεεigned the NRRL Accession No. B-21459. The rchd534 gene also shares homology with the Droεophila Mad gene. Mad genes have been shown to play a role in the TGF-/3 signalling pathway (Sekelsky et al., 1995, Genetics 139: 1347-1358; Chen et al. , 1996, Nature 383: 691-696; Serra, et al., 1996, Nature Medicine 2: 390-391). TGF-β signalling is conεidered to be beneficial to atheroεcleroεiε and reεtenoεiε (Border et al. , 1995, Nature Medicine 1: 1000; Grainger, et al., 1995, Nature Medicine 1: 1067-1073; Kojima, et al., 1991, J. Cell Biol. 113: 1439- 1445; Nikol, et al. , 1992, J. Clin. Inveεt. 90: 1582-1592). The data deεcribed below demonεtrate that the rchd534 and fchd540 proteinε interact with one another; and this interaction may lead to the inhibition of TGF-β signalling. Furthermore, the expreεεion of theεe two genes, as described below, iε εpecific 'to endothelial cellε. Becauεe theεe two geneε "1) are both expressed specifically in endothelial cells, 2) are both up-regulated in endothelial cells under certain conditions, 3) encode MAD proteins that interact with one another in endothelial cells, and 4) inhibit TGF-β signalling (which iε conεidered to be beneficial to atheroεcleroεiε) , rchd534 and fchd540 proteinε are attractive targetε for therapeutic intervention in cardiovaεcular diεeaεe. In particular, treatment regi enε that inhibit the interaction or activity of the rchd534 and fchd540 proteins can be beneficial for the treatment cardiovascular diεease.

Further analyseε demonεtrated that the rchd534 protein ir.teractε with itεelf to form a homodimer. Thuε, treatment regimenε that inhibit the interaction of the rchd534 protein with itεelf can be beneficial for the treatment cardiovaεcular diεeaεe.

In addition, the analyεeε deεcribed below demonεtrated novel interactions of both the rchd534 and fchd540 proteins with other proteinε known to be involved in the TGF-β εignalling pathway. The protein memberε of the TGF-3 εignalling pathway teεted included MADR1 (Hoodleεε εt al., 1996, Cell 85:489-500), MADR2 (Eppert et al. , 1996, Cell 86: 543-552), DPC4 (Raftery et al., 1988, Geneticε 139: 241-254), T3RI, TSR1, ActRIb, ALK3 , and ALK6 (Wieεer et al., 1995, EMBO J. 14: 2199-2208). For example, the rchd534 protein interactε εtrongly in endothelial cellε with MADRl, MADR2, DPC4, and weakly in 293 (human embryonic kidney) cellε with activated formε of receptorε T/3RI and ActRI. The fchd540 protein interactε strongly in 293 cellε with activated forms of receptors T3RI and ALK6.

In the absence of transfected rchd543 and fchd540 genes, transfected MADRl or transfected MADR2 mediated a 20-fold induction of a TGF-β inducible promoter in BAECs. Co- expreεεion of either tranεfected rchd534 or transfected rchd540 in this εyεtem eliminated the induction, and alεo prevented the localization of MADR2 in the nucleuε in reεponεe to TGF-β εignalling. Therefore, treatment regimens that inhibit the interaction of the rchd534 and fchd540 proteins with other proteins involved in the TGF-β pathway also can be beneficial for the treatment cardiovascular of diεease. As described above, the expression of rchd534 and fchd540 is εpecific, within arterial tissue, to endothelial cells. Accordingly, the rchd534 and rchd540 genes may be tarrjets for intervention in a variety of inflammatory .d fibroproliferative diεorderε that involve endothelial cellε, including, but not limited to, cancer, angiogeneεiε, inflammation, and fibrosis.

11.1. MATERIALS AND METHODS

11.1.1. YEAST STRAINS, MEDIA, AND MICROBIOLOGICAL TECHNIQUES

Standard yeast media including synthetic complete medium lacking L-leucine, L-tryptophan, and L-hiεtidine were prepared and yeaεt genetic manipulations were performed aε deεcribed (Sherman, 1991, Meth. Enzymol., 194:3-21). Yeaεt tranεformations were performed using εtandard protocolε (Gietz et al., 1992, Nucleic Acidε Res., 2J):1425. Ito et al., 1983, J. Bacteriol., 153:163-168) . Plasmid DNAs were iεolated from yeaεt εtrainε by a εtandard method (Hoffman and Winston, 1987, Gene, 57:267-272).

11.1.2. PLASMID AND YEAST STRAIN CONSTRUCTION

The coding region of human fchd540 was amplified by PCR and cloned in frame into pGBT9 (Bartel et al., 1993, Cellular Interactions in Development, pp. 153-159) resulting in plasmid pGBT9-fchd540. pGBT9-fchd540 was transformed into two-hybrid εcreening εtrain HF7c and one reεulting tranεformant waε designated TB35.

11.1.3. TWO-HYBRID SCREENING

Two-hybrid εcreening was carried out esεentially aε deεcribed (Bartel et al., 1993, εupra) uεing TB35 aε the recipient εtrain and a human breaεt two-hybrid library. 11.1.4. PAPER FILTER BETA-GALACTOSIDASE ASSAYS The "paper filter beta-galactoεidase (beta-g.1) assay was performed essentially as previously described (Brill et al., 1994, Mol. Biol. Cell 5: 297-312).

11.2. RESULTS

11.2.1. STRONG PHYSICAL INTERACTION OF RCH 534 AND FCHD540 MEASURED BY TWO-HYBRID ASSAY

The fchd540 coding sequence waε amplified by PCR and cloned into pGBT9 creating a GAL4 DNA-binding domain-fchd540 fuεion gene. « The εcreening strain HF7c waε tranεformed with thiε conεtruct. The rchd534 coding εequence was cloned into pG^"AD424 (Bartel et al., 1993, εupra) creating a GAL4 transcriptional activation domain-rchd534 fuεion gene, which waε then uεed to tranεform εtrain Y187.

Yeaεt expression plasmids encoding the GAL4 DNA-binding domain either alone or fused in frame to fchd540, rchd534, Droεophilia MAD, DPC , or p53 were transformed into MATa two- hybrid screening strain HF7c. Yeast expresεion plaεmidε encoding the GAL4 tranεcriptional activation domain alone and GAL4 activation domain fuεions to rchd534 and SV40 were transformed into MATα two-hybrid screening εtrain Y187. p53 and SV40 interact with each other and εhould not interact with the experimental proteins. The HF7c transformantε were propagated aε εtripes on semisolid synthetic complete medium lacking L-tryptophan and the Y187 transformantε were grown aε εtripeε on εemiεolid εynthetic complete medium lacking L- leucine. Both εetε of stripes were replica plated in the form of a grid onto a single rich YPAD plate and the haploid strainε of oppoεite mating typeε were allowed to mate overnight at 30°C. The yeaεt εtrainε on the mating plate were then replica plated to a εynthetic complete plate lacking L-leucine and L-tryptophan to select for diploidε and incubated at 30°C overnight. Diploid εtrains on the εynthetic complete plate lacking L-leucine and L-tryptophan were replica plated to a εynthetic complete plate lacking L- luucine, L-tryptophan, and L-hiεtidine to asεay HIS3 expreεεion and a paper filter on a synthetic complete plate lacking L-leucine and L-tryptophan. The next day the paper filter waε εubjected to the paper filter beta-galactosidase aεεay to meaεure expreεεion of the lacZ reporter gene. HIS3 expresεion waε εcored after 3 dayε of growth at 30°C. The reεultε are εhown in Table 3.

The rchd534 fiεh protein waε found to interact εtrongly with the fchd540 bait protein and not to interact with the rchd534, "MAD, DPC , p53, and GAL4 DNA binding domain bait proteinε. Thiε reεult demonεtrated that rchd534 and fchd540 εtrongly phyεically interact with each other with εignificant εpecificity.

11.2.2. IDENTIFICATION OF PROTEINS THAT

PHYSICALLY INTERACT WITH FCHD540

The fchd540 coding εequence waε amplified by PCR and cloned into pGBT9 (Bartel et al., 1993, εupra) creating a GAL4 DNA- binding domain-fchd540 fuεion gene. HF7c waε transformed with this construct resulting in strain TB35. TB35 grew on synthetic complete medium lacking L-tryptophan but not on synthetic complete medium lacking L-tryptophan and L- hiεtidine demonεtrating that the GAL4 DNA-binding domain- fchd540 fuεion doeε not have intrinεic tranεcriptional activation activity.

TB35 waε tranεformed with the human breaεt two-hybrid library and 5 million tranεformantε were obtained. The transformants were plated on synthetic complete medium lacking L-leucine, L-tryptophan, and L-hiεtidine and yeaεt colonieε that both grew on synthetic complete medium lacking L-leucine, L-tryptophan, and L-histidine and expressed the beta-galactosidase reporter gene were identified. The 30 strains with the strongeεt beta-galactoεidaεe induction were characterized. Library plaεmidε were iεolated from these strains, and the 5' ends of all of the cDNA insertε were εequenced. 11.2.3. RETRANSFORMATION AND SPECIFICITY TESTING OF TCHV03A AND TCHVR4A

Two of the plaεmidε that encoded the εtrongeεt interactors were found to contain rchd534 cDNAs. Plaεmid tchv03A was _c found to encode amino acids 17-235 of rchd534 and plaεmid tchvR4A was found to encode amino acids 25-235 of rchd534.

It waε confirmed that theεe rchd534 cDNAε encode proteins that phyεically interact εpecifically with fchd540. Yeast expresεion plaε idε encoding the GAL4 DNA-binding domain either alone or fuεed in frame to fchd540, rchd534, Droεophila MAD, DPC4 , and p53 were tranεformed into MATa two- hybrid εcreening εtrain HF7c. Yeaεt expreεεion plaεmidε encoding the GAL4 tranεcriptional activation domain (GAL4 AD) alone and GAL4 activation domain fuεionε to tchv03a, tchvR4A ^ and SV40 were transformed into MAT two-hybrid screening εtrain Y187. p53 and SV40 interact with each other and εhould not interact with the experimental proteinε. The HF7c tranεformantε were propagated aε stripes on semi-εolid synthetic complete medium lacking L-leucine. Both setε of εtripeε were replica plated in the form of a grid onto a εingle rich YPAD plate and the haploid εtrainε of oppoεite mating typeε were allowed to mate overnight at 30°C. The yeaεt εtrains on the mating plate were then replica plated to a synthetic complete plate lacking L-leucine and L-tryptophan to εelect for diploidε and incubated at 30°C overnight. Diploid εtrainε on the synthetic complete plate lacking L- leucine and L-tryptophan were replica plated to a εynthetic complete plate lacking L-leucine, L-tryptophan, and L- hiεtidine to aεεay HIS3 expreεεion and a paper filter on a ₀ synthetic complete plate lacking L-leucine and L-tryptophan. The next day the paper filter waε εubjected to the paper filter beta-galactoεidase .asεay to meaεure expreεεion of the lacZ reporter gene. HIS3 expreεεion waε εcored after 3 dayε of growth at 30°C. The reεultε are εhown in the table below. ₅ The εtrength or abεence of physical interaction between each combination of test proteins is liεted. Strong interactions are defined aε interactions that cauεe the activation of both the HIS3 and lacZ reporter geneε.

TABLE 3

CDNA-GAT4 Activation Domain Fusion Tested rchd534 tchv03A tchvR4A SV40 GAL4 AD alone

GAL4 DNA- Binding Domain Fusionε fchd5-40 Strong Strong Strong None None rchd534 None None None None None

Droε. MAD .. None None None None None

DPC4 None None None None None p53 None None None Strong None

GAL4 DNA- None None None None None Binding Domain alone

The tchv03A and tchvR4A fish proteins were found tc interact εtrongly with the fchd540 bait protein and to not interact with the rchd534, MAD, DPC4, p53, and GAL4 DNA binding domain bait proteins. Theεe results confirm the result that the rchd534 and fchd540 proteins interact strongly with each other.

11.3. FURTHER ANALYSIS OF RCHD534 AND FCHD540 FUNCTION The εignificance of the rchd534/fchd540 protein interaction was confirmed by examination of their expresεion and activity in human cells and animal models.

11.3.1. CHROMOSOMAL LOCALIZATION The rchd534 gene was localized to chromoεome 15 and the fchd540 gene waε localized to chromosome 18, regions of_the human genome that contain other MAD homologues. These regions of the human genome have alεo been implicated in the pathogenesiε of εeveral human malignancieε.

11.3.2. TISSUE EXPRESSION PATTERNS The expression patterns were examined using in situ hybridization techniques. Fluorescently labeled DNA probeε of both the rchd534 and fchd540 geneε were uεed to probe human carotid endartectomy samples. The expresεion" of rchd534 and fchd540 waε εpecific to endothelial cellε lining the luminal εurface of the cirotid artery. In addition, a rabbit polyclonal antiεerum generated againεt the rchd534 gene product prominently and selectively stained the endothelium present in large vesεelε εuch aε human coronary arterieε aε well aε εmaller veεεelε preεent within human myocardium. Neither gene εbowed expresεion in any other cell type preεent in the arterial tiεεue εample, including smooth muεcle cells and macrophages.

Expresεion patternε of both geneε were alεo examined in reεponεe to certain εtimuluε. Both geneε are selectively upregulated under the steady laminar εhear εtreεε (LSS) paradigm, but not under the turbulent shear streεε paradigm or in response to stimuluε by the cytokineε rhIL-13, TNFα, IFNγ or active TGF/3 aε meaεured in HUVEC cellε. Thuε, the rchd534 and the fchd540 geneε appear to be εelectively responsive to a LSS stimulus, manifesting no response to a non-laminar fluid mechanical stimuluε, nor any other humoral εtimuli tested. Thus, given that theεe two geneε are: (1) localized to a region of the human genome that haε been implicated in the pathogeneεis of several human malignancies; (2) specifically expressed in a cell-type that is found only in vascular tisεue, including atheroεclerotic plaques; (3) up-regulated under the steady laminar shear εtreεε cardiovaεcular diεeaεe paradigm; and (4) εpecifically inhibit TGF-β εignalling indicate that rchd534 and fchd540 are excellent and εpecific targetε for therapeutic intervention in the treatment of fibroproliferative and oncogenic diεorderε including tumor growth and vascularization. 11. 3 . 3 . CELLULAR LOCALIZATION

The cellular localization of the rchd534 and fchd540 proteinε in bovine aortic endothelial cellε (BAECs) waε examined in relationεhip to other proteinε involved in the TGF-β εignalling pathway. In all experiments, the rchd534 and fchd540 proteinε were located in the cytoplaεm. M*.DR2 waε ^located in the cytoplaεm when tranεfected alone <..... in the nucleuε when co-tranεfected with activated TβRI or when TGF-β waε added to the culture medium. Co-tranεfection of rchd534 or fchd540 with MADR2 prevented the localization of MADR2 in the nucleuε in reεponse to TGF-β εignalling.

11.3.4. PROTEIN INTERACTIONS IN HUMAN CELLS The interaction of the rchd 34 and fchd540 proteinε, obεerved in yeaεt cellε aε deεcribed above, waε teεted in mammalian endothelial cell tiεεue culture. Either bovine aortic endothelial cellε (BAECε) or 293 cellε (human embryonic kidney cellε, ATCC Accession No. CRL-1573) were transfected with conεtructs encoding both the rchd534 and fchd540 proteins, each fused to a different flag peptide allowing for specific immunoprecipitation. The rchd534 and fchd540 proteins were found to co-immunoprecipitate as heterodimerε in extractε produced from both 293 cellε and BAECε. The co-immunoprecipitation of rchd534 and fchd540 further εupports that these proteins interact in -human cellε that are phyεiologically relevant to cardiovaεcular disease.

The ability of the rchd534 and fchd540 proteins to interact with themselveε and with other protein memberε of the TGF-β signalling pathway (MADRl, MADR2, DPC4, TbRl, TSR1, ActRlb, ALK3 , ALK6) , waε teεted uεing thiε co-immunoprecipitation method. Each gene waε tranεfected alone and in variouε combinations with other TGF-β pathway genes in either 293 cellε or BAECε. The rchd534 protein formed homodimerε in 293 cellε and BAECε. The fchd540 protein did not form homodimers in 293 cells or BAECs. Aε mentioned above, the rchd534 and fchd540 proteinε formed heterodimerε in 293 cellε and BAECε. This interaction iε about 50 fold εtronger in BAECs than 293 cellε baεed on equal amountε of protein. However, the rchd52"4-fchd540 protein interaction waε εigniiicantly leεε avid than the rchd534 protein'ε interaction with itself.

The rchd534 protein interacted with MADRl, MADR2, and DPC4 in 293 cellε and BAECε. The εtrength of MADRl and MADR2 interactionε waε about the εame between 293 cellε and BAECε and much greater in BAECε for DPC4. The fchd540 protein interacted very weakly with MADRl, MADR2, and DPC4 in 293 cellε. The rchd534 protein interacted εtrongly with activated formε of TβRI and ActRI and weakly with activated ALK6 in 293 cells. The fchd540 protein interacted strongly with activated TβRI and ALK6 receptors, and weakly with activated forms of TSRI, ALK3 , and ActRIb in 293 cells. Thus, in addition to the interaction of the rchd534 and fchd540 proteins, the interaction of the rchd534 protein with itself, as well as the interaction of the rchd534 protein and the fchd540 protein with the other proteins in the TGF-β pathway described above are excellent targets for therapeutic intervention.

11.3.5. EFFECT OF EXPRESSION ON TGF-B SIGNALLING The effect of both rchd534 and fσhd540 on the TGF-β signalling pathway was tested in vitro. Primary BAECs were tranεfected with a conεtruct called p3TP-Lux, containing a TGF-β reεponεive promoter fuεed to a reporter gene (Wrana et al., 19944, Nature 370: 341-347). The rchd534 gene or the fchd540 gene in pCI expreεsion vectors (Promega) was transfected with and without MADRl (pCMV5MADRl-Flag, Hoodleεε et al. 1996 Cell 85: 489-500) or MADR2 (pCMV5MADR2-Flag, Eppert et al. 1996 Cell 86: 543-552) . The TGF-β reεponεe waε induced 20-fold by either MADRl or MADR2. Co-expression of either rchd534 or fchd540 completely eliminated this induction. Thus, the rchd534 and fchd540 proteins inhibited MADRl- and MADR2-mediated TGF-β εignalling in endothelial cells. To confirm the specificity of this inhibitory effect, site specific mutants of both rchd534 or fchd540 were constructed, baεed on known mutations identified in D_Oεophila homologueε, that would be predicted to diεrupt MAD-like εignaling functionε (Sekelεky et al., 1995, Genetics 139:1347-58; Raftery, 1995, Genetics 139:241-54; Newfeld et al., 1996, Development 122:2099-108; Wierεdorff et al., 1996, Development 122:2153-62). Unlike wild type rchd534 and fchd540, theεe mutant proteins were unable to inhibit the activation of the p3TP promoter in reεponse to TGF-β. The expreεεion levels of the mutant and wild-type proteins were . comparable indicating the loss of function^" was not due to εecondary instability.

Interestingly, Smad3, the C. eleganε homolog to MAD3 which also functions in TGFβ signalling is over 90% identical to Smad2,~the C. eleganε MAD2 homolog, in the MH2 domain. Although this has not yet been directly investigated, it iε likely that Smad7, the C. elegans homolog of the fchd540 gene, may function similarly to its inhibition to prevent association and activation of Smad3 by the TGFβ receptor, that is, to inhibit the phosphorylation of Smad3 and its association with protein components of the TGF-β εignalling pathway.

These results further demonstrate that the interactions of either the rchd534 protein or the fchd540 protein with MADR2 or with activated TβRI are excellent targets for therapeutic intervention. Aε deεcribed above, the expreεsion of rchd534 and fchd540 is specific, within arterial tisεue, to endothelial cellε. Accordingly, the rchd534 and rchd540 geneε may be targetε for intervention in a variety of inflammatory and fibroproliferative diεorderε that involve endothelial cellε, including, but not limited to, cancer angiogeneεiε, inflammation, and fibrosis.

12. EXAMPLE: ANTISENSE AND RIBOZYME MOLECULES FOR

INHIBITION OF RCHD534 AND FCHD540 EXPRESSION

The principles presented in Section 5.6.1.1, above, can be used to deεign oligonucleotides for uεe in inhibiting the expreεsion of target genes, such as the rchd534 or fchd540 genes. The following antisense molecules can be uεed to inhibit the expreεεion of the rchd534 gene:

Antisense: a) 5'-CATTTCATTTCATACAA-3' which is complementary to nucleotides -14 to +3 of rchd534 in FIG. 6A-6D.

b) 5'-CATTTCATTTCATACAATATATG-3' which iε complementary to nucleotideε -20 to +3 of rchd534 in FIG. 6A-6D.

c) 5'-CATTTCATTTCATACAATATATGGCCTTT-3' which iε complementary to nucleotideε -26 to +3 of rchd534 in FIG. 6A-6D.

d) 5'-CATTTCATTTCATACAATATATGGCCTTTTGTGGC-3' which iε complementary to nucleotideε -32 to +3 of rchd534 in FIG. 6A-6D.

e) 5'-GGACATTTCATTTCATACAATATATGGCCTTTTGT-3' which iε complementary to nucleotides -29 to +6 of rchd534 in

FIG. 6A-6D.

f) 5'-TTCATTTCATACAATATATGGCCTTTTGT-3 ' which is complementary to nucleotideε -29 to -1 of rchd534 in FIG. 6A-6D.

g) 5'-TCATACAATATATGGCCTTTTGT-3 ' which iε complementary to nucleotideε -29 to -7 of rchd534 in FIG. 6A-6D.

h) 5'-AATATATGGCCTTTTGT-3' which iε complementary to nucleotideε -29 to -13 of rchd534 in FIG. 6A-6D.

The following antisense molecules can be used to inhibit the expresεion of the fchd540 gene: a) 5'-CATGCGGGGCGAGGAGG-3' which iε complementary to nucleotides -14 to +3 of fchd540 in FIG. 2A-2E. b} 5'-CATGCGGGGCGAGGAGGCGAGGA-3' which iε complementary to nucleotideε -20 to +3 of fchd540-in FIG. 2A-2E.

c) 5 ' -CATGCGGGGCGAGGAGGCGAGGAGAAAAG-3 ' which iε complementary to nucleotideε -26 to +3 of fchd540 in FIG. 2A-2E.

d) 5 ' -CATGCGGGGCGAGGAGGCGAGGAGAAAAGTCGTTT-3 ' which iε complementary to nucleotides -32 t +3 of fchd540 in FIG. 2A-2E.

e) 5 ' -GAACATGCGGGGCGAGGAGGCGAGGAGAAAAGTCG-3 ' which iε complementary to nucleotides -29 to +6 of fchd540 in FIG. 2A-2E.

f) 5 ' -GCGGGGCGAGGAGGCGAGGAGAAAAGTCG-3 ' which iε complementary to nucleotideε -29 to -1 of fchd540 in FIG. 2A-2E.

g) 5'-CGAGGAGGCGAGGAGAAAAGTCG-3' which iε complementary to nucleotideε -29 to -7 of fchd540 in FIG. 2A-2F.

h) 5'-GGCGAGGAGAAAAGTCG-3' which iε complementary to nucleotideε -29 to -13 of fchd540 in FIG. 2A-2E.

Ribozymeε:

The central, catalytic portion of a hammerhead ribozyme molecule conεiεt of the following εequence: 5'-CAAAGCNGNXXXXNCNGAGNAGUC-3 ' ; wherein the 5 '-proximal CA baεeε hybridize to a complementary 5'-UG-3' in the target mRNA. The firεt four underlined baεeε form a εtem by baεe pairing with the εecond εet of underlined baεeε, with the intervening baεeε, εhown aε X'ε, forming a non-pairing loop. In order to hybridize to a target mRNA, a hammerhead ribozyme contains additional baseε flanking each end of the central εegment εhown above. The 5' ribozyme flanking εegment iε complementary to the respective flanking εequeπceε immediately 3' to the target UG; ana the 3' flanking εegment iε complementary to the reεpective flanking sequence beginning two bases upstream of the target U, and extending 5 '-ward (in effect, skipping the first base upstream of the target U) . Cleavage occurs between firεt and εecond bases upstream of (i.e., 5' to) the U in the target 5'-UG-3' site.

The following ribozyme molecules can be uεed to inhibit the expreεεion of the rchd534 gene:

a) 5 ' -GGUGGAGCCCCAGGGCAUUACCUCAAAGCNGNXXXXNCNGAGNAGUCGUGG GCAAGGUGGGCACUCAGGUGGG-3 ' which will cleave the rchd534 mRNA between nucleotideε 716 and 717 in FIG. 6A-6D. b) 5'-GUGUCUCUAUGGGUUUGCCCAAAGCNGNXXXXNCNGAGNAGUCUCUGGACA UUUCAUUUCAUAC-3 ' which will cleave the rchd534 mRNA between nucleotideε 1040 and 1041 in FIG. 6A-6D. C) 5 '-GGCCCUCUCGCCGUCGGGCUCCUUGCUGAGCAAAGCNGNXXXXNCNGAGNA

GUCGAUGCCGAAGCCGAUCUUGCUGCGCG-3' which will cleave between nucleotides 1421 and 1422 in FIG. 6A-6D.

The following ribozyme molecules can be uεed to inhibit the expreεεion of the fchd540 gene:

a) 5' -CGUUUGCCUGCUAAGGAGCGAACAAAGCNGNXXXXNCNGAGNAGUCGAUGU UUCUUUGUGAGUCGGGCGCCG-3 ' , which will cleave the fchd540 mRNA between nucleotideε -53 and -52 in FIG. 2A-2E. b) 5'-CGCCGGACGAGCGCAGAUCGUUUGGUCCUGAACAAAGCNGNXXXXNCNGAG NAGUCCGGGGCGAGGAGGCGAGGAGAAAAGUCG-3 ' , which will cleave the fchd540 mRNA between nucleotideε -1 and +1 in FIG. 2A-2E. c) 5 '-GGAGUAAGGAGGGGGGGGAGACUCUAGUUCGCAAAGCNGNXXXXNCNGAGN

AGUCAGUCGGCUAAGGUGAUGGGGGUUGCAGCACACC-3 ' which will cleave the fchd540 mRNA between nucleotideε +602 and +603 in FIG. 2A-2E.

13. EXA1.PLE: EXPRESSION OF FCHD540 ENHANCES

TUMORIGENICITY IN PANCREATIC CANCER

The reεultε presented below demonεtrate that the fchd540 protein inhibitε the TGFβ responεe and leads to incre? d cell proliferation. Inhibition of activity or expresεion of negative regulatory proteins εuch aε fchd540 or rchd534, are, therefore, uεeful in activating or enhancing the TGFβ- reεponεe and therefore, controlling fibroproliferative and oncogenic diεeaεe.

Results below demonεtrate that fchd540 mRNA levels are increaεed in human pancreatic cancer by compariεon with the

₁₅ normal pancreaε. Additionally, in εitu hybridization experimentε demonstrate that fchd540 is overexpresεed in the pancreatic cancer cellε within the tumor mass. Stable transfection of COLO-357 and PANC-1 human pancreatic cancer cell lines with a full-length fchd540 expression construct

_₀ leads to complete losε of the TGFβ reεponεe. Unlike untranεfected cellε, thoεe overexpreεεing fchd540 continued to proliferate in the preεence of TGFβ.

13.1. MATERIALS AND METHODS

₂₅ 13.1.1. MATERIALS

The following materialε were purchaεed: FBS, DMEM and RPMI medium, trypεin εolution, penicillin-streptomycin solution, and Geneticin (G418) from Irvine Scientific (Santa Ana, California) ; Geneεcreen membraneε from New England Nuclear

₃₀ (Boεton, Maεεachuεettε) ; restriction enzymes, random primed labeling kits, Genius 3 non-radioactive nucleic acid detection kits, and Geniuε 4 RNA labeling kitε from Boebringer Mannheim (Indianapoliε, Illinoiε) ; Immobilon P membraneε from Millipore (Bedford, Maεεachusetts) ; and p21^clpl

₃₅ (AB-3) antibodies from NeoMarkerε (Fremont, California) ; Sequenaεe Verεion 2.0 DNA Sequencing from USB (Cleveland, Ohio) ; [α-³²P]dCTP, [α-^3Ss]dATP, and Hybond N+ membranes-from Amerεham (Arlington Heights, Illinoiε)-; Lipofectamine from Gibco .BRL (Gaitherεburg, Maryland) . All other reagentε were from Sigma (St. Louiε, Miεεouri) . PANC-1, MIA PaCa-2 , ASPC-1, CAPAN-1 human pancreatic cell lines were obtained from ATCC (Rockville, aryland) .

13.1.2. TISSUE SAMPLES

Normal human pancreatic tiεεue εamples_ (N=12; 7- male, 5 female donors; median age- 41.8 yearε; range 14-68 yearε) and human pancreatic cancer tiεεueε (N=16; 10 male, 6 female; median age 62.6 yearε; range 53-83 yearε) were obtained through an organ donor program and from pancreatic cancer patientε undergoing --εurgery. According to the TNM claεεification of the Union Intenationale Contre le Cancer (UICC) 6 tumorε were εtage 1, 1 εtage 2, asd 9 εtage 3 ductal adenocarcinoma. Freεhly removed tiεεue samples were fixed in 10% formaldehyde εolution for 12-24 hr and paraffin-embedded for hiεtological analyεiε. In addition, tissue samples were frozen in liquid nitrogen immediately upon surgical removal and maintained at -80°C until uεe for RNA extraction.

13.1.3. RNA EXTRACTION AND NORTHERN BLOT ANALYSIS Total RNA waε extracted by the εingle step acid guadinium thiocyanate phenol chloroform method and poly(A) + RNA was prepared by affinity chromatography on oligo-dT cellulose (Baldwin, et al., 1996, Int . J. Cancer 6:283-288) Size fractioned RNA was electrotransferred onto nylon membranes. Blots were hybridized with a ³²P labeled fchd540 cDNA probe and exposed at -80C to Kodak Biomax MS films. A 190 bp 7S cDNA probe, and a 150 bp β-actin cDNA probe were uεed to confirm equivalent loading of total, and poly(A) + RNA loading, reεpectively (Baldwin, et al., 1996, Jnt. J. Cancer 6:283-288; Korc, et al., 1992, J. Clin . Inveεt . 90: 1352- 1360) . Specifically, total RNA (20 μg/lane) from 6 normal pancreatic tiεsues, and 8 pancreatic cancerε waε εubjected to Northern blot analyεiε uεing a labeled fchd540 cDNA probe (500,000 cpm/ml) . A 7S riboεomal cDNA probe (50,000 cpm/ml) wi-s uεed as a loading and tranεfer control. Expoεure times were 2 dayε for fchd540 and 6 hr for 7S. For cell line analyεis, poly(A)+ RNA (2 μg/lane) waε iεolated from ASPC-1, CAPAN-1, COLO-357, MIA-PaCa-2, PANC-1, and T3M4 pancreatic 5 cancer cell lineε and from human placenta were analyzed by Northern blotting uεing a ³²P-labeled fchd540 cDNA probe (500,000 cpm/ml). A human β-actin cDNA probe (50,000 cpm/ml) waε used as a loading and tranεfer control. Expoεure timeε were 1 day for fchd540 and 2 hourε for β-actin. Total RNA

10 (20 μg/lane) from parental, εham-tranεfected, and 4 fchd540 tranεfected cloneε aε indicated of COLO-357 and PANC-1 pancreatic cancer cellε was εubjected to Northern blot analyεiε uεing a ³²P-labeled fchd540 cDNA?probe (500,000 cpm/ml). Exposure time was 2 dayε. Equal loading of laneε

15 waε confirmed by ethidium bromide εtaining of the RNA.

13.1.4. IN SITU HYBRIDIZATION Tiεεue εectionε (4 μm thick) were placed on 3-aminopropyl- methoxyεilane-coated εlideε, deparaffinized and incubated at

20 23°C for 20 minuteε with 0.2N HCI and at 37°C for 15 min with 40 μg/ml proteinaεe K. The εectionε were then poεt-fixed for 5 min in phoεphate buffered εaline (PBS) containing 4% paraformaldehyde, incubated briefly twice with PBS containing 2 mg/ml glycine and once in 50% (V/V) fomamide/2x SSC for 1

25 hour prior to initiation of the hybridization with 100 μl hybridization buffer, containing digoxigenin-labeled fchd540 riboprobe.

The probeε were labeled with digoxigenin-UTP by 17 or 5P6 RNA polyrneraεe uεing the Geniuε 4 RNA labeling kit.

30 Hybridization waε performed in a moiεt chamber for 16 hr at 42C. The εectionε were then incubated for 60 minutes at 23°C with 1% (w/v) blocking reagents, and for 30 minutes at 23 °C with a 1:2000 dilution of an alkaline phosphatase conjugated polyclonal sheep anti-digoxigenin Fab fragment antibody uεing

35 the Geniuε 3 nonradioactive nucleic acid detection kit. Following a 2-3 hourε incubation with color solution containing nitrobl e tetrazolium and X-phosphate in a dark box, the εeσtionε were mounted in aqueouε mounting medium.

13.1.5. CELL CULTURE 5 Human pancreatic cancer cells were routinely grown in DMEM (COLO-357, MIA-PaCa-2, PANC-1) or RPMI (ASPC-1, CAPAN-1, T3M4) supplemented with 10% FES, 100 U/ml penicillin, and 100

_* p.g/ml streptomycin (complete medium) . Growth asεayε were performed by incubating cellε in serum-free medium (DMEM

10 containing 0.1% BSA, 5 μg/m¹ tranεferrin, 5 ng/ml εodiu εelenite, and antibioticε) in the abεence or preεence of TGFβl, followed by addition of 3- (4 , 5-methylthiazol-2-yl) - 2,5-diphenyl-tetrazo_.lium bromide (MTT, 62.5 μg/well) for 4 hourε (Raitano, et al., 1993, J. Biol . Chem . 265: 10466-

15 10472) . In pancreatic cancer cellε the reεultε of the MTT aεεay correlate with results obtained by cell counting with a hemocymeter and by monitoring [³H] -thymidine incorporation (Raitano, et al., 1993, J. Biol . Chem . 265: 10466-10472; Baldwin et al., 1993, Growth Factorε , 8: 23-34). Basal

20 anchorage-independent growth was assessed by a double soft agar asεay (Kornmann et al., 1998, J. Clin . Inveεt . 101, 344- 352) . After 14 dayε colonieε were counted by microεcopy and εtained with MTT εolution for 12 hourε.

25 13.1.6. GROWTH IN NUDE MICE

Sham tranεfected or fchd540 tranεfected COLO-357 cellε (2xl0⁶) were injected subcutaneously into 2 sides of 4-6 weeks old, female, athymic (nude) mice. The animals were monitored for tumor formation every week for 6 to 12 weeks. Tumor

30 volume waε calculated aε τr/4 x width x height x length of the tumor (Kornmann et al., 1998, J. Clin . Inveεt . 101, 344-352).

13.1.7. STABLE AND TRANSIENT TRANSFECTIONS Transfection of fchd540.pCI.neo into COLO-357 and PANC-1 35 cells was carried out by the lipofectarmine method. After reaching confluence, cellε were split 1:10 in selection medium (complete medium supplemented with 0.4 mg/ml and 1 irj/ml G418 for COLO-357 and PANC-1 cells, respectively). Single clones were isolated after 2-4 weeks. After clonal expansion, cells from each individual clone were εcreened for expresεion of fchd540 by Northern blot analyεiε. Parental COLO-357 and PANC-I cellε were alεo tranέfected with a control expression vector containing the neomycin resistant gen-ϊ (pRSVneo) , and the reεultant cloneε termed εham tranεfected. Poεitive cloneε were routinely grown in εelection medium. For tranεient tranεfection, cellε were plated overnight at a denεity of 25,000 cellε/well in 24-well plateε and tranεiently tranεfected with the p3TP-Lux plaεmid using 1.5 μl/well lipofectamine and 0.5 μg plasmid/well in 500 μl serum free medium. Following a 5 hourε incubation, equal volumes of medium containing 20% FBS were added for 12 hours. Cellε were then incubated for 24 hourε in complete medium and εubεequently incubated for 12 hourε in serum free medium prior to the addition of TGFβl. Cells were solubilized in lyεiε buffer containing 25mM Triε HCI (pH 7.8), 2 mM DTT, 2 mM EGTA, 10% glycerol and 1% Triton X-100. Luciferaεe was asεayed with a luminometer (Monolight 2010B: Analytical Lumineεcence Laboratory, San Diego, California) for 10 εecondε after addition of the substrate solution containing 20 mM tricine, 1.07 mM magneεium-bicarbonate, 2.67 mM magneεium-εulfate, 0.1 mM EDTA, 530 μM ATP, 470 μM luciferin, 270 μM Coenzyme A (εodium εalt) , and 33.3 mM DTT.

13.1.8. IMMUNOBLOTTING Cellε were εolubilized in lyεiε buffer containing 50 mM TRIS, 150 mM NaCI, 1 mM EDTA, 1 μg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride (PMSF) , and 1% Triton X-100. Proteinε were εubjected to SDS polyacrylamide gel electrophoreεiε (SDS-PAGE) , transferred to Immobilon P membranes, incubated for 90 minutes with the indicated antibody, and for 60 minutes with a secondary antibody against mouse or rabbit IgG. Visualization was performed by enhanced chemiluminescence. 13.1.9. STATISTICS Student's t-test waε uεed for εtatiεtical analyεiε, P<0.05 waε taken as the level of εignificance. Reεultε of MTT cell growth aεεayε and luciferaεe reporter gene aεεayε are 5 expreεεed aε mean ± εtandard error of mean (SEM) of at leaεt 3 εeparate experimentε.

13.2. RESULTS

13.2.1. fchd540 "EXPRESSION IN HUMAN PANCREATIC

TISSUES AND PANCREATIC CANCER CELL LINES

10 —^■

In order to examine the expreεεion patternε of fchd540,

Northern blot analyεiε waε performed to compare normal tiεsue with pancreatic cancer tissue. Northern bolt analysiε revealed a faint 4.4 kb fchd540 mRNA transcript in 3 of 12 normal pancreatic tiεεue εampleε, and moderate to high levelε of thiε mRNA moiety in 7 of 16 pancreatic cancer εampleε.

Two approaches were taken next to determine whether pancreatic cancer cell lineε expreεε fchd540 mRNA. Firεt,

Northern blot analyεiε of poly (A) + RNA iεolated from 6 pancreatic cancer cell lineε revealed moderate levelε of this mRNA moiety in ASPC-1, CAPAN-I, COLO-357, and PANC-1 cells, and relatively low levels in MIA-PaCa-2 and T3M4 pancreatic cancer cellε. In order to further examine the localization of fchd540 in these tisεueε, in εitu hybridization was

__ carried out in those cancer samples that overexpressed fchd540 by Northern blot analysis. fchd540 mRNA was abundant in the cancer cells within the tumor mass, and was also present in the endothelial lining of the associated blood vesεelε. In εitu hybridization with sense probes did not

30 produce any εpecific εignal.

13.2.2. EFFECTS OF OVEREXPRESSING fchd540 IN

COLO-357 AND PANC-1 PANCREATIC CANCER CELLS

To inveεtigate the functional effectε of fchd540 overexpreεεion in pancreatic cancer cell lineε, COLO-357 and

35 PANC-1 were εtably tranεfected with a full length fchd540 conεtruct. Overall, 20 independent COLO-357 and 10 PANC-1 clones were εelected after 4 weekε of growth in εelection medium. Subεequent experimentε were carried out in 4 cloneε from each cell line. Theεe clones were εelected because they diεplayed increaεed fchd540 mRNA levelε by Northern blot analyεis of total RNA.

TGFβl inhibited the growth of wild-type and sham (control) transfected COLO-357 cellε after 72 hourε of incubati . , with maximal effectε occurring at a concentration of 1 nM TGFβl (—41%; p<0.01). In contraεt, under the εame conditions, TGFβl did^" not inhibit the growth of fchd540 overexpresεing COLO-357 cloneε (FIG. 7A) .

TGF-βl inhibited the growth of wild-type and εham tranεfected PANC-1 cellε, maximal effects- occurring at a concentration of 1 nM TGFβl (-29%; p<ύ.01). In contrast, under the same conditions TGFβl did not inhibit the growth of fchd540 overexpressing PANC-1 clones (FIG. 7B) .

Wild-type and sham transfected COLO-357 cells displayed exponential doubling times of 29 to 31 hours. The fchd540 overexpresεing COLO-357 clones exhibited similar doubling times (30 to 38 hours) . No significant difference in the doubling times waε obεerved between parental, εham tranεfected, and fchd540 tranεfected PANC-1 cloneε (24 to 26 h) . However, wild-type and εham tranεfected COLO-357 cellε diεplayed colony forming efficiencieε of —1.0%, whereaε fchd540 tranεfected cloneε displayed significantly (p<0.001) higher colony forming efficiencieε of 12% - 14% (FIG. 8A) . Parental and wild- type PANC-1 cellε exhibited relatively high colony forming efficiencieε of —28%, which did not significantly differ from those observed in the fchd540 tranεfected PANC-1 cloneε (FIG. 8B) .

To determine whether the enhanced anchorage-independent growth of fchd540 expreεsing COLO-357 clones resulted in enhanced tumorigenicity in vivo, 2xl0⁶ sham transfected or fchd540 transfected COLO-357 cells were injected subcutaneouεly in athymic (nude) mice at each site and tumor growth was measured weekly. fchd540 expresεing COLO-357 cloneε exhibited earlier and more rapid tumor growth in compariεon to εham transfected cellε (FIG. 8C| .

Taken together, reεults presented in this Example demonstrate that fchd540 mRNA levels were elevated in a significant parcentage of human pancreatic cancers in comparison with the normal pancreaε. Expreεεion of fchd540 waε detected in the cancer cellε within the tumor mass. Additionally, cultured human pancreatic cancer cell¹ lines demonstrated fchd540 expreεεed at low levelε. Finally overexpreεεion of fchd540 in two TGFβ-εenεitive cell lines reεulted in complete loεε of TGFβ reεponεe. This observation indicates that enhanced expression of fchd540 can abrogate the TGFβ responεe in TGFβ-related oncogenic diεorderε, εuch aε pancreatic cancer, thus demonεtrating a εpecific target for therapeutic intervention for fibroproliferative and oncogenic diseaεe stateε involving aberrant TGFβ εignaling.

13. DEPOSIT OF MICROORGANISMS The following microorganiεmε were deposited with the

American Type Culture Collection (ATCC) , Manasεaε, Virginia, on the indicated dateε and aεεigned the indicated acceεεion numberε:

Microorganiεm ATCC Acceεεion No. Date of Depoεit

PFCHD531 69983 February 7, 1996

PFCHD540 69984 February 7, 1996 fchd545 69974 January 5, 1996

The following microorganiεm waε depoεited with the Agricultural Research Service Culture Collection (NRRL) , Peoria, Illinois, on the indicated date and aεεigned the indicated acceεεion number:

Microorganiεm NRRL Accession No. Date of Deposit FCHD534 B-21459 June 6, 1995 The preεent invention iε not to be limited in εcope by the εpecific embodimentε deεcribed herein, which are intended aε εingle illustrationε of individual aεpectε of the invention, and functionally equivalent methodε and componentε are within the εcope of the invention. Indeed, variouε modifications of the invention, in addition to thoεe εhown and deεcribed herein will become apparent to thoεe εkilled in the art from the foregoing deεcription and accompanying drawings. Such modificationε are intended to fall within the εcope of the appended claimε.

1 53.1 -

International Application No: PCT/

MICROORGANISMS

Optional Sheet in connection with the microorganism referred to on page 15 , lines Q-₃7 of the description '

A. IDENTIFICATION OF DEPOSIT '

Further deposits are identified on an additional sheet

Name of depositary institution '

Agricultural Research Culture Collection (NRRL)

Address of depositary institution (including postal code and country)

1815 N. University Street

Peoria, IL 61604

US

Date of deposit ' June 6, 1995 Accession Number ' B-21459

B. ADDITIONAL INDICATIONS ' (leave blank if not applicable). This information u continued on a separate attached sheet

C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ' era* ,*_*,«..*...»*_««*$-«.,

D. SEPARATE FURNISHING OF INDICATIONS ' (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later ' (Specify the general nature of the indications e.g , "Accession Number of Deposit")

E. D This sheet was received with the International application when filed (to be checked by the receiving Office)

^Λ τ^^^i^_^

(Authorized Officer)

D The date of receipt (from the applicant) by the Intemational Bureau '

(Authorized Officer)

Form PCT/RO/134 (January 1981 ) - 153.2

International Application No: PCT/

Form PCT/RO/134 (cont.)

American Type Culture Collection

10801 University Blvd., Manassas, VA 20110-2209 US

Accession No. Date of Deposit 69974 January 5, 1996 69983 February 7, 1996 69984 February 7, 1996

Claims

WHAT IS CLAIMED IS:

1. A method for identifying a substance to be tested for an ability to ameliorate εymptoms of a fibroproliferative disease or oncogenic related disorder comprising, assaying the ability of the substance to modulate the expression of, or the activity of the encoded protein product of, the rchd534 gene or the fchd5 0 gene.

2. The method of Claim 1 in which the fibroproliferative disease is diabetic retinopathy.

3. The method of- Claim 1 in which the oncogenic related disorder is a tumor growth.

4. The method of Claim 1 in which the oncogenic related disorder is angiogenesis.

5. The method of Claim 1, wherein the oncogenic related disorder is pancreatic cancer.

6. The method of Claim 1, wherein the expression of, or the activity of the encoded protein product of, the rchd534 gene or the fchd540 is reduced.

7. The method of Claim 1 in which the -expression of the gene is assayed by:

(a) exposing a sample of cells to a test substance;

(b) assaying the expression of said gene in the εample of cells; and

(c) comparing the expression level of the gene in the sample exposed to the substance to the expression level of the gene in a control sample of cells.

8. The method of Claim 7 in which the subεtance is an oligonucleotide complementary to the 5' region of the gene and blocks transcription via triple helix formation.

- 154 -

9. The method of Claim 7 in which the εubstance is an antisense or ribozyme molecule that blocks -translation of the gene.

10. The method of claim 1 in which the substance is a small organic or inorganic molecule that modulates the activity of the protein product by binding to the pr v- in product.

11. The method of claim 10 in which the reduces the activity of the protein product by binding to the protein product.

12. The method of claim 1 in which the substance is an antibody t.at modulates the activity of the protein product by binding to the protein product.

13. The method of claim 12 in which the εubstance reduces the activity of the protein product by binding to the protein product.

14. A method for enhancing TGF-β signalling in a cell, comprising contacting a cell expressing the fchd540 gene with a substance that reduces the expression of, or the activity of the encoded protein product of, the fchd540 gene, such that TGF-jS signalling in the cell is enhanced.

15. The method of claim 14, wherein the substance inhibits interaction between the fchd540 protein and a protein member of the TGF-β signalling pathway.

16. The method of claim 15, wherein the member of the TGF- β signalling pathway is activated T3R1.

17. The method of claim 14, wherein the substance inhibits the interaction between the rchd534 protein and the fchd540 protein.

- 155 -

18. The method of Claim 14 in which the substance is an antiεonse or ribozyme molecule that blocks translation of the gene.

19. The method of Claim 14 in which the substance iε complementary to the 5' region of the gene and blocks transcription via triple helix formation.

20. The method of Claim 14 in which the substance is an antibody that inhibitε the activity of the protein product.

21. A method for ameliorating symptoms of fibroproliferative disease or an oncogenic related disorder comprising administering a substance that reduces the expression of, or the activity of the encoded protein product of, the fchd540 gene, to a patient exhibiting such εymptoms.

22. The method of claim 21, wherein the subεtance inhibitε interaction between the fchd540 protein and a protein member of the TGF-β εignalling pathway.

23. The method of claim 22, wherein the member of the TGF- β signalling pathway is activated T/3R1.

24. The method of claim 21, wherein the substance inhibits the interaction between the rchd534 protein and the fchd540 protein.

25. The method of Claim 22 in which the substance is an antisense or ribozyme molecule that blocks translation of the gene.

26. The method of Claim 22 in which the subεtance iε complementary to the 5' region of the gene and blockε tranεcription via triple helix formation.

- 156 -

27. The method of Claim 22 in which the substance is an antibody that inhibits the activity of the protein product.

28. A method for enhancing TGF-/3 signalling in a cell, comprising contacting a cell expresεing the rchd534 gene with a εubεtance that reduces the expreεεion of, or the activity of the encoded protein product of, the rchd534 gene, _...j that TGF-/3 εignalling in the cell iε enhanced.

29. The method of claim 28, wherein the εubstance inhibits interaction between the rchd534 protein and a protein member of the TGF-β εignalling pathway.

30. The method of claim 29, wherein the member of the TGF- β signalling pathway is activated T3R1.

31. The method of claim 28, wherein the substance inhibits the interaction between the rchd534 protein and the fchd540 protein.

32. The method of Claim 28 in which the subεtance iε an antiεense or ribozyme molecule that blockε tranεlation of the gene.

33. The method of Claim 28 in which the εubεtance iε complementary to the 5' region of the gene and blocks tranεcription via triple helix formation.

34. The method of Claim 28 in which the εubεtance iε an antibody that inhibits the activity of the protein product.

35. A method for ameliorating symptomε of fibroproliferative disease or an oncogenic related disorder comprising administering a substance that reduces the expreεsion of, or the activity of the encoded protein product of, the rchd534 gene, to a patient exhibiting such symptoms.

- 157 -

36. The method of claim 35, wherein the substance inhibits interaction between the rchd534 protein and a protein member of the TGF-β signalling pathway.

37. The m thod of claim 36, wherein the member of the TGF- β signalling pathway is activated T/5R1.

38. The method of claim 35, wherein the_ substance inhibits the interaction between the rchd534 protein and the fchd540 protein.

39. The method of Claim 35 in which the substance iε an an.tiεenεe or ribozyme molecule that blocks translation of the gene.

40. The method of Claim 35 in which the substance is complementary to the 5' region of the gene and blocks tranεcription via triple helix formation.

41. The method of Claim 35 in which the substance is an antibody that inhibits the activity of the protein product.

42. A method for diagnosing a fibroproliferative diseaεe or an oncogenic related disorder comprising detecting the level of fchd540 tranεcript or rchd534 tranεcript preεent in a patient sample.

43. The method of Claim 42, wherein the fchd540 or the rchd534 transcript level detected is differential to that of a corresponding control sample.

44. The method of Claim 43, wherein the fchd540 or the rchd534 transcript level detected is higher than that of a corresponding control sample.

45. The method of Claim 42 wherein the oncogenic related diεorder is pancreatic cancer.

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46. A method for diagnosing a fibroproliferative disease or an oncogenic related disorder comprising detecting fchd540 or rchd534 protein level or protein activity present in a patient εample.

47. The method of Claim 45, wherein the fchd540 or the rchd534 protein level or activity detected iε differential to that of a correεponding control sample.

48. The method of Claim 46, wherein the fchd540 or the rchd534 protein level or activity detected is higher than that of a corresponding control εample.

49. The method of Claim 45 wherein the oncogenic related disorder is pancreatic cancer.

50. A method for characterizing a fibroproliferative disease or oncogenic related disorder comprising, asεaying, in a patient sample, overexpression of a gene encoding the fchd531 protein, the fchd540 protein or the rchd534 protein relative to expresεion in a control εample.

51. The method of claim 51, wherein the oncogenic related diεorder iε pancreatic cancer.

52. A method for monitoring the efficacy of a compound in clinical trials for the treatment of a fibroproliferative disease or oncogenic related disorder comprising, assaying, in a patient sample, overexpression of a gene encoding the fchd540 protein or the rchd534 protein relative to expreεsion in a control sample.

53. The method of claim 52 , wherein the oncogenic related diεorder iε pancreatic cancer.

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