U.S. patent application number 09/986718 was filed with the patent office on 2002-11-28 for compositions and methods for the treatment and diagnosis of cardiovascular disease using rchd534 as a target.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Falb, Dean A., Gimbrone, Michael A. JR..
Application Number | 20020178458 09/986718 |
Document ID | / |
Family ID | 27011613 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020178458 |
Kind Code |
A1 |
Falb, Dean A. ; et
al. |
November 28, 2002 |
Compositions and methods for the treatment and diagnosis of
cardiovascular disease using rchd534 as a target
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 efficacy 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.
Inventors: |
Falb, Dean A.; (Wellesley,
MA) ; Gimbrone, Michael A. JR.; (Jamaica Plain,
MA) |
Correspondence
Address: |
PENNIE & EDMONDS LLP
1667 K STREET NW
SUITE 1000
WASHINGTON
DC
20006
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
27011613 |
Appl. No.: |
09/986718 |
Filed: |
November 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09986718 |
Nov 9, 2001 |
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09176330 |
Oct 22, 1998 |
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09176330 |
Oct 22, 1998 |
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08485573 |
Jun 7, 1995 |
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5968770 |
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08485573 |
Jun 7, 1995 |
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08386844 |
Feb 10, 1995 |
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6156500 |
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Current U.S.
Class: |
800/9 ; 435/183;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
9/10 20180101; C12N 9/0065 20130101; A61P 9/12 20180101; C12N
9/0083 20130101; A61K 38/00 20130101; C12N 9/0089 20130101; C07K
14/47 20130101; A61P 9/08 20180101 |
Class at
Publication: |
800/9 ; 435/69.1;
435/320.1; 435/183; 435/325; 536/23.2 |
International
Class: |
A01K 067/00; C07H
021/04; C12N 009/00; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated nucleic acid containing the following nucleotide
sequence: rchd005 (SEQ ID NO.:1), rchd024 (SEQ ID NO.:2), rchd032
(SEQ ID NO.:3), rchd036 (SEQ ID NO.:4), rchd502 (SEQ ID NO.:5),
rchd523 (SEQ ID NO.:6), rchd528 (SEQ ID NO.:7), or rchd534 (SEQ ID
NO.:36). or the nucleotide sequence of a gene or gene fragment
contained in the following clone as deposited with the NRRL:
pRCHD005 (in NRRL Accession No. B-21376), pRCHD024 (in NRRL
Accession No. B-21377), pRCHD032 (in NRRL Accession No. B-21378),
pRCHD036 (in NRRL Accession No. B-21379), PRCHD502 (in NRRL
Accession No. B-21380), PRCHD523 (in NRRL Accession No. B-21381),
pFCHD523 (in NRRL Accession No. ), PRCHD528 (in NRRL Accession No.
B-21382), or pFCHD534 (in NRRL Accession No. ).
2. An isolated nucleic acid which hybridizes under stringent
conditions to the nucleotide sequence of claim 1 or its complement,
or to the gene or gene fragment contained in the clone of claim 1
as deposited with the NRRL.
3. An isolated nucleic acid which encodes an amino acid sequence
encoded by the nucleotide sequence of claim 1 or its complement, or
the gene or gene fragment contained in the clone of claim 1 as
deposited with the NRRL.
4. A nucleotide vector containing the nucleotide sequence of claim
1, 2 or 3.
5. An expression vector containing the nucleotide sequence of claim
1, 2 or 3 in operative association with a nucleotide regulatory
element that controls expression of the nucleotide sequence in a
host cell.
6. A genetically engineered host cell containing the nucleotide
sequence of claim 1, 2 or 3.
7. A genetically engineered host cell containing the nucleotide
sequence of claim 1, 2 or 3 in operative association with a
nucleotide regulatory element that controls expression of the
nucleotide sequence in the host cell.
8. A substantially pure gene product encoded by the nucleic acid of
claim 1, 2, or 3.
9. An antibody that immunospecifically binds the gene product of
claim 8.
10. A transgenic animal in which the nucleic acid of claim 1, 2 or
3 is an expressed transgene contained in the genome of the
animal.
11. A transgenic animal in which expression of genomic sequences
encoding the gene product of claim 8 is prevented or
suppressed.
12. A method for diagnosing cardiovascular disease, comprising
detecting, in a patient sample, a gene or its gene product which is
differentially expressed in cardiovascular disease states.
13. The method of claim 12 in which the cardiovascular disease is
atherosclerosis.
14. The method of claim 12 in which the cardiovascular disease is
ischemia/reperfusion.
15. The method of claim 12 in which the cardiovascular disease is
hypertension.
16. The method of claim 12 in which the cardiovascular disease is
restenosis.
17. The method of claim 12 in which the gene is up-regulated in
individuals genetically predisposed to cardiovascular disease.
18. The method of claim 17 in which the gene encodes a Na--K--Cl
cotransporter protein homologue, an rchd024 protein, and rchd032
protein, an rchd036 protein, a homolog of rat matrin F/G protein,
an endoperoxide synthase type II protein, an rchd523 protein, an
rchd528 protein, or an rchd534 protein.
19. The method of claim 12 in which the gene is down-regulated in
individuals genetically predisposed to cardiovascular disease.
20. The method of claim 19 in which the gene encodes a glutathione
peroxidase protein or a Bcl-2 protein.
21. The method of claim 12 in which the gene is up-regulated by
treatment with IL-1.
22. The method of claim 21 in which the gene encodes an Na--K--Cl
cotransporter protein homologue, an rchd024 protein, an rchd032
protein, an rchd036 protein, or an endoperoxide synthase type II
protein.
23. The method of claim 12 in which the gene is up-regulated by
treatment with shear stress.
24. The method of claim 23 in which the gene encodes an Na--K--Cl
cotransporter protein homologue, an rchd024 protein, a rat matrin
F/G protein homologue, an endoperoxide synthase type II protein, an
rchd523 protein, an rchd528 protein, or an rchd534 protein.
25. The method of claim 12 wherein the gene is down-regulated by
treatment of individuals with a high fat/high cholesterol diet.
26. The method of claim 25 in which the gene encodes a glutathione
peroxidase protein or a Bcl-2 protein.
27. A method for treating cardiovascular disease, comprising
administering a compound that modulates the synthesis or expression
of a target gene, or the activity of a target gene product to a
patient in need of such treatment.
28. The method of claim 27 in which the cardiovascular disease is
atherosclerosis.
29. The method of claim 27 in which the cardiovascular disease is
ischemia/reperfusion.
30. The method of claim 27 in which the cardiovascular disease is
hypertension.
31. The method of claim 27 in which the cardiovascular disease is
restenosis.
32. The method of claim 27 in which the compound inhibits the
expression of the target gene, or the synthesis or activity of the
target gene product.
33. The method of claim 32 in which the gene encodes a Na--K--Cl
cotransporter protein homologue, an rchd024 protein, and rchd032
protein, an rchd036 protein, a homolog of rat matrin F/G protein,
an endoperoxide synthase type II protein, an chd523 protein, an
rchd528 protein, or an rchd534 protein.
34. The method of claim 27 in which the compound is an ntisense or
ribozyme molecule that blocks translation of the arget gene.
35. The method of claim 34 in which the gene encodes a Na--K--Cl
cotransporter protein homologue, an rchd024 protein, and rchd032
protein, an rchd036 protein, a homologue of rat atrin F/G protein,
an endoperoxide synthase type II protein, and rchd523 protein, an
rchd528 protein, or an rchd534 protein.
36. The method of claim 27 in which the compound is complementary
to the 5' region of the target gene and blocks transcription via
triple helix formation.
37. The method of claim 36 in which the gene encodes a Na--K--Cl
cotransporter protein homologue, an rchd024 protein, and rchd032
protein, an rchd036 protein, a homologue of rat matrin F/G protein,
an endoperoxide synthase type II protein, and rchd523 protein, an
rchd528 protein, or an rchd534 protein.
38. The method of claim 27 in which the compound is an antibody
that neutralizes the activity of the target gene product.
39. The method of claim 38 in which the gene product is a Na--K--Cl
cotransporter protein homologue, an rchd024 protein, and rchd032
protein, an rchd036 protein, a homologue of rat matrin F/G protein,
an endoperoxide synthase type II protein, and rchd523 protein, an
rchd528 protein, or an rchd534 protein.
40. The method of claim 27 in which the compound enhances the
expression of the target gene, or the synthesis or activity the
target gene product.
41. The method of claim 40 in which the target gene encodes Bcl-2
or glutathione peroxidase.
42. A method for treating cardiovascular disease, comprising
administering nucleic acid encoding an active target gene product
to a patient in need of such treatment.
43. The method of claim 42 in which the nucleic acid encodes Bcl-2
or glutathione peroxidase.
44. A method for treating cardiovascular disease, comprising
administering an effective amount of a target gene product to a
patient in need of such therapy.
45. The method of claim 44 in which the gene product is Bcl-2 or
glutathione peroxidase.
46. A method of monitoring the efficacy of a compound in clinical
trials for the treatment of cardiovascular disease, comprising
detecting, in a patient sample, a gene or its gene product which is
differentially expressed in cardiovascular disease states.
47. The method of claim 46 in which the cardiovascular disease is
atherosclerosis.
48. The method of claim 46 in which the cardiovascular disease is
ischemia/reperfusion.
49. The method of claim 46 in which the cardiovascular disease is
hypertension.
50. The method of claim 46 in which the cardiovascular disease is
restenosis.
51. The method of claim 46 in which the gene is up-regulated in
individuals genetically predisposed to cardiovascular disease.
52. The method of claim 51 in which the gene encodes a Na--K--Cl
cotransporter protein homologue, an rchd024 protein, and rchd032
protein, an rchd036 protein, a homolog of rat matrin F/G protein,
an endoperoxide synthase type II protein, and rchd523 protein, an
rchd528 protein, or an rchd534 protein.
53. The method of claim 46 in which the gene is down-regulated in
individuals genetically predisposed to cardiovascular disease.
54. The method of claim 53 in which the gene encodes a glutathione
peroxidase protein or a Bcl-2 protein.
55. The method of claim 46 in which the gene is up-regulated by
treatment with IL-1.
56. The method of claim 55 in which the gene encodes an Na--K--Cl
cotransporter protein homologue, an rchd024 protein, an rchd032
protein, an rchd036 protein, or an endoperoxide synthase type II
protein.
57. The method of claim 46 in which the gene is up-regulated by
treatment with shear stress.
58. The method of claim 57 in which the gene encodes an Na--K--Cl
cotransporter protein homologue, an rchd024 protein, a rat matrin
F/G protein homologue, an endoperoxide synthase type II protein, an
rchd523 protein, an rchd528 protein, or an rchd534 protein.
59. The method of claim 46 wherein the gene is down-regulated by
treatment of individuals with a high fat/high cholesterol diet.
60. The method of claim 59 in which the gene encodes a glutathione
peroxidase protein or a Bcl-2 protein.
61. A method for identifying a compound that modulates the activity
of a multiple transmembrane domain receptor target gene product,
comprising: contacting a first cell expressing the multiple
transmembrane domain receptor target gene product wtith a test
compound and an activator of the multiple transmembrane domain
receptor target gene product, measuring the level of intracellular
calcium release within the first cell and comparing the level to
that of a second multiple transmembrane domain receptor target gene
product-expressing cell which has been contacted with the activator
but not with the test compound so that if the level of
intracellular calcium release within the first cells differs from
that of the second cell, a compound which modulates the activity of
a multiple transmembrane domain receptor target gene product has
been identified.
62. The method of claim 61 wherein the multiple transmembrane
domain receptor target gene product is an rchd523 gene product.
63. The method of claim 61 wherein the cell is a Xenopus oocyte
cell.
64. The method of claim 61 wherein the cell is a myeloma cell.
65. The method of claim 18 in which the gene encodes an rchd523
protein.
66. The method of claim 18 in which the gene encodes an rchd534
protein.
Description
[0001] This application is a continuation-in-part of co-pending
application Ser. No. 08/386,844, filed Feb. 10, 1995, which is
hereby incorporated by reference in its entirety.
1. INTRODUCTION
[0002] 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. Genes which are differentially expressed in
cardiovascular disease states, relative to their expression in
normal, or non-cardiovascular disease states are identified. Genes
are also identified via the ability of their gene products to
interact with other gene products involved in cardiovascular
disease. The genes identified may be used diagnostically or as
targets for therapeutic intervention. In this regard, the present
invention provides methods for the identification and therapeutic
use of compounds in the treatment and diagnosis of cardiovascular
disease. Additionally, methods are provided for the diagnostic
monitoring of patients undergoing clinical evaluation for the
treatment of cardiovascular disease, for monitoring the efficacy of
compounds in clinical trials, and for identifying subjects who may
be predisposed to cardiovascular disease.
2. BACKGROUND OF THE INVENTION
[0003] 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 principle
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.
[0004] 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.
[0005] 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.
[0006] Ischemia is a condition characterized by a lack of oxygen
supply in tissues of organs due to inadequate perfusion. Such
inadequate perfusion can have number of natural causes, including
atherosclerotic or restenotic lesions, anemia, or stroke, to name a
few. Many medical interventions, such as the interruption of 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.
[0007] 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.
[0008] 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 thrombosis.
[0009] Very recently, a modified 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.
[0010] 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.
[0011] Cell Biol. 111: 2149); in adherent cultures of human
monocyte-derived macrophages treated with oxidized LDL (Malden 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).
[0012] Other studies have focused on the expression of
adhesion-molecules on the surface of activated endothelial cells
which mediate monocyte adhesion. These adhesion 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, ELAM 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. (Kume et al., 1992, J. Clin. Invest. 90:
1138-1144). VCAM-1, 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).
[0013] Recently, 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.
[0014] 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 identifying those which may serve as therapeutic targets
for the diagnosis and treatment of various forms of cardiovascular
disease.
3. SUMMARY OF THE INVENTION
[0015] 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. "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.
[0016] 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.
[0017] 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.
[0018] The present invention encompasses methods for prognostic and
diagnostic evaluation of cardiovascular 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 disease.
[0019] 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 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.
[0020] In the working examples described herein, eight novel human
genes are identified that are demonstrated to be differentially
expressed in different cardiovascular disease states Additionally,
the differential expression of three previously identified human
genes is described. The identification of these genes and the
characterization of their expression in particular disease states
provide newly identified roles in cardiovascular disease for both
the novel genes and the known genes.
[0021] Bcl-2 and glutathione peroxidase are the products of known
genes that are shown herein to be down regulated in monocytes of
patients exposed to an atherogenic high fat/high cholesterol diet.
Furthermore, counteracting the down-regulation of bcl-2 under
atherogenic conditions, as described herein, may ameliorate
atherosclerosis. Accordingly, methods are provided for the
diagnosis, monitoring in clinical trials, and treatment of
cardiovascular disease based upon the discoveries herein regarding
the expression patterns of bcl-2 and glutathione peroxidase.
Because these two genes were known to be involved in preventing
apoptosis, the discovery of their down-regulation under atherogenic
conditions provides a novel, positive correlation between apoptosis
and atherogenesis. Accordingly, methods provided herein for
diagnosing, monitoring, and treating cardiovascular disease may
also be based on a number of genes involved in the apoptotic
pathway, including but not limited to ICE (IL-1 converting enzyme);
Bad; BAG-1 (Bcl-2 associated athanogene 1, Takayama et al., 1995,
Cell 80: 279-284); BAX (Bcl-2associated X protein, Oltvai et al.,
1993, Cell 74: 609-619); BclXL (Boise, et al., 1993, Cell 74:
597-608); BAK (Bcl-2 antagonist killer, Farrow et al., 1995. Nature
374: 631-733); and Bcl-Xs (Tsujmoto et al., 1984, Science 226:
1097-1099). The cardiovascular diseases that may be so diagnosed,
monitored in clinical trials, and treated include but are not
limited to atherosclerosis, ischemia/reperfusion, and
restenosis.
[0022] rchd005, rchd024, rchd032, and rchd036 are newly identified
genes that are each up-regulated in endothelial cells treated with
IL-1. Accordingly, methods are provided for the diagnosis,
monitoring in clinical trials, and treatment of cardiovascular
disease based upon the discoveries herein regarding the expression
patterns of rchd005, rchd024, rchd032, and rchd036.
[0023] Endoperoxide synthase is a known gene, and rchd502, rchd523,
rchd528, and rchd534 are newly identified genes that are each
up-regulated in endothelial cells subjected to shear stress.
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 endoperoxide synthase,
rchd502, rchd523, rchd528, and rchd534.
[0024] More specifically, because each of these genes is
up-regulated either by IL-1 (rchd005, rchd024, rchd032, and
rchd036) or by shear stress (endoperoxide synthase, rchd502,
rchd523, rchd528, and rchd534), treatment methods can be designed
to reduce or eliminate their expression, particularly in
endothelial cells. Alternatively, treatment methods include
inhibiting the activity of the protein products of these genes. In
addition, 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 diagnosed, monitored in
clinical trials, and treated include but are not limited to
atherosclerosis, ischemia/reperfusion, hypertension, restenosis,
and arterial inflammation.
[0025] The rchd523 gene can be a particularly useful target for
treatment methods as well as diagnostic and clinical monitoring
methods. As a transmembrane protein, the rchd523 gene product is
accessible from the cell surface. Accordingly, natural ligands,
derivatives of natural ligands, and antibodies that bind to the
rchd523 gene product can be utilized to inhibit its activity, or
alternatively, to target the specific destruction of cells that are
in the disease state. Furthermore, the extracellular domains of the
rchd523 gene product provide especially efficient screening systems
for identifying compounds that bind to the rchd523 gene product.
Compounds that bind the receptor domain of the rchd523 gene
product, for example, can be identified by their ability to
mobilize Ca.sup.2+ and thereby produce a fluorescent signal, as
described in Section 5.5.1, below.
[0026] Such an assay system can also be used to screen and identify
antagonists of the interaction between the rchd523gene product and
ligands that bind to the rchd523 gene product. For example, the
compounds can compete with the endogenous (i.e., natural) ligand
for the rchd523 gene product. The resulting reduction in the amount
of ligand-bound rchd523 gene transmembrane protein will modulate
the activity of disease state cells, such as endothelial cells.
Soluble proteins or peptides, such as peptides comprising one or
more of the extracellular domains, or portions and/or analogs
thereof of the rchd523 gene product, including, for example,
soluble fusion proteins such as Ig-tailed fusion proteins, can be
particularly useful for this purpose.
[0027] Similarly, antibodies that are specific to one or more of
the extracellular domain of the rchd523 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
rchd523 gene product is up-regulated in endothelial cells under
shear stress, its detection positively corresponds with
cardiovascular disease.
[0028] The examples presented in Sections 6-9, below, demonstrate
the use of the cardiovascular disease paradigms of the invention to
identify cardiovascular disease target genes.
[0029] The example presented in Section 10, 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.
[0030] The example presented in Section 11, below, demonstrates the
use of fingerprint genes, particularly rchd523, in the imaging of a
diseased cardiovascular tissue. The example presented in Section
12, below, demonstrates the use of target genes, particularly
rchd523, in screening for ligands of target gene product redptor
domains, as well as antagonists of the ligand-receptor
interaction.
4. DESCRIPTION OF THE FIGURES
[0031] FIG. 1. In vivo cholesterol differential display. mRNA
prepared from human monocytes isolated from the blood of patients
on different diets. cDNA prepared from one patient on a high fat
diet/high serum cholesterol (lanes 1,2) and low fat diet/low serum
cholesterol (lanes 3,4) was displayed using the forward primer
T.sub.11XG and the reverse primer OP014 (agcatggctc). The DNA
corresponding to marked band (#14) was excised and amplified for
sequence analysis.
[0032] FIG. 2. Band #14 Northern blot analysis. A random
primer-labeled band #14 probe was hybridized with a Northern blot
prepared from the same patient's monocytes used in differential
display. An 8 kb band was seen in the low fat/low cholesterol
conditions, and not in the high fat/high cholesterol
conditions.
[0033] FIG. 3. Quantitative RT-PCR analysis of mouse bcl-2 mRNA
levels in apoE-deficient mice. Monocyte RNA from apoE-deficient and
control mice was compared using primers for mouse bcl-2
(for-cacccctggcatcttctccttcc/r- ev-atcctcccccagttcaccccatcc) shown
in the upper panel and mouse Actin
(for-cctgatagatgggcactgtgt/rev-gaacacggcattgtcactaact) shown in the
lower panel. A 1:3 dilution series of each input cDNA was done in
pairs with the left band in each pair deriving from wild-type cDNA
and the right band from apoE-deficient cDNA.
[0034] FIG. 4. RT-PCR quantification of human glutathione
peroxidase (HUMGPXP1) cDNA from human clinical samples cDNA
prepared from RNA derived from blood monocytes of the same patient
under a high fat diet (serum cholesterol level=200; top panel) and
a low fat diet (serum cholesterol level=170; bottom panel).
Dilution series of amplification products using GPX1.3 primers
derived from HUMGPXP1 sequences 1121-1142
(for-aagtcgcgcccgcccctgaaat) and 1260-1237
(rev-gatccctggccaccgtccgtctga) is shown in the left portion of each
panel. Dilution series of amplification products using human actin
primers (for-accctgaagtacccat/re- v-tagaagcatttgcggtg) is shown in
the right portion of each panel. The HUMGPXP1 band decreased in
intensity under a high fat diet (compare top left to bottom left),
whereas the actin control band was equally intense under each diet
(compare top right to bottom right).
[0035] FIG. 5. IL-1 activated HUVEC differential display. mRNA
prepared from control HUVEC (lanes 9,10), 1 hr. of 10 units/ml IL-1
treatment (lanes 7,8), or 6 hr. treatment (lanes 11,12), was used
in differential display reactions with the forward primer OPE7
(agatgcagcc) and reverse primer T.sub.11XA, which is an equimolar
mix of oligonucleotides where X is G, C, or A. The DNA
corresponding to marked band, rchd005, was excised and amplified
for Northern analysis and subcloning.
[0036] FIG. 6. Northern blot analysis of endothelial IL-1inducible
rchd005. 2 .mu.g of total RNA from control, 1 hr. and 6 hr. samples
was eluted on an agarose gel, blotted, and incubated with a
.sup.32P labeled probe prepared from amplified rchd005 sequences.
The indicated band migrated with markers corresponding to
approximately 7.5 kb.
[0037] FIG. 7. A Northern blot prepared from shear stressed RNA and
hybridized with the same rchd005 probe detects a 7.5 kb band
up-regulated most strongly at 1 hr.
[0038] FIG. 8. Band rchd005 DNA sequence. The sequence was
determined by sequencing the insert of pRCHD005, resulting from the
ligation of amplified rchd005 sequences into the TA cloning
vector.
[0039] FIG. 9. IL-1 activated HUVEC differential display. mRNA
prepared from control HUVEC (lanes 3,4), 1 hr. of 10 units/ml IL-1
treatment (lanes 1,2), or 6 hr. treatment (lanes 5,6), was used in
differential display reactions with the forward primer OPG20
(tctccctcag) and reverse primer T.sub.11XC, which is an equimolar
mix of oligonucleotides where X is G, C, or A. The DNA
corresponding to marked band, rchd024, was excised and amplified
for Northern analysis and subcloning.
[0040] FIG. 10. Northern blot analysis of endothelial IL-1inducible
band rchd024. 2 .mu.g of total RNA from control, 1 hr. and 6 hr.
samples was eluted on an agarose gel, blotted, and incubated with a
.sup.32P labeled probe prepared from amplified band rchd024
sequences. The indicated band migrated with markers corresponding
to approximately 10 kb.
[0041] FIG. 11. Shear stress Northern blot analysis of endothelial
IL-1 inducible band rchd024. A Northern blot prepared from shear
stressed RNA and hybridized with the same rchd024 probe detected a
10 kb band up-regulated most strongly at 6 hr.
[0042] FIG. 12. Band rchd024 DNA sequence. The sequence was
determined by sequencing the insert of pRCHD024, resulting from the
ligation of amplified rchd024 sequences into the TA cloning
vector.
[0043] FIG. 13. IL-1 activated HUVEC differential display for
rchd032. mRNA prepared from control HUVEC (lanes 3,4), 1 hr. of 10
units/ml IL-1 treatment (lanes 1,2), or 6 hr. treatment (lanes
5,6), was used in differential display reactions with the forward
primer OPI9 (tggagagcag) and reverse primer T.sub.11XA, which is an
equimolar mix of oligonucleotides where X is G, C, or A. The DNA
corresponding to marked band, rchd032, was excised and amplified
for Northern analysis and subcloning.
[0044] FIG. 14. RT-PCR quantification of rchd032 cDNA from IL-1
activated HUVEC's cDNA prepared from RNA derived from control, 1
hr., and 6 hr. IL-1 activated HUVEC's. Shown in lanes 1,2, and 3
are a 5 fold dilution series of input cDNA amplified in the upper
panel with rchd032 primers
(for-atttataaaggggtaattcatta/rev-ttaaagccaatttcaaaataat), and in
the lower panel with human actin primers
(for-accctgaagtaccccat/rev-tagaagcat- ttgcggtg). A band at the
1:125 dilution in lane 3 is visible in the 6 hr. sample but not in
the control.
[0045] FIG. 15. Band rchd032 DNA sequence. The sequence was
determined by sequencing the insert of pRCHD0321 resulting from the
ligation of amplified rchdO32 sequences into the TA cloning
vector.
[0046] FIG. 16. IL-1 activated HUVEC differential display for
rchd036. mRNA prepared from control HUVEC (lanes 3,4), 1 hr. of 10
units/ml IL-1 treatment (lanes 1,2), or 6 hr. treatment (lanes
5,6), was used in differential display reactions with the forward
primer OPI17 (ggtggtgatg) and reverse primer T.sub.11XC, which is
an equimolar mix of oligonucleotides where X is G, C, or A. The DNA
corresponding to marked band, rchd036, was excised and amplified
for Northern analysis and subcloning.
[0047] FIG. 17. Northern blot analysis of endothelial IL-1inducible
band rchd036. 2 .mu.g of total RNA from control (lane 1), 1 hr.
(lane 2), and 6 hr. (lane 3) samples was eluted on an agarose gel,
blotted, and incubated with a .sup.32P labeled probe prepared from
amplified band rchd036 sequences. The indicated band migrated with
markers corresponding to approximately 8 kb.
[0048] FIG. 18. Band rchd036 DNA sequence. The sequence was
determined by sequencing the insert of pRCHD036, resulting from the
ligation of amplified rchd036 sequences into the TA cloning
vector.
[0049] FIG. 19. Laminar shear stress HUVEC differential display.
mRNA prepared from control HUVEC (lanes 3,4), 1 hr. (lanes 1,2) of
10 dyn/cm2 laminar shear stress treatment or 6 hr. treatment (lanes
5,6), was used in differential display reactions with the forward
primer OPE7 (agatgcagcc) and reverse primer T.sub.11XA, which is an
equimolar mix of oligonucleotides where X is G, C, or A. The DNA
corresponding to marked band, rchd502, was excised and amplified
for Northern analysis and subcloning.
[0050] FIG. 20. Northern blot analysis of shear stress inducible
band rchd502. 2 .mu.g of total RNA from control, 1 hr. and 6 hr.
shear stressed samples was eluted on an agarose gel, blotted, and
incubated with a .sup.32P labeled probe prepared from amplified
band rchd502 sequences. The indicated band migrates with markers
corresponding to approximately 4.5 kb.
[0051] FIG. 21. Northern blot analysis of shear stress inducible
band rchd502 on IL-1 blot. 2 .mu.g of total RNA from control (lane
1), 1 hr. (lane 2), and 6 hr. (lane 3) IL-1 induced HUVEC samples
was eluted on an agarose gel, blotted, and incubated with a
.sup.32P labeled probe prepared from amplified band rchd502
sequences. A 4.5 kb band is seen which was not up-regulated by
IL-1.
[0052] FIG. 22. Band rchd502 DNA sequence. The sequence was
determined by sequencing the insert of pRCHD502, resulting from the
ligation of amplified rchd502 sequences into the TA cloning
vector.
[0053] FIG. 23. Laminar shear stress HUVEC differential display for
rchd505. mRNA prepared from control HUVEC (lanes 3,4), 1 hr. (lanes
1,2) or 6 hr. (lanes 5,6) of 10 dyn/cm2 laminar shear stress
treatment was used in differential display reactions with the
forward primer OPE2 (ggtgcgggaa) and reverse primer T.sub.11XA,
which is an equimolar mix of oligonucleotides where X is G,C, or A.
The DNA corresponding to marked band, rchd505, was excised and
amplified for Northern analysis and subcloning.
[0054] FIG. 24. Northern blot analysis of shear stress inducible
band rchd505. 2 .mu.g of total RNA from control, 1 hr. and 6 hr.
shear stressed samples was eluted on an agarose gel, blotted, and
incubated with a .sup.32P labeled probe prepared from amplified
band rchd505 sequences. The indicated band migrated with markers
corresponding to approximately 5.0 kb.
[0055] FIG. 25. Northern blot analysis of shear stress inducible
band rchd505 on IL-1 blot. 2 .mu.g of total RNA from control (lane
1), 1 hr. (lane 2), and 6 hr. (lane 3) IL-1 induced HUVEC samples
was eluted on an agarose gel, blotted, and incubated with a
.sup.32P labeled probe prepared from amplified band rchd505
sequences. A 5.0 kb inducible band is seen.
[0056] FIG. 26. Laminar shear stress HUVEC differential display for
rchd523. mRNA prepared from control HUVEC (lanes 3,4), 1 hr. (lanes
1,2) or 6 hr. (lanes 5,6) of 10 dyn/cm2 laminar shear stress
treatment was used in differential display reactions with the
forward primer OPI11 (acatgccgtg) and reverse primer T.sub.11XC,
which is an equimolar mix of oligonucleotides where X is G,C, or A.
The DNA corresponding to marked band, rchd523, was excised and
amplified for Northern analysis and subcloning.
[0057] FIG. 27. RT-PCR quantification of rchd523 cDNA from shear
stressed endothelial cell cDNA prepared from RNA derived from
control, 1 hr., and 6 hr. shear stressed HUVEc's. Shown in lanes
1,2, and 3 are a 5-fold dilution series of input cDNA amplified in
the upper panel with rchd523 primers
(for-atgccgtgtgggttagtc/rev-attttatgggaaggtttttaca), and in lanes 4
and 5, a 5-fold dilution series using human actin primers
(for-accctgaagtaccccat/rev-tagaagcatttgcggtg). A band at the 1:5
dilution in lane 2 is visible in the 6 hr. sample but not in the
control.
[0058] FIG. 28. DNA and encoded amino acid sequence of the rchd523
gene.
[0059] FIG. 29. Laminar shear stress HUVEC differential display for
rchd528. mRNA prepared from control HUVEC (lanes 3,4), 1 hr. (lanes
1,2) or 6 hr. (lanes 5,6) of 10 dyn/cm2 laminar shear stress
treatment was used in differential display reactions with the
forward primer OPI19 (aatgcgggag) and reverse primer T.sub.11XG,
which is an equimolar mix of oligonucleotides where X is G,C, or A.
The DNA corresponding to marked band, rchd528, was excised and
amplified for Northern analysis and subcloning.
[0060] FIG. 30. Northern blot analysis of shear stress inducible
band rchd528. 2 .mu.g of total RNA from control (lane 1), 1 hr.
(lane 2), and 6 hr. (lane 3) shear stressed samples was eluted on
an agarose gel, blotted, and incubated with a .sup.32P labeled
probe prepared from amplified band rchd528 sequences. The indicated
band migrated with markers corresponding to approximately 5.0
kb.
[0061] FIG. 31. Band rchd528 DNA sequence. The sequence was
determined by sequencing the insert of pRCHD528, resulting from the
ligation of amplified rchd528 sequences into the TA cloning
vector.
[0062] FIG. 32. Restriction map of plasmid pScR-bcl2. FIG. 33.
Northern blot analysis of expression of rchd036 mRNA under shear
stress. RNA was prepared from HUVEC's that were untreated (control)
and treated with shear stress for 1 hr. and 6 hr. The blot was
probed with labeled rchdO36 DNA.
[0063] FIG. 34. Northern blot analysis of expression of rchd534
mRNA under shear stress. RNA was prepared from HUVEC's that were
untreated (control) and treated with shear stress for 1 hr. and 6
hr. The blot was probed with labeled rchd534 DNA.
[0064] FIG. 35. DNA and encoded amino acid sequence of the rchd534
gene.
5. DETAILED DESCRIPTION OF THE INVENTION
[0065] Methods and compositions for the diagnosis and treatment of
cardiovascular disease, including but not limited to
atherosclerosis, ischemia/reperfusion, hypertension, restenosis,
and arterial inflammation, are 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.
[0066] 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 genes" 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.
[0067] 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 to which such gene
products may contribute, in Section 5.4.4.
[0068] Methods for the identification of compounds which modulate
the expression of genes or the activity of gene products involved
in cardiovascular disease 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.
[0069] 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.
[0070] 5.1. Identification of Differentially Expressed Genes
[0071] 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.
[0072] "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.
[0073] 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 degree to which 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.
[0074] 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 compounds useful for the treatment
of cardiovascular disease. A fingerprint gene may also have the
characteristics of a target gene.
[0075] "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.
[0076] 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.
[0077] 5.1.1. Paradigms for the Identification of Differentially
Expressed Genes
[0078] One strategy for identifying genes that are involved in
cardiovascular disease is to detect genes that are expressed
differentially under conditions associated with the disease versus
non-disease conditions. The sub-sections below describe a number of
experimental systems, called aradigms, which may be used to detect
such differentially expressed genes. In general, the paradigms
include at least one experimental condition in which subjects or
samples are treated in a manner associated with cardiovascular
disease, in addition to at least one experimental control condition
lacking such disease associated treatment. Differentially expressed
genes are detected, as described herein, below, by comparing the
pattern of gene expression between the experimental and control
conditions.
[0079] 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 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.
[0080] 5.1.1.1. Foam Cell Paradigm--1
[0081] 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 formation. Such
paradigms may serve to identify genes involved in the
differentiation of this cell type, or their uptake of oxidized
LDL.
[0082] One embodiment of such a paradigm, hereinafter referred to
as Paradigm A. First, human blood is drawn and peripheral monocytes
are isolated by methods routinely practiced in the art. These human
monocytes 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 cells are then treated
for various lengths of time with agents thought to be involved in
foam cell formation. These agents 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 cells are harvested and analyzed for
differential expression 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 expressed in treated versus control cells.
[0083] 5.1.1.2. Foam Cell Paradigm--2
[0084] Alternative paradigms involving monocytes for detecting
differentially expressed genes associated with atherosclerosis
involve the simulation of the phenomenon of transmigration. When
monocytes encounter arterial injury, they adhere to the vascular
endothelial layer, transmigrate across this layer, and locate
between the endothelium and the layer of smooth muscle cells 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 system, 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
expression can then be compared between these foam cells and
untreated monocytes.
[0085] 5.1.1.3. Foam Cell Paradigm--3
[0086] Yet another system includes the third cell type, smooth
muscle cell, that plays a critical role in atherogenesis (Navab et
al., 1988, J. Clin. Invest., 82: 1853). In this system, a
multilayer of human aortic smooth uscle cells was grown on a
micropore filter covered with a gel layer of native collagen, and a
monolayer of human aortic endothelial cells was grown on top of the
collagen layer. Exposure of this coculture to human monocytes in
the presence of chemotactic factor rFMLP resulted in monocyte
attachment to the endothelial cells followed by migration across
the endothelial monolayer into the collagen layer of the
subendothelial space. This type of culture can also be treated with
LDL to generate foam cells. The foam cells can then be harvested
and their pattern of gene expression compared to that of untreated
cells as explained below in sub-section 5.1.2.
[0087] 5.1.1.4. in vivo Monocyte Paradigm
[0088] 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, monocytes are isolated according to the methods routinely
practiced in the art, and RNA is purified, as described below, in
sub-section 5.1.2. These same patients are subsequently 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 cholesterol and monocyte RNA may be
purified once again. The order in which patients receive the diets
may be varied. The RNA derived from patients maintained on two of
the diets, or on all three diets, may then be compared and analyzed
for differential gene expression as, explained below in sub-section
5.1.2. The Example presented in Section 7, below, demonstrates the
use of such an in vivo monocyte paradigm to identify genes which
are expressed differentially in monocytes of patients maintained on
an atherogenic diet versus their expression under a control diet.
Such a paradigm may also be used in conjunction with an in vitro
preliminary detection system, as described in Section 7, below.
[0089] 5.1.1.5. Endothelial Cell--IL-1 Paradigm
[0090] In addition to the detection of differential gene expression
in monocytes, paradigms focusing on endothelial cells may be used
to detect genes involved in cardiovascular disease. In one such
paradigm, hereinafter referred to as Paradigm C, human umbilical
vein endothelial cells (HUVEC's) are grown in vitro. Experimental
cultures are treated with human IL-1.beta., a factor known to be
involved in the inflammatory response, in order to mimic the
physiologic conditions involved in the atherosclerotic state.
Alternatively experimental HUVEC cultures may be treated with
lysophosphatidylcholine, a major phospholipid component of
atherogenic lipoproteins or oxidized human LDL. Control cultures
are grown in the absence of these compounds.
[0091] After a certain period of exposure treatment, experimental
and control cells are harvested and analyzed for differential gene
expression as described in sub-section 5.1.2, below. The Example
presented in Section 8, below, demonstrates the use of such an IL-1
induced endothelial cell paradigm to identify sequences which are
differentially expressed in treated versus control cells.
[0092] 5.1.1.6. Endothelial Cell--Shear Stress Paradigm
[0093] In another paradigm involving endothelial cells, hereinafter
referred to as Paradigm D, cultures are exposed to fluid shear
stress which is thought to be responsible for the prevalence of
atherosclerotic lesions in areas of unusual circulatory flow.
Unusual blood flow also plays a role in the harmful effects of
ischemia/reperfusion, wherein an organ receiving inadequate blood
supply is suddenly reperfused with an overabundance of blood when
the obstruction is overcome.
[0094] Cultured HUVEC monolayers are exposed to laminar sheer
stress 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
stress, experimental and control cells are harvested and analyzed
for differential gene expression as described in sub-section 5.1.2,
below. The Example presented in Section 9, below, demonstrates the
use of such a shear stressed endothelial cell paradigm to identify
sequences which are differentially expressed in exposed versus
control cells.
[0095] In all such paradigms designed to identify genes which are
involved in cardiovascular disease, including but not limited to
those described above in Sections 5.1.1.1 through 5.1.1.6,
compounds such as drugs known to have an ameliorative effect on the
disease symptoms may be incorporated into the experimental system.
Such compounds may include known therapeutics, as well as compounds
that are not useful as therapeutics due to their harmful side
effects. Test cells that are cultured as explained in the paradigms
described in Sections 5.1.1.1 through 5.1.1.6, for example, may be
exposed 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 process by these
compounds.
[0096] Test cells may also be compared to unrelated cells (e.g.,
fibroblasts) 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
methods, the genes and gene products upon which these compounds act
can be identified and used in the assays described below to
identify novel therapeutic compounds for the treatment of
cardiovascular disease.
[0097] 5.1.2. Analysis of Paradigm Material
[0098] In order to identify differentially expressed genes, RNA,
either total or mRNA, may be isolated from one or more tissues of
the subjects utilized in paradigms such as those described earlier
in this Section. RNA samples are obtained from tissues of
experimental subjects and from corresponding tissues of control
subjects. Any RNA isolation technique which does not select against
the isolation of mRNA may be utilized for the purification of such
RNA samples. See, for example, Sambrook et al., 1989, Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and
Ausubel, F. M. et al., eds., 1987-1993, Current Protocols in
Molecular Biology, John Wiley & Sons, Inc. New York, both of
which are incorporated herein by reference in their entirety.
Additionally, large numbers of tissue samples may readily be
processed using techniques well known to those of skill in the art,
such as, for example, the single-step RNA isolation process of
Chomczynski, P. (1989, U.S. Pat. No. 4,843,155), which is
incorporated herein by reference in its entirety.
[0099] Transcripts within the collected RNA samples which represent
RNA produced by differentially expressed genes may be identified by
utilizing a variety of methods which are well known to those of
skill in the art. For example, differential screening (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. Pat. 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
expressed.
[0100] Differential screening 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 is screened
with a total cDNA probe corresponding to the mRNA population of a
second cell type. For example, one cDNA probe may correspond to a
total cell cDNA probe of a cell type derived from a control
subject, 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 represent clones derived from genes
differentially expressed in the cell type of interest in control
versus experimental subjects.
[0101] Subtractive hybridization techniques generally involve the
isolation of mRNA taken from two different sources, e.g., control
and experimental tissue, the hybridization of the mRNA or
single-stranded cDNA reverse-transcribed from the isolated mRNA,
and the removal of all hybridized, and therefore double-stranded,
sequences. The remaining non-hybridized, single-stranded cDNAs,
potentially represent clones derived from genes that are
differentially expressed in the two mRNA sources. Such
single-stranded cDNAs are then used as the starting material for
the construction of a library comprising clones derived from
differentially expressed genes. The differential display technique
describes a procedure, utilizing the well known polymerase chain
reaction (PCR; the experimental embodiment set forth in Mullis, K.
B., 1987, U.S. Pat. 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 transcripts present within any given cell. Utilizing
different pairs of primers allows each of the mRNA transcripts
present in a cell to be amplified. Among such amplified transcripts
may be identified those which have been produced from
differentially expressed genes.
[0102] The reverse oligonucleotide primer of the primer pairs may
contain an oligo dT stretch of nucleotides, 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 nucleotides at its 3' end.
Because, statistically, only a subset of the mRNA derived sequences
present in the sample of interest will hybridize to such primers,
the additional nucleotides allow the primers to amplify only a
subset of the mRNA derived sequences 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.
[0103] The forward primer may contain a nucleotide sequence
expected, statistically, to have the ability to hybridize to cDNA
sequences derived from the tissues 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.
[0104] PCR reaction conditions should be chosen which optimize
amplified product yield and specificity, and, additionally, produce
amplified products of lengths which may be resolved 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 discussed above, and annealing and
elongation step temperatures and reaction times.
[0105] 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 indicate potentially
differentially expressed genes. nce potentially differentially
expressed gene sequences have been identified via bulk techniques
such as, for example, those described above, the differential
expression of such putatively differentially expressed genes should
be corroborated. Corroboration may be accomplished via, for
example, such well known techniques as Northern analysis and/or
RT-PCR.
[0106] Upon corroboration, the differentially expressed genes may
be further characterized, and may be identified as target and/or
fingerprint genes, as discussed, below, in Section 5.3.
[0107] Also, amplified sequences of differentially expressed genes
obtained through, for example, differential display may be used to
isolate full length clones of the corresponding gene. The full
length coding portion of the gene may readily be isolated, without
undue experimentation, by molecular biological techniques well
known in the art. For example, the isolated differentially
expressed amplified fragment may be labeled and used to screen a
cDNA library. Alternatively, the labeled fragment may be used to
screen a genomic library.
[0108] PCR technology may also be utilized to isolate full length
cDNA sequences. As described, above, in this Section, the isolated,
amplified gene fragments obtained through differential display have
5' terminal ends at some random 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
differential display) may be obtained using, for example,
RT-PCR.
[0109] In one embodiment of such a procedure for the identification
and cloning of full length gene sequences, RNA may be isolated,
following standard procedures, from an appropriate tissue or
cellular source. A reverse transcription 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
guanines using a standard terminal transferase reaction, the hybrid
may be digested with RNAase H, and second strand synthesis may then
be primed with a poly-C primer. Using the two primers, the 5'
portion of the gene is amplified using PCR. Sequences obtained may
then be isolated and recombined with previously isolated sequences
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 Ausubel et al., 1989, supra.
[0110] 5.2. Identification of Pathway Genes
[0111] This section describes methods for the identification of
genes, termed "pathway genes", involved in cardiovascular disease.
"Pathway gene", as used herein, refers to a gene whose gene product
exhibits the ability to interact with gene products involved in
cardiovascular disease. A pathway gene may be differentially
expressed and, therefore, may additionally have the characteristics
of a target and/or fingerprint gene.
[0112] Any method suitable for detecting protein-protein
interactions may be employed for identifying pathway gene products
by identifying interactions 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 such known gene products represent
pathway gene products and the genes which encode them represent
pathway genes.
[0113] Among the traditional methods which may be employed are
co-immunoprecipitation, crosslinking and co-purification through
gradients or chromatographic columns. Utilizing procedures such as
these allows for the identification of pathway gene products. Once
identified, a pathway gene product may be used, in conjunction with
standard techniques, to identify its corresponding pathway gene.
For example, at least a portion of the amino acid sequence of the
pathway gene product may be ascertained using techniques well known
to those of skill 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 sequence obtained may be used as a guide for the
generation of oligonucleotide mixtures that can be used to screen
for pathway gene sequences. Screening made 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).
[0114] Additionally, methods may be employed which result in the
simultaneous identification of pathway genes which encode the
protein interacting with a protein involved in cardiovascular
disease. These methods include, for example, probing expression
libraries with labeled protein known or suggested to be involved in
cardiovascular disease, using this protein in a manner similar to
the well known technique of antibody probing of .lambda.gt.sub.11
libraries.
[0115] One such method which detects protein interactions in vivo,
the two-hybrid system, is described in detail for illustration only
and not by way of limitation. One version of this system has been
described (Chien et al., 1991, Proc. Natl. Acad. Sci. USA,
88:9578-9582) and is commercially available from Clontech (Palo
Alto, Calif.).
[0116] Briefly, utilizing such a system, 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 consists of the activator protein's
activation domain fused to an unknown protein that is encoded by a
cDNA which has been recombined into this plasmid as part of a cDNA
library. The plasmids are transformed into a strain of the yeast
Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ)
whose regulatory region contains the activator's binding sites.
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
because it cannot localize to the activator's binding sites.
Interaction of the two proteins reconstitutes the functional
activator protein and results in expression of the reporter gene,
which is detected by an assay for the reporter gene product.
[0117] The two-hybrid system 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 sequences may be
fused 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 transformants may be screened for those
that express the reporter gene. These colonies 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.
[0118] 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 genes involved in
cardiovascular disease, 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 genes for
bFGF, IGF-I, VEGF, IL-1, M-CSF, TGF.beta., TGF.alpha., TNFA,
HB-EGF, PDGF, IFN-.gamma., and GM-CSF, to name a few.
[0119] A cDNA library of the cell line from which proteins that
interact with bait gene are to be detected can be made using
methods routinely practiced in the art. According to the particular
system described herein, for example, the cDNA fragments may be
inserted into a vector such that they are translationally fused to
the activation domain of GAL4. This library may be co-transformed
along with the bait gene-GAL4 fusion plasmid into a yeast strain
which contains a lacZ gene driven by a promoter which contains the
GAL4 activation sequence. A cDNA encoded protein, fused to the GAL4
activation domain, that interacts with bait gene will reconstitute
an active GAL4 protein and thereby drive expression of the lacZ
gene. Colonies which express lacZ may be detected by their blue
color in the presence of X-gal. The cDNA may then be purified from
these strains, and used to produce and isolate the bait
gene-interacting protein using techniques routinely practiced in
the art.
[0120] Once a pathway gene has been identified and isolated, it may
be further characterized as, for example, discussed below, in
Section 5.3.
[0121] 5.3. Characterization of Differentially Expressed and
Pathway Genes
[0122] Differentially expressed genes, such as those identified via
the methods discussed, above, in Section 5.1.1, pathway genes, such
as those identified via the methods discussed, above, in Section
5.2, as well as genes identified by alternative means, may be
further characterized by utilizing, for example, methods such as
those discussed herein. Such genes will be referred to herein as
"identified genes".
[0123] Analyses such as those described herein will yield
information regarding the biological function of the identified
genes. An assessment of the biological function of the
differentially expressed genes, in addition, will allow for their
designation as target and/or fingerprint genes. Specifically, any
of the differentially expressed genes whose further
characterization indicates that a modulation of the gene's
expression or a modulation of the gene product's activity may
ameliorate cardiovascular disease will be designated "target
genes", as defined, above, in Section 5.1. Such target genes and
target gene products, along with those discussed below, will
constitute the focus of the compound discovery strategies
discussed, below, in Section 5.5.
[0124] Any of the differentially expressed genes whose further
characterization indicates that such modulations may not positively
affect cardiovascular disease, but whose expression pattern
contributes to a gene expression "fingerprint pattern" correlative
of, for example, a cardiovascular disease condition will be
designated a "fingerprint gene". "Fingerprint patterns" will be
more fully discussed, below, in Section 5.8. It should be noted
that each of the target genes may also function as fingerprint
genes, as may all or a subset of the pathway genes.
[0125] It should further be noted that the pathway genes may also
be characterized according to techniques such as those described
herein. Those pathway genes which yield information indicating that
they are differentially expressed and that modulation of the gene's
expression or a modulation of the gene product's activity may
ameliorate cardiovascular disease will be also be designated
"target genes". Such target genes and target gene products, along
with those discussed above, will constitute the focus of the
compound discovery strategies discussed, below, in Section 5.5.
[0126] 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, nonetheless, ameliorate cardiovascular disease
symptoms. In such cases, these genes and gene products would also
be considered a focus of the compound discovery strategies of
Section 5.5, below.
[0127] In instances wherein a pathway gene's characterization
indicates that modulation of gene expression or gene product
activity may not positively affect cardiovascular disease, but
whose expression is differentially expressed and which contributes
to a gene expression fingerprint pattern correlative of, for
example, a cardiovascular disease state, such pathway genes may
additionally be designated as fingerprint genes.
[0128] 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 techniques well
known to those of skill in the art, may be used to further
characterize such genes. For example, the sequence of the
identified genes may reveal homologies to one or more known
sequence motifs which may yield information regarding-the
biological function of the identified gene product.
[0129] 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 analyses and RT-PCR.
Such analyses provide information as to whether the identified
genes are expressed in tissues expected to contribute to
cardiovascular disease. Such analyses may also provide quantitative
information regarding steady state mRNA regulation, yielding data
concerning which of the identified genes exhibits a high level of
regulation in, preferably, tissues which may be expected to
contribute to cardiovascular disease.
[0130] Such analyses may also be performed on an isolated 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
information regarding the biological function of an identified gene
relative to cardiovascular disease in instances wherein only a
subset of the cells within the tissue is thought to be relevant to
cardiovascular disease.
[0131] Third, the sequences of the identified genes may be used,
utilizing standard techniques, to place the genes onto genetic
maps, e.g., mouse (Copeland & Jenkins, 1991, Trends in Genetics
7: 113-118) and human genetic maps (Cohen, et al., 1993, Nature
366: 698-701). Such mapping information may yield information
regarding the genes' importance to human disease by, for example,
identifying genes which map near genetic regions to which known
genetic cardiovascular disease tendencies map.
[0132] Fourth, the biological function of the identified genes may
be more directly assessed by utilizing relevant in vivo and in
vitro systems. In vivo systems may include, but are not limited to,
animal systems which naturally exhibit cardiovascular disease
predisposition, or ones which have been engineered to exhibit such
symptoms, including but not limited to the apoE-deficient
atherosclerosis mouse model (Plump et al., 1992, Cell 71: 343-353).
Such systems are discussed in Section 5.4.4.1, below.
[0133] The use of such an in vivo system is described in detail in
the example provided in Section 7, below, confirming the role of
the target gene bcl-2 (see Table 1, in Section 5.4.1, below).
Briefly, bcl-2 expression first was shown to be down-regulated in
the apoE-deficient atherosclerosis mouse model. Then, a transgenic
mouse was engineered bearing the human bcl-2 gene under the control
of a promoter which is induced in monocyte foam cells under
atherogenic conditions. To test the effect of the induction of
bcl-2 under such conditions, the transgenic mouse is crossed with
the apoE-deficient mouse. apoE-deficient progeny bearing the highly
expressible bcl-2 gene are then examined for plaque formation and
development. Reduction in plaque formation and development in these
progeny confirms the effectiveness of intervening in cardiovascular
disease through this target gene.
[0134] In vitro systems may include, but are not limited to,
cell-based systems comprising cell types known or suspected of
involvement in cardiovascular disease. Such systems are discussed
in detail, below, in Section 5.4.4.2.
[0135] 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
underexpressed, and the subsequent 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 system of
interest, and its subsequent effect then assayed.
[0136] The information obtained through such characterizations may
suggest relevant methods for the treatment of cardiovascular
disease involving the gene of interest. For example, treatment may
include a modulation of gene expression and/or gene product
activity. Characterization procedures such as those described
herein may indicate where such modulation should involve an
increase or a decrease in the expression or activity of the gene or
gene product of interest. Such methods of treatment are discussed,
below, in Section 5.5.4.
[0137] 5.4. Differentially Expressed and Pathway Genes
[0138] Identified genes, which include but are not limited to
differentially expressed genes such as those identified in Section
5.1.1, above, and pathway genes, such as those identified in
Section 5.2, above, are described herein. Specifically, the nucleic
acid sequences and gene products of such identified genes are
described herein. Further, antibodies directed against the
identified genes' products, and cell- and animal-based models by
which the identified genes may be further characterized and
utilized are also discussed in this Section.
[0139] 5.4.1. Differentially Expressed and Pathway Gene
Sequences
[0140] The differentially expressed and pathway genes of the
invention are 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.
[0141] Table 1 lists differentially expressed genes identified
through, for example, the paradigms discussed, above, in Section
5.1.1, and below, in the examples presented in Sections 6 through
9. Table 1 also summarizes information regarding the further
characterization of such genes.
[0142] First, the paradigm used initially to detect the
differentially expressed gene is described under the column headed
"Paradigm of Original Detection". The expression patterns of those
genes which have been shown to be differentially expressed, for
example, under one or more of the paradigm conditions described in
Section 5.1.1 are summarized under the column headed "Paradigm
Expression Pattern". For each of the tested genes, the paradigm
which was used and the difference in the expression of the gene
among the samples generated is shown. "" indicates that gene
expression is up-regulated (i.e., there is an increase in the
amount of detectable mRNA) among the samples generated, while ""
indicates that gene expression is down-regulated (i.e., there is a
decrease in the amount of detectable mRNA) among the samples
generated. "Detectable" as used herein, refers to levels 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.
[0143] 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 genes contain nucleotide sequences similar
or homologous to sequences found in nucleic acid databases,
references to such similarities are listed.
[0144] The genes listed in Table 1 may be obtained using cloning
methods well known to those skilled in 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 is incorporated by
reference herein in its entirety). Probes for the novel sequences
reported herein may be obtained directly from the isolated clones
deposited with the NRRL, as indicated in Table 2, below.
Alternatively, oligonucleotide probes for the novel genes may be
synthesized based on the DNA sequences disclosed herein in FIGS. 8,
12, 15, 18, 22, 28, 31, and 35. Such synthetic oligonucleotides may
be similarly produced based on the sequences provided for the
previously known genes described in the following references:
Cleary et al., 1986, Cell 47: 19-28 (bcl-2); Takahashi et al.,
1990, J. Biochem 108: 145-148 (glutathione peroxidase); and Jones
et al., 1993, J. Biol. Chem. 268: 9049-9054 (prostaglandin
endoperoxide synthase II), each of which is incorporated herein in
its entirety.
[0145] 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; Barnes, 1994, Proc. Natl. Acad. Sci. USA
91: 2216-2220; and Cheng et al., Proc. Natl. Acad. Sci. USA 91:
5695-5699). Oligonucleotides can be designed based on the sequence
obtained from the partial clone that can amplify a reverse
transcribed mRNA encoding the entire coding sequence. This method
was used, as described in the example in Section 9, below, to
obtain the entire coding region of the rchd523 gene.
[0146] Alternatively, probes can be used 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 7.1.1, below. In
fact, as described in detail in the example in Section 9, below,
this method was applied in order to obtain the entire coding region
of the rchd534 gene. Briefly, the up-regulation of this gene was
detected, under Paradigm D, in HUVEC's subjected to shear stress.
Then, amplified partial sequence of the rchd534 gene was subcloned.
The insert was then isolated and used to probe a cDNA library
prepared from shear stress treated HUVEC's. A cDNA clone containing
the entire rchd534 coding region was detected, isolated, and
sequenced. The genes described herein that were detected in
endothelial cells may also be cloned from a cDNA library
constructed from endothelial cells isolated as described in
Progress in Hemostasis and Thrombosis, Vol. 3, P. Spaet, editor,
Grune & Stratton Inc., New York, 1-28.
[0147] Alternatively, the genes may be retrieved from a human
placenta cDNA library (Clontech Laboratories, Palo Alto, CA),
according to Takahashi et al., 1990, supra; a HUVEC cDNA library as
described in Jones et al. 1993, supra; or an acute lymphoblastic
leukemia (SUP-B2) cDNA library as described in Cleary et al., 1986,
supra, for example. Genomic DNA libraries can be prepared from any
source.
1TABLE 1 Differentially Expressed and Pathway Genes Paradigm
Paradigm of Expr. Cell Type Chromosomal Gene Seq. ID # Original
Detection Pattern Detected in Location Ref Seq. Band 14: B
Monocytes 1 bcl-2 (Section 5.1.1.4) Glutathione B Monocytes 2
peroxidase rchd005 1 C Endothelial New 3 FIG. 8 (Section 5.1.1.5)
rchd024 2 C Endothelial 4 New rchd032 3 C Endothelial New rchd036 4
C Endothelial 15 New rchd502 5 D Endothelial New 4 (Section
5.1.1.6) rchd505: D Endothelial 5 Endoperoxide synthase rchd523 6 D
Endothelial 7 New rchd528 7 D Endothelial New rchd534 36 D
Endothelial 15 New 1 Cleary et al., 1986, Cell 47: 19-28. 2
Takahashi et al., 1990, J. Biochem. 108: 145-148. 3 Shark Na--K--Cl
cotransporter, Xu et al., 1994 Proc. Natl. Acad. Sci. U.S.A. 91:
2201-2205. 4 Rat matrin F/G, Hakes et al., 1991 Proc. Natl. Acad.
Sci. U.S.A. 88: 6186-6190. 5 Jones et al., 1993, J. Biol. Chem.
268: 9049-9054.
[0148] Table 2, below, lists isolated clones that contain sequences
of the novel genes listed in Table 1. Such clones were produced
from amplified sequences of the indicated differential display band
which were subcloned into the TA cloning vector (Invitrogen, San
Diego, Calif.), as described in Section 6.1, below. Also listed in
Table 2, below, are the strains deposited with the NRRL which
contain each such clone. Such strains were produced by transforming
E. coli strain INV.alpha.F' (Invitrogen) with the indicated
plasmid, as described in Section 6.1, below. The names of the
plasmids containing the entire coding region of a novel gene bear
the prefix pFCHD, and the names of the strains carrying these
plasmids bear the prefix FCHD.
2 TABLE 2 Plasmid Clone Strain Deposited Contained within GENE with
NPRL Deposited Strain rchd005 RCHD005 pRCHD005 rchd024 RCHD024
pRCHD024 rchd032 RCHD032 pRCHD032 rchd036 RCHD036 pRCHD036 rchd502
RCHD502 pRCHD502 rchd523 RCHD523 pFCHD523 RCDH523 pRCHD523 rchd528
RCHD528 pRCHD528 rchd534 FCHD534 pFCHD534
[0149] As used 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 disclosed herein (as
shown in FIGS. 8, 12, 15, 18, 22, 28, 31, and 35), or contained in
the clones listed in Table 2, as deposited with the NRRL; (b) any
DNA sequence that encodes the amino acid sequence encoded by the
DNA sequences disclosed herein (as shown in FIGS. 8, 12, 15, 18,
22, 28, 31, and 35), contained in the clones, listed in Table 2, as
deposited with the NRRL or contained within the coding region of
the gene to which the DNA sequences disclosed herein (as shown in
FIGS. 8, 12, 15, 18, 22, 28, 31, and 35) or contained in the clones
listed in Table 2, as deposited with the NRRL, belong; (c) any DNA
sequence that hybridizes to the complement of the coding sequences
disclosed herein, contained in the clones listed in Table 2, as
deposited with the NRRL, or contained within the coding region of
the gene to which the DNA sequences disclosed herein (as shown in
FIGS. 8, 12, 15, 18, 22, 28, 31, and 35) or contained in the clones
listed in Table 2, as deposited with the NRRL, belong, under highly
stringent conditions, e.g., hybridization to filter-bound DNA in
0.5 M NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at
65.degree. C., and washing in 0.1.times. SSC/0.1% SDS at 68.degree.
C. (Ausubel F. M. et al., eds., 1989, Current Protocols in
Molecular Biology, Vol. I, Green Publishing Associates, Inc., and
John Wiley & sons, Inc., New York, at p. 2.10.3) and encodes a
gene product functionally equivalent to a gene product encoded by
sequences contained within the clones listed in Table 2; and/or (d)
any DNA sequence that hybridizes to the complement of the coding
sequences disclosed herein, (as shown in FIGS. 8, 12, 15, 18, 22,
28, 31, and 35) contained in the clones listed in Table 2, as
deposited with the NRRL or contained within the coding region of
the gene to which DNA sequences disclosed herein (as shown in FIGS.
8, 12, 15, 18, 22, 28, 31, and 35) or contained in the clones,
listed in Table 2, as deposited with the NRRL, belong, under less
stringent conditions, such as moderately stringent conditions,
e.g., washing in 0.2.times. SSC/0.1% SDS at 42.degree. C. (Ausubel
et al., 1989, supra), yet which still encodes a functionally
equivalent gene product. The invention also includes nucleic acid
molecules, preferably DNA molecules, that hybridize to, and are
therefore the complements of, the DNA sequences (a) through (c), in
the preceding paragraph. Such hybridization conditions may be
highly stringent or less highly stringent, as described above. In
instances wherein the nucleic acid molecules are
deoxyoligonucleotides ("oligos"), highly stringent conditions may
refer, e.g., to washing in 6.times. SSC/0.05% sodium pyrophosphate
at 37.degree. C. (for 14-base oligos), 48.degree. C. (for 17-base
oligos), 55.degree. C. (for 20-base oligos), and 60.degree. C. (for
23-base oligos). These 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 sequences. Further, such sequences may
be used as part of ribozyme and/or triple helix sequences, also
useful for target gene regulation. Still further, such molecules
may be used as components of diagnostic methods whereby the
presence of a cardiovascular disease-causing allele, may be
detected.
[0150] The invention also encompasses (a) DNA vectors that contain
any of the foregoing coding sequences and/or their complements
(i.e., antisense); (b) DNA expression vectors that contain any of
the foregoing coding sequences 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 expression of the coding
sequences in the host cell. As used herein, regulatory elements
include but are not limited to inducible and non-inducible
promoters, enhancers, operators and other elements known to those
skilled in the art that drive and regulate expression. The
invention includes fragments of any of the DNA sequences disclosed
herein.
[0151] In addition to the gene sequences described above,
homologues of such sequences, as may, for example be present in
other species, may be identified and may be readily isolated,
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 also be identified via similar techniques. For
example, the isolated differentially expressed gene sequence may be
labeled and used to screen a cDNA library constructed from mRNA
obtained from the organism of interest. 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 screen a genomic library derived from the organism
of interest, again, using appropriately stringent conditions. Such
low stringency conditions will be well known to those 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 see, for example,
Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold
Springs Harbor Press, N.Y.; and Ausubel et al., 1989, Current
Protocols in Molecular Biology, Green Publishing Associates and
Wiley Interscience, N.Y.
[0152] Further, a previously unknown differentially expressed or
pathway gene-type sequence may be isolated by performing PCR using
two degenerate oligonucleotide primer pools designed on the basis
of amino acid sequences within the gene of interest. The template
for the reaction may be cDNA obtained by reverse transcription of
mRNA prepared from human or non-human cell lines or tissue known or
suspected to express a differentially expressed or pathway gene
allele.
[0153] The PCR product may be subcloned and sequenced to insure
that the amplified sequences represent the sequences of a
differentially expressed or pathway gene-like nucleic acid
sequence. The PCR fragment may then be used to isolate a full
length cDNA clone by a variety of methods. For example, the
amplified fragment may be labeled and used to screen a
bacteriophage cDNA library. Alternatively, the labeled fragment may
be used to screen a genomic library.
[0154] 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 source.
A reverse transcription reaction may be performed on the RNA using
an oligonucleotide primer specific for the most 5' end of the
amplified fragment for the priming of first strand synthesis. The
resulting RNA/DNA hybrid may then be "tailed" with guanines using a
standard terminal transferase reaction, the hybrid may be digested
with RNAase H, and second strand synthesis may then be primed With
a poly-C primer. Thus, cDNA sequences upstream of the amplified
fragment may easily be isolated. For a review of cloning strategies
which may be used, see e.g., Sambrook et al., 1989, supra.
[0155] In cases where the differentially expressed or pathway gene
identified is the normal, or wild type, gene, this gene may be used
to isolate mutant alleles of the gene. Such an isolation is
preferable in processes and disorders which are known or suspected
to have a genetic basis. Mutant alleles may be isolated from
individuals either known or suspected to have a genotype which
contributes to cardiovascular disease symptoms. Mutant alleles and
mutant allele products may then be utilized in the therapeutic and
diagnostic assay systems described below.
[0156] A cDNA of the mutant gene may be isolated, 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 synthesized by
hybridizing an oligo-dT oligonucleotide to mRNA isolated from
tissue known or suspected to be expressed in an individual
putatively carrying the mutant allele, and by extending the new
strand with reverse transcriptase. The second strand of the cDNA is
then synthesized using an oligonucleotide that hybridizes
specifically to the 5' end of the normal gene. Using these two
primers, the product is then amplified via PCR, cloned into a
suitable vector, and subjected to DNA sequence analysis 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(s) responsible for the loss or alteration of function of
the mutant gene product can be ascertained.
[0157] Alternatively, a genomic or cDNA library can be constructed
and screened using DNA or RNA, respectively, from a tissue known to
or suspected of expressing the gene of interest in an individual
suspected of or known to carry the mutant allele. The normal gene
or any suitable fragment thereof may then be labeled and used as a
probed td identify the corresponding mutant allele in the library.
The clone containing this gene may then be purified through methods
routinely practiced in the art, and subjected to sequence analysis
as described, above, in this Section.
[0158] Additionally, an expression library can be constructed
utilizing DNA isolated from or cDNA synthesized from a tissue known
to or suspected of expressing the gene of interest in an individual
suspected of or known to carry the mutant allele. In this manner,
gene products made by the putatively mutant tissue may be expressed
and screened using standard antibody screening techniques in
conjunction with antibodies raised against the normal gene product,
as 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 expressed gene product
with altered function (e.g., as a result of a missense mutation), a
polyclonal set of antibodies are likely to cross-react with the
mutant gene product. Library clones detected via their reaction
with such labeled antibodies can be purified and subjected to
sequence analysis as described in this Section, above.
[0159] 5.4.2. Differentially Expressed and Pathway Gene
Products
[0160] Differentially expressed and pathway gene products include
those proteins encoded by the differentially expressed and pathway
gene sequences described in Section 5.4.1, above. Specifically,
differentially expressed and pathway gene products may include
differentially expressed and pathway gene polypeptides encoded by
the differentially expressed and pathway gene sequences contained
in the clones listed in Table 2, above, as deposited with the NRRL,
or contained in the coding regions of the genes to which DNA
sequences disclosed herein (in FIGS. 8, 12, 15, 18, 22, 28, 31, and
35) or contained in the clones, listed in Table 2, as deposited
with the NRRL, belong, for example.
[0161] In addition, differentially expressed and pathway gene
products may include proteins that represent functionally
equivalent gene products. Such an equivalent differentially
expressed or pathway gene product may contain deletions, additions
or substitutions of amino acid residues within the amino acid
sequence encoded by the differentially expressed or pathway gene
sequences described, above, in Section 5.4.1, but which result in a
silent change, thus producing a functionally equivalent
differentially expressed on pathway gene product. Amino acid
substitutions may be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues involved.
[0162] For example, nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and methionine; polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine; positively charged (basic) amino acids include arginine,
lysine, and histidine; and negatively charged (acidic) amino acids
include aspartic acid and glutamic acid. "Functionally equivalent",
as utilized herein, refers to a protein capable of exhibiting a
substantially similar in vivo activity as the endogenous
differentially expressed or pathway gene products encoded by the
differentially expressed or pathway gene sequences described in
Section 5.4.1, above. Alternatively, when utilized as part of
assays such as those described, below, in Section 5.5,
"functionally equivalent" may refer to peptides capable of
interacting with other cellular or extracellular molecules in a
manner substantially similar to the way in which the corresponding
portion of the endogenous differentially expressed or pathway gene
product would.
[0163] The differentially expressed or pathway gene products may be
produced by recombinant DNA technology using techniques well known
in the art. Thus, methods for preparing the differentially
expressed or pathway gene polypeptides and peptides of the
invention by expressing nucleic acid encoding differentially
expressed or pathway gene sequences are described herein. Methods
which are well known to those skilled in the art can be used to
construct expression vectors containing differentially expressed or
pathway gene protein coding sequences and appropriate
transcriptional/translational control signals. These methods
include, for example, in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Sambrook et al., 1989, supra, and Ausubel et al., 1989, supra.
Alternatively, RNA capable of encoding differentially expressed or
pathway gene protein sequences may be chemically synthesized using,
for example, synthesizers. See, for example, the techniques
described in "Oligonucleotide Synthesis", 1984, Gait, M. J. ed.,
IRL Press, Oxford, which is incorporated by reference herein in its
entirety. A variety of host-expression vector systems may be
utilized to express the differentially expressed or pathway gene
coding sequences of the invention. Such host-expression systems
represent vehicles by which the coding sequences of interest may be
produced and subsequently purified, but also represent cells which
may, when transformed or transfected with the appropriate
nucleotide coding sequences, exhibit the differentially expressed
or pathway gene protein of the invention in situ. These include but
are not limited to microorganisms such as bacteria (e.g., E. coli,
B. subtilis) transformed with recombinant bacteriophage DNA,
plasmid DNA or cosmid DNA expression vectors containing
differentially expressed or pathway gene protein coding sequences;
yeast (e.g. Saccharomyces, Pichia) transformed with recombinant
yeast expression vectors containing the differentially expressed or
pathway gene protein coding sequences; insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus)
containing the differentially expressed or pathway gene protein
coding sequences; plant cell systems infected with recombinant
virus expredion vectors (e.g., cauliflower mosaic virus, 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 systems (e.g. COS, CHO, BHK, 293, 3T3) harboring recombinant
expression constructs containing promoters derived from the genome
of mammalian cells (e.g., metallothionein promoter) or from
mammalian viruses (e.g., the adenovirus late promoter; the vaccinia
virus 7.5K promoter).
[0164] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
differentially expressed or pathway gene protein being expressed.
For example, when a large quantity of such a protein is to be
produced, for the generation of antibodies or to screen peptide
libraries, for example, vectors which direct the expression of high
levels of fusion protein products that are readily purified may be
desirable. Such vectors include, but are not limited, to the E.
coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
2:1791), in which the differentially expressed or pathway gene
protein coding sequence may be ligated individually into the vector
in frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids
Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
264:5503-5509); and the like. pGEX vectors may also be used to
express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption 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 sites so that the cloned target gene
protein can be released from the GST moiety.
[0165] In an insect system, Autographa califormica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The
differentially expressed or pathway gene coding sequence may be
cloned individually into non-essential regions (for example the
polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter). Successful
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). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed. (E.g., see Smith et
al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).
[0166] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the differentially expressed 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 sequence. This chimeric gene may then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing 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
expressed or pathway gene, including its own initiation codon and
adjacent sequences, 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, exogenous
translational control signals, including, perhaps, the ATG
initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., 1987, Methods in Enzymol.
153:516-544).
[0167] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins. Appropriate cell lines or host systems can be chosen
to 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 cells include but are not limited
to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.
[0168] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the differentially expressed or pathway gene
protein may be engineered. Rather than using expression vectors
which contain viral origins of replication, host cells can be
transformed with DNA controlled by appropriate expression control
elements (e.g., promoter, enhancer, sequences, transcription
terminators, polyadenylation sites, etc.), and a selectable marker.
Following the introduction of the foreign DNA, engineered cells may
be allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the differentially expressed 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 expressed or pathway gene
protein.
[0169] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance 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 confers resistance to mycophenolic acid (Mulligan & Berg,
1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers
resistance to the aminoglycoside G-418 (Colberre-Garapin, et al.,
1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to
hygromycin (Santerre, et al., 1984, Gene 30:147) genes.
[0170] An alternative fusion protein system allows for the ready
purification of non-denatured fusion proteins expressed in human
cell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:
8972-8976). In this system, the gene of interest is subcloned into
a vaccinia recombination plasmid such that the gene's open reading
frame is translationally fused to an amino-terminal tag consisting
of six histidine residues. Extracts from cells infected with
recombinant vaccinia virus are loaded onto Ni.sup.2+ nitriloacetic
acid-agarose columns and histidine-tagged proteins are selectively
eluted with imidazole-containing buffers.
[0171] When used as a component in assay systems such as those
described, below, in Section 5.5, the differentially expressed or
pathway gene protein may be labeled, either directly or indirectly,
to facilitate detection of a complex formed between the
differentially expressed or pathway gene protein and a test
substance. Any of a variety of suitable labeling systems may be
used including but not limited to radioisotopes such as .sup.125I;
enzyme labelling systems that generate a detectable calorimetric
signal or light when exposed to substrate; and fluorescent
labels.
[0172] Where recombinant DNA technology is used to produce the
differentially expressed or pathway gene protein for such assay
systems, it may be advantageous to engineer fusion proteins that
can facilitate labeling, immobilization and/or detection.
[0173] Indirect labeling involves the use of a protein, such as a
labeled antibody, which specifically binds to either a
differentially expressed or pathway gene product. Such antibodies
include but are not limited to polyclonal, monoclonal, chimeric,
single chain, Fab fragments and fragments-produced by an Fab
expression library.
[0174] 5.4.3. Differentially Expressed or Pathway Gene Product
Antibodies
[0175] Described herein are methods for the production of
antibodies capable of specifically 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').sub.2 fragments,
fragments produced by a Fab expression library, anti-idiotypic
(anti-Id) antibodies, 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. Thus, such antibodies may be utilized as part
of cardiovascular disease treatment methods, and/or may be used as
part of diagnostic techniques whereby patients may be tested for
abnormal levels of fingerprint, target, or pathway gene proteins,
or for the presence of abnormal forms of the such proteins.
[0176] For the production of antibodies to a differentially
expressed or pathway gene, various host animals may be immunized by
injection with a differentially expressed or pathway gene protein,
or a portion thereof. Such host animals may include but are not
limited to rabbits, mice, and rats, to name but a few. Various
adjuvants may be used to increase the immunological response,
depending on the host species, including but not limited to
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanin, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and Corynebacterium
parvum.
[0177] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen, such as target gene product, or an antigenic functional
derivative thereof. For the production of polyclonal antibodies,
host animals such as those described above, may be immunized by
injection with differentially expressed or pathway gene product
supplemented with adjuvants as also described above.
[0178] 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. Pat. 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
Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing the
mAb of this invention may be cultivated in vitro or in vivo.
Production of high titers of mabs in vivo makes this the presently
preferred method of production.
[0179] In addition, techniques developed for the production of
"chimeric antibodies" (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 splicing the genes
from a mouse antibody molecule of appropriate antigen specificity
together with genes from a human antibody molecule of appropriate
biological activity can be used. A chimeric antibody is a molecule
in which different portions are derived from different animal
species, such as those having a variable region derived from a
murine mAb and a human immunoglobulin constant region.
[0180] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778;Bird, 1988,
Science 242:423-426; Huston et al., 1988, Proc.
[0181] Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989,
Nature 334:544-546) can be adapted to produce differentially
expressed or pathway gene-single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide.
[0182] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, such fragments include
but are not limited to: the F(ab').sub.2 fragments which can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries may be constructed (Huse et al., 1989, Science,
246:1275-1281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity.
[0183] 5.4.4. Cell- and Animal-Based Model Systems
[0184] Described herein are cell- and animal-based systems which
act as models for cardiovascular disease. These systems may be used
in a variety of applications. For example, the cell- and
animal-based model systems may be used to further characterize
differentially expressed and pathway genes, as described, above, in
Section 5.3. Such further characterization may, for example,
indicate that a differentially expressed gene is a target gene.
Second, such assays may be utilized as part of screening strategies
designed to identify compounds which are capable of ameliorating
cardiovascular disease symptoms, as described, below, in Section
5.5.4. Thus, the animal- and cell-based models may be used to
identify drugs, pharmaceuticals, 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 models
may be used to determine the LD.sub.50, and the ED.sub.50 in animal
subjects, and such data can be used to determine the in vivo
efficacy of potential cardiovascular disease treatments.
[0185] 5.4.4.1. Animal-Based Systems
[0186] Animal-based model systems of cardiovascular disease may
include, but are not limited to, non-recombinant and engineered
transgenic animals.
[0187] Non-recombinant animal models for cardiovascular disease may
include, for example, genetic models. Such genetic cardiovascular
disease models may include, for example, apoB or apoR deficient
pigs (Rapacz, et al., 1986, Science 234:1573-1577) and Watanabe
heritable hyperlipidemic (WHHL) rabbits (Kita et al., 1987, Proc.
Natl. Acad. Sci USA 84: 5928-5931).
[0188] Non-recombinant, non-genetic animal models of
atherosclerosis may include, for example, pig, rabbit, or rat
models in which the animal has been exposed to either chemical
wounding through dietary supplementation of LDL, or mechanical
wounding through balloon catheter angioplasty, for example.
[0189] Additionally, animal models exhibiting cardiovascular
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 sequences may be introduced into, and
overexpressed in, the genome of the animal of interest, or, if
endogenous target gene sequences are present, they may either be
overexpressed or, alternatively, be disrupted in order to
underexpress or inactivate target gene expression, such as
described for the disruption of apoE in mice (Plump et al., 1992,
Cell 71: 343-353).
[0190] 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 expression in the animal
and cell type of interest. Such regulatory regions will be well
known to those of skill in the art, and may be utilized in the
absence of undue experimentation.
[0191] The use of such a genetically engineered animal-based system
is described in detail in the example provided in Section 7, below,
for the target gene bcl-2 (see Table 1, in Section 5.4.1, above).
Briefly, bcl-2 expression first was shown to be down-regulated in
the apoE-deficient atherosclerosis mouse model. Then, a transgenic
mouse was engineered bearing the human bcl-2 gene under the control
of a promoter which is induced under atherogenic conditions. To
test the effect of the induction of bcl-2 under such conditions,
the transgenic mouse is crossed with the apoE-deficient mouse.
apoE-deficient progeny bearing the highly expressible bcl-2 gene
are then examined for plaque formation and development. Reduction
in plaque formation and development in these progeny confirms the
effectiveness of intervening in cardiovascular disease through this
target gene.
[0192] For underexpression of an endogenous target gene sequence,
such a sequence may be isolated and engineered such that when
reintroduced into the genome of the animal 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
animal's genome. Gene targeting is discussed, below, in this
Section.
[0193] Animals of any species, including, but not limited to, mice,
rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human
primates, e.g., baboons, monkeys, and chimpanzees may be used to
generate cardiovascular disease animal models.
[0194] Any technique known in the art may be used to introduce a
target gene transgene into animals 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 transfer into
germ lines (Van der Putten et al., 1985, Proc. Natl. Acad. Sci.,
USA 82:6148-6152); gene targeting in embryonic stem 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 is incorporated by reference
herein in its entirety.
[0195] The present invention provides for transgenic animals that
carry the transgene in all their cells, as well as animals which
carry the transgene in some, but not all their cells, i.e., mosaic
animals. The transgene may be integrated as a single transgene or
in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
The transgene may also be selectively 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 endogenous target gene,
gene targeting is preferred. Briefly, when such a technique is to
be utilized, vectors containing some nucleotide sequences
homologous to the endogenous target gene of interest are designed
for the purpose of integrating, via homologous recombination with
chromosomal sequences, 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 sequences required
for such a cell-type specific inactivation will depend upon the
particular cell type of interest, and will be apparent to those of
skill in the art.
[0196] Once transgenic animals have been generated, the expression
of the recombinant target gene and protein may be assayed utilizing
standard techniques. Initial screening may be accomplished by
Southern blot analysis or PCR techniques to analyze animal tissues
to assay whether integration of the transgene has taken place. The
level of mRNA expression of the transgene in the tissues of the
transgenic animals may also be assessed using techniques which
include but are not limited to Northern blot analysis of tissue
samples obtained from the animal, in situ hybridization analysis,
and RT-PCR. Samples of target gene-expressing tissue, may also be
evaluated immunocytochemically using antibodies specific for the
target gene transgene gene product of interest.
[0197] The target gene transgenic animals that express target gene
mRNA or target gene transgene peptide (detected
immunocytochemically, using antibodies directed against the target
gene product's epitopes) at easily detectable levels should then be
further evaluated to identify those animals which display
characteristic cardiovascular disease symptoms. Such symptoms may
include, for example, increased prevalence and size of fatty
streaks and/or cardiovascular disease plaques.
[0198] Additionally, specific cell types within the transgenic
animals may be analyzed and assayed for cellular phenotypes
characteristic of cardiovascular disease. In the case of monocytes,
such phenotypes may include but are not limited to increases in
rates of LDL uptake, adhesion to endothelial cells, transmigration,
foam cell formation, fatty streak formation, and production of foam
cell specific products. Cellular phenotype assays are discussed in
detail in Section 5.4.4.2, below. Further, such cellular phenotypes
may include a particular cell type's fingerprint pattern of
expression as compared to known fingerprint expression profiles of
the particular cell type in animals exhibiting cardiovascular
disease symptoms. Fingerprint profiles are described in detail in
Section 5.8.1, below. Such transgenic animals serve as suitable
model systems for cardiovascular disease.
[0199] Once target gene transgenic founder animals are produced,
they may be bred, inbred, outbred, or crossbred to produce colonies
of the particular animal. Examples of such breeding strategies
include but are not limited to: outbreeding of founder animals with
more than one integration site 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
expression of each target gene transgene; crossing of heterozygous
transgenic animals to produce animals homozygous for a given
integration site in order both to augment expression and eliminate
the possible need for screening of animals by DNA analysis;
crossing of separate homozygous lines to produce compound
heterozygous or homozygous lines; breeding animals to different
inbred genetic backgrounds so as to examine effects of modifying
alleles on expression of the target gene transgene and the
development of cardiovascular disease symptoms. one such approach
is to cross the target gene transgenic founder animals with a wild
type strain to produce an Fl generation that exhibits
cardiovascular disease symptoms. The Fl generation may then be
inbred in order to develop a homozygous line, if it is found that
homozygous target gene transgenic animals are viable.
[0200] 5.4.4.2. Cell-Based Assays
[0201] Cells that contain and express target gene sequences which
encode target gene protein, and, further, exhibit cellular
phenotypes associated with cardiovascular disease, may be utilized
to identify compounds that exhibit anti-cardiovascular disease
activity. In the case of monocytes, such phenotypes may include but
are not limited to increases in 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.beta., TGF.alpha.,
TNFA, HB-EGF, PDGF, IFN-.gamma., and GM-CSF. Transmigration 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 across the endothelial monolayer and into
the collagen layer of the subendothelial space.
[0202] Such cells may include non-recombinant cell lines, such as
U937 (ATCC# CRL1593) and THP-1 (TIB202). Further, such cells may
include recombinant, transgenic cell lines. For example, the
cardiovascular disease animal models of the invention, discussed,
above, in Section 5.4.4.1, may be used to generate cell lines,
containing one or more cell types involved in cardiovascular
disease, that can be used as cell culture models for this disorder.
While primary cultures derived from the cardiovascular disease
transgenic animals of the invention may be utilized, the generation
of continuous cell lines is preferred. For examples of techniques
which may be used to derive a continuous cell line from the
transgenic animals, see Small et al., 1985, Mol. Cell Biol.
5:642-648.
[0203] Alternatively, cells of a cell type known to be involved in
cardiovascular disease may be transfected with sequences capable of
increasing or decreasing the amount of target gene expression
within the cell. For example, target gene sequences may be
introduced into, and overexpressed in, the genome of the cell of
interest, or, if endogenous target gene sequences are present, they
may be either overexpressed or, alternatively disrupted in order to
underexpress or inactivate target gene expression.
[0204] 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 expression in the cell
type of interest. Such regulatory regions will be well known to
those of skill in the art, and may be utilized in the absence of
undue experimentation.
[0205] For underexpression of an endogenous target gene sequence,
such 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. Target gene introduction is discussed, above, in Section
5.4.4.1.
[0206] Transfection of target gene sequence nucleic acid may be
accomplished by utilizing standard techniques See, for example,
Ausubel, 1989, supra. Transfected cells should be evaluated for the
presence of the recombinant target gene sequences, for expression
and accumulation of target gene mRNA, and for the presence of
recombinant target gene protein production. In instances wherein a
decrease in target gene expression is desired, standard techniques
may be used to demonstrate whether a decrease in endogenous target
gene expression and/or in target gene product production is
achieved.
[0207] 5.5. Screening Assays for Compounds that Interact with the
Target Gene Product
[0208] The following assays are designed to identify compounds that
bind to target gene products, 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. For example, in the case
of the rchd523 gene product, which is a transmembrane receptor-type
protein, such techniques can identify ligands for such a receptor.
An rchd523 gene product ligand can, for example, act as the basis
for amelioration of such cardiovascular diseases as
atherosclerosis, ischemia/reperfusion, hypertension, restenosis,
and arterial inflammation, given that rchd523 up-regulation is
specific to endothelial cells. Such compounds may include, but are
not limited to peptides, antibodies, or small 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.
[0209] Compounds identified via assays such as those described
herein may be useful, for example, in elaborating the biological
function of the target gene product, and for ameliorating
cardiovascular disease. In instances whereby a cardiovascular
disease condition results from an overall lower level of target
gene expression and/or target gene product in a cell or tissue,
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 symptoms.
[0210] In other instances mutations within the target gene may
cause aberrant types or excessive amounts of target gene proteins
to be made which have a deleterious effect that leads to
cardiovascular disease. Similarly, physiological conditions may
cause an excessive increase in target gene expression leading to
cardiovascular disease. In such cases, compounds that bind target
gene protein may be identified that inhibit the activity of the
bound target gene protein. Assays for testing the effectiveness of
compounds, identified by, for example, techniques such as those
described in this Section are discussed, below, in Section
5.5.4.
[0211] 5.5.1. in vitro Screening Assays for Compounds that Bind to
the Target Gene Product
[0212] In vitro systems may be designed to identify compounds
capable of binding the target gene of the invention. Such compounds
may include, but are not limited to, peptides made of D-and/or
L-configuration amino acids (in, for example, the form of random
peptide libraries; see e.g., Lam, K. S. et al., 1991, Nature
354:82-84), phosphopeptides (in, for example, the form of random or
partially degenerate, directed phosphopeptide libraries; see, e.g.,
Songyang, Z. et al., 1993, Cell 72:767-778), antibodies, and small
organic or inorganic molecules. Compounds identified may be useful,
for example, in modulating the activity of target gene proteins,
preferably mutant target gene proteins, 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 themselves disrupt such
interactions.
[0213] The principle of the assays used to identify compounds that
bind to the target gene protein involves preparing a reaction
mixture of the target gene protein and the test compound under
conditions and for a time sufficient to allow the two components to
interact and bind, thus forming a complex which can be removed
and/or detected in the reaction mixture. These assays can be
conducted in a variety of ways. For example, one method to conduct
such an assay would involve anchoring the target gene or the test
substance onto a solid phase and detecting target gene/test
substance 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 solid surface, and the test
compound, which is not anchored, may be labeled, either directly or
indirectly.
[0214] In practice, microtitre plates are conveniently utilized.
The anchored component 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, preferably a
monoclonal antibody, specific for the protein may be used to anchor
the protein to the solid surface. The surfaces may be prepared in
advance and stored.
[0215] In order to conduct the assay, the nonimmobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously nonimmobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
nonimmobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the previously nonimmobilized
component (the antibody, in turn, may be directly labeled or
indirectly labeled with a labeled anti-Ig antibody).
[0216] Alternatively, a reaction can be conducted in a liquid
phase, the reaction products separated from unreacted components,
and complexes detected; e.g., using an immobilized antibody
specific for target gene product or the test compound to anchor any
complexes formed in solution, and a labeled antibody specific for
the other component of the possible complex to detect anchored
complexes.
[0217] Compounds that are shown to bind to a particular target gene
product through one of the methods described above can be further
tested for their ability to elicit a biochemical response from the
target gene protein. A particular embodiment is described herein
for receptor proteins involved in signal transduction, including
but not limited to the rchd523 gene product. Compounds that
interact with a target gene product receptor domain, can be
screened for their ability to function as ligands, i.e., to bind to
the receptor protein in a manner that triggers the signal
transduction pathway. Useful receptor fragments or analogs in the
invention are those which interact with ligand. The receptor
component can be assayed functionally, i.e., for its ability to
bind ligand and mobilize Ca.sup.++ (see below). These assays
include, as components, ligand and a recombinant target gene
product (or a suitable fragment or analog) configured to permit
detection of binding.
[0218] 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. Cells, preferably
myeloma cells or Xenopus oocytes, transfected with a target gene
expression vector (constructed according to the methods described
in Section 5.4.2, above) are loaded with FURA-2 or INDO-1 by
standard techniques. Mobilization of Ca.sup.2+ induced by ligand is
measured by fluorescence spectroscopy as previously described
(Grynkiewicz et al., 1985, J. Biol. Chem. 260:3440). Ligands that
react with the target gene product receptor domain, therefore, can
be identified by their ability to produce a fluorescent signal.
Their receptor binding activities can be quantified and compared by
measuring the level of fluorescence produced over background.
[0219] The rchd523 gene product consists of a G protein-coupled
receptor with multiple transmembrane domains. The
Ca.sup.2+mobilization assay, therefore, can be used to screen
compounds that are ligands of the rchd523 receptor. This screening
method is described in detail with respect to rchd523 in the
example in Section 12, below. Identification of rchd523 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 antagonists are useful in the treatment
of cardiovascular disease.
[0220] 5.5.2. Assays for Cellular or Extracellular Proteins that
Interact with the Target Gene Product
[0221] 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., supra, 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.
[0222] 5.5.3. Assays for Compounds that Interfere with Interaction
Between Target Gene Product and Other Compounds
[0223] The target gene proteins of the invention may, in vivo,
interact with one or more cellular or extracellular proteins. Such
proteins may include, but are not limited to, those proteins
identified via methods such as those described, above, in Section
5.5.2. For the purposes of this discussion, target gene products
and such cellular and extracellular proteins are referred to herein
as "binding partners". Compounds that disrupt such interactions may
be useful 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 described in Section 5.5.1. above.
[0224] The basic principle of the assay 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 involves preparing a reaction mixture
containing the target gene protein and the binding partner under
conditions and for a time sufficient to allow the two proteins to
interact and bind, thus forming a complex. In order to test a
compound for inhibitory activity, the reaction mixture is prepared
in the presence and absence of the test compound. The test compound
may be initially included in the reaction mixture or may be added
at a time subsequent to the addition of target gene and its
cellular or extracellular binding partner. Control reaction
mixtures are incubated without the test 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 also be compared to complex
formation within reaction mixtures containing the test compound and
mutant target gene protein. This comparison may be important in
those cases wherein it is desirable to identify compounds that
disrupt interactions of mutant but not normal target gene
proteins.
[0225] The assay for compounds that interfere with the interaction
of the binding partners can be conducted in a heterogeneous or
homogeneous format. Heterogeneous assays involve anchoring one of
the binding partners onto a solid phase and detecting complexes
anchored on the solid phase at the end of the reaction. In
homogeneous assays, the entire reaction is carried out in a liquid
phase. In either approach, the order of addition of reactants can
be varied to obtain different information about the compounds being
tested. For example, test compounds that interfere with the
interaction between the binding partners, e.g., by competition, can
be identified by conducting the reaction in the presence of the
test substance; i.e., by adding the test substance to the reaction
mixture prior to or simultaneously with the target gene protein and
interactive cellular or extracellular protein. Alternatively, test
compounds that disrupt preformed complexes, e.g. compounds with
higher binding constants 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 described briefly below.
[0226] In a heterogeneous assay system, either the target gene
protein or the interactive cellular or extracellular binding
partner protein, is anchored onto a solid 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
solid surface. The surfaces may be prepared in advance and
stored.
[0227] In order to conduct the assay, the binding partner of the
immobilized species is exposed to the coated surface 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 solid 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 is
not pre-labeled, an indirect label can be used to detect complexes
anchored on the surface; e.g., using a labeled antibody specific
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 components, test
compounds which inhibit complex formation or which disrupt
preformed complexes can be detected.
[0228] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific for one
binding partner to anchor any complexes 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 compounds which inhibit complex
or which disrupt preformed complexes can be identified.
[0229] In an alternate embodiment of the invention, a homogeneous
assay can be used. In this 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 is
labeled, but the signal generated by the label is quenched due to
complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein
which utilizes this approach for immunoassays). The addition of a
test substance that competes with and displaces one of the binding
partners from the preformed complex will result in the generation
of a signal above background. In this way, test substances which
disrupt target gene protein-cellular or extracellular protein
interaction can be identified.
[0230] In a particular embodiment, the target gene protein can be
prepared for immobilization using recombinant DNA techniques
described in Section 5.4.2, supra. For example, the target gene
coding region can be fused to a glutathione-S-transferase (GST)
gene, using a fusion vector such as PGEX-5X-1, in such a manner
that its binding activity is maintained in the resulting fusion
protein. The interactive cellular or extracellular protein can be
purified and used to raise a monoclonal antibody, using methods
routinely practiced in the art and described above, in Section
5.4.3. This antibody can be labeled with the radioactive isotope
.sup.125I, for example, by methods routinely practiced in the art.
In a heterogeneous assay, e.g., the GST-target gene fusion 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 system 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 successful inhibition of the interaction by the test
compound will result in a decrease in measured radioactivity.
[0231] 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 beads. The test compound can be added either
during or after the binding partners are allowed to interact. This
mixture can then be added to the glutathione-agarose beads and
unbound material is washed away. Again the extent of inhibition of
the binding partner interaction can be detected by adding the
labeled antibody and measuring the radioactivity associated with
the beads.
[0232] In another embodiment of the invention, these 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 proteins. Any number of
methods routinely practiced in the art can be used to identify and
isolate the protein's binding site. These methods include, but are
not limited to, mutagenesis of one of the genes encoding the
proteins and screening for disruption of binding in a
co-immunoprecipitation assay. Compensating mutations in the target
gene can be selected. Sequence analysis of the genes encoding the
respective proteins will reveal the mutations that correspond to
the region of the protein involved in interactive binding.
Alternatively, one protein can be anchored to a solid surface using
methods described in this Section above, and allowed to interact
with and bind to its labeled binding partner, which has been
treated with a proteolytic enzyme, such as trypsin. After washing,
a short, labeled peptide comprising the binding domain may remain
associated with the solid material, which can be isolated and
identified by amino acid sequencing. Also, once the gene coding for
the for the cellular or extracellular protein is obtained, short
gene segments can be engineered to express peptide fragments of the
protein, which can then be tested for binding activity and purified
or synthesized.
[0233] For example, and not by way of limitation, target gene can
be anchored to a solid material as described above in this 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 isotope, such as .sup.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 washing 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., using 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, supra at pp. 50-60) or, if the gene has already
been isolated, by using recombinant DNA technology, as described in
Section 5.4.2, supra.
[0234] A particular embodiment of the invention features a method
of screening candidate compounds for their ability to antagonize
the interaction between ligand and the receptor domain of a target
gene product, including but not limited to the receptor domain of
the rchd523 gene product. The rchd523 gene product, which is a G
protein-coupled receptor protein containing multiple transmembrane
domains, is especially useful in screening for antagonists of
ligand-receptor interactions. The method involves: a) mixing a
candidate antagonist compound with a first compound which includes
a recombinant target gene product comprising 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 first and second compounds bind; and c)
identifying antagonistic compounds as those which interfere with
the binding of the first compound to the second compound and/or
which reduce the ligand-mediated release of intracellular
Ca.sup.++.
[0235] By an "antagonist" is meant a molecule which inhibits 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.sup.++). Preferred therapeutics
include antagonists, e.g., peptide fragments (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.
[0236] Because the receptor component of the target gene product
can be produced by recombinant techniques and because candidate
antagonists may be screened in vitro, the instant invention
provides a simple and rapid approach to the identification of
useful therapeutics.
[0237] Specific receptor fragments of interest include any portions
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 antagonists (as 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 regions may be identified by
comparison with related proteins of similar structure (e.g., other
members of the G-protein-coupled receptor superfamily); useful
regions are those exhibiting homology to the extracellular domains
of well-characterized members of the family.
[0238] Alternatively, from the primary amino acid sequence, the
secondary protein structure and, therefore, the extracellular
domain regions may be deduced semi-empirically using a
hydrophobicity/hydrophilicity calculation such as the Chou-Fasman
method (see, e.g., Chou and Fasman, Ann. Rev. Biochem. 47:251,
1978). Hydrophilic domains, particularly ones surrounded by
hydrophobic stretches (e.g., transmembrane domains) present
themselves as strong candidates for extracellular domains. Finally,
extracellular domains may be identified experimentally using
standard enzymatic digest analysis, e.g., tryptic digest
analysis.
[0239] Candidate fragments (e.g., all or part of the transmembrane
segments or any extracellular fragment) are tested for interaction
with ligand by the assays described herein (e.g., the assay
described above). Such fragments are also tested for their ability
to antagonize the interaction between ligand and its endogenous
receptor using the assays described herein. Analogs of useful
receptor fragments (as described above) may also be produced and
tested for efficacy as screening components or antagonists (using
the assays described herein); such analogs are also considered to
be useful in the invention.
[0240] Of particular interest are receptor fragments 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 as antagonists, particularly
if the loops cooperate with the amino-terminal domain to facilitate
ligand binding. Alternatively, such loops and extracellular
N-terminal domain (as well as the full length target gene product)
provide immunogens for producing anti-target gene product
antibodies.
[0241] Binding of ligand to its receptor may be assayed by any of
the methods described above in Section 5.5.1. Preferably, cells
expressing recombinant target gene product (or a suitable target
gene product fragment or analog) are immobilized on a solid
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 association with the
receptor component (and, therefore, in association with the solid
substrate). Binding of labelled ligand to receptor-bearing cells is
used as a "control" against which antagonist assays are measured.
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 antagonist useful in the invention specifically
interferes with labelled ligand binding to the immobilized
receptor-expressing cells.
[0242] 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 transduction normally
mediated by the receptor. To test this using a functional assay,
stably transfected cell lines containing the target gene product
can be produced as described herein and reporter compounds such as
the calcium binding agent, FURA-2, loaded into the cytoplasm by
standard techniques. Stimulation of the heterologous target gene
product with ligand or another agonist leads to intracellular
calcium release and the concomitant fluorescence of the
calcium-FURA-2 complex. This provides a convenient means for
measuring agonist activity. Inclusion of potential antagonists
along with ligand allows for the screening and identification of
authentic receptor antagonists as those which effectively block
ligand binding without producing fluorescence (i.e., without
causing the mobilization of intracellular Ca.sup.++). Such an
antagonist may be expected to be a useful therapeutic agent for
cardiovascular disorders.
[0243] Appropriate candidate antagonists include target gene
product fragments, particularly fragments containing a
ligand-binding portion adjacent to or including one or more
transmembrane segments or an extracellular domain of the receptor
(described above); such fragments would preferably including five
or more amino acids. Other candidate antagonists include analogs of
ligand and other peptides as well as non-peptide compounds and
anti-target gene product antibodies designed or derived from
analysis of the receptor.
[0244] This screening method is described in detail with respect to
the rchd523 gene in the example in Section 12, below. Because the
rchd523 gene product is a G protein-coupled receptor, antagonists
of the interaction between the rchd523 gene product and its natural
ligand provide excellent candidates for compounds effective in the
treatment of cardiovascular disease.
[0245] 5.5.4. Assays for Amelioration of Cardiovascular Disease
Symptoms
[0246] Any of the binding compounds, including but not limited to
compounds such as those identified in the foregoing assay systems,
may be tested for the ability to ameliorate cardiovascular disease
symptoms. Cell-based and animal model-based assays for the
identification of compounds exhibiting such an ability to
ameliorate cardiovascular disease symptoms are described below.
[0247] First, cell-based systems such as those described, above, in
Section 5.4.4.2., may be used to identify compounds which may act
to ameliorate cardiovascular disease symptoms. For example, such
cell systems may be exposed to a compound, suspected of exhibiting
an ability to ameliorate cardiovascular disease symptoms, at a
sufficient concentration and for a time sufficient to elicit such
an amelioration of cardiovascular disease symptoms in the exposed
cells. After exposure, the cells are examined to determine whether
one or more of the cardiovascular disease cellular phenotypes has
been altered to resemble a more normal or more wild type,
non-cardiovascular disease 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 decreased 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.beta., TGF.alpha., TNF.alpha., HB-EGF, PDGF, IFN-.gamma., and
GM-CSF. Transmigration 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 across
the endothelial monolayer and into the collagen layer of the
subendothelial space.
[0248] In addition, animal-based cardiovascular disease systems,
such as those described, above, in Section 5.4.4.1, may be used to
identify compounds capable of ameliorating cardiovascular disease
symptoms. Such animal models may be used as test substrates for the
identification of drugs, pharmaceuticals, therapies, and
interventions which may be effective in treating cardiovascular
disease. For example, animal models may be exposed to a compound,
suspected of exhibiting an ability to ameliorate cardiovascular
disease symptoms, at a sufficient concentration and for a time
sufficient to elicit such an amelioration of cardiovascular disease
symptoms in the exposed animals. The response of the animals to the
exposure may be monitored by assessing the reversal of disorders
associated with cardiovascular disease, for example, by counting
the number of atherosclerotic plaques and/or measuring their size
before and after treatment.
[0249] With regard to intervention, any treatments which reverse
any aspect of cardiovascular disease symptoms should be considered
as candidates for human cardiovascular disease therapeutic
intervention. Dosages of test agents may be determined by deriving
dose-response curves, as discussed in Section 5.7.1, below.
[0250] Additionally, gene expression patterns may be utilized to
assess the ability of a compound to ameliorate cardiovascular
disease symptoms. For example, the expression pattern of one or
more fingerprint genes may form part of a "fingerprint profile"
which may be then be used in such an assessment. "Fingerprint
profile", as used herein, refers to the pattern of mRNA expression
obtained for a given tissue or cell type under a given set of
conditions. Such conditions may include, but are not limited to,
atherosclerosis, 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 discussed, above, in
Section 5.1.2, Northern analysis and/or RT-PCR. Any of the gene
sequences described, above, in Section 5.4.1. may be used as probes
and/or PCR primers for the generation and corroboration of such
fingerprint profiles.
[0251] Fingerprint profiles may be characterized for known states,
either cardiovascular disease or normal, within the cell- and/or
animal-based model systems. Subsequently, these known fingerprint
profiles may be compared to ascertain the effect a test compound
has to modify such fingerprint profiles, and to cause the profile
to more closely resemble that of a more desirable fingerprint.
[0252] For example, administration of a compound may cause the
fingerprint profile of a cardiovascular disease model system to
more closely resemble the control system. Administration of a
compound may, alternatively, cause the fingerprint profile of a
control system to begin to mimic a cardiovascular disease state.
Such a compound may, for example, be used in further characterizing
the compound of interest, or may be used in the generation of
additional animal models.
[0253] 5.5.5. Monitoring of Effects During Clinical Trials
[0254] Monitoring the influence of compounds on cardiovascular
disease states may be applied not only in basic drug screening, but
also in clinical trials. In such clinical trials, the expression of
a panel of genes that have been discovered in one of the paradigms
described in Section 5.1.1.1 through 5.1.1.6 may be used as a "read
out" of a particular drug's effect on a cardiovascular disease
state.
[0255] For example, and not by way of limitation, Paradigm A
provides for the identification of fingerprint genes that are
up-regulated in monocytes treated with oxidized LDL. Thus, to study
the effect of anti-oxidant drugs, for example, in a clinical trial,
blood may be drawn from patients before and at different stages
during treatment with such a drug. Their monocytes may then be
isolated and RNA prepared and analyzed by differential display as
described in Sections 6.1.1 and 6.1.2. The levels of expression 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 methods described in
Section 5.8.2. In this way, the fingerprint profiles may serve as
surrogate markers indicative of the physiological response 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 described in further detail
in the example in Section 10, below.
[0256] This method may also be applied to the other paradigms
disclosed herein. For example, and not by way of limitation, the
fingerprint profile of Paradigm B reveals that bcl-2 and
glutathione peroxidase are both down-regulated in the monocytes of
patients exposed to a high lipid diet, e.g. cholesterol or fat,
that leads to high serum LDL levels. Drugs may be tested, for
example, for their ability to ameliorate the effects of
hypercholesterolemia in clinical trials. Patients with high LDL
levels may have their monocytes isolated before, and at different
stages after, drug treatment. The drug's efficacy may be measured
by determining the degree of restored expression of bcl-2 and
glutathione peroxidase, as described above for the Paradigm A
fingerprint profile.
[0257] 5.6. Compounds and Methods for Treatment of Cardiovascular
Disease
[0258] Described below are methods and compositions whereby
cardiovascular disease symptoms may be ameliorated. Certain
cardiovascular diseases are brought about, at least in part, by an
excessive level of gene product, or by the presence of a gene
product exhibiting an abnormal or excessive activity. As such, the
reduction in the level and/or activity of such gene products would
bring about the amelioration of cardiovascular disease symptoms.
Techniques for the reduction of target gene expression levels or
target gene product activity levels are discussed in Section 5.6.1,
below.
[0259] Alternatively, certain other cardiovascular diseases are
brought about, at least in part, by the absence or reduction of the
level of gene expression, or a reduction in the level of a gene
product's activity. As such, an increase in the level of gene
expression and/or the activity of such gene products would bring
about the amelioration of cardiovascular disease symptoms.
Techniques for increasing target gene expression levels or target
gene product activity levels are discussed in Section 5.6.2,
below.
[0260] 5.6.1. Compounds that Inhibit Expression, Synthesis or
Activity of Mutant Target Gene Activity
[0261] As discussed above, target genes involved in cardiovascular
disease disorders can cause such disorders via an increased level
of target gene activity. As summarized in Table 1, above, and
detailed in the examples in Sections 8 and 9, below, a number of
genes are now known to be up-regulated in endothelial cells under
disease conditions. Specifically, rchd005, rchd024, rchd032, and
rchd036 are all up-regulated in endothelial cells treated with
IL-1.Furthermore, rchd502, rchd523, rchd528, rchd534, and
endoperoxide synthase are all up-regulated in endothelial cells
subjected to shear stress. A variety of techniques may be utilized
to inhibit the expression, synthesis, or activity of such target
genes and/or proteins.
[0262] For example, compounds such as those identified through
assays described, above, in Section 5.5, which exhibit inhibitory
activity, may be used in accordance with the invention to
ameliorate cardiovascular disease symptoms. As discussed in Section
5.5, above, such molecules may include, but are not limited to
small organic molecules, peptides, antibodies, and the like.
Inhibitory antibody techniques are described, below, in Section
5.6.1.2.
[0263] For example, compounds can be administered that compete with
endogenous ligand for the rchd523 gen-product. The resulting
reduction in the amount of ligand-bound rchd523 gene transmembrane
protein will modulated endothelial cell physiology. Compounds that
can be particularly useful for this purpose include, for example,
soluble proteins or peptides, such as peptides comprising one or
more of the extracellular domains, or portions and/or analogs
thereof, of the rchd523 gene product, including, for example,
soluble fusion proteins such as Ig-tailed fusion proteins. (For a
discussion of the production of Ig-tailed fusion proteins, see, for
example, U.S. Pat. No. 5,116,964.). Alternatively, compounds, such
as ligand analogs or antibodies, that bind to the rchd523 gene
product receptor site, but do not activate the protein, (e.g.,
receptor-ligand antagonists) can be effective in inhibiting rchd523
gene product activity.
[0264] Further, antisense and ribozyme molecules which inhibit
expression of the target gene may also be used 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,
also as described, below, in Section 5.6.1.1, triple helix
molecules may be utilized in inhibiting the aberrant target gene
activity.
[0265] 5.6.1.1. Inhibitory Antisense, Ribozyme and Triple Helix
Approaches
[0266] Among the compounds which may exhibit the ability to
ameliorate cardiovascular disease symptoms are antisense, ribozyme,
and triple helix molecules. Such molecules 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.
[0267] Anti-sense RNA and DNA molecules act to directly block the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. With respect to antisense DNA,
oligodeoxyribonucleotides derived from the translation initiation
site, e.g., between the -10 and +10 regions of the target gene
nucleotide sequence of interest, are preferred.
[0268] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. The mechanism of ribozyme action
involves sequence specific hybridization of the ribozyme molecule
to complementary target RNA, followed by an endonucleolytic
cleavage. The composition of ribozyme molecules must include one or
more sequences complementary to the target gene mRNA, and must
include the well known catalytic sequence responsible for mRNA
cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is
incorporated by reference herein in its entirety. As such within
the scope of the invention are engineered hammerhead motif ribozyme
molecules that specifically and efficiently catalyze
endonucleolytic cleavage of RNA sequences encoding target gene
proteins.
[0269] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the molecule of
interest for ribozyme cleavage sites which include the following
sequences, GUA, GUU and GUC. Once identified, short RNA sequences
of between 15 and 20ribonucleotides corresponding to the region of
the target gene containing the cleavage site may be evaluated for
predicted structural features, such as secondary structure, that
may render the oligonucleotide sequence unsuitable. The suitability
of candidate sequences may also be evaluated by testing their
accessibility to hybridization with complementary oligonucleotides,
using ribonuclease protection assays.
[0270] Nucleic acid molecules to be used in triple helix formation
for the inhibition of transcription should be single stranded and
composed of deoxyribonucleotides. The base composition of these
oligonucleotides must be designed to promote triple helix formation
via Hoogsteen base pairing rules, which generally require sizeable
stretches of either purines or pyrimidines to be present on one
strand of a duplex. Nucleotide sequences may be pyrimidine-based,
which will result in TAT and CGC.sup.+ triplets across the three
associated strands of the resulting triple helix. The
pyrimidine-rich molecules provide base complementarity to a
purine-rich region of a single strand of the duplex in a parallel
orientation to that strand. In addition, nucleic acid molecules may
be chosen that are purine-rich, for example, containing a stretch
of G residues. These molecules will form a triple helix with a DNA
duplex that is rich in GC paris, in which the majority of the
purine residues are located on a single strand of the targeted
duplex, resulting in GGC triplets across the three strands in the
triplex.
[0271] Alternatively, the potential sequences that can be targeted
for triple helix formation may be increased 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 first one strand of a duplex and then the other,
eliminating the necessity for a sizeable stretch of either purines
or pyrimidines to be present on one strand of a duplex.
[0272] It is possible that the antisense, ribozyme, and/or triple
helix molecules described herein may reduce or inhibit the
transcription (triple helix) and/or translation (antisense,
ribozyme) of mRNA produced by both normal and mutant target gene
alleles. In order to ensure that substantially normal levels of
target gene activity are maintained, nucleic acid molecules that
encode and express target gene polypeptides exhibiting normal
activity may be introduced into cells via gene therapy methods such
as those described, below, in Section 5.7. that do not contain
sequences susceptible to whatever antisense, ribozyme, or triple
helix treatments are being utilized. Alternatively, it may be
preferable to coadminister normal target gene protein into the cell
or tissue in order to maintain the requisite level of cellular or
tissue target gene activity.
[0273] Anti-sense RNA and DNA, ribozyme, and triple helix molecules
of the invention may be prepared by any method known in the art for
the synthesis of DNA and RNA molecules. These include techniques
for chemically synthesizing oligodeoxyribonucleotides and
oligoribonucleotides well known in the art such as for example
solid phase phosphoramidite chemical synthesis. Alternatively, RNA
molecules may be generated by in vitro and in vivo transcription of
DNA sequences encoding the antisense RNA molecule. Such DNA
sequences may be incorporated into a wide variety of vectors which
incorporate suitable RNA polymerase promoters such as the T7 or SP6
polymerase promoters. Alternatively, antisense cDNA constructs that
synthesize antisense RNA constitutively or inducibly, depending on
the promoter used, can be introduced stably into cell lines.
[0274] Various well-known modifications to the DNA molecules may be
introduced as a means of increasing intracellular stability and
half-life. Possible modifications include but are not limited to
the addition of flanking sequences of ribonucleotides or
deoxyribonucleotides to the 5' and/or 3' ends of the molecule or
the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages within the oligodeoxyribonucleotide
backbone.
[0275] 5.6.1.2. Antibodies for Target Gene Products
[0276] Antibodies that are both specific for target gene protein
and interfere with its activity may be used to inhibit target gene
function. Such antibodies may be generated using standard
techniques described in Section 5.4.3., supra, against the proteins
themselves or against peptides corresponding to portions of the
proteins. Such antibodies include but are not limited to
polyclonal, monoclonal, Fab fragments, single chain antibodies,
chimeric antibodies, etc.
[0277] In instances where the target gene protein is intracellular
and whole antibodies are used, internalizing antibodies may be
preferred. However, lipofectin liposomes may be used to deliver the
antibody or a fragment of the Fab region which binds to the target
gene epitope into cells. Where fragments of the antibody are used,
the smallest inhibitory fragment which binds to the target
protein's binding domain is preferred. For example, peptides having
an amino acid sequence 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 synthesized chemically or produced via
recombinant DNA technology using methods well known in the art
(e.g., see Creighton, 1983, supra; and Sambrook et al., 1989,
supra). Alternatively, single chain neutralizing antibodies which
bind to intracellular target gene epitopes may also be
administered. Such single chain antibodies may be administered, for
example, by expressing nucleotide sequences encoding single-chain
antibodies within the target cell population by utilizing, for
example, techniques such as those described in Marasco et al.
(Marasco, W. et al., 1993, Proc. Natl. Acad. Sci. USA
90:7889-7893).
[0278] In some instances, the target gene protein is extracellular,
or is a transmembrane protein, such as the rchd523 gene product.
Antibodies that are specific for one or more extracellular domains
of the rchd523 gene product, for example, and that interfere with
its activity, are particularly useful in treating cardiovascular
disease. Such antibodies are especially efficient because they can
access the target domains directly from the bloodstream. Any of the
administration techniques described, below in Section 5.7which are
appropriate for peptide administration may be utilized to
effectively administer inhibitory target gene antibodies to their
site of action.
[0279] 5.6.2. Methods for Restoring Target Gene Activity
[0280] Target genes that cause cardiovascular disease may be
underexpressed within cardiovascular disease situations. As
summarized in Table 1, above, and detailed in the example in
Sections 7, below, several genes are now known to be down-regulated
in monocytes under disease conditions. Specifically, bcl-2 and
glutathione peroxidase gene expression is down-regulated in the
monocytes of patients exposed to a high lipid diet, e.g.
cholesterol or fat, that leads to high serum LDL levels.
Alternatively, the activity of target gene products may be
diminished, leading to the development of cardiovascular disease
symptoms. Described in this Section are methods whereby the level
of target gene activity may be increased to levels wherein
cardiovascular disease symptoms 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.
[0281] For example, a target gene protein, at a level sufficient to
ameliorate cardiovascular disease symptoms may be administered to a
patient exhibiting such symptoms. Any of the techniques discussed,
below, in Section 5.7, may be utilized for such administration. One
of skill in the art will readily know how to determine the
concentration of effective, non-toxic doses of the normal target
gene protein, utilizing techniques such as those described, below,
in Section 5.7.1.
[0282] Additionally, RNA sequences encoding target gene rotein may
be directly administered to a patient exhibiting ardiovascular
disease symptoms, at a concentration sufficient to produce a level
of target gene protein such that cardiovascular disease symptoms
are ameliorated. Any of he techniques discussed, below, in Section
5.7, which achieve intracellular administration of compounds, such
as, for example, liposome administration, may be utilized for the
administration of such RNA molecules. The RNA molecules may be
produced, for example, by recombinant techniques such as those
described, above, in Section 5.4.2.
[0283] Further, patients may be treated by gene replacement
therapy. One or more copies of a normal target gene, or a portion
of the gene that directs the production of a normal target gene
protein with target gene function, may be inserted into cells using
vectors which include, but are not limited to adenovirus,
adeno-associated virus, and retrovirus vectors, in addition to
other particles that introduce DNA into cells, such as liposomes.
Additionally, techniques such as those described above may be
utilized for the introduction of normal target gene sequences into
human cells.
[0284] Cells, preferably, autologous cells, containing normal
target gene expressing gene sequences may then be introduced or
reintroduced into the patient at positions which allow for the
amelioration of cardiovascular disease symptoms. Such cell
replacement techniques may be preferred, for example, when the
target gene product is a secreted, extracellular gene product.
[0285] 5.7. Pharmaceutical Preparations and Methods of
Administration
[0286] The identified compounds that inhibit target gene
expression, synthesis and/or activity can be administered to a
patient at therapeutically effective doses to treat or ameliorate
cardiovascular disease. A therapeutically effective dose refers to
that amount of the compound ufficient to result in amelioration of
symptoms of ardiovascular disease.
[0287] 5.7.1. Effective Dose
[0288] Toxicity and therapeutic efficacy of such compounds an be
determined by standard pharmaceutical procedures in ell cultures or
experimental animals, e.g., for determining he LD.sub.50 (the dose
lethal to 50% of the population) and the ED.sub.50 (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 expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large-therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissue in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0289] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0290] 5.7.2. Formulations and Use
[0291] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
[0292] Thus, the compounds and their physiologically acceptable
salts and solvates may be formulated for administration by
inhalation or insufflation (either through the mouth or the nose)
or oral, buccal, parenteral or rectal administration.
[0293] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g. sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and
[0294] preservatives (e.g., methyl or propyl-p-hydroxybenzoates or
sorbic acid). The preparations may also contain buffer salts,
flavoring, coloring and sweetening agents as appropriate.
[0295] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0296] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0297] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0298] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0299] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0300] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0301] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0302] 5.8. Diagnosis of Cardiovascular Disease Abnormalities
[0303] A variety of methods may be employed, utilizing reagents
such as fingerprint gene nucleotide sequences described in Section
5.4.1, and antibodies directed against differentially expressed and
pathway gene peptides, as described, above, in Sections 5.4.2.
(peptides) and 5.4.3. (antibodies). Specifically, such reagents may
be used, for example, for the detection of the presence of target
gene mutations, or the detection of either over or under expression
of target gene mRNA.
[0304] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
specific fingerprint gene nucleic acid or anti-fingerprint gene
antibody reagent described herein, which may be conveniently used,
e.g., in clinical settings, to diagnose patients exhibiting
cardiovascular disease symptoms or at risk for developing
cardiovascular disease.
[0305] Any cell type or tissue, preferably monocytes, endothelial
cells, or smooth muscle cells, in which the fingerprint gene is
expressed may be utilized in the diagnostics described below.
[0306] 5.8.1. Detection of Fingerprint Gene Nucleic Acids
[0307] DNA or RNA from the cell type or tissue to be analyzed may
easily be isolated using procedures which are well known to those
in the art. Diagnostic procedures may also be performed "in situ"
directly upon tissue sections (fixed and/or frozen) of patient
tissue obtained from biopsies or resections, such that no nucleic
acid purification is necessary. Nucleic acid reagents such as those
described in Section 5.1. may be used as probes and/or primers for
such in situ procedures (see, for example, Nuovo, G. J., 1992, PCR
in situ hybridization: protocols and applications, Raven Press,
NY).
[0308] Fingerprint gene nucleotide sequences, either RNA or DNA,
may, for example, be used in hybridization or amplification assays
of biological samples to detect cardiovascular disease-related gene
structures and expression. Such assays may include, but are not
limited to, Southern or Northern analyses, single stranded
conformational polymorphism analyses, in situ hybridization assays,
and polymerase chain reaction analyses. Such analyses may reveal
both quantitative aspects of the expression pattern of the
fingerprint gene, and qualitative aspects of the fingerprint gene
expression and/or gene composition. That is, such aspects may
include, for example, point mutations, insertions, deletions,
chromosomal rearrangements, and/or activation or inactivation of
gene expression.
[0309] Preferred diagnostic methods for the detection of
fingerprint gene-specific nucleic acid molecules may involve for
example, contacting and incubating nucleic acids, derived from the
cell type or tissue 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 these reagents
to their complementary sequences within the nucleic acid molecule
of interest. Preferably, the lengths of these nucleic acid reagents
are at least 9 to 30 nucleotides. After incubation, all
non-annealed nucleic acids are removed from the nucleic
acid:fingerprint molecule hybrid. The presence of nucleic acids
from the fingerprint tissue which have hybridized, if any such
molecules exist, is then detected. Using such a detection scheme,
the nucleic acid from the tissue or cell type of interest may be
immobilized, for example, to a solid support such as a membrane, or
a plastic surface such as that on a microtitre plate or polystyrene
beads. In this case, after incubation, non-annealed, labeled
fingerprint nucleic acid reagents of the type described in Section
5.1. are easily removed. Detection of the remaining, annealed,
labeled nucleic acid reagents is accomplished using standard
technique well-known to those in the art.
[0310] Alternative diagnostic methods for the detection of
fingerprint gene specific nucleic acid molecules may involve their
amplification, e.g., by PCR (the experimental embodiment set forth
in Mullis, K. B., 1987, U.S. Pat. No. 4,683,202), ligase chain
reaction (Barany, F., 1991, Proc. Natl. Acad. Sci. USA 88:189-193),
self sustained sequence replication (Guatelli, J.C. et al., 1990,
Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional
amplification system (Kwoh, D. Y et al., 1989, Proc. Natl. Acad.
Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al.,
1988, Bio/Technology 6:1197), or any other nucleic acid
amplification method, followed by the detection of the amplified
molecules using techniques well known to those of skill in the art.
These detection schemes are especially useful for the detection of
nucleic acid molecules if such molecules are present in very low
numbers.
[0311] In one embodiment of such a detection scheme, a cDNA
molecule is obtained from an RNA molecule of interest (e.g., by
reverse transcription of the RNA molecule into cDNA). Cell types or
tissues from which such RNA may be isolated include any tissue in
which wild type fingerprint gene is known to be expressed,
including, but not limited, to monocytes, endothelium, and/or
smooth muscle. A fingerprint sequence within the cDNA is then used
as the template for a nucleic acid amplification reaction, such as
a PCR amplification reaction, or the like. The nucleic acid
reagents used as synthesis initiation reagents (e.g., primers) in
the reverse transcription and nucleic acid amplification steps 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 least 15-30 nucleotides. For detection
of the amplified product, the nucleic acid amplification may be
performed using radioactively or non-radioactively labeled
nucleotides. Alternatively, enough amplified product may be made
such that the product may be visualized by standard ethidium
bromide staining or by utilizing any other suitable nucleic acid
staining method.
[0312] In addition to methods which focus primarily on the
detection of one nucleic acid sequence, fingerprint profiles, as
discussed in Section 5.5.4, may also be assessed in such detection
schemes. Fingerprint profiles may be generated, for example, by
utilizing a differential display procedure, as discussed, above, in
Section 5.1.2, Northern analysis and/or RT-PCR. Any of the gene
sequences described, above, in Section 5.4.1. may be used as probes
and/or PCR primers for the generation and corroboration of such
fingerprint profiles.
[0313] 5.8.2. Detection of Fingerprint Gene Peptides
[0314] Antibodies directed against wild type or mutant fingerprint
gene peptides, which are discussed, above, in Section 5.4.3, may
also be used as cardiovascular disease diagnostics and prognostics,
as described, for example, herein. Such diagnostic methods, may be
used to detect abnormalities in the level of fingerprint gene
protein expression, or abnormalities in the structure and/or
tissue, cellular, or subcellular location of fingerprint gene
protein. Structural differences may include, for example,
differences in the size, electronegativity, or antigenicity of the
mutant fingerprint gene protein relative to the normal fingerprint
gene protein.
[0315] Protein from the tissue or cell type to be analyzed may
easily be isolated using techniques which are well known to those
of skill in the art. The protein isolation methods employed herein
may, for example, be such as those described in Harlow and Lane
(Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual",
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.),
which is incorporated herein by reference in its entirety.
[0316] Preferred diagnostic methods for the detection of wild type
or mutant fingerprint gene peptide molecules may involve, for
example, immunoassays wherein fingerprint gene peptides are
detected by their interaction with an anti-fingerprint gene
specific peptide antibody.
[0317] For example, antibodies, or fragments of antibodies, such as
those described, above, in Section 5.4.3, useful in the present
invention may be used to quantitatively or qualitatively detect the
presence of wild type or mutant fingerprint gene peptides. This can
be accomplished, for example, by immunofluorescence techniques
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 expressed on the cell surface.
[0318] The antibodies (or fragments thereof) useful in the present
invention may, additionally, be employed histologically, as in
immunofluorescence or immunoelectron microscopy, for in situ
detection of fingerprint gene peptides. In situ detection may be
accomplished by removing a histological specimen 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 sample. Through
the use of such a procedure, it is possible to determine not only
the presence of the fingerprint gene peptides, but also their
distribution in the examined tissue. Using the present invention,
those of ordinary skill will readily perceive that any of a wide
variety of histological methods (such as staining procedures) can
be modified in order to achieve such in situ detection.
[0319] Immunoassays for wild type or mutant fingerprint gene
peptides typically comprise incubating a biological sample, such as
a biological fluid, a tissue extract, freshly harvested cells, or
cells which have been incubated in tissue 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.
[0320] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers followed by treatment with
the detectably labeled fingerprint gene specific antibody. The
solid phase support may then be washed with the buffer a second
time to remove unbound antibody. The amount of bound label on solid
support may then be detected by conventional means.
[0321] By "solid phase support or carrier" is intended any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, 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.
[0322] 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 assay conditions for each
determination by employing routine experimentation.
[0323] One of the ways in which the fingerprint gene
peptide-specific antibody can be detectably labeled is by linking
the same to an enzyme and use in an enzyme immunoassay (EIA)
(Voller, "The Enzyme Linked Immunosorbent Assay (ELISA)",
Diagnostic Horizons 2:1-7, 1978,Microbiological Associates
Quarterly Publication, Walkersville, Md.; Voller, et al., J. Clin.
Pathol. 31:507-520 (1978); Butler, Meth. Enzymol. 73:482-523
(1981); Maggio, (ed.) Enzyme Immunoassay, CRC Press, Boca Raton,
Fla., 1980; Ishikawa, et al., (eds.) Enzyme Immunoassay, Kgaku
Shoin, Tokyo, 1981). The enzyme which is bound to the antibody will
react with an appropriate substrate, preferably a chromogenic
substrate, in such a manner as to produce a chemical moiety which
can be detected, for example, by spectrophotometric, fluorimetric
or by visual means. Enzymes which can be used to detectably label
the antibody include, but are not limited to, malate dehydrogenase,
staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose
phosphate isomerase, horseradish peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease,
urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase
and acetylcholinesterase. The detection can be accomplished by
colorimetric methods which employ a chromogenic substrate for the
enzyme. Detection may also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0324] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect
fingerprint gene wild type or mutant peptides through the use of a
radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles
of Radioimmunoassays, Seventh Training Course 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.
[0325] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0326] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0327] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0328] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0329] 5.8.3. Imaging Cardiovascular Disease Conditions
[0330] In some cases, differentially expressed gene products
identified herein may be up-regulated under cardiovascular disease
conditions and expressed on the surface of the affected tissue.
Such target gene products allow for the non-invasive imaging of
damaged or diseased cardiovascular tissue for the purposed of
diagnosis and directing of treatment of the disease. For example,
such differentially expressed gene products may include but are not
limited to atherosclerosis specific adhesion molecules responsible
for atherogenesis, or monocyte scavenger receptors that are
up-regulated in response to oxidized LDL, which are discussed in
Section 2, above. Alternatively, other such surface proteins may be
specifically up-regulated in tissues suffering from
ischemia/reperfusion or other tissues with atherosclerotic or
restenotic lesions.
[0331] As described in the example in Section 9, below, the rchd523
gene is a gene that is up-regulated in endothelial cells under
shear stress. Furthermore, the rchd523 gene encodes a novel G
protein-coupled receptor, containing an extracellular amino
terminal domain, in addition to multiple transmembrane domains. The
rchd523 gene product, therefore, provides an excellent tool for
imaging cardiovascular disease conditions. An example illustrating
the use of this method in accordance with the invention is provided
in Section 11, below.
[0332] Monoclonal antibodies, as described in Section 5.6.1.2,
above, which specifically bind to such surface proteins, such as
the rchd523 gene product, may be used for the diagnosis of
cardiovascular disease by in vivo tissue imaging techniques. An
antibody specific for a target gene product, or preferably an
antigen binding fragment thereof, is conjugated to a label (e.g., a
gamma emitting radioisotope) which generates a detectable signal
and administered to a subject (human or animal) suspected of having
cardiovascular disease. After sufficient time to allow the
detectably-labeled antibody to localize at the diseased or damaged
tissue site (or sites), the signal generated by the label is
detected by a photoscanning device. The detected signal is then
converted to an image of the tissue. This image makes it possible
to localize the tissue in vivo. This data can then be used to
develop an appropriate therapeutic strategy.
[0333] Antibody fragments, rather than whole antibody molecules,
are generally preferred for use in tissue imaging. Antibody
fragments accumulate at the tissue(s) more rapidly because they are
distributed more readily than are entire antibody molecules. Thus
an image can be obtained in less time than is possible using whole
antibody. These fragments are also cleared more rapidly from
tissues, resulting in a lower background signal. See, e.g., Haber
et al., U.S. Pat. No. 4,036,945; Goldenberg et al., U.S. Pat. No.
4,331,647. The divalent antigen binding fragment (Fab').sub.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 suitable for conjugation to
a monoclonal antibody for diseased or damaged tissue localization
include, but are not limited to radiolabels (i.e., radioisotopes),
fluorescent labels and biotin labels.
[0334] Among the radioisotopes that can be used to label antibodies
or antibody fragments, gamma-emitters, positron-emitters,
X-ray-emitters and fluorescence-emitters are suitable for
localization. Suitable radioisotopes for labeling antibodies
include Iodine-131, Iodine-123, Iodine-125, Iodine-126, Iodine-133,
Bromine-77, Indium-111, Indium-113m, Gallium-67, Gallium-68,
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 less interchangeably as labels since
halogen-labeled antibodies and/or normal immunoglobulins would have
substantially the same kinetics and distribution and similar
metabolism.
[0335] The gamma-emitters Indium-111 and Technetium-99m are
preferred because these radiometals are detectable with a gamma
camera and have favorable half lives for imaging in vivo. Antibody
can be labelled with Indium-111 or Technetium-99m via a conjugated
metal chelator, such as DTPA (diethlenetriaminepentaacetic acid).
See Krejcarek et al., 1977, Biochem. Biophys. Res. Comm. 77:581;
Khaw et al., 1980,Science 209:295; Gansow et al., U.S. Pat. No.
4,472,509; Hnatowich, U.S. Pat. No. 4,479,930, the teachings of
which are incorporated herein by reference.
[0336] Fluorescent compounds that are suitable for conjugation to a
monoclonal antibody include fluorescein sodium, 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 ascertain with no more than
routine experimentation, other fluorescent compounds that are
suitable for labeling monoclonal antibodies.
6. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN
RESPONSE TO PARADIGM A: IN VITRO FOAM CELL PARADIGM
[0337] According to the invention, differential display may be used
to detect genes that are differentially expressed in monocytes that
were treated so as to simulate the conditions under which foam
cells develop during atherogenesis.
[0338] 6.1. Materials and Methods
[0339] 6.1.1. Cell Isolation and Culturing
[0340] Blood (.about.200 ml) was drawn into chilled 20 ml
vacutainer tubes to which 3 ml of citrate phosphate dextrose
(Sigma) was added. Blood was then pooled into 50 ml tubes and spun
in the Beckman GS-6R at 1250 RPM for 15 minutes at 4.degree. C. The
upper clear layer (.about.25 ml) was then removed with a pipette
and discarded and replaced with the same volume of 4.degree. C.
PBS. The blood was then mixed, and spun again at 2680RPM for 15
minutes at 4.degree. C. The upper layer was then removed and
discarded, and the buffy coat at the interface was removed in
.about.5 ml and placed in a separate 50 ml tube, and the pipette
was washed with 20 ml PBS. Cells were added to a T flask and stored
at 4.degree. C. for 16 hours. A small aliquot of the cells were
then removed and counted using a hemacytometer. The final red blood
cell concentration in the buffy coat population was then adjusted
to 1.5.times.10.sup.9/ml with PBS, the cells were added to
Leucoprep tubes (Becton Dickinson) after being allowed to come to
room temperature, and spun at 2300RPM for 25 minutes at 25.degree.
C. The upper clear layer was removed and discarded and the turbid
layer over the gel was removed and pooled in 50 ml tubes. Samples
were then diluted 1 to 50 ml with PBS (25.degree. C.) and spun at
1000 RPM for 10 minutes. The supernatant was then removed, and the
pellet was resuspended in 50 ml PBS. This procedure was repeated 3
more times. After the last spin, the cells were resuspended in a
small volume of PBS and counted.
[0341] Tissue culture dishes were coated with bovine collagen
before monocytes were plated out. 1/6 volume of 7.times. RPMI (JRH
Biosciences) was added to Vitrogen 100 collagen (Celtrix) which was
then diluted 1:10 with RPMI to a final concentration of 0.35 mg/ml.
Collagen mixture was then added to plates (2.5 ml/100 mm dish) and
placed at 37.degree. C. for at least one hour to allow for gel
formation. After gel formation has taken place, the RPMI was
removed and cells were added in RPMI/10% plasma derived serum
(PDS). PDS was prepared by drawing blood into chilled evacuated
tubes containing {fraction (1/10)}th volume 3.8% sodium citrate.
Blood was then transferred into new Sorvall tubes and spun at
14,000-16,000 RPM for 20 minutes at 4.degree. C. Plasma layer was
removed and pooled in new tubes to which {fraction (1/50)}th volume
1M CaCl.sub.2 was added. Plasma was mixed and aliquoted into new
Sorvall tubes and incubated at 37% for 2 hours 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.degree. C. Supernatant was collected, pooled, and heat
inactivated at 56.degree. C. prior to sterile filtration and
freezing.
[0342] 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, acetylated LDL (all
LDL at 50 .mu.g/ml), lysophosphatidylcholine (Sigma, 37.5 .mu.M),
or homocysteine (Sigma, 1 mM). After incubation with these reagents
for periods ranging from 2 hours up to 3 days, the media was
withdrawn and the cells were dissolved in RNA lysis buffer and RNA
was prepared as described, above, in Section 6.1.
[0343] Lipoproteins
[0344] For oxidation, human LDL (Sigma) was first diluted to 1
mg/ml with PBS and then dialyzed against PBS at 4.degree. C.
overnight. LDL was then diluted to 0.3 mg/ml with PBS.
CuSO.sub.4.5H.sub.2O was then added to 5 .mu.M final concentration,
and the solution was incubated in a T flask in a 37.degree. C.
incubator for 24 hr. LDL solution was then dialyzed at 4.degree. C.
against 0.15M NaCl/0.3 mM EDTA for 2 days with several changes,
before being removed and concentrated using an Amicon spin column
by spinning for 1 hr. 4000 RPM at 4.degree. C.
[0345] 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.degree. C. 8 .mu.l of acetic anhydride was added 2 .mu.l at a
time over 1 hr. LDL was then dialyzed for 48 hr. against 0.15M
NaCl/0.3 mM EDTA at 4.degree. 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 OD.sub.280,
with 0.15M NaCl/0.3 mM EDTA used as diluent in all cases.
[0346] 6.1.2. Analysis of Paradigm Material
Differential Display
[0347] Removal of DNA:
[0348] The RNA pellet was resuspended in H.sub.2O and quantified by
spectrophotometry at OD.sub.260. Approximately half of the sample
was then treated with DNAse I to remove contaminating chromosomal
DNA. RNA was amplified by PCR using the following procedure. 50 ul
RNA sample (10-20 .mu.g), 5.7 .mu.l 10.times. PCR buffer
(Perkin-Elmer/Cetus), 1 .mu.l RNAse inhibitor (40 units/.mu.l)
(Boehringer Mannheim, Germany) were mixed together, vortexed, and
briefly spun. 2 .mu.l DNAse I (10 units/.mu.l) (Boehringer
Mannheim) was added to the reaction which was incubated for 30 min.
at 37.degree. C. The total volume was brought to 200 .mu.l with
DEPC H.sub.2O, extracted once with phenol/chloroform, once with
chloroform, and precipitated by adding 20 .mu.l 3M NaOAc, pH 4.8,
(DEPC-treated), 500 .mu.l absolute ETOH and incubating for 1 hour
on dry ice or -20.degree. C. overnight. The precipitated sample was
centrifuged for 15 min., and the pellet was washed with 70% ETOH.
The sample was re-centrifuged, the remaining liquid was aspirated,
and the pellet was resuspended in 100 .mu.l H.sub.2O. The
concentration of RNA was measured by reading the OD.sub.260.
[0349] First Strand cDNA Synthesis:
[0350] For each RNA sample duplicate reactions were carried out in
parallel. 400 ng RNA plus DEPC H.sub.2O in a total volume of 10
.mu.l were added to 4 .mu.l T.sub.11XX reverse primer (10 .mu.M)
(Operon). The specific primers used in each experiment are provided
in the Description of the Figures in Section 4, above. The mixture
was incubated at 70.degree. C. for 5 min. to denature the RNA and
then placed at r.t. 26 .mu.l of reaction mix containing the
following components was added to each denatured RNA/primer sample:
8 .mu.l 5.times. First Strand Buffer (Gibco/BRL, Gaithersburg,
Md.), 4 .mu.l 0.1M DTT (Gibco/BRL), 2 .mu.l RNAse inhibitor (40
units/.mu.l) (Boehringer Mannheim), 4 .mu.l 200 .mu.M dNTP mix, 6
.mu.l H.sub.2O, 2 .mu.l Superscript reverse transcriptase (200
units/.mu.l) (Gibco/BRL). The reactions were mixed gently and
incubated for 30 min. at 42.degree. C. 60 .mu.l of H.sub.2O (final
volume=100 .mu.l) were then added and the samples were denatured
for 5 min. at 85.degree. C. and stored at -20.degree. C.
[0351] PCR Reactions:
[0352] 13 .mu.l of reaction mix was added to each 5tube of a 96
well plate on ice. The reaction mix contained 6.4 .mu.l H.sub.2O, 2
.mu.l 10.times. PCR Buffer (Perkin-Elmer), 2 .mu.l 20 .mu.M dNTP's,
0.4 .mu.l .sup.35S dATP (12.5 .mu.Ci/.mu.l; 50 .mu.Ci total)
(Dupont/NEN), 2 .mu.l forward primer (10 .mu.M) (Operon), and 0.2
.mu.l AmpliTaq Polymerase (5 units/.mu.l) (Perkin-Elmer). Next, 2
.mu.l of reverse primer (T.sub.11XX, 10 .mu.M) were added to the
side of each tube followed by 5 .mu.l of cDNA also to the sides of
the tubes, which were still on ice. The specific primets used in
each experiment are provided in the Description of the Figures in
Section 4, above. Tubes 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: 1 *= X40 94 .degree. C . 2 min . * 94 .degree. C . 15
sec . * 40 .degree. C . 2 min . * ramp 72 .degree. C . 72 .degree.
C . 1 min . * 72 .degree. C . 30 sec . 72 .degree. C . 5 min . 4
.degree. C . hold
[0353] When the PCR machine reached 94.degree. C., the plate was
removed from ice and placed directly into the Perkin-Elmer 9600 PCR
machine. Following PCR, 15 .mu.l 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.degree. C. for 5 min., cooled on ice, centrifuged, and placed
on ice. Approximately 4 .mu.l 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 (Wha-tman Paper, England) and dried under
vacuum. Bands were visualized by autoradiography.
[0354] Band Isolation and Amplification:
[0355] Differentially expressed bands were excised from the dried
gel with a razor blade and placed into a microfuge tube with 100
.mu.l H.sub.2O and heated at 100.degree. C. for 5 min., vortexed,
heated again to 100.degree. C. for 5 min., and vortex again. After
cooling, 100 .mu.l H.sub.2O, 20 .mu.l 3M NaOAc, 1 .mu.l glycogen
(20 mg/ml), and 500 .mu.l ethanol were added and chilled. After
centrifugation, the pellet was washed and resuspended in 10 .mu.l
H.sub.2O.
[0356] The isolated differentially expressed bands were then
amplified by PCR using the following reaction conditions:
3 58 .mu.l H.sub.2O 10 .mu.l 10 x PCR Buffer 10 .mu.l 200 .mu.m
dNTP's 10 .mu.l 10 .mu.M reverse primer 10 .mu.l 10 .mu.M forward
primer 1.5 .mu.l amplified band 0.5 .mu.l AmpliTaq polymerase (5
units/.mu.l) (Perkin Elmer)
[0357] PCR was performed using the program described in this
Section, above, for differential display. After PCR, glycerol
loading dyes were added and samples were loaded onto a 2%
preparative TAE/Biogel (Bio101, La Jolla, Calif.) agarose gel and
eluted. Bands were then excised from the gel with a razor blade and
vortexed for 15 min. at r.t., and purified using the Mermaid kit
from Bio101 by adding 3 volumes of Mermaid high salt binding
solution and 8 .mu.l of resuspended glassfog in a microfuge tube.
Glassfog was then pelleted, washed 3 times with ethanol wash
solution, and then DNA was eluted twice in 10 .mu.l at 50.degree.
C.
[0358] Subcloning:
[0359] The TA cloning kit (Invitrogen, San Diego, Calif.) was used
to subclone the amplified bands. The ligation reaction typically
consisted of 4 .mu.l sterile H.sub.2O, 1 .mu.l ligation buffer, 2
.mu.l TA cloning vector, 2 .mu.l PCR product, and 1 .mu.l T4 DNA
ligase. The volume of PCR product can vary, but the total volume of
PCR product plus H.sub.2O was always 6 .mu.l. Ligations (including
vector alone) were incubated overnight at 12.degree. C. before
bacterial transformation. TA cloning kit competent bacteria
(INV.alpha.F': enda1, recAl, hsdR17(r-k, m+k), supE44, .lambda.-,
thi-1, gyrA, relA1, .phi.80lacZ.alpha..DELTA.M15.DELTA-
.(1acZYA-argF), deoR+, F') were thawed on ice and 2 .mu.l of 0.5 M
.beta.-mercaptoethanol were added to each tube. 2 .mu.l from each
ligation were added to each tube of competent cells (50 .mu.l),
mixed without vortexing, and incubated on ice for 30 min. Tubes
were then placed in 42.degree. C. bath for exactly 30 sec., before
being returned to ice for 2 min. 450 .mu.l of SOC media (Sambrook
et al., 1989, supra) were then added to each tube which were then
shaken at 37.degree. C. for 1 hr. Bacteria were then pelleted,
resuspended in .about.200 .mu.l SOC and plated on Luria broth agar
plates containing X-gal and 60 .mu.g/ml ampicillin and incubated
overnight at 37.degree. C. White colonies were then picked and
screened for inserts using PCR.
[0360] A master mix containing 2 .mu.l 10.times. PCR buffer, 1.6
.mu.l 2.5 mM dNTP's, 0.1 .mu.l 25 mM MgCl.sub.2, 0.2 .mu.l M13
reverse primer (100 ng/.mu.l), 0.2 .mu.l M13 forward primer (100
ng/pl), 0.1 .mu.l AmpliTaq (Perkin-Elmer), and 15.8 .mu.l H.sub.2O
was made. 40 .mu.l of the master 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: 2 *= X35 94 .degree. C . 2 min . *
94 .degree. C . 15 sec . * 47 .degree. C . 2 min . * ramp 72
.degree. C . 72 .degree. C . 30 sec . * 72 .degree. C . 30 sec . 72
.degree. C . 10 min . 4 .degree. C . hold
[0361] Reaction products were eluted on a 2% agarose gel and
compared to vector control. Colonies with vectors containing
inserts were purified by streaking onto LB/Amp plates. Vectors were
isolated from such strains and subjected to sequence analysis,
using an Applied Biosystems Automated Sequencer-(Applied
Biosystems, Inc. Seattle, Wash.).
[0362] Northern Analysis:
[0363] Northern analysis was performed to confirm the differential
expression of the genes corresponding to the amplified bands. The
probes used to detect mRNA were synthesized as follows: typically 2
Al amplified band (.about.30 ng), 7 .mu.l H.sub.2O, and 2 .mu.l
10.times. Hexanucleotide mix (Boehringer-Mannheim) were mixed and
heated to 95.degree. C. for 5 min., and then allowed to cool on
ice. The volume of the amplified band can vary, but the total
volume of the band plus H.sub.2O was always 9 .mu.l. 3 .mu.l
dATP/dGTP/dTTP mix (1:1:1 of 0.5 mM each), 5 .mu.l .alpha..sup.32P
dCTP 3000 Ci/MM (50 .mu.Ci total) (Amersham, Arlington Heights,
Ill.), and 1 .mu.l Klenow (2 units) (Boehringer-Mannheim) were
mixed and incubated at 37.degree. C. After 1 hr., 30 .mu.l TE were
added and the reaction was loaded onto a Biospin-6.TM. column
(Biorad, Hercules, Calif.), and centrifuged. A 1 .mu.l aliquot of
eluate was used to measure incorporation in a scintillation counter
with scintillant to ensure that 10.sup.6cpm/.mu.l of incorporation
was achieved.
[0364] The samples were loaded onto a denaturing agarose gel. A 300
ml 1% gel was made by adding 3 g of agarose (SeaKem.TM. LE, FMC
BioProducts, Rockland, Me.) and 60 ml of 5.times. MOPS buffer to
210 ml sterile H.sub.2O. 5.times. MOPS buffer (0.1M MOPS (pH 7.0),
40 mM NaOAc, 5 mM EDTA (pH 8.0)) was made by adding 20.6 g of MOPS
to 800 ml of 50 mM NaOAc (13.3 ml of 3M NaOAc pH 4.8 in 800 ml
sterile H.sub.2O); then adjusting the pH to 7.0 with 10M NaOH;
adding 10 ml of 0.5M EDTA (pH8.0); and adding H.sub.2O to a final
volume of 1L. The mixture was heated until melted, then cooled to
50.degree. C., at which time 5 .mu.l ethidium bromide (5mg/ml) and
30 ml of 37% formaldehyde of gel were added. The gel was swirled
quickly to mix, and then poured immediately.
[0365] 2 .mu.g RNA sample, lx final 1.5.times. RNA loading dyes
(60% formamide, 9% formaldehyde, 1.5.times. MOPS, 0.075% XC/BPB
dyes) and H.sub.2O were mixed to a final volume of 40 .mu.l. The
tubes were heated at 65.degree. C. for 5 min. and then cooled on
ice. 10 .mu.g of RNA MW standards (New England Biolabs, Beverly,
Mass.) were also denatured-with dye and loaded onto the gel. The
gel was run overnight at 32V in MOPS running buffer.
[0366] The gel was then soaked in 0.5 .mu.g/ml Ethidium Bromide for
45 min., 50 mM NaOH/0.1 M NaCl for 30 min., 0.1 M Tris pH 8.0 for
30 min., and 20.times. SSC for 20 min. Each soaking step was done
at r.t. with shaking. The gel was then photographed along with a
fluorescent ruler before blotting with Hybond-N membrane
(Amersham), according to the methods of Sambrook et al., 1989,
supra, in 20.times. SSC overnight.
[0367] For hybridization, the blot was placed into a roller bottle
containing 10 ml of prehybridization solution consisting of 50%
formamide and 1.times. Denhardt's solution, and placed into
65.degree. C. incubator for 30 min. The probe was then heated to
95.degree. C., chilled on ice, and added to 10 ml of hybridization
solution, consisting of 50% formamide, 1.times. Denhardt's
solution, 10% dextransulfate, to a final concentration of 10.sup.6
cpm/ml. The prehybridization solution was then replaced with the
probe solution and incubated overnight at 42.degree. C. The
following day, the blot was washed three times for 30 min. in
2.times. SSC/0.1% SDS at room temperature before being covered in
plastic wrap and put down for exposure.
[0368] RT-PCR Analysis:
[0369] RT-PCR was performed to detect differentially expressed
levels of mRNA from the genes corresponding to amplified bands.
First strand synthesis was conducted by mixing 20 .mu.l DNased RNA
(.about.2 .mu.g), 1 .mu.l oligo dT (Operon) (1 .mu.g), and 9.75
.mu.l H.sub.2O. The samples were heated at 70.degree. C. for 10
min., and then allowed to cool on ice. 10 .mu.l first strand buffer
(Gibco/BRL), 5 .mu.l 0.1M DTT, 1.25 .mu.l 20 mM dNTP's (500 .mu.M
final), 1 .mu.l RNAsin (40 units/.mu.l) (Boehringer Mannheim), and
2 .mu.l Superscript Reverse Transcriptase (200 units/.mu.l)
(Gibco/BRL) were added to the reaction, incubated at 42.degree. C.
for 1 hr., and then placed at 85.degree. C. for 5 min., and stored
at -20.degree. C.
[0370] PCR was performed on the reverse transcribed samples. Each
reaction contained 2 .mu.l 10.times. PCR buffer, 14.5 .mu.l
H.sub.2O, 0.2 .mu.l 20 mM dNTP's (200 .mu.M final), 0.5 .mu.l 20
.mu.M forward primer (0.4 .mu.M final), 0.5 .mu.l 20 .mu.M reverse
primer (0.4 .mu.M final), 0.3 .mu.l AmpliTaq polymerase
(Perkin-Elmer/Cetus), 2 .mu.l cDNA dilution or positive control (40
pg). The specific primers used in each experiment are provided in
the Description of the Figures in Section 4, above. Samples were
placed in the PCR 9600 machine at 94.degree. C. (hot start), which
was programmed as follows: 3 *= 35 x 94 .degree. C . 2 min . (
samples loaded ) * 94 .degree. C . 45 sec . * 55 .degree. C . 45
sec . * 72 .degree. C . 2 min . 72 .degree. C . 5 min . 4 .degree.
C . hold
[0371] Reactions were carried out on cDNA dilution series and tubes
were removed at various cycles from the machine during 72.degree.
C. step. Reaction products were eluted on a 1.8% agarose gel and
visualized with ethidium bromide.
[0372] 6.1.3. Chromosomal Localization of Target Genes
[0373] Once the nucleotide sequence has been determined, the
presence of the gene on a particular chromosome is detected.
Oligonucleotide primers based on the nucleotide sequence of the
target gene are used in PCR reactions using individual human
chromosomes as templates. Individual samples of each the
twenty-three human chromosomes are commercially available (Coriel
Institute for Medical Research, Camden, N.J.). The chromosomal DNA
is amplified according to the following conditions: long
chromosomal DNA, 2.mu.l 10.times. PCR buffer, 1.6.mu.l 2.5 mM
dNTP's, 0.1 .mu.l 25 mM MgCl.sub.2, 0.2 .mu.l reverse primer
(100ng/.mu.l), 0.2.mu.l forward primer (100 ng/.mu.l), 0.1 .mu.l
Taq polymerase, and 15.8.mu.l H.sub.2O. Samples are placed in the
PCR 9600 machine at 94.degree. C. (hot start), which is programmed
as follows: 4 *= 35 x 94 .degree. C . 2 min . ( samples loaded ) *
94 .degree. C . 20 sec . * 55 .degree. C . 30 sec . * 72 .degree. C
. 30 sec . 72 .degree. C . 5 min . 4 .degree. C . hold
7. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN
RESPONSE TO PARADIGM B: IN VIVO MONOCYTES
[0374] In an alternative embodiment of the invention, genes
differentially expressed in monocytes were detected under highly
physiologically relevant, in vivo conditions. According to Paradigm
B, human subjects were held in a clinical setting and the
fat/cholesterol content of their diets was controlled. Monocytes
were isolated at different stages of treatment, and their gene
expression pattern was compared to that of control groups.
[0375] By use of Paradigm B, the human bcl-2 gene was identified.
Its expression decreases in response to the atherogenic conditions
of high fat/high cholesterol (FIG. 1). The Apo E-/- mouse is the
first mouse model of atherosclerosis with pathology similar to that
of human plaque development (Plump et al., 1992, Cell 71: 343-353).
Serum cholesterol levels in these mice on a chow diet is five times
higher than those of control littermates. To address whether the
regulation of the mouse bcl-2 gene is also affected by serum
cholesterol levels, monocytes from apoE-deficient mice and
littermate wild-type controls were purified and mouse bcl-2 mRNA
levels were compared using quantitative RT-PCR. By this method,
mouse bcl-2 MRNA levels were significantly lower in the
apoE-deficient mice relative to the wild-type controls (FIG.
3).
[0376] The differential expression pattern of the human glutathione
peroxidase gene (HUMGPXP1) was also discovered. The differential
expression of HUMGPXP1 was initially detected in a preliminary
detection system, described, below, in Section 7.1.2. Once HUMGPXP1
sequences were obtained, the gene's differential expression pattern
was verified and characterized under the physiologically relevant
conditions of Paradigm B. Glutathione peroxidase is known to be
involved in the removal of toxic peroxides that form in the course
of growth and metabolism under normal aerobic conditions and under
oxidative stress. Human plasma glutathione peroxidase gene was
originally isolated from a human placenta cDNA library (Takahashi
et al., 1990, J. Biochem. 108: 145-148). It has been shown to be
expressed in two human cell lines of the myeloid lineage (Porter et
al., 1992, Clinical Science 83: 343-345). Other studies have also
linked reduced levels of this enzyme with heart attack risk (Guidi,
et al., 1986, J. Clin. Lab Invest. 46: 549-551; Wang et al., 1981,
Klin. Wochenschr. 59: 817-818; Kok et al., 1989, J. Am. Med. Assoc.
261: 1161-1164; and Gromadzinska & Sklodowska, 1990, J. Am.
Med. Assoc. 263: 949-950). Glutathione peroxidase has not been
previously known to be down-regulated in human monocytes under
cardiovascular disease conditions, as described herein.
[0377] Interestingly, bcl-2 has been recognized as playing a key
role in preventing apoptosis, and expression of glutathione
peroxidase in the absence of bcl-2 is able to compensate for this
loss by preventing apoptosis (Hockenbery et al., 1993, Cell 75:
241-251). These findings regarding bcl-2 and HUMGPXP1, described
herein in this section, suggested a novel role for the monocyte in
plaque formation which involves apoptosis induction caused by high
LDL concentrations inside the cell, or perhaps by oxidative stress
in the cell mediated by oxidized LDL.
[0378] To confirm this relationship between apoptosis and
atherosclerosis, the ability of bcl-2 expression to ameliorate
atherosclerosis is tested. Because bcl-2 is normally down-regulated
under atherogenic conditions, a transgenic mouse strain is
engineered in which the human bcl-2 gene is expressed under the
control of the scavenger receptor promoter, which is induced in
monocyte foam cells under atherogenic conditions. This transgenic
mouse is then crossed with an apoE-deficient atherosclerotic mouse
model. The ability of the increased expression of the bcl-2 target
gene to ameliorate atherosclerosis is demonstrated by a decrease in
initiation and progression of plaque formation observed in the
transgenic apoE-deficient mouse.
[0379] The identification of the differential expression of these
genes, therefore, provides targets for the treatment and diagnosis
of cardiovascular disease. Intervening in the apoptotic pathway
through Bcl-2 and glutathione peroxidase, may lead to lesion
regression or prevention of plaque formation, or both. Furthermore,
the discovery of a connection between the apoptotic pathway and
atherosclerosis demonstrates the effectiveness of the methods
described herein in identifying the full panoply of gene products
that are involved in the atherosclerotic disease process.
Furthermore, the down-regulation of bcl-2 and HUMGPXP1 under
Paradigm B provides a fingerprint for the study of the effect of
excess LDL on monocytes.
[0380] 7.1. Materials and Methods
[0381] 7.1.1. in vivo Cholesterol Studies
[0382] Patients were held in a clinical setting for a total of 9
weeks during which time their lipid intake was very tightly
controlled. There were a total of 3 diets, and each patient was
held on each diet for 3 weeks. Patients were healthy young (third
decade of life) individuals with no history or symptoms of heart
disease or dislipidemias. The 3 diets are described below:
4 American Heart Association Diet II fat 25% cholesterol 80 mg/1000
kCal polyunsaturated/saturated fat 1.5 Average American Diet fat
43% cholesterol 200 mg/1000 kCal polyunsaturated/saturated fat 0.34
Combination Diet fat 43% cholesterol 80 mg/1000 kCal
polyunsaturated/saturated fat 0.34
[0383] The 3 diets were isocaloric, and the individual components
of each diet may vary with the participant's preference as long as
the lipid levels in the diet were maintained.
[0384] Cell Isolation
[0385] At the end of each 3 week diet period, blood was drawn from
each patient after a 12 hour period of fasting and monocytes were
purified. 50 ml of blood was drawn into 5 evacuated tubes
containing 1.4 ml each of citrate phosphate dextrose to prevent
coagulation. Blood was pooled into 50 ml tubes and spun at 400 g
(1250 RPM/Sorvall RC3B) for 15 minutes at 4.degree. C. The upper
serum layer (.about.25 ml) was then removed with a pipette and
replaced with phosphate buffered saline (PBS) at 4.degree. C. The
blood was mixed and then spun at 1850 .times. g (2680 RPM) for 15
minutes at 4.degree. C. Most of the clear upper layer was removed
with a pipette, before the buffy coat at the interface was taken in
.about.5 ml. The buffy coat was placed into a separate 50 ml tube,
and the pipette used to remove it was washed with 20 ml PBS. A
small aliquot of these cells was then diluted 1:1000 in PBS and
counted under a microscope using a hemacytometer. Red blood cell
concentration was then adjusted with PBS to a final concentration
of 1.5.times.10.sup.9/ml, and 10 ml aliquots were added to
Leucoprep Becton Dickinson) tubes for monocyte isolation. Tubes
were spun for 25 minutes at 25.degree. C. in a Sorvall RT6000 with
the brake off. Most of the clear upper layer was discarded, and the
turbid layer above the gel was saved and pooled in 50 ml tubes. The
volume of each tube was then increased to 50 ml with 25.degree. C.
PBS, and spun at 1000 RPM (Sorvall RC3B) for 10 minutes at
4.degree. C. The liquid was then discarded, the pellet was
resuspended in 50 ml PBS, and spun again. This process was repeated
3 more times. The final cell pellet was then resuspended in 2 ml
RNA lysis buffer (Sambrook et al., 1989, supra) and frozen for
subsequent RNA isolation as described above in Section 6.1.1.
[0386] Differential display, Northern analysis, RT-PCR, subcloning,
and DNA sequencing were carried out as described, above, in Section
6.1.2.
[0387] 7.1.2. Preliminary Detection System
[0388] The preliminary detection system described in this section
was used to identify sequences that are differentially expressed in
a readily assayed, in vitro system. Sequences that showed some
homology to those thought to be involved in cardiovascular disease
were then used as specific primers or probes, or both, in Paradigm
B, wherein the differential expression was ascertained under
physiologically relevant conditions, as described in section 7.1.1,
above.
[0389] Cell Culture
[0390] Blood (.about.100 ml) was drawn from healthy human donors
into vacutainer tubes containing heparin (Becton Dickinson). Blood
was diluted 1:1 with PD (Phosphate buffered saline (PBS) without Ca
or Mg, plus 0.3 mM EDTA), and layered onto Ficoll (Lymphocyte
Separation Media--Organon Teknikon) as 30 ml of blood/7 ml ficoll
in a 50 ml blue-capped Falcon tube, and centrifuged at 2000 RPM for
25 min. at room temperature (r.t.). The buffy coat was removed with
a pipette, transferred to another 50 ml tube, diluted to 30 ml with
PD, and centrifuged at 1200 RPM for 10 min. at r.t. The pellet was
resuspended in 30 ml PD and the previous centrifugation step was
repeated. The pellet was resuspended in 40 ml RPMI (2 mM
1-Glutamine+penicillin/streptomycin), plated onto 4 plates, and
incubated at 37.degree. C. for 2 hours. Supernatant was removed,
and the plates were washed 3.times. with PBS at 37.degree. C.
Plates were finally resuspended in 10 ml each with RPMI/20% human
AB serum (Sigma, St. Louis, MO.). On day 5, the media was changed
and 100 units/ml of human .gamma.-IFN (Genzyme) were added. On day
7, the media was removed and replaced with RPMI/20% human
LDL-deficient serum +100 units/ml of human .gamma.-IFN. Native,
oxidized, and acetylated LDL were each added to one plate with the
fourth plate serving as control. After the specified incubation
time (5 hr. or 24 hr.) the media was removed and the cells were
resuspended in 2 ml guanidine isothiocyanate RNA lysis buffer
(Sambrook et al., 1989, supra). Lysed cells were then syringed with
23 G. needle, layered over 5.7M CsCl, and centrifuged for 20 hr. at
35K RPM. RNA was isolated according to the method of Sambrook et
al., 1989, supra.
[0391] Lipoproteins were prepared as described, above, in section
6.1.1. Differential display, Northern analysis, RT-PCR, subcloning,
and DNA sequencing were carried out as described, above, in Section
6.1.2. For differential display, the primers used were T.sub.11CC
(reverse) and OPE4 (forward), consisting of 5'GTGACATGCC3'. For
RT-PCR, the first strand cDNA was primed with T.sub.11CC, and PCR
reactions were carried out with rfhma15 primers
(for-catgcctgtagaaaaaggtt/rev-cttcatagaatctaagccta), and mouse
.gamma.actin primers
(for-cctgatagatgggcactgtgt/rev-gaacacggcattgtc- actaact).
[0392] 7.1.3. Transgenic ApoE-Deficient Mouse Expressing Human
bcl-2
[0393] Transgenic mice bearing a construct (FIG. 32) with the mouse
scavenger receptor regulatory element (5 kb) (M. Freeman, et al.,
1995, unpublished results) driving expression of the human bcl-2
gene (hbcl-2) were produced. The scavenger receptor regulatory
element (ScR) is known to activate reporter gene expression in
peritoneal macrophages in transgenic mice (M. Freeman, 1995,
unpublished results). This 5 kb fragment is linked to the human
bcl-2 cDNA (Cleary, et al., 1986, supra) via a NotI restriction
site. Human growth hormone (hGH) sequences (Mayo, et al., 1983,
Nature 306: 86-88) are then ligated onto the 3' end of this
Construct through filled-in BamHI and EcoRV sites to provide
message stability. This construct is then digested with XhoI and
the 9 kb ScR-hbcl2-hGH sequences are purified away from vector
sequences. Another plasmid sample is digested with KpnI to yield a
fragment with only 1.5 kb of scavenger receptor regulatory
sequences which provide a lower level of expression. These
fragments are then injected independently into mouse embryos
derived from the FVB and C57BL/6 mouse strains according to
standard protocols (Hogan, et al., Manipulating the Mouse Embryo,
1994, Cold Spring Harbor Laboratory Press). Following birth, tail
sections are cut from mice derived from injected embryos and
analyzed for the presence of transgene sequences using hbcl-2
sequences as probes on Southern blots.
[0394] Transgenic mice bearing the ScR-hbcl2-hGH construct are then
bred to wild-type mice of the same respective strain, and then the
offspring are backcrossed to produce homozygous lines of mice.
These mice are then bred to apoE-deficient mice. offspring are
analyzed for presence of the ScR-hbcl2-hGH by preparing tail
sections and probing with hbcl-2 sequences on Southern blots.
Offspring are then analyzed for lesion formation and progression
according to the methods of Plump, et al., 1992, supra.
[0395] 7.2. Results
[0396] Differential display analysis was carried out on monocyte
RNA derived from the blood of patients whose serum cholesterol
levels were manipulated through fat/cholesterol intake in their
diets. FIG. 1 shows band #14 which was present in the low dietary
fat/low serum cholesterol conditions and goes away in the high
dietary fat/high serum cholesterol conditions. When a radioactively
labeled probe was prepared from band #14 and hybridized with a
Northern blot prepared from RNA from the same patient (FIG. 2), an
8 kb band was seen which was present in low serum cholesterol and
disappeared in high serum cholesterol conditions. When band #14
sequences were subcloned, sequenced, and compared with the sequence
database a 98% (203/207 bp) sequence similarity with the human
bcl-2 gene (Cleary et al., 1986, Cell 47, 19-28) was obtained,
indicating that band #14 is bcl-2.
[0397] Glutathione peroxidase (HUMGPXP1) in expression in monocytes
was examined to determine its physiological relationship to bcl-2.
Differential expression of HUMGPXP1 was first detected in a
preliminary detection system using monocytes cultured in vitro.
Human monocytes were prepared as described above in subsection
7.1.2. Cells were lysed after 5 hours and RNA was prepared.
Differential display analysis was carried out, and regulated bands
were isolated and characterized. The DNA sequence was determined
from a number of independent subclones of amplified sequences of
one such regulated band designated band 15. Using the BLAST program
(Altschul, et al., 1990, J. Mol. Biol. 215: 403-410), a 176/177
(99%) sequence similarity was found between band 15 a sequence for
human plasma glutathione peroxidase exon 1 (HUMGPXP1). This
sequence occurs upstream of the reported transcription start site.
Nonetheless, RT-PCR analysis confirmed that the band 15 sequences
are in fact within the same transcription unit as sequences
downstream of the reported transcription start site.
[0398] Based on this preliminary result, the gene expression
pattern of glutathione peroxidase (HUMGPXP1) was further analyzed
for verification and characterization in physiologically relevant
samples according to Paradigm B. Monocytes derived from human blood
under atherogenic conditions (high serum cholesterol) and healthy
conditions (low serum cholesterol) were examined with RT-PCR. As
shown in FIG. 4, there appears to be 2-3 fold less cDNA amplified
by the HUMGPXP1 primers from the high fat/cholesterol monocytes
than in the low fat/cholesterol monocytes, while the actin control
bands are the same.
[0399] Monocytes from apoE-deficient mice and littermate wild-type
controls were purified and mouse bcl-2 mRNA levels were compared
using quantitative RT-PCR. By this method, mouse bcl-2 mRNA levels
were significantly lower in the apoE-deficient mice relative to the
wild-type controls (FIG. 3).
[0400] These results demonstrate that bcl-2 is an excellent target
gene for intervening in lesion formation and development. It was
previously known that, under normal conditions, bcl-2 expression
prevents apoptosis. The observed down-regulation of bcl-2 caused by
atherogenic conditions, therefore, provides an explanation of how
such atherogenic conditions may lead to plaque formation. By
down-regulating the normally protective bcl-2 gene, high serum
cholesterol triggers a series of events, entailing the induction of
the apoptotic pathway, which results in programmed cell death,
which in turn causes an inflammatory response and subsequent plaque
formation.
[0401] This model may be tested by counteracting the observed
down-regulation of bcl-2. The human bcl-2 gene is placed in the
ScR-hbcl2-hGH construct in which it is transcribed by a promoter
that is activated in monocyte foam cells under atherogenic
conditions. This construct is then introduced into an
apoE-deficient mouse that otherwise serves as a model for
atherosclerosis. The effect of bcl-2 expression on atherosclerosis
is evidenced by the reduction in plaque initiation and development
in the apoE-deficient mice bearing the construct. Amelioration of
atherosclerosis may, therefore, be accomplished by such
intervention in the down-regulation of the bcl-2 target gene.
8. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN
RESPONSE TO PARADIGM C: IL-1 INDUCTION OF ENDOTHELIAL CELLS
[0402] According to the invention, differential display was used to
detect four novel genes that are differentially expressed in
endothelial cells that were treated in vitro with IL-1. Three of
these genes, rchdO24, rchdO32, and rchdO36, are not homologous to
any known gene. The fourth gene, rchdoo5, is 70% homologous to a
cloned shark gene called bumetanide-sensitive Na--K--Cl cotransport
protein. A human homolog of this gene has been reported, but the
sequence has not yet been published (1994, Proc. Natl. Acad. Sci.
USA 91: 2201-2205). The discovery of the up-regulation of these
four genes provides a fingerprint profile of IL-1 induced
endothelial cells. This fingerprint profile can be used in the
treatment and diagnosis of cardiovascular iseases, including but
not limited to atherosclerosis, ischemia/reperfusion, hypertension,
restenosis, and arterial inflammation.
[0403] 8.1. Materials and Methods
[0404] Primary cultures of HUVEC's were established from normal
term umbilical cords as described (In Progress in Hemostasis 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 passaged 1-3 times
before activation.
[0405] For activation, cells were cultured with 10 units/ml of
human IL-1.beta. for 1 or 6 hr. before lysis in guanidinium
isothiocyanate RNA lysis buffer (Sambrook et al., 1989, supra).
Lysed cells were then syringed with a 23 G. needle, layered over
5.7M CsCl, and centrifuged for 20 hr. at 35K.
[0406] Alternatively, cells were induced in the presence of 100
.mu.M lysophosphatidylcholine, or 50 .mu.g/ml oxidized human LDL
(Sigma) for periods of 1 or 6 hr. RNA was isolated as described,
above, in Section 6.1. Differential display, Northern analysis,
RT-PCR, subcloning, and DNA sequencing were carried out as
described, above, in Section 6.1.2, except that Northern blot
hybridizations were carried out as follows: for pre-hybridization,
the blot was placed into roller bottle containing 10 ml of
rapid-hyb solution (Amersham), and placed into 65.degree. C.
incubator for at least 1 hr. For hybridization, 1.times.10.sup.7
cpm of the probe was then heated to 95.degree. C., chilled on ice,
and added to 10 ml of rapid-hyb solution. The prehybridization
solution was then replaced with probe solution and incubated for 3
hr at 65.degree. C. The following day, the blot was washed once for
20 min. at r.t. in 2.times. SSC/0.1% SDS and twice for 15 min. at
65.degree. C. in 0.1.times. SSC/0.1% SDS before being covered in
plastic wrap and put down for exposure.
[0407] Chromosomal locations were determined according to the
method described in Section 6.1.3, above. For rchdO24, the primers
used were for-cccatagactaggctcatag, and
rev-tttaaagagaaattcaaatc.
[0408] 8.2. Results
[0409] HUVEC's were activated with 10 units/ml IL-1.beta. for 1 or
6 hours and compared to resting HUVEC's using differential display.
As shown in FIG. 5, a band marked rchd005 is present in lanes 11
and 12 (IL-1, 6 hr.) but not in lanes 9 and 10 (control), or lanes
7 and 8 (IL-1, 1 hr.). This band, rchd005, was isolated and
subcloned and sequenced. When a probe prepared form this band was
used to screen a Northern blot, expression was seen at 6 hr., but
not at 1 hr. or in the control (FIG. 6). However, when this same
probe was hybridized to a Northern blot prepared from shear
stressed RNA, according to Paradigm D described in Section 9,
below, a different pattern of up-regulation was also seen (FIG. 7).
Expression was up at 1 hr. and then nearly disappeared by 6 hr.
Amplified rchdOo5 DNA was subcloned and sequenced. Sequence
analysis revealed an approximately 360 bp insert (FIG. 8) with 70%
sequence similarity to a cloned shark gene called
bumetanide-sensitive Na--K--Cl cotransport protein.
[0410] Another IL-1 inducible band, rchd024, is shown in FIG. 9.
Northern analysis on IL-1 up-regulated RNA reveals a kb message
present at 6 hr. (FIG. 10) that also shows a low level of
up-regulation under shear stress at 6 hr. (FIG. 11). The DNA
sequence was obtained from subclones of amplified DNA (FIG. 12).
Database searching revealed no significant sequence similarities. A
PCR amplification experiment determined that the rchd024 gene is
located on human chromosome 4.
[0411] Band rchd032 was isolated on the basis of its differentially
increased expression after 6 hr. treatment with IL-1 (FIG. 13),
which was confirmed by RT-PCR analysis (FIG. 14). Amplified rchd032
sequences were subcloned and sequenced (FIG. 15). No significant
homology to any known gene was found.
[0412] Band rchd036 was also isolated on the basis of its
differential expression 6 hr. after IL-1 treatment (FIG. 16).
Northern analysis (FIG. 17) revealed an 8 kb band which was
up-regulated 6 hr. after IL-1 treatment. Another Northern analysis
was performed testing rchd036 under the shear stress condition of
Paradigm D, which are described in the example in Section 9, below.
Interestingly, rchd036 is not induced by shear stress, as indicated
by the lack of any band after either 1 hr. or 6 hr. of treatment
(FIG. 33). This tesult provides an example of an IL-1-inducible
endothelial cell gene that is not regulated by shear stress,
indicating that these induction pathways can be separated, and may
provide for drugs with greater specificity for the treatment of
inflammation and atherosclerosis. The DNA sequence was obtained
from subclones of amplified DNA (FIG. 18), and a search of the
database revealed no sequence similarities. A PCR amplification
experiment determined that the rchd036 gene is located on human
chromosome 15.
9. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN
RESPONSE TO PARADIGM D: ENDOTHELIAL CELL SHEAR STRESS
[0413] According to the invention, differential display was used to
detect genes that are differentially expressed in endothelial cells
that were subjected to fluid shear stress in vitro. Shear stress is
thought to be responsible for the prevalence of atherosclerotic
lesions in areas of unusual circulatory flow. Using the method of
Paradigm D, four bands with novel DNA sequences were identified.
rchd528 does not share homology with any known gene. rchd502, on
the other hand is homologous to rat matrin F/G mRNA sequence. This
rat gene encodes a protein that is found in the nuclear matrix and
contains the zinc finger DNA binding motif, (Hakes, et al., 1991,
Proc. Natl. Acad. Sci. U.S.A. 88:6186-6190). In fact, the sequences
in rchd502 encode part of the zinc finger portion of the protein.
Given that rchd502 is up-regulated by a mechanical force and the
rat matrin protein is a nuclear structural protein that also binds
to DNA, rchd502 may be involved in translating a physical force on
the cell into a program of gene expression. Furthermore, rchd502 is
first gene demonstrated to be up-regulated by shear-stress but not
by IL-1. It therefore provides an excellent novel tool for
diagnosis and treatment of cardiovascular disease.
[0414] The complete sequence of the rchd523 gene reveals that it
encodes a novel G protein-coupled receptor protein, consisting of
375 amino acids and a multiple transmembrane domain sequence motif.
The discovery of such a novel protein is particularly useful in
designing treatments as well as diagnostic and monitoring systems
for cardiovascular disease. In carrying out signal transduction, G
proteins play an important early role in the pathways that cause
changes in cellular physiology. The rchd523 gene product,
therefore, provides an excellent target for intervention in the
treatment of cardiovascular disease. Furthermore, as a
transmembrane protein, the rchd523 gene product can be readily
accessed (e.g., by inhibitory compounds during treatment) or
detected on the endothelial cell surface. It, therefore, also
provides an excellent target for detection of cardiovascular
disease states in diagnostic systems, as well as in the monitoring
of the efficacy of compounds in clinical trials. Furthermore, the
extracellular domains of the rchd523 gene product provide
especially efficient screening systems for identifying compounds
that bind to the rchd523 gene product. Such compounds can be useful
in treating cardiovascular disease by inhibiting rchd523 gene
product activity.
[0415] The sequence of the complete coding region of the rchd534
gene was also obtained. The rchd534 gene encodes a novel protein
consisting of 235 amino acids.
[0416] Also using the method of Paradigm D, the previously
identified human prostaglandin endoperoxide synthase type II was
isolated. This gene was previously known to be involved in
inflammation, and to be up-regulated by IL-1 (Jones et al., 1993,
J. Biol. Chem. 268: 9049-9054), but its up-regulation by shear
stress was previously unknown. This result confirmed the general
effectiveness of the techniques used according to the invention in
the detection of genes involved cardiovascular disease.
[0417] Furthermore, the up-regulation of these five genes in shear
stressed endothelial cells provides a fingerprint for the study of
cardiovascular diseases, including but not limited to
atherosclerosis, ischemia/reperfusion, hypertension, and
restenosis. The fact that one of these genes, rchd502, is not
up-regulated under Paradigm C (IL-1 induction) provides an
extremely useful means of distinguishing and targeting
physiological phenomena specific to shear stress.
[0418] 9.1. Materials and Methods
[0419] Primary cultures of HUVEC's were established from normal
term umbilical cords as described (In Progress in Hemostasis 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 passaged 1-3 times
before shear stress induction.
[0420] For induction, second passage HUVEC's were plated on tissue
culture-treated polystyrene and subjected to 10 dyn/cm2 laminar
flow for 1 and 6 hr. as described (1994, J. Clin. Invest. 94:
885-891) or 3-10 dyn/cm.sup.2 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
display, Northern analysis, RT-PCR, subcloning, and DNA sequencing
were carried out as described, above, in Section 6.1.2, except that
Northern blot hybridizations were carried out as described, above,
in Section 8.1.
[0421] For rchd523, the RACE procedure kit was used to obtain the
entire coding region of the rchd523 gene. The procedure was carried
out according to the manufacturer's instructions (Clonetech, Palo
Alto, CA; see also: Chenchik, et al., 1995, CLONTECHniques (X) 1:
5-8; Barnes, 1994, Proc. Natl. Acad. Sci. USA 91: 2216-2220; and
Cheng et al., Proc. Natl. Acad. Sci. USA 91: 5695-5699). Primers
were designed based on amplified rchd523 sequences. Template mRNA
was isolated from shear stressed HUVEC's.
[0422] For rchd534, amplified sequences, which contained a portion
of the gene, were subcloned and then used to retrieve the entire
coding region of the rchd534 gene. Prob's were prepared by
isolating the subcloned insert DNA from vector DNA, and labeling
with 32p as described above in Section 6.1.2. Labeled insert DNA
was used to probe a cDNA library, prepared from mRNA which was
isolated from shear stressed HUVEC's as described in this section,
above. The cDNA library was produced and screened according to
well-known methods (Sambrook et al., 1989, supra), using the
bacteriophage X-ZAP vector (Stratagene, LaJolla, Calif.). Plaques
that were detected by the rchd534 probe were isolated and
sequenced.
[0423] Determination of chromosomal location was carried out
according to the method described in Section 6.1.3, above. The
primers used for rchd523 were (for-atgccgtgtgggttagtc) and
(rev-attttatgggaaggtttttaca); and for rchd534 were
(for-cttttctgcgtctcccat) and (rev-agacatcagaaactccaa- cc).
[0424] 9.2. Results
[0425] HUVEC's were subjected to laminar shear stress for 1 or 6
hr. and compared to static control cells in differential display.
As shown in FIG. 19, a band (rchd5o2) is identified which is found
in lanes 5,6 (6 hr.) but not in lanes 1,2 (control). This band was
excised, amplified, and sequenced. Northern analysis using
amplified rchd5o2 sequences revealed a 4.5 kb band that is
up-regulated at 6 hr. compared to controls (FIG. 20). When rchd502
probe was hybridized to a Northern blot prepared from IL-1 induced
endothelial cells, up-regulation of a 4.5 kb band is not seen (FIG.
21). This result provides the first example of a shear
stress-inducible endothelial cell gene that is not regulated by
IL-1, indicating that these induction pathways can be separated,
and may provide for drugs with greater specificity for the
treatment of inflammation and atherosclerosis. Sequencing was done,
and the resulting sequence is shown in FIG. 22. When this sequence
was compared to the sequence database, an 84% (183/217) sequence
similarity with Rat matrin F/G mRNA sequence was obtained.
[0426] Shear stress band rchd505 decreased 1 hr. and 6 hr. after
shear stress, as compared to untreated control cells (FIG. 23).
Northern analysis revealed differential expression except that
rchd505 was up-regulated after 1 hr. and 6 hr. shear stress
treatment (FIG. 24). This same band was similarly up-regulated in
cells treated with IL-1 according to Paradigm C (FIG. 25). Sequence
analysis revealed that rchd505 is the previously characterized
human endoperoxide synthase type II. rchd523 was detected under
differential display as a band up-regulated after 1 hr. and 6 hr.
shear stress treatment (FIG. 26). The 6 hr. up-regulation of
rchd523 was confirmed by RT-PCR (FIG. 27). Amplified rchd523
sequences were subcloned, and an isolate was sequenced and
designated pRCHD523. The RACE procedure was used to obtain a 2.5 kb
cDNA containing the entire coding sequence of the rchd523 gene. The
cDNA isolate containing the complete coding sequence of rchd523 is
designated pFCHD523. Sequence analysis revealed that the rchd523
gene product encodes a novel G protein-coupled receptor, consisting
of 375 amino acids and a multiple transmembrane domain sequence
motif. A PCR amplification experiment determined that the rchd523
gene is located on human chromosome 7. rchd528 was also detected as
an up-regulated band after 1 hr. and 6 hr. shear stress treatment
(FIG. 29). This result was confirmed by Northern analysis in which
probes of rchd528 amplified sequence detected an approximately 5.0
kb message that was up-regulated moderately after 1 hr., and
up-regulated very strongly after 6 hr. (FIG. 30). The amplified
sequences were subcloned and sequenced (FIG. 31). Comparison with
sequences in the database revealed no homologies between rchd528
and any known DNA sequence.
[0427] rchd534 also was detected as being up-regulated in response
to shear stress. Northern analysis revealed that rchd534 is
strongly induced after 6 hours of shear stress treatment (FIG. 34).
The amplified sequences were subcloned, sequenced, and re-isolated
for use as a probe for retrieving full-length rchd534 cDNA. A 3.3
kb X-ZAP clone was sequenced to reveal full-length rchd534 cDNA
(FIG. 35). This clone containing the entire coding region the
rchd534 gene was designated pFCHD534. The encoded protein consists
of 235 amino acids. Comparison with sequences in the database
revealed no homologies between rchd534 and any known DNA sequences.
A PCR amplification experiment determined that the rchd523 gene is
located on human chromosome 15. rchd534 was also shown not to be
regulated by IL-1 when tested under the conditions of Paradigm C,
as described in Section 8, above. Just like rchd502, rchd534 is an
example of a shear stress-inducible endothelial cell gene that is
not regulated by IL-1, confirming that these induction pathways can
be separated, and may provide for drugs with greater specificity
for the treatment of inflammation and atherosclerosis.
10. EXAMPLE: USE OF GENES UNDER PARADIGM A AS SURROGATE MARKERS IN
CLINICAL TRIALS
[0428] 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 used to monitor clinical trials of drugs in human
patients. The fingerprint profile, described generally in Section
5.5.4, above, indicates the characteristic pattern of differential
gene regulation corresponding to a particular disease state.
Paradigm A, described in Section 5.1.1.1, and illustrated in the
example in Section 6, above, for example, provides the fingerprint
profile of monocytes under oxidative stress. This profile gives an
indicative reading, therefore, 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 monocytes of patients
undergoing clinical tests.
[0429] 10.1. Treatment of Patients and Cell Isolation
[0430] Test patients may be administered compounds suspected of
having anti-oxidant activity. Control patients may be given a
placebo.
[0431] Blood may be drawn from each patient after a 12 hour period
of fasting and monocytes may be purified as described, above, in
Section 7.1.1. RNA may be isolated as described in Section 6.1.1,
above.
[0432] 10.2. Analysis of Samples
[0433] RNA may be subjected to differential display analysis as
described in Section 6.1.2, above. A decrease in the physiological
response state of the monocytes is indicated by a decreased
intensity of those bands that were up-regulated by oxidized LDL
under Paradigm A, and an increased intensity of those bands that
were down-regulated by oxidized LDL under Paradigm A, as described
in Section 6.2, above.
11. EXAMPLE: IMAGING OF A CARDIOVASCULAR DISEASE CONDITION
[0434] According to the invention, differentially expressed gene
products which are localized on the surface of affected tissue may
be used as markers for imaging the diseased or damaged tissue.
Conjugated antibodies that are specific to the differentially
expressed gene product may be administered to a patient or a test
animal intravenously. This method provides the advantage of
allowing the diseased or damaged tissue to be visualized
non-invasively.
[0435] 11.1. Monoclonal Conjugated Antibodies
[0436] The differentially expressed surface gene product, such as
the rchd523 gene product, is expressed in a recombinant host and
purified using methods described in Section 5.4.2, above.
Preferably, a protein fragment comprising one or more of the
extracellular domains of the rchd523 product is produced. Once
purified, it is be used to produce F(ab').sub.2 or Fab fragments,
as described in Section 5.4.3, above. These fragments are then
labelled with technetium-99m (.sup.99mTc) using a conjugated metal
chelator, such as DTPA as described in section 5.8.3, above.
[0437] 11.2. Administration and Detection of Imaging Agents
[0438] Labeled MAb may be administered intravenously to a patient
being diagnosed for atherosclerosis, restenosis, or
ischemia/reperfusion. Sufficient time is allowed for the
detectably-labeled antibody to localize at the diseased or damaged
tissue site (or sites), and bind to the rchd523 gene product. The
signal generated by the label is detected by a photoscanning
device. The detected signal is then converted to an image of the
tissue, revealing cells, such as endothelial cells, in which
rchd523 gene expression is up-regulated.
12. EXAMPLE: SCREENING FOR LIGANDS OF THE rchd 523 GENE PRODUCT AND
ANTAGONISTS OF rchd523 GENE PRODUCT-LIGAND INTERACTION
[0439] The rchd523 gene product is a member of the G
protein-coupled receptor protein family, containing multiple
transmembrane domains. The receptor binding activity of this
protein family is detected by assaying for Ca.sup.2+ mobility
through the membrane of cells in which the receptor gene is
expressed. This assay, described below, is used to identify ligands
that bind to the rchd523 gene product receptor. Establishing this
ligand-receptor activity then provides for a screen in which
antagonists of the ligand-receptor interaction are identified. An
antagonist is detected by its ability to inhibit the Ca.sup.2
mobility induced by ligand-receptor binding. Such antagonists,
therefore, provide compounds that are useful in the treatment of
cardiovascular disease, by counteracting the activity of the
product of this target gene which is up-regulated in the disease
state.
[0440] Binding of ligand to the rchd523 gene product is measured as
follows. The cDNA containing the entire coding region of the
rchd523 gene is removed from pFCHD523 and placed under the control
of a promoter that is highly expressed in mammalian cells in an
appropriate expression vector. The resulting construct is
transfected into myeloma cells, which are then loaded with FURA-2
or INDO-1 by standard techniques. Ligands are added to the cell
culture to test their ability to bind to the rchd523 receptor in a
manner that triggers signal transduction, as measured by Ca.sup.2+
mobilization across the cell membrane. Mobilization of Ca.sup.2
induced by ligand is measured by fluorescence spectroscopy as
described in Grynkiewicz et al., 1985, J. Biol. Chem. 260:3440.
Ligands that react with the target gene product receptor domain are
identified by their ability to produce a fluorescent signal. Their
receptor binding activities are quantified and compared by
measuring the level of fluorescence produced over background.
[0441] Candidate antagonists are then screened for their ability to
interfere with ligand-receptor binding. Myeloma transfectants
expressing rchd523 gene product are treated with ligand alone, and
ligand in the presence of candidate antagonist. Candidate
antagonists that cause a reduction in the fluorescence signal are
designated antagonists of the ligand-rchd523 receptor
interaction.
13. DEPOSIT OF MICROORGANISMS
[0442] The following microorganisms were deposited with the
Agricultural Research Service Culture Collection (NRRL), Peoria,
Illinois, on January 11, 1995 and assigned the indicated-accession
numbers:
5 Microorganism NRRL Accession No. RCHD005 B-21376 RCHD024 B-21377
RCHD032 B-21378 RCHD036 B-21379 RCHD502 B-21380 RCHD523 B-21381
RCHD528 B-21382
[0443] The following microorganisms were deposited with the
Agricultural Research Service Culture Collection (NRRL), Peoria,
Ill., on Jun. 6, 1995 and assigned the indicated accession
numbers:
6 Microorganism NRRL Accession No. FCHD523 FCHD534
[0444] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
Sequence CWU 1
1
* * * * *