U.S. patent application number 08/870434 was filed with the patent office on 2002-03-21 for compositions and methods for the treatment and diagnosis of cardiovascular disease.
Invention is credited to FALB, DEAN A., Gimbrone, Michael A. JR..
Application Number | 20020034736 08/870434 |
Document ID | / |
Family ID | 26682786 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020034736 |
Kind Code |
A1 |
FALB, DEAN A. ; et
al. |
March 21, 2002 |
COMPOSITIONS AND METHODS FOR THE TREATMENT AND DIAGNOSIS OF
CARDIOVASCULAR DISEASE
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
1155 AVENUE OF THE AMERICAS
NEW YORK
NY
100362711
|
Family ID: |
26682786 |
Appl. No.: |
08/870434 |
Filed: |
June 6, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08870434 |
Jun 6, 1997 |
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08799910 |
Feb 13, 1997 |
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60011787 |
Feb 16, 1996 |
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Current U.S.
Class: |
435/6.16 ;
424/130.1; 435/7.1; 514/1.9; 514/12.2; 514/13.3; 514/15.1;
514/15.7; 514/16.4; 514/19.3; 514/44A; 514/8.9; 530/350; 530/387.1;
536/23.1 |
Current CPC
Class: |
C12N 2310/15 20130101;
A61K 38/00 20130101; C12N 15/113 20130101; A61P 9/00 20180101; A61P
9/12 20180101; C12N 2310/121 20130101; A01K 2217/05 20130101; C12N
2310/12 20130101; A61P 9/10 20180101; C07K 14/47 20130101 |
Class at
Publication: |
435/6 ; 514/12;
424/130.1; 536/23.1; 514/44; 435/7.1; 530/387.1; 530/350 |
International
Class: |
A61K 039/395; C07K
017/00; C07K 016/00; C07K 014/00; G01N 033/53; C12Q 001/68; C07H
021/04; A01N 043/04 |
Claims
What is claimed is:
1. An isolated polynucleotide containing the following nucleotide
sequence: fchd531 (SEQ ID NO.:1), fchd540 (SEQ ID NO.:2), fchd545
(SEQ ID NO.:3), fchd602 (SEQ ID NO.:4) or, fchd605 (SEQ ID NO.:5).
or the nucleotide sequence of a gene or gene fragment contained in
the following clone as deposited with the ATCC: pFCHD531 (in ATCC
Accession No. 69983), pFCHD540 (in ATCC Accession No. 69984), or
fchd545 (in ATCC Accession No. 69974).
2. An isolated polynucleotide 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 ATCC.
3. An isolated polynucleotide which encodes an amino acid sequence
encoded by the nucleotide sequence of claim 1 or its complement, or
encoded by the gene or gene fragment contained in the clone of
claim 1 as deposited with the ATCC.
4. A polynucleotide vector containing the nucleotide sequence of
claim 1, 2 or 3.
5. A polynucleotide 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 polynucleotide
of claim 1, 2 or 3.
7. A genetically engineered host cell containing the polynucleotide
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 polynucleotide
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 polynucleotide 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
assaying, in a patient sample, the differential expression of a
gene encoding the fchd531 protein, the fchd540 protein, the fchd545
protein, the fchd602 protein, or the fchd605 protein.
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.
18. The method of claim 17 in which the gene encodes the fchd540,
fchd602, or fchd605 protein.
19. The method of claim 12 in which the gene is down-regulated.
20. The method of claim 19 in which the gene encodes the fchd531 or
fchd545 protein.
21. A method for treating cardiovascular disease, comprising
administering a compound that modulates the expression of, or the
activity of the encoded protein product of, the fchd531 gene, the
fchd540 gene, the fchd545 gene, the fchd602 gene, or the fchd605
gene to a patient in need of such treatment.
22. The method of claim 21 in which the cardiovascular disease is
atherosclerosis.
23. The method of claim 21 in which the cardiovascular disease is
ischemia/reperfusion.
24. The method of claim 21 in which the cardiovascular disease is
hypertension.
25. The method of claim 21 in which the cardiovascular disease is
restenosis.
26. The method of claim 21 in which the compound inhibits the
expression of the gene or the activity of the protein product.
27. The method of claim 26 in which the gene is the fchd540,
fchd602, or fchd605 gene.
28. The method of claim 27 in which the compound is an antisense or
ribozyme molecule that blocks translation of the gene.
29. The method of claim 27 in which the compound is complementary
to the 5' region of the gene and blocks transcription via triple
helix formation.
30. The method of claim 26 in which the compound is an antibody
that inhibits the activity of the protein product.
31. The method of claim 21 in which the compound enhances the
expression of the gene or the activity the protein product.
32. The method of claim 31 in which the gene is the fchd531 or
fchd545 gene.
33. A method for treating cardiovascular disease, comprising
administering a polynucleotide encoding the fchd531 or fchd545
protein to a patient in need of such treatment.
34. A method for treating cardiovascular disease, comprising
administering an effective amount of the fchd531 or fchd545 protein
to a patient in need of such therapy.
35. A method for monitoring the efficacy of a compound in clinical
trials for the treatment of cardiovascular disease, comprising
assaying, in a patient sample, the differential expression of a
gene encoding the fchd531 protein, the fchd540 protein, the fchd545
protein, the fchd602 protein, or the fchd605 protein.
36. The method of claim 35 in which the cardiovascular disease is
atherosclerosis.
37. The method of claim 35 in which the cardiovascular disease is
ischemia/reperfusion.
38. The method of claim 35 in which the cardiovascular disease is
hypertension.
39. The method of claim 35 in which the cardiovascular disease is
restenosis.
40. The method of claim 35 in which the gene is up-regulated.
41. The method of claim 40 in which the gene encodes the fchd540,
fchd602, or fchd605 protein.
42. The method of claim 35 in which the gene is down-regulated.
43. The method of claim 42 in which the gene encodes the fchd531 or
fchd545 protein.
44. A method for identifying a substance for treating
cardiovascular disease comprising assaying the ability of the
substance to modulate the expression of, or the activity of the
encoded protein product of, the fchd531 gene, the fchd540 gene, the
fchd545 gene, the fchd602 gene, or the fchd605 gene.
45. The method of claim 44 in which the cardiovascular disease is
atherosclerosis.
46. The method of claim 44 in which the cardiovascular disease is
ischemia/reperfusion.
47. The method of claim 44 in which the cardiovascular disease is
hypertension.
48. The method of claim 44 in which the cardiovascular disease is
restenosis.
49. The method of claim 44 in which the modulation of expression of
said gene is assayed by: (a) exposing a sample of cells to a test
substance; (b) assaying the expression of said gene in the sample
of cells; and (c) comparing the expression level of the gene in the
sample exposed to the substance to the expression level of the gene
in a control sample of cells, in which a difference between the
expression level of the gene in the sample exposed to the substance
and the control indicates the modulation of expression of the
gene.
50. The method of claim 49 in which the gene is down-regulated by
the test substance.
51. The method of claim 50 in which the substance is an
oligonucleotide complementary to the 5' region of the gene and
blocks transcription via triple helix formation.
52. The method of claim 50 in which the substance is an antisense
or ribozyme molecule that blocks translation of the gene.
53. The method of claim 49 in which the gene is up-regulated by the
test substance.
54. The method of claim 44 in which the substance is a small
organic or inorganic molecule that modulates the activity of the
protein product by binding to the protein product.
55. The method of claim 44 in which the substance is an antibody
that modulates the activity of the protein product by binding to
the protein product.
56. An assay for identifying a substance that binds to the fchd531
protein, the fchd540 protein, the fchd545 protein, the fchd602
protein, or the fchd605 protein, comprising: (a) contacting a
protein or peptide containing an amino acid sequence corresponding
to the binding site of the protein with a test substance, under
conditions and for a time sufficient to permit binding and
formation of a complex between the protein or peptide and the test
substance, and (b) detecting the formation of a complex, in which
the ability of the test substance to bind to the protein is
indicated by the presence of the test substance in the complex.
57. An assay for identifying a substance that inhibits the
interaction between the rchd534 protein and the fchd540 protein
comprising: (a) contacting a protein or peptide containing an amino
acid sequence corresponding to the binding site of the rchd534
protein with a protein or peptide containing an amino acid sequence
corresponding to the binding site of the fchd540 protein, under
conditions and for a time sufficient to permit binding and
formation of a complex, in the presence of a test substance, and
(b) detecting the formation of a complex, in which the ability of a
test substance to inhibit the interaction between the rchd534
protein and fchd540 protein is indicated by a decrease in complex
formation as compared to the amount of complex formed in the
absence of the test substance.
58. An assay for identifying a substance that inhibits the
interaction between two rchd534 protein molecules comprising: (a)
contacting a first protein or peptide containing an amino acid
sequence corresponding to the binding site of the rchd534 protein
with a second protein or peptide containing an amino acid sequence
corresponding to the binding site of the rchd534 protein, under
conditions and for a time sufficient to permit binding and
formation of a complex, in the presence of a test substance, and
(b) detecting the formation of a complex, in which the ability of a
test substance to inhibit the interaction between two rchd534
protein molecules is indicated by a decrease in complex formation
as compared to the amount of complex formed in the absence of the
test substance.
59. An assay for identifying a substance that inhibits the
interaction between the rchd534 protein and a protein member of the
TGF-.beta. signalling pathway comprising: (a) contacting a protein
or peptide containing an amino acid sequence corresponding to the
binding site of the rchd534 protein with a protein or peptide
containing an amino acid sequence corresponding to the binding site
of the protein member of the TGF-.beta. signalling pathway, under
conditions and for a time sufficient to permit binding and
formation of a complex, in the presence of a test substance, and
(b) detecting the formation of a complex, in which the ability of a
test substance to inhibit the interaction between the rchd534
protein and the protein member of the TGF-.beta. signalling pathway
is indicated by a decrease in complex formation as compared to the
amount of complex formed in the absence of the test substance.
60. The assay of claim 59 wherein the protein member of the
TGF-.beta. signalling pathway is MADR1, MADR2, DPC4, activated
T.beta.R1, activated ActR1b, or activated ALK6.
61. An assay for identifying a substance that inhibits the
interaction between the fchd540 protein and a protein member of the
TGF-.beta. signalling pathway comprising: (a) contacting a protein
or peptide containing an amino acid sequence corresponding to the
binding site of the fchd540 protein with a protein or peptide
containing an amino acid sequence corresponding to the binding site
of the protein member of the TGF-.beta. signalling pathway, under
conditions and for a time sufficient to permit binding and
formation of a complex, in the presence of a test substance, and
(b) detecting the formation of a complex, in which the ability of
the test substance to inhibit the interaction between the fchd540
protein and the protein member of the TGF-.beta. signalling pathway
is indicated by a decrease in complex formation as compared to the
amount of complex formed in the absence of the test substance.
62. The assay of claim 61 wherein the protein member of the
TGF-.beta. signalling pathway is MADR1, MADR2, DPC4, activated
T.beta.R1, activated ALK6, activated TSR1, activated ALK3, or
activated ActR1.beta..
63. A method for treating cardiovascular disease comprising
administering a compound that inhibits the interaction between the
rchd534 protein and the fchd540 protein.
64. A method for treating cardiovascular disease comprising
administering a compound that inhibits the interaction between two
rchd534 protein molecules.
65. A method for treating cardiovascular disease comprising
administering a compound that inhibits the interaction between the
rchd534 protein and a protein member of the TGF-.beta. signalling
pathway.
66. The method of claim 65 wherein the protein member of the
TGF-.beta. signalling pathway is MADR1, MADR2, DPC4, activated
T.beta.R1, activated ActR1b, or activated ALK6.
67. A method for treating cardiovascular disease comprising
administering a compound that inhibits the interaction between the
fchd540 protein and a protein member of the TGF-.beta. signalling
pathway.
68. The method of claim 67 wherein the protein member of the
TGF-.beta. signalling pathway is MADR1, MADR2, DPC4, activated
T.beta.R1, activated ALK6, activated TSR1, activated ALK3, or
activated ActR1.beta..
69. A method for identifying a substance that enhances the
TGF-.beta. signalling response comprising: (a) contacting a
genetically engineered cell with a test substance, said cell
comprising 1) a reporter gene in operative association with an
inducible TGF-.beta. regulatory element; 2) a recombinant gene
encoding the rchd534 protein or a recombinant gene encoding the
fchd540 protein; and 3) a recombinant gene encoding the MADR1
protein or a recombinant gene encoding the MADR2 protein; and (b)
detecting expression of said reporter gene in which ability of the
test substance to enhance the TGF-.beta. signalling response is
indicated by an increase in expression of the reporter gene as
compared to the amount of expression in the absence of the test
substance.
70. A method for identifying a substance for treating
fibroproliferative disease or oncogenic related disorders
comprising assaying the ability of the substance to modulate
expression of, or the activity of the encoded protein product of,
the rchd534 gene or the fchd540 gene.
71. The method of claim 70 in which the fibroproliferative disease
is diabetic retinopathy.
72. The method of claim 70 in which the oncogenic related disorder
is a tumor growth.
73. The method of claim 70 in which the oncogenic related disorder
is angiogenesis.
74. A method for treating fibroproliferative disease or oncogenic
related disorders comprising administering a compound that inhibits
the interaction between the rchd534 protein and a protein member of
the TGF-.beta. signalling pathway.
75. A method for treating fibroproliferative disease or compound
that inhibits the interaction between the rchd534 protein and the
fchd540 protein.
76. A method for treating fibroproliferative disease or oncogenic
related disorders comprising administering a compound that inhibits
the interaction between the fchd540 protein and a protein member of
the TGF-.beta. signalling pathway.
Description
[0001] This application is a continuation-in-part of application
Ser. No. 08/799,910, filed Feb. 13, 1997, which claims the benefit
under 35 U.S.C. .sctn.119(e) of co-pending provisional Application
No. 60/011,787 filed Feb. 16, 1996.
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. The present invention further relates to screening
methods to identify compositions and their therapeutic use for the
treatment of fibro-proliferative and oncogenic disorders, including
diabetic retinopathy, artherosclerosis, angiogenesis, inflammation,
fibrosis, tumor growth and vascularization. Genes which are
differentially expressed in cardiovascular or oncogenic disease
states, relative to their expression in normal, or non-disease
states are identified. Genes are also identified via the ability of
their gene products to interact with other gene products involved
in cardiovascular or oncogenic disease. The genes identified may be
used diagnostically or as targets for therapeutic intervention. 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 principal
cause of death in the United States. Atherosclerosis is a complex
disease involving many cell types and molecular factors (for a
detailed review, see Ross, 1993, Nature 362: 801-809). The process,
in normal circumstances a protective response to insults to the
endothelium and smooth muscle cells (SMCs) of the wall of the
artery, consists of the formation of fibrofatty and fibrous lesions
or plaques, preceded and accompanied by inflammation. The advanced
lesions of atherosclerosis may occlude the artery concerned, and
result from an excessive inflammatory-fibroproliferative response
to numerous different forms of insult. For example, shear stresses
are thought to be responsible for the frequent occurrence of
atherosclerotic plaques in regions of the circulatory system where
turbulent blood flow occurs, such as branch points and irregular
structures.
[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] 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.
[0010] 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.
[0011] For example, the expression of PDGF (platelet derived growth
factor) or its receptor was studied: in rats during repair of
arterial injury (Majesky et al., 1990, J. Cell Biol. 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-I, ICAM-1, and class II major histocompatibility
antigens were reported to be induced in response to injury to
rabbit aorta (Tanaka, et al., 1993, Circulation 88: 1788-1803).
[0013] 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 identify 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.
[0016] The present invention further relates to screening methods
to identify compositions and their therapeutic use for the
treatment of fibroproliferative and oncogenic disorders, including
diabetic retinopathy, cancer, tumorigenesis, vascularization of
tumors, angiogenesis artherosclerosis inflammation and
fibrosis.
[0017] "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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] The invention also provides methods for the identification
of compounds that modulate the expression of genes or the activity
of gene products involved in fibroproliferative or oncogenic
disorders, including tumorigenesis and the vascularization of
tumors.
[0023] 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.
[0024] In the working examples described herein, five novel human
genes are identified that are demonstrated to be differentially
expressed in different cardiovascular disease states. The
identification of these genes and the characterization of their
expression in particular disease states provide newly identified
roles in cardiovascular disease for these genes.
[0025] Specifically, fchd531, fchd540, and fchd545 are novel genes
that are each differentially regulated in endothelial cells
subjected to shear stress. fchd531 and fchd545 are each
down-regulated, whereas fchd540 is up-regulated by shear stress.
fchd602 and fchd605 are novel genes that are each up-regulated in
monocytes treated with oxidized LDL. Accordingly, methods are
provided for the diagnosis, monitoring in clinical trials,
screening for therapeutically effective compounds, and treatment of
cardiovascular disease based upon the discoveries herein regarding
the expression patterns of fchd531, fchd540, fchd545, fchd602, and
fchd605.
[0026] Both fchd540 and rchd534 are up-reguated in response to
laminar shear stress and are specifically expressed in vascular
tissue. These findings combined with the observations that both
fchd540 and rchd534 specifically inhibit TGF-.beta. signalling and
that these genes are located in an area of the human genome
implicated in the pathogenesis of several human malignancies
indicates that they are excellent and specific targets for
therapeutic intervention in the treatment of fibroproliferative and
oncogenic disorders including tumorigenesis and
vascularization.
[0027] The characteristic up-regulation of genes fchd540, fchd602,
and fchd605 can be used to design cardiovascular disease treatment
strategies. For those up-regulated genes that have a causative
effect on the disease conditions, treatment methods can be designed
to reduce or eliminate their expression, particularly in
endothelial cells or monocytes. Alternatively, treatment methods
include inhibiting the activity of the protein products of these
genes. For those up-regulated genes that have a protective effect,
treatment methods can be designed for enhancing the activity of the
products of such genes.
[0028] In either situation, detecting expression of these genes in
excess of normal expression provides for the diagnosis of
cardiovascular disease. Furthermore, in testing the efficacy of
compounds during clinical trials, a decrease in the level of the
expression of these genes corresponds to a return from a disease
condition to a normal state, and thereby indicates a positive
effect of the compound. The cardiovascular diseases that may be so
diagnosed, monitored in clinical trials, and treated include but
are not limited to atherosclerosis, ischemia/reperfusion,
hypertension, restenosis, and arterial inflammation.
[0029] The characteristic down-regulation of fchd531 and fchd545
can also be used to design cardiovascular disease treatment
strategies. For those genes whose down-regulation has a pathogenic
effect, treatment methods can be designed to restore or increase
their expression, particularly in endothelial cells. Alternatively,
treatment methods include increasing the activity of the protein
products of these genes. For those genes whose down-regulation has
a protective effect, treatment methods can be designed for
decreasing the amount or activity of the products of such
genes.
[0030] In either situation, detecting expression of these genes in
below normal expression provides for the diagnosis of
cardiovascular disease. Furthermore, in testing the efficacy of
compounds during clinical trials, an increase in the level of the
expression of these genes corresponds to a return from a disease
condition to a normal state, and thereby indicates a positive
effect of the compound. The cardiovascular diseases that may be so
diagnosed, monitored in clinical trials, and treated include but
are not limited to atherosclerosis, ischemia/reperfusion,
hypertension, restenosis, and arterial inflammation.
[0031] The invention encompasses methods for screening compounds
and other substances for treating cardiovascular disease by
assaying their ability to modulate the expression of the target
genes disclosed herein or activity of the protein products of the
target genes. The invention further encompasses methods for
screening compounds and other substances for treating
fibroproliferative disorders and oncogenic disorders by assaying
their ability to modulate the expression of the target genes
disclosed herein or activity of the protein products of the target
genes. Such screening methods include, but are not limited to,
assays for identifying compounds and other substances that interact
with (e.g., bind to) the target gene protein products.
[0032] In addition, the invention encompasses methods for treating
cardiovascular disease by administering compounds and other
substances that modulate the overall activity of the target gene
products. Compounds and other substances can effect such modulation
either on the level of target gene expression or target protein
activity.
[0033] The invention is based in part on the identification of
novel protein-protein interactions of the rchd534 protein with
itself and with the fchd540 protein, as well as interactions of the
rchd534 protein or the fchd540 protein with other protein members
of the TGF-.beta. signalling pathway. The rchd534 gene was
described in Applicant's co-pending Application No. 08/485,573,
filed Jun. 7, 1995, which is hereby incorporated by reference in
its entirety. Screening methods are provided for identifying
compounds and other substances for treating cardiovascular disease
by assaying their ability to inhibit these interactions.
Furthermore, methods are provided for identifying compounds and
other substances that enhance the TGF-.beta. response by modulating
the activity of the expression of the rchd534 or fchd540 genes or
the activity of their gene products. In addition, methods are
provided for treating cardiovascular disease by administering
compounds and other substances that inhibit these protein
interactions.
[0034] The invention is based in part on the identification of the
endothelial cell specific expression pattern of two genes, rchd534
and fchd540, whose protein products inhibit the TGF-.beta.
response. The fchd540 gene has been mapped to regions of the human
genome that have been implicated in the pathogenesis of several
human malignancies. The invention is further based on the finding
that these genes and mutants thereof may be used to modulate
TGF-.beta. induced signalling in endothelial cells. Accordingly,
the rchd534 and rchd540 genes may be targets for intervention in a
variety of inflammatory and fibroproliferative disorders that
involve endothelial cells, including, but not limited to, oncology
related disorders, disorders related to vascularization, such as
cancer angiogenesis, inflammation, and fibrosis.
[0035] Membrane bound target gene products containing extracellular
domains can be a particularly useful target for treatment methods
as well as diagnostic and clinical monitoring methods. The fchd602
gene, for example, encodes a transmembrane protein, which contains
multiple transmembrane domains and, therefore, can be readily
contacted by other compounds on the cell surface. Accordingly,
natural ligands, derivatives of natural ligands, and antibodies
that bind to the fchd602 gene product can be utilized to inhibit
its activity, or alternatively, to target the specific destruction
of cells that express the gene. Furthermore, the extracellular
domains of the fchd602 gene product provide targets which allow for
the design of especially efficient screening systems for
identifying compounds that bind to the fchd602 gene product.
[0036] Such an assay system can also be used to screen and identify
antagonists of the interaction between the fchd602 gene product and
ligands that bind to the fchd602 gene product. For example, the
compounds can act as decoys by binding to the endogenous (i.e.,
natural) ligand for the fchd602 gene product. The resulting
reduction in the amount of ligand-bound fchd602 gene transmembrane
protein will modulate the activity of disease state cells, such as
monocytes. Soluble proteins or peptides, such as peptides
comprising one or more of the extracellular domains, or portions
and/or analogs thereof of the fchd602 gene product, including, for
example, soluble fusion proteins such as Ig-tailed fusion proteins,
can be particularly useful for this purpose.
[0037] Similarly, antibodies that are specific to one or more of
the extracellular domains of the fchd602 product provide for the
ready detection of this target gene product in diagnostic tests or
in clinical test monitoring. Accordingly, endothelial cells can be
treated, either in vivo or in vitro, with such a labeled antibody
to determine the disease state of endothelial cells. Because the
fchd602 gene product is up-regulated in monocytes in the disease
state, its detection positively corresponds with cardiovascular
disease.
[0038] Such methods for treatment, diagnosis, and clinical test
monitoring which use the fchd602 gene product as described above
can also be applied to other target genes that encode transmembrane
gene products, including but not limited to the fchd545 gene, which
encodes multiple transmembrane domains and extracellular
domains.
[0039] The examples presented in Sections 6 and 7, below,
demonstrate the use of the cardiovascular disease paradigms of the
invention to identify cardiovascular disease target genes.
[0040] The example presented in Section 8, below, demonstrates the
use of fingerprint genes in diagnostics and as surrogate markers
for testing the efficacy of candidate drugs in basic research and
in clinical trials.
[0041] The example presented in Section 9, below, demonstrates the
use of fingerprint genes, particularly fchd545, in the imaging of a
diseased cardiovascular tissue.
[0042] The example presented in Section 11, below, demonstrates the
interaction of two target gene products, the rchd534 and fchd540
proteins, and the further characterization of their roles in
oncology, angiogenesis, cardiovascular disease and the TGF-.beta.
signalling pathway.
4. DESCRIPTION OF THE FIGURES
[0043] FIG. 1. Nucleotide sequence and encoded amino acid sequence
of the fchd531 gene.
[0044] FIG. 2. Nucleotide sequence and encoded amino acid sequence
of the fchd540 gene.
[0045] FIG. 3. Nucleotide sequence and encoded amino acid sequence
of the fchd545 gene.
[0046] FIG. 4. Nucleotide sequence and encoded amino acid sequence
from the fchd602 gene.
[0047] FIG. 5. Nucleotide sequence and encoded amino acid sequence
from the fchd605 gene.
[0048] FIG. 6. Nucleotide sequence and encoded amino acid sequence
of the rchd534 gene.
5. DETAILED DESCRIPTION OF THE INVENTION
[0049] 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. Methods and compositions
for the treatment of oncogenic related disorders, including
tumorigenesis and the vascularization of tumors, are also
described. The invention is based, in part, on the evaluation of
the expression and role of all genes that are differentially
expressed in paradigms that are physiologically relevant to the
disease condition. This permits the definition of disease pathways
and the identification of targets in the pathway that are useful
both diagnostically and therapeutically.
[0050] 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.
[0051] Further, the gene products of such fingerprint, target, and
pathway genes are described in Section 5.4.2, antibodies to such
gene products are described in Section 5.4.3, as are cell- and
animal-based models of cardiovascular disease and oncogenic related
disorders to which such gene products may contribute, in Section
5.4.4.
[0052] Methods for the identification of compounds which modulate
the expression of genes or the activity of gene products involved
in cardiovascular disease and fibroproliferative and oncogenic
related disorders including tumorigenesis are described in Section
5.5. Methods for monitoring the efficacy of compounds during
clinical trials are described in Section 5.5.4. Additionally
described below, in Section 5.6, are methods for the treatment of
cardiovascular disease and oncogenic related disorders.
[0053] Also discussed below, in Section 5.8, are methods for
prognostic and diagnostic evaluation of cardiovascular disease,
including the identification of subjects exhibiting a
predisposition to this disease, and the imaging of cardiovascular
disease conditions.
5.1. IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES
[0054] 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.
[0055] "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.
[0056] 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.
[0057] 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.
[0058] "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.
[0059] A variety of methods may be utilized for the identification
of genes which are involved in cardiovascular disease. These
methods include but are not limited to the experimental paradigms
described, below, in Section 5.1.1. Material from the paradigms may
be characterized for the presence of differentially expressed gene
sequences as discussed, below, in Section 5.1.2.
5.1.1. PARADIGMS FOR THE IDENTIFICATION OF DIFFERENTIALLY EXPRESSED
GENES
[0060] 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 paradigms, which may be used to detect
such differentially expressed genes. In general, the paradigms
include at least one experimental condition in which subjects or
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.
[0061] 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.
5.1.1.1. FOAM CELL PARADIGM-1
[0062] 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.
[0063] One embodiment of such a paradigm, hereinafter referred to
as Paradigm A, is carried out as follows: 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.
5.1.1.2. FOAM CELL PARADIGM-2
[0064] 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.
5.1.1.3. FOAM CELL PARADIGM-3
[0065] 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 muscle 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.
5.1.1.4. IN VIVO MONOCYTE PARADIGM
[0066] 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.
5.1.1.5. ENDOTHELIAL CELL-IL-1 PARADIGM
[0067] 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. After a certain period
of treatment, experimental and control cells are harvested and
analyzed for differential gene expression as described in
sub-section 5.1.2, below.
5.1.1.6. ENDOTHELIAL CELL-SHEAR STRESS PARADIGM
[0068] 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.
[0069] Cultured HUVEC monolayers are exposed to laminar shear
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 7, below, demonstrates the
use of such a shear stressed endothelial cell paradigm to identify
sequences which are differentially expressed in exposed versus
control cells.
[0070] 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.
[0071] 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.
[0072] 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.
5.1.2. ANALYSIS OF PARADIGM MATERIAL
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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. 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.
[0080] 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.
[0081] Once 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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.
5.2. IDENTIFICATION OF PATHWAY GENES
[0086] 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.
[0087] 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.
[0088] 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 may be accomplished, for
example by standard hybridization or PCR techniques. Techniques for
the generation of oligonucleotide mixtures and screening are
well-known. (See, e.g., Ausubel, supra., and PCR Protocols: A Guide
to Methods and Applications, 1990, Innis, M. et al., eds. Academic
Press, Inc., New York).
[0089] 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.gt11
libraries.
[0090] 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.).
[0091] 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.
[0092] 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.
[0093] 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., TNF.alpha.,
HB-EGF, PDGF, IFN-.gamma., and GM-CSF, to name a few.
[0094] 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.
[0095] Once a pathway gene has been identified and isolated, it may
be further characterized as, for example, discussed below, in
Section 5.3.
[0096] A preferred embodiment of the use of the yeast two-hybrid
system is described in detail in the example in Section 12, below.
As described in Section 12, the yeast two-hybrid system was used to
detect the interaction between the protein products of two target
genes, rchd534 and fchd540.
5.3. CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED AND PATHWAY
GENES
[0097] 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".
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] For example, genes which are up-regulated under disease
conditions may be involved in causing or exacerbating the disease
condition. Treatments directed at down-regulating the activity of
such harmfully expressed genes will ameliorate the disease
condition. On the other hand, the up-regulation of genes under
disease conditions may be part of a protective response by affected
cells. Treatments directed at increasing or enhancing the activity
of such up-regulated gene products, especially in individuals
lacking normal up-regulation, will similarly ameliorate disease
conditions. Such methods of treatment are discussed, below, in
Section 5.6.
5.4. DIFFERENTIALLY EXPRESSED AND PATHWAY GENES
[0112] 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.
5.4.1. DIFFERENTIALLY EXPRESSED AND PATHWAY GENE SEQUENCES
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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 ATCC, 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.
1-5.
[0118] 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.
[0119] 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 6.1.1, below.
[0120] 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. Alternatively, the genes may be retrieved
from a human placenta cDNA library (Clontech Laboratories, Palo
Alto, Calif.), 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 of
Paradigm Cell Type Gene Seq. ID # Original Detection Expr. Pattern
Detected in Ref Seq. fchd531 1 D (Section 5.1.1.6) .dwnarw.
Endothelial New,1 fchd540 2 D .Arrow-up bold. Endothelial New,2
fchd545 3 D .dwnarw. Endothelial New,3 fchd602 4 A (Section
5.1.1.1) .Arrow-up bold. Monocytes New,4 fchd605 5 A .Arrow-up
bold. Monocytes New,5 FIG.5 .sup.1GenBank accession number 005343.
.sup.2Drosohila Mothers against dpp (Mad), Sekelsky et al., 1995,
Genetics 139; 1347-1358. .sup.3Human Voltage-dependent Anion
Channel, Blachly-Dyson, E., et al., 1993, J. Biol. Chem. 268;
1835-1841; and EST T24012 .sup.4Rat Cl-6, Diamond, R,H., et al.,
1993, J. Biol. Chem, 268: 15185-15192. .sup.6Mouse gly96, Charles,
C.H., et al., 1993, Oncogene 8; 797-801: and EST T49532.
[0121] Table 2, below, lists the strains of E. coli deposited with
the ATCC that contain plasmids bearing the novel genes listed in
Table 1.
2 TABLE 2 Strain Deposited GENE with ATCC fchd531 pFCHD531 fchd540
pFCHD540 fchd545 fchd545
[0122] 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. 1-5), or contained in the clones listed in Table 2,
as deposited with the ATCC; (b) any DNA sequence that encodes the
amino acid sequence encoded by the DNA sequences disclosed herein
(as shown in FIGS. 1-5), contained in the clones, listed in Table
2, as deposited with the ATCC or contained within the coding region
of the gene to which the DNA sequences disclosed herein (as shown
in FIGS. 1-5) or contained in the clones listed in Table 2, as
deposited with the ATCC, 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 ATCC, or contained within the coding region of the gene to
which the DNA sequences disclosed herein (as shown in FIGS. 1-5) or
contained in the clones listed in Table 2, as deposited with the
ATCC, 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. 1-5) contained in the clones listed in
Table 2, as deposited with the ATCC or contained within the coding
region of the gene to which DNA sequences disclosed herein (as
shown in FIGS. 1-5) or contained in the clones, listed in Table 2,
as deposited with the ATCC, 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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 to 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.
[0133] 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.
5.4.2. DIFFERENTIALLY EXPRESSED AND PATHWAY GENE PRODUCTS
[0134] 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 ATCC,
or contained in the coding regions of the genes to which DNA
sequences disclosed herein (in FIGS. 1-5) or contained in the
clones, listed in Table 2, as deposited with the ATCC, belong, for
example.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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 expression 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).
[0139] 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.
[0140] In a preferred embodiment, full length cDNA sequences are
appended with in-frame Bam HI sites at the amino terminus and Eco
RI sites at the carboxyl terminus using standard PCR methodologies
(Innis et al., 1990, supra) and ligated into the pGEX-2TK vector
(Pharmacia, Uppsala, Sweden). The resulting cDNA construct contains
a kinase recognition site at the amino terminus for radioactive
labelling and glutathione S-transferase sequences at the carboxyl
terminus for affinity purification (Nilsson, et al., 1985, EMBO J.
4: 1075; Zabeau and Stanley, 1982, EMBO J. 1: 1217.
[0141] In an insect system, Autographa californica 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).
[0142] 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).
[0143] In a preferred embodiment, cDNA sequences encoding the
full-length open reading frames are ligated into pCMV.beta.
replacing the .beta.-galactosidase gene such that cDNA expression
is driven by the CMV promoter (Alam, 1990, Anal. Biochem. 188:
245-254; MacGregor & Caskey, 1989, Nucl. Acids Res. 17: 2365;
Norton & Corrin, 1985, Mol. Cell. Biol. 5: 281).
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
5.4.3. DIFFERENTIALLY EXPRESSED OR PATHWAY GENE PRODUCT
ANTIBODIES
[0151] 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.
[0152] 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.
[0153] In a preferred embodiment, peptide sequences corresponding
to amino sequences of target gene products were selected and
submitted to Research Genetics (Huntsville, AL) for synthesis and
antibody production. Peptides were modified as described (Tam, J.
P., 1988, Proc. Natl. Acad. Sci. USA 85: 5409-5413; Tam, J. P., and
Zavala, F., 1989, J. Immunol. Methods 124: 53-61; Tam, J. P., and
Lu, Y. A., 1989, Proc. Natl. Acad. Sci. USA 86: 9084-9088),
emulsified in an equal volume of Freund's adjuvant and injected
into rabbits at 3 to 4 subcutaneous dorsal sites for a total volume
of 1.0 ml (0.5 mg peptide) per immunization. The animals were
boosted after 2 and 6 weeks and bled at weeks 4, 8, and 10. The
blood was allowed to clot and serum was collected by
centrifugation. The generation of polyclonal antibodies against the
fchd545 gene product is described in detail in the example in
Section 10, below.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 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. 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.
[0158] 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.
5.4.4. CELL- AND ANIMAL-BASED MODEL SYSTEMS
[0159] 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.
5.4.4.1. ANIMAL-BASED SYSTEMS
[0160] Animal-based model systems of cardiovascular disease may
include, but are not limited to, non-recombinant and engineered
transgenic animals.
[0161] 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).
[0162] 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.
[0163] 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).
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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. Recombinant methods for expressing target genes
are described in Section 5.4.2, above.
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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.
5.4.4.2. CELL-BASED ASSAYS
[0173] 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.
[0174] Such cells may include non-recombinant monocyte cell lines,
such as U937 (ATCC# CRL-1593), THP-1 (ATCC# TIB-202), and P388D1
(ATCC# TIB-63); endothelial cells such as HUVEC's and bovine aortic
endothelial cells (BAEC's); as well as generic mammalian cell lines
such as HeLa cells and COS cells, e.g., COS-7 (ATCC# CRL-1651).
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.
[0175] 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.
[0176] 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. Recombinant methods for expressing target
genes are described in Section 5.4.2, above.
[0177] 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. Transfection of host cells with target genes is discussed,
above, in Section 5.4.4.1.
[0178] Cells treated with compounds or transfected with target
genes can be examined for phenotypes associated with cardiovascular
disease. In the case of monocytes, such phenotypes 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.,
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.
[0179] Similarly, HUVEC's can be treated with test compounds or
transfected with genetically engineered target genes described in
Section 5.4.2, above. The HUVEC's can then be examined for
phenotypes associated with cardiovascular disease, including, but
not limited to changes in cellular morphology, cell proliferation,
cell migration, and mononuclear cell adhesion; or for the effects
on production of other proteins involved in cardiovascular disease
such as ICAM, VCAM, PDGF-.beta., and E-selectin.
[0180] 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.
5.5. SCREENING ASSAYS FOR COMPOUNDS THAT INTERACT WITH THE TARGET
GENE PRODUCT AND/OR MODULATE TARGET GENE EXPRESSION
[0181] 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. Such compounds can act as
the basis for amelioration of such cardiovascular diseases as
atherosclerosis, ischemia/reperfusion, hypertension, restenosis,
and arterial inflammation by modulating the activity of the protein
products of target genes. Such compounds may also act as the basis
for the amelioration of fibroproliferative and oncogenic related
disorders, including tumorigenesis and the vascularization of
tumors. 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.
[0182] 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.
[0183] In some cases, a target gene observed to be up-regulated
under disease conditions may be exerting a protective effect.
Compounds that enhance the expression of such up-regulated genes,
or the activity of their gene products, would also ameliorate
disease symptoms, especially in individuals whose target gene is
not normally up-regulated.
[0184] 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.
5.5.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO THE
TARGET GENE PRODUCT
[0185] 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. For instance, the example in Section 12, below,
describes the interaction between the rchd534 protein and the
fchd540 protein. Compounds that disrupt the interaction between
these two proteins may be useful in the treatment of cardiovascular
disease.
[0186] 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.
[0187] 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.
[0188] 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).
[0189] 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.
[0190] 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. 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.
[0191] 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.
Identification of ligand, and measuring the activity of the
ligand-receptor complex, leads to the identification of antagonists
of this interaction, as described in Section 5.5.3, below. Such
antagonists are useful in the treatment of cardiovascular
disease.
5.5.2. ASSAYS FOR CELLULAR OR EXTRACELLULAR PROTEINS THAT INTERACT
WITH THE TARGET GENE PRODUCT
[0192] 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.
[0193] The example presented in Section 12, below, demonstrates the
use of this method to detect the interaction between the rchd534
protein and the fchd540 protein, which both had been identified as
target proteins.
5.5.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH INTERACTION BETWEEN
TARGET GENE PRODUCT AND OTHER COMPOUNDS
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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. 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.++.
[0206] 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.
[0207] 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.
[0208] 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, useful
regions are those exhibiting homology to the extracellular domains
of well-characterized members of the family.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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.
5.5.3. ASSAYS FOR AMELIORATION OF CARDIOVASCULAR DISEASE
SYMPTOMS
[0215] 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.
[0216] 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.
[0217] 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.
[0218] Further, both cell-based systems and animal-based systems as
described herein may be used to identify compounds which act to
ameliorate symptoms of fibroproliferative and oncogenic related
disorders, including tumorigenesis and the vascularization of
tumors. Such cell-based and animal-based systems may be exposed to
a compound, suspected of exhibiting an ability to ameliorate
fibroproliferative disease symptoms, at a sufficient concentration
and for a time sufficient to elicit such an amelioration of
fibroproliferative disease symptoms in the exposed system. The
response may be monitored by assessing the reversal of disorders
associated with fibroproliferative disease, for example by
measuring the size and growth of tumors or vascularization of
tumors before and after treatment.
[0219] 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.
[0220] 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.
[0221] 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.
[0222] 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.
5.5.4. MONITORING OF EFFECTS DURING CLINICAL TRIALS
[0223] 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.
[0224] 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, drua treatment. This method is described in further detail
in the example in Section 8, below. Specifically, the up-regulation
of fchd602 and fchd605 under treatment with oxidized LDL provides a
fingerprint profile for monocytes under oxidative stress. The
fchd602 and fchd605 genes can serve, therefore, as surrogate
markers during clinical treatment of cardiovascular disease.
Accordingly, the influence of anti-oxidant drugs on oxidative
potential is measured by recording the differential display of
fchd602 and fchd605 in the monocytes of patients undergoing
clinical treatment.
5.5.5. ASSAYS FOR COMPOUNDS THAT MODULATE EXPRESSION OF TARGET
GENES
[0225] Compounds and other substances that modulate expression of
target genes can be screened using in vitro cellular systems. In a
manner analogous to the monitoring of compounds clinical samples
described in Section 5.5.5, above, a sample of cells, such as a
tissue culture is exposed to a test substance. Appropriate tissue
culture cells include, but are not limited to, human umbilical vein
endothelial cells (HUVECs), bovine aortic endothelial cells
(BAECs), and 293 cells (embryonic human kidney cells). The RNA is
then extracted from the cells. The level of transcription of a
specific target gene can be detected using, for example, standard
RT-PCR amplification techniques and/or Northern analysis (as
described in the example in Section 6.1.2, below). Alternatively,
the level of target protein production can be assayed by using
antibodies that detect the target gene protein, as described in
Section 5.5.1, above. The level of expression is compared to a
control cell sample which was not exposed to the test
substance.
[0226] Compounds that can be screened for modulation of expression
of the target gene include, but are not limited to, small inorganic
or organic molecules, peptides, such as peptide hormones analogs,
steroid hormones, analogs of such hormones, and other proteins.
Compounds that down-regulate expression include, but are not
limited to, oligonucleotides that are complementary to the 5'-end
of the mRNA of the target gene and inhibit transcription by forming
triple helix structures, and ribozymes or antisense molecules which
inhibit translation of the target gene mRNA. Techniques and
strategies for designing such down-regulating test compounds are
described in detail in Section 5.6, below.
5.6. COMPOUNDS AND METHODS FOR TREATMENT OF CARDIOVASCULAR AND
FIBROPROLIFERATIVE DISEASE
[0227] Described below are methods and compositions whereby
cardiovascular disease symptoms may be ameliorated. The methods and
compositions described below may also be applied for the
amelioration of symptoms associated with fibroproliferative and
oncogenic disorders, but as a way of example will be discussed in
the subsections below in terms of cardiovascular disease disorders.
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.
[0228] 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.
[0229] In some cases, the up-regulation of a gene in a disease
state reflects a protective role for that gene product in
responding to the disease condition. Enhancement of such a target
gene's expression, or the activity of the target gene product, will
reinforce the protective effect it exerts. Some cardiovascular
disease states may result from an abnormally low level of activity
of such a protective gene. In these cases also, 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.
5.6.1. COMPOUNDS THAT INHIBIT EXPRESSION, SYNTHESIS OR ACTIVITY OF
MUTANT TARGET GENE ACTIVITY
[0230] 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 6 and 7, below, a number of
genes have been demonstrated to be up-regulated in monocytes and
endothelial cells under disease conditions. Specifically, fchd602
and fchd605 are each up-regulated in monocytes treated with
oxidized LDL. Furthermore, fchd540 is up-regulated in endothelial
cells subjected to shear stress. In some cases, such up-regulation
may have a causative or exacerbating effect on the disease state. A
variety of techniques may be utilized to inhibit the expression,
synthesis, or activity of such target genes and/or proteins.
[0231] 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.
[0232] For example, compounds can be administered that compete with
endogenous ligand for a transmembrane target gene product. The
resulting reduction in the amount of ligand-bound target gene
transmembrane protein will modulate cell physiology. Compounds that
can be particularly useful for 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 target 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.).
[0233] Alternatively, compounds, such as ligand analogs or
antibodies, that bind to the target gene product receptor site, but
do not activate the protein, (e.g., receptor-ligand antagonists)
can be effective in inhibiting target gene product activity.
[0234] 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.
5.6.1.1. INHIBITORY ANTISENSE, RIBOZYME, TRIPLE HELIX, AND GENE
INACTIVATION APPROACHES
[0235] 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.
[0236] Antisense RNA and DNA molecules act to directly block the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation.
[0237] Antisense approaches involve the design of oligonucleotides
(either DNA or RNA) that are complementary to target gene mRNA. The
antisense oligonucleotides will bind to the complementary target
gene mRNA transcripts and prevent translation. Absolute
complementarity, although preferred, is not required. A sequence
"complementary" to a portion of an RNA, as referred to herein,
means a sequence having sufficient complementarity to be able to
hybridize with the RNA, forming a stable duplex; in the case of
double-stranded antisense nucleic acids, a single strand of the
duplex DNA may thus be tested, or triplex formation may be assayed.
The ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid.
Generally, the longer the hybridizing nucleic acid, the more base
mismatches with an RNA it may contain and still form a stable
duplex (or triplex, as the case may be). One skilled in the art can
ascertain a tolerable degree of mismatch by use of standard
procedures to determine the melting point of the hybridized
complex.
[0238] Oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have recently shown to be effective
at inhibiting translation of mRNAs as well. See generally, Wagner,
R., 1994, Nature 372:333-335. Thus, oligonucleotides complementary
to either the 5'- or 3'- non-translated, non-coding regions of the
target gene could be used in an antisense approach to inhibit
translation of endogenous target gene mRNA. Oligonucleotides
complementary to the 5' untranslated region of the mRNA should
include the complement of the AUG start codon. Antisense
oligonucleotides complementary to mRNA coding regions are less
efficient inhibitors of translation but could be used in accordance
with the invention. Whether designed to hybridize to the 5'-, 3'-
or coding region of target gene mRNA, antisense nucleic acids
should be at least six nucleotides in length, and are preferably
oligonucleotides ranging from 6 to about 50 nucleotides in length.
In specific aspects the oligonucleotide is at least 10 nucleotides,
at least 17 nucleotides, at least 25 nucleotides or at least 50
nucleotides.
[0239] Regardless of the choice of target sequence, it is preferred
that in vitro studies are first performed to quantitate the ability
of the antisense oligonucleotide to inhibit gene expression. It is
preferred that these studies utilize controls that distinguish
between antisense gene inhibition and nonspecific biological
effects of oligonucleotides. It is also preferred that these
studies compare levels of the target RNA or protein with that of an
internal control RNA or protein. Additionally, it is envisioned
that results obtained using the antisense oligonucleotide are
compared with those obtained using a control oligonucleotide. It is
preferred that the control oligonucleotide is of approximately the
same length as the test oligonucleotide and that the nucleotide
sequence of the oligonucleotide differs from the antisense sequence
no more than is necessary to prevent specific hybridization to the
target sequence.
[0240] The oligonucleotides can be DNA or RNA or chimeric mixtures
or derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, hybridization, etc. The
oligonucleotide may include other appended groups such as peptides
(e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across the cell membrane (see, e.g.,
Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;
Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT
Publication No. WO88/09810, published Dec. 15, 1988) or the
blood-brain barrier (see, e.g., PCT Publication No. W089/10134,
published Apr. 25, 1988), hybridization-triggered cleavage agents.
(See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or
intercalating agents. (See, e.g., Zon, 1988, Pharm. Res.
5:539-549). To this end, the oligonucleotide may be conjugated to
another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[0241] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including but
not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomet-
hyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,
2-dimethylguanine, 2-methyladenine, 2-methylguanine,
3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0242] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0243] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group consisting of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0244] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., 1987, Nucl.
Acids Res. 15:6625-6641). The oligonucleotide is a
2'-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.
15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987,
FEBS Lett. 215:327-330).
[0245] Oligonucleotides of the invention may be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(1988, Nucl. Acids Res. 16:3209), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451), etc.
[0246] While antisense nucleotides complementary to the target gene
coding region sequence could be used, those complementary to the
transcribed untranslated region are most preferred.
[0247] Specific antisense oligonucleotides for the rchd534 gene and
fchd540 gene are described in the Example in Section 13, below.
[0248] The antisense molecules should be delivered to cells which
express the target gene in vivo, e.g., endothelial cells. A number
of methods have been developed for delivering antisense DNA or RNA
to cells; e.g., antisense molecules can be injected directly into
the tissue site, or modified antisense molecules, designed to
target the desired cells (e.g., antisense linked to peptides or
antibodies that specifically bind receptors or antigens expressed
on the target cell surface) can be administered systemically.
[0249] However, it is often difficult to achieve intracellular
concentrations of the antisense sufficient to suppress translation
of endogenous mRNAs. Therefore a preferred approach utilizes a
recombinant DNA construct in which the antisense oligonucleotide is
placed under the control of a strong pol III or pol II promoter.
The use of such a construct to transfect target cells in the
patient will result in the transcription of sufficient amounts of
single stranded RNAs that will form complementary base pairs with
the endogenous target gene transcripts and thereby prevent
translation of the target gene mRNA. For example, a vector can be
introduced in vivo such that it is taken up by a cell and directs
the transcription of an antisense RNA. Such a vector can remain
episomal or become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA. Such vectors can
be constructed by recombinant DNA technology methods standard in
the art. Vectors can be plasmid, viral, or others known in the art,
used for replication and expression in mammalian cells. Expression
of the sequence encoding the antisense RNA can be by any promoter
known in the art to act in mammalian, preferably human cells. Such
promoters can be inducible or constitutive. Such promoters include
but are not limited to: the SV40 early promoter region (Bernoist
and Chambon, 1981, Nature 290:304-310), the promoter contained in
the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al.,
1980, Cell 22:787-797), the herpes thymidine kinase promoter
(Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445),
the regulatory sequences of the metallothionein gene (Brinster et
al., 1982, Nature 296:39-42), etc. Any type of plasmid, cosmid, YAC
or viral vector can be used to prepare the recombinant DNA
construct which can be introduced directly into the tissue site;
e.g., atherosclerotic vascular tissue. Alternatively, viral vectors
can be used which selectively infect the desired tissue, in which
case administration may be accomplished by another route (e.g.,
systemically).
[0250] 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. Ribozyme molecules designed to catalytically cleave
target gene mRNA transcripts can also be used to prevent
translation of target gene mRNA and expression of target gene.
(See, e.g., PCT International Publication WO90/11364, published
Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225). While
ribozymes that cleave mRNA at site specific recognition sequences
can be used to destroy target gene mRNAs, the use of hammerhead
ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at
locations dictated by flanking regions that form complementary base
pairs with the target mRNA. The sole requirement is that the target
mRNA have the following sequence of two bases: 5'-UG-3'. The
construction and production of hammerhead ribozymes is well known
in the art and is described more fully in Haseloff and Gerlach,
1988, Nature, 334:585-591. For example, there are hundreds of
potential hammerhead ribozyme cleavage sites within the nucleotide
sequence of rchd534 and fchd540 cDNA. Preferably the ribozyme is
engineered so that the cleavage recognition site is located near
the 5' end of the target mRNA; i.e., to increase efficiency and
minimize the intracellular accumulation of non-functional mRNA
transcripts.
[0251] Specific hammerhead ribozymes molecules for the rchd534 and
fchd540 genes are described in the Example in Section 13,
below.
[0252] The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the
one which occurs naturally in Tetrahymena Thermophila (known as the
IVS, or L-19 IVS RNA) and which has been extensively described by
Thomas Cech and collaborators (Zaug, et al., 1984, Science,
224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et
al., 1986, Nature, 324:429-433; published International patent
application No. WO 88/04300 by University Patents Inc.; Been and
Cech, 1986, Cell, 47:207-216). The Cech-type ribozymes have an
eight base pair active site which hybridizes to a target RNA
sequence whereafter cleavage of the target RNA takes place. The
invention encompasses those Cech-type ribozymes which target eight
base-pair active site sequences that are present in target
gene.
[0253] As in the antisense approach, the ribozymes can be composed
of modified oligonucleotides (e.g. for improved stability,
targeting, etc.) and should be delivered to cells which express the
target gene in vivo, e.g., endothelial cells. A preferred method of
delivery involves using a DNA construct "encoding" the ribozyme
under the control of a strong constitutive pol III or pol II
promoter, so that transfected cells will produce sufficient
quantities of the ribozyme to destroy endogenous target gene
messages and inhibit translation. Because ribozymes, unlike
antisense molecules, are catalytic, a lower intracellular
concentration is required for efficiency.
[0254] 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.
[0255] 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.
[0256] 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.
[0257] Endogenous target gene expression can also be reduced by
inactivating or "knocking out" the target gene or its promoter
using targeted homologous recombination. (E.g., see Smithies et
al., 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell
51:503-512; Thompson et al., 1989 Cell 5:313-321; each of which is
incorporated by reference herein in its entirety). For example, a
mutant, non-functional target (or a completely unrelated DNA
sequence) flanked by DNA homologous to the endogenous target gene
(either the coding regions or regulatory regions of the target
gene) can be used, with or without a selectable marker and/or a
negative selectable marker, to transfect cells that express target
in vivo. Insertion of the DNA construct, via targeted homologous
recombination, results in inactivation of the target gene. Such
approaches can be adapted for use in humans provided the
recombinant DNA constructs are directly administered or targeted to
the required site in vivo using appropriate viral vectors, e.g.,
vectors for delivery vascular tissue.
[0258] Alternatively, endogenous target gene expression can be
reduced by targeting deoxyribonucleotide sequences complementary to
the regulatory region of the target gene (i.e., the target promoter
and/or enhancers) to form triple helical structures that prevent
transcription of the target gene in target cells in the body. (See
generally, Helene, C. 1991, Anticancer Drug Des., 6(6):569-84;
Helene, C., et al., 1992, Ann, N.Y. Acad. Sci., 660:27-36; and
Maher, L. J., 1992, Bioassays 14(12):807-15).
[0259] In yet another embodiment of the invention, the activity of
a target can be reduced using a "dominant negative" approach to
effectuate reduction in cardiovascular disease symptoms. For
example, if two gene products interact, such as the rchd534 and
fchd540 proteins, then the presence of a mutant version of one or
both of these proteins in the cell can reduce the overall pool of
complexes consisting of entirely wild-type proteins. In this
manner, the overall level of activity resulting from the
rchd534/fchd540 protein interaction can be reduced.
5.6.1.2. ANTIBODIES FOR TARGET GENE PRODUCTS
[0260] 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.
[0261] 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).
[0262] In some instances, the target gene protein is extracellular,
or is a transmembrane protein, such as the fchd545 and fchd602 gene
products. Antibodies that are specific for one or more
extracellular domains of these gene products, 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 5.7 which are appropriate for peptide administration may
be utilized to effectively administer inhibitory target gene
antibodies to their site of action.
5.6.2. METHODS FOR RESTORING OR ENHANCING TARGET GENE ACTIVITY
[0263] 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
Section 7, below, several genes are now known to be down-regulated
in endothelial cells under disease conditions. Specifically,
fchd531 and fchd545 are down-regulated in endothelial cells
subjected to shear stress. Alternatively, the activity of target
gene products may be decreased, leading to the development of
cardiovascular disease symptoms. Such down-regulation of target
gene expression or decrease of target gene product activity might
have a causative or exacerbating effect on the disease state.
[0264] In some cases, target genes that are up-regulated in the
disease state might be exerting a protective effect. As summarized
in Table 1, above, and detailed in the examples in Sections 6 and
7, below, a number of genes are now known to be up-regulated in
monocytes and endothelial cells under disease conditions.
Specifically, fchd602 and fchd605 are each up-regulated in
monocytes treated with oxidized LDL. Furthermore, fchd540 is
up-regulated in endothelial cells subjected to shear stress. A
variety of techniques may be utilized to increase the expression,
synthesis, or activity of such target genes and/or proteins, for
those genes that exert a protective effect in response to disease
conditions.
[0265] 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.
[0266] 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.
[0267] Additionally, RNA sequences encoding target gene protein may
be directly administered to a patient exhibiting cardiovascular
disease symptoms, at a concentration sufficient to produce a level
of target gene protein such that cardiovascular disease symptoms
are ameliorated. Any of the 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.
[0268] 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.
[0269] 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.
5.7. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION
[0270] 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 sufficient to result in amelioration of
symptoms of cardiovascular disease.
5.7.1. EFFECTIVE DOSE
[0271] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the 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.
[0272] 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.
5.7.2. FORMULATIONS AND USE
[0273] 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.
[0274] 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.
[0275] 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 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.
[0276] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0277] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0278] 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.
[0279] 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.
[0280] 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.
[0281] 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.
[0282] 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.
5.8. DIAGNOSIS OF CARDIOVASCULAR DISEASE ABNORMALITIES
[0283] 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.
[0284] 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.
[0285] 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.
5.8.1. DETECTION OF FINGERPRINT GENE NUCLEIC ACIDS
[0286] 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).
[0287] 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.
[0288] 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
techniques well-known to those in the art.
[0289] 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.
[0290] 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.
[0291] 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.
5.8.2. DETECTION OF FINGERPRINT GENE PEPTIDES
[0292] 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.
[0293] Protein from the tissue or cell type to be analyzed may
easily be detected or isolated using techniques which are well
known to those of skill in the art, including but not limited to
western blot analysis. For a detailed explanation of methods for
carrying out western blot analysis, see Sambrook et al, 1989,
supra, at Chapter 18. The protein detection and isolation methods
employed herein may also be such as those described in Harlow and
Lane, for example, (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.
[0294] 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.
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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.
[0299] 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.
[0300] 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.
[0301] 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,
FL, 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
calorimetric 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.
[0302] 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.
[0303] 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.
[0304] 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).
[0305] 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.
[0306] 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.
5.8.3. IMAGING CARDIOVASCULAR DISEASE CONDITIONS
[0307] 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.
[0308] As described in the example in Section 6, below, fchd602 is
a gene that is up-regulated in monocytes under disease conditions.
Furthermore, the fchd602 gene encodes a novel protein containing
multiple transmembrane domains. Not only is the fchd602 gene
expressed in monocytes, which play a role in the initiation and
progression of atherosclerotic lesions, it is also upregulated in
monocytes under such disease conditions. The fchd602 gene product,
therefore, provides and excellent tool for imaging cardiovascular
disease conditions.
[0309] This method can be applied in a similar manner to other
transmembrane target gene products, such as the fchd545 gene
product. As described in the example in Section 7, below, the
fchd545 gene encodes a novel anion channel, containing multiple
transmembrane domains. Because the fchd545 gene product might be
more readily detected in normal tissue, as opposed to tissue in the
disease state, it also provides an excellent tool for imaging
cardiovascular disease conditions.
[0310] An example illustrating the use of this method in accordance
with the invention is provided in Section 9, below. Monoclonal and
polyclonal antibodies, as described in Section 5.6.1.2, above,
which specifically bind to such surface proteins, such as the
fchd602 and fchd545 gene products, can be used for the diagnosis of
cardiovascular disease by in vivo tissue imaging techniques. Such
antibodies raised against the fchd545 gene product are described in
detail in the example in Section 10, below. 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.
[0311] 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.
[0312] 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.
[0313] 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.
[0314] 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
[0315] 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. By use of Paradigm A, the novel
genes fchd602 and fchd605 were identified. Both fchd602 and fchd605
are up-regulated under the disease condition of treatment with
oxidized LDL.
[0316] The fchd602 gene product contains multiple transmembrane
domains, and has sequence similarity to the rat C1-6 gene, which is
induced in regenerating rat liver, is insulin inducible, and also
contains multiple transmembrane domains (Diamond, R. H., et al.,
1993, J. Biol. Chem. 268: 15185-15192). The fchd605 gene product
has sequence similarity to the mouse gly96 gene (Charles, C. H., et
al., 1993, Oncogene 8: 797-801), and to EST T49532.
[0317] The discovery of the up-regulation of these two genes
provides a fingerprint profile, e.g., markers, for monocytes in the
process of foam cell formation. This profile can be used in the
treatment and diagnosis of cardiovascular disease, including but
not limited to atherosclerosis, ischemia/reperfusion, hypertension,
restenosis, and arterial inflammation.
[0318] Furthermore, as a transmembrane protein, the fchd602 gene
product can be readily accessed or detected on the monocyte cell
surface by other compounds. It provides, therefore, 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 this
gene product provide targets which allow for the design of
especially efficient screening systems for identifying compounds
that bind to them. Such compounds can be useful in treating
cardiovascular disease by modulating the activity of the
transmembrane gene product.
6.1. MATERIALS AND METHODS
6.1.1. CELL ISOLATION AND CULTURING
[0319] 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 2680 RPM 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 2300 RPM 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 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.
[0320] Tissue culture dishes were coated with bovine collagen
before monocytes were plated out. 1/6 volume of 7X 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.
[0321] 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.
[0322] Lipoproteins 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 5uM 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/0.3mM 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.
[0323] 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/0.3mM EDTA used as diluent in all cases.
6.1.2. ANALYSIS OF PARADIGM MATERIAL
Differential Display:
[0324] Removal of DNA: 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 10x
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.
[0325] First strand cDNA synthesis: 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 5x 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 AM 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.
[0326] PCR reactions: 13 .mu.l of reaction mix was added to each
tube of a 96 well plate on ice. The reaction mix contained 6.4
.mu.l H.sub.2O, 2 .mu.l 10x 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 (for-) primer (10 .mu.M)
(operon), and 0.2 .mu.l AmpliTaq Polymerase (5 units/.mu.l)
(Perkin-Elmer). Next, 2 .mu.l of reverse (rev-) 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 primers used in each experiment were as follows:
[0327] fchd602: rev-T.sub.11XC and for-GTGAGGCGTC
[0328] fchd605: rev-T.sub.11XC and for-TGGACCGGTG
[0329] 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:
3 94.degree. C. 2 min. *94.degree. C. 15 sec. *40.degree. C. 2 min.
*ramp 72.degree. C. 1 min. *72.degree. C. 30 sec. 72.degree. C. 5
min. 4.degree. C. hold * = X40
[0330] 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 3 MM paper (Whatman Paper, England) and dried under
vacuum. Bands were visualized by autoradiography.
[0331] Band isolation and amplification: 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.
[0332] The isolated differentially expressed bands were then
amplified by PCR using the following reaction conditions:
4 58 .mu.l H.sub.2O 10 .mu.l 10x 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)
[0333] 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 BiolOl 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.
[0334] Subcloning: 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, recA1, hsdR17(r-k, m+k),
supE44, .lambda.-, thi-1, gyrA, relA1,
.phi.801acZ.alpha..DELTA.M15- .DELTA.(lacZYA-argF), deoR+, F') were
thawed on ice and 2 .mu.l of 0.5M .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.
[0335] A master mix containing 2 .mu.l 10x 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/.mu.l),
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:
5 94.degree. C. 2 min. *94.degree. C. 15 sec. *47.degree. C. 2 min.
*ramp 72.degree. C. 30 sec. *72.degree. C. 30 sec. 72.degree. C. 10
min. 4.degree. C. hold * = X35
[0336] 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.).
[0337] Northern analysis: Northern analysis was performed to
confirm the differential expression of the genes corresponding to
the amplified bands. The probes used to detect mRNA were
synthesized as follows: typically 2 .mu.l amplified band (.about.30
ng), 7 .mu.l H.sub.2O, and 2 .mu.l 10x 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 .parallel.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.6 cpm/pl of incorporation was achieved.
[0338] 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 5x MOPS buffer to 210 ml
sterile H2O. 5x 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 (5 mg/ml) and 30 ml of 37%
formaldehyde of gel were added. The gel was swirled quickly to mix,
and then poured immediately.
[0339] 2 .mu.g RNA sample, 1x final 1.5x RNA loading dyes (60%
formamide, 9% formaldehyde, 1.5X MOPS, .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.
[0340] 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.
[0341] 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.
[0342] RT-PCR Analysis: 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.
[0343] PCR was performed on the reverse transcribed samples. Each
reaction contained 2 .mu.l 10x 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 (.about.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:
6 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 * = 35x
[0344] 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.
[0345] Gene Retrieval: Amplified sequences, which contained
portions of the genes, were subcloned and then used individually to
retrieve a cDNA encoding the corresponding gene. Probes were
prepared by isolating the subcloned insert DNA from vector DNA, and
labeling with .sup.32P as described above in Section 6.1.2. Labeled
insert DNA containing fchd602 sequences was used to probe a cDNA
library prepared from human macrophage cell line U937. Labeled
insert DNA containing fchd605 sequences was used to probe a cDNA
library prepared from human primary blood monocytes. The cDNA
libraries were prepared and screened according to methods routinely
practiced in the art (see Sambrook et al., 1989, supra). Plaques
from the libraries that were detected by the probes were isolated
and the cDNA insert within the phage vector was sequenced.
[0346] The RACE procedure kit was used either as an alternative to
cDNA library screening, or, when the cDNA library did not yield a
clone encoding the full-length gene, to obtain adjacent sequences
of the gene. The procedure was carried out according to the
manufacturer's instructions (Clontech, Palo Alto, Calif.; 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
either on amplified sequences, or on sequences obtained from
isolates from the cDNA libraries. Template mRNA for fchd605 was
isolated from human primary blood monocytes.
6.1.3. CHROMOSOMAL LOCALIZATION OF TARGET GENES
[0347] 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 10x 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:
7 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 * = 35x
6.2. RESULTS
[0348] Differential display was performed on monocytes treated with
oxidized LDL and untreated monocytes. Bands corresponding to
fchd602 and fchd605 were detected as up-regulated by oxidized LDL,
as compared with the untreated monocytes. The up-regulation was
confirmed by northern blot analysis.
[0349] The fchd602 gene produced a 2.5 kb mRNA that was
up-regulated after 5 hours of treatment with oxidized LDL,
minimally oxidized LDL, and lysophosphatidylcholine. No message was
detected in untreated or native LDL treated control monocytes. The
amplified DNA sequence was used to recover a cDNA of approximately
875 bp comprising an open reading frame encoding approximately 182
amino acids. The DNA sequence and encoded amino acid sequence of
this cDNA from the fchd602 gene is shown in FIG. 4. The open
reading frame has 88% sequence similarity to the rat C1-6 gene,
which is induced in regenerating rat liver, is insulin inducible,
and also contains multiple transmembrane domains (Diamond, R. H.,
et al., 1993, J. Biol. Chem. 268: 15185-15192). The fchd605 gene
produced a 1.5 kb mRNA that is up-regulated after 5 hours treatment
with oxidized LDL, and to a lesser degree with native LDL, as
compared to untreated monocytes. The amplified DNA was sequenced
and used to recover a cDNA of approximately 2.2 kb, which was
sequenced to reveal a partial open reading frame of approximately
258 bp, encoding approximately 86 amino acids. The DNA sequence and
encoded amino acid sequence from the fchd605 gene is shown in FIG.
5. The sequence has similarity to the mouse gly96 gene, which
encodes a cytokine inducible glycosylated protein expressed in
mouse lung, testes, and uterus.
7. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN
RESPONSE TO PARADIGM D: ENDOTHELIAL CELL SHEAR STRESS
[0350] 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, three novel DNA sequences were identified.
[0351] The fchd531 gene is down-regulated in endothelial cells
under both turbulent and laminar shear stress, as compared to the
static control. The fchd531 gene encodes a novel 570 amino acid
polypeptide, and has 94% sequence similarity to the mouse penta
zinc finger gene (Pzf), which has not been published, but is
contained in the GenBank sequence data base under accession no
U05343.
[0352] The fchd540 gene is up-regulated in endothelial cells under
laminar shear stress, but is not up-regulated by IL-1 treatment.
The fchd540 gene encodes a novel intracellular protein which has
sequence similarity to the Drosophila Mad protein (Sekelsky et al.,
1995, Genetics 139: 1347-1358).
[0353] The fchd545 gene is down-regulated in endothelial cells
under laminar shear stress as compared to endothelial cells under
turbulent shear stress and static control endothelial cells. The
fchd545 gene encodes an 848 amino acid polypeptide which has 73%
sequence similarity to the human Voltage-dependent Anion Channel
protein (Blachly-Dyson, E., et al., 1993, J. Biol. Chem. 268:
1835-1841.). The fchd545 gene is also expressed in the human heart,
smooth muscles, and testes.
[0354] The up-regulation of the fchd540 gene and down-regulation of
the fchd531 and fchd545 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,
fchd540, 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.
[0355] Furthermore, as a transmembrane protein, the fchd545 gene
product can be readily accessed or detected on the endothelial cell
surface by other compounds. It provides, therefore, 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 this
gene product provide targets which allow for designing especially
efficient screening systems for identifying compounds that bind to
them. Such compounds can be useful in treating cardiovascular
disease by modulating the activity of the transmembrane gene
product.
7.1. MATERIALS AND METHODS
[0356] 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.
[0357] For induction, second passage HUVEC's were plated on tissue
culture-treated polystyrene and subjected to 10 dyn/cm.sup.2
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. Specific
primers used in differential display were as follows:
[0358] fchd531: for-T.sub.11XA and rev-AGACGTCCAC
[0359] fchd540: for-T.sub.11XA and rev-ACTTCGCCAC
[0360] fchd545: for-T.sub.11XC and rev-TCGGACGTGA
[0361] Amplified sequences, which contained portions of the genes,
were subcloned and then used individually to retrieve a cDNA
encoding the corresponding gene. Probes 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 cDNA library prepared from shear stress induced endothelial
cells. The library was prepared and probed using methods routinely
practiced in the art (see Sambrook et al., 1989, supra). Plaques
from the libraries that were detected by the probes were isolated
and the cDNA insert within the phage vector was sequenced.
[0362] The RACE procedure kit was used either as an alternative to
cDNA library screening, or, when the cDNA library did not yield a
clone encoding the full-length gene, to obtain adjacent sequences
of the gene. The procedure was carried out according to the
manufacturer's instructions (Clontech, Palo Alto, Calif.; 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
either on amplified sequences, or on sequences obtained from
isolates from the cDNA libraries. Template mRNA was isolated from
shear stressed HUVEC's.
[0363] Northern blot analysis of RNA extracted from various human
organs and tissues was performed using commercially available
pre-blotted filters (Clontech, Palo Alto, Calif.).
7.2. RESULTS
[0364] An amplified fchd531 fragment obtained from differential
display was subcloned and sequenced, and used to obtain a 1.9 kb
cDNA containing the entire fchd531 coding region. The DNA sequence
and encoded amino acid sequence of the novel fchd531 gene is shown
in FIG. 1. The fchd531 gene encodes a 570 amino acid polypeptide,
and has 94% sequence similarity to the mouse penta zinc finger gene
(Pzf) (GenBank accession number U05343). Northern analysis of
HUVEC's which were subjected turbulent and laminar shear stress
demonstrated that the fchd531 gene produces an approximately 5 kb
message which is down-regulated under laminar shear stress, but not
turbulent shear stress, compared with the static control.
[0365] The fchd540 gene was detected as an up-regulated message
under shear stress. The amplified fragment was used to probe a
Northern blot containing samples from HUVECs treated with laminar
shear stress. A 4.4 kb fchd540 mRNA is up-regulated after 6 hours
treatment with laminar shear stress. The fchd540 gene is not
induced by IL-1 by the method of Paradigm C, (Section 5.1.1.5,
above). The amplified fragment was sequenced and used to obtain a
2.7 kb cDNA containing the entire fchd540 coding region. The DNA
sequence and encoded amino acid sequence from the fchd540 gene is
shown in FIG. 2. The fchd540 gene encodes a 426 amino acid
polypeptide and has sequence similarity to the Drosophila Mad gene
(Sekelsky et al., 1995, Genetics 139: 1347-1358).
[0366] The fchd545 gene was detected as a down-regulated message
under shear stress. Northern analysis revealed that the fchd545
gene produces a 1.4kb message which is down regulated by turbulent
shear stress, but not by laminar shear stress, as compared with
static control. The amplified fragment was sequenced and used to
isolate a 1.4kb cDNA containing the complete fchd545 coding
sequence. The DNA sequence and encoded amino acid sequence of the
fchd545 gene is shown in FIG. 3. The fchd545 gene encodes a 283
amino acid polypeptide which has 73% sequence similarity to the
human Voltage-dependent Anion Channel (Blachly-Dyson, E., et al.,
1993, J. Biol. Chem. 268: 1835-1841). Northern analysis of a
commercially available (Clontech, Palo Alto, Calif.) northern blot
revealed that the fchd545 gene is expressed in human heart, smooth
muscle, and testes.
8. EXAMPLE: USE OF GENES UNDER PARADIGM A AS SURROGATE MARKERS IN
CLINICAL TRIALS
[0367] 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. The target genes,
therefore, serve as surrogate markers by giving an indicative
reading of the physiological response of monocytes to the uptake of
oxidized LDL. Accordingly, the influence of anti-oxidant drugs on
the oxidative potential may be measured by performing differential
display on the monocytes of patients undergoing clinical tests.
8.1. TREATMENT OF PATIENTS AND CELL ISOLATION
[0368] Test patients may be administered compounds suspected of
having anti-oxidant activity. Control patients may be given a
placebo.
[0369] 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. Primers may then be designed for amplification based on the
DNA sequence of target genes identified as up-regulated, such as
fchd602 and fchd605, or down-regulated under Paradigm A.
8.2. ANALYSIS OF SAMPLES
[0370] 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 corresponding to fchd602 and fchd605,
which were up-regulated by oxidized LDL under Paradigm A, as
described in Section 6.2, above.
9. EXAMPLE: IMAGING OF A CARDIOVASCULAR DISEASE CONDITION
[0371] 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.
[0372] For the purposes of illustration, this method is described
in detail for the fchd6o2 gene product. The principles and
techniques can be applied to any transmembrane target gene product,
including, for example, the fchd545 gene product.
9.1. MONOCLONAL CONJUGATED ANTIBODIES
[0373] The differentially expressed surface gene product, such as
the fchd602 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 fchd602 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.
9.2. ADMINISTRATION AND DETECTION OF IMAGING AGENTS
[0374] 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 fchd602 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 monocytes, in which fchd602 gene
expression is up-regulated.
10. POLYCLONAL ANTIBODIES TO TARGET GENE PEPTIDE SEQUENCES
[0375] Peptide sequences corresponding to the indicated amino
sequences of cDNAs were selected and submitted to Research Genetics
(Huntsville, AL) for synthesis and antibody production. Peptides
were modified as described (Tam, J. P., 1988, Proc. Natl. Acad.
Sci. USA 85: 5409-5413; Tam, J. P., and Zavala, F., 1989, J.
Immunol. Methods 124: 53-61; Tam, J. P., and Lu, Y. A., 1989, Proc.
Natl. Acad. Sci. USA 86: 9084-9088), emulsified in an equal volume
of Freund's adjuvant and injected into rabbits at 3 to 4
subcutaneous dorsal sites for a total volume of 1.0 ml (0.5 mg
peptide) per immunization. The animals were boosted after 2 and 6
weeks and bled at weeks 4, 8, and 10. The blood was allowed to clot
and serum was collected by centrifugation.
[0376] The peptides used are summarized below:
8 fchd545 Peptide Antigens Name Position Sequence fchd545.1 48-63
YTDTGKASGNLETKYK fchd545.2 107-121 TGKKSGKLKASYKRD
11. EXAMPLE: THE RCHD534 AND FCHD540 GENE PRODUCTS INTERACT
[0377] The novel fchd540 gene and its nucleotide sequence is
described in Section 7, above. The fchd540 gene shares homology
with the Drosophila Mad gene. The rchd534 gene (described in
Applicant's co-pending Application No. 08/485,573, filed Jun. 7,
1995, which is incorporated by reference in its entirety herein) is
another gene that is up-regulated in endothelial cells by shear
stress. The DNA and encoded amino acid sequence of the rchd534 gene
is shown in FIG. 6. The rchd534 gene was deposited in the
Agricultural Research Service Culture Collection (NRRL) in
microorganism FCHD534 on Jun. 6, 1995 and assigned the NRRL
Accession No. B-21459. The rchd534 gene also shares homology with
the Drosophila Mad gene. Mad genes have been shown to play a role
in the TGF-.beta. signalling pathway (Sekelsky et al., 1995,
Genetics 139: 1347-1358; Chen et al., 1996, Nature 383: 691-696;
Serra, et al., 1996, Nature Medicine 2: 390-391). TGF-.beta.
signalling is considered to be beneficial to atherosclerosis and
restenosis (Border et al., 1995, Nature Medicine 1: 1000; Grainger,
et al., 1995, Nature Medicine 1: 1067-1073; Kojima, et al., 1991,
J. Cell Biol. 113: 1439-1445; Nikol, et al., 1992, J. Clin. Invest.
90: 1582-1592).
[0378] The data described below demonstrate that the rchd534 and
fchd540 proteins interact with one another; and this interaction
may lead to the inhibition of TGF-.beta. signalling. Furthermore,
the expression of these two genes, as described below, is specific
to endothelial cells. Because these two genes 1) are both expressed
specifically in endothelial cells, 2) are both up-regulated in
endothelial cells under certain conditions, 3) encode MAD proteins
that interact with one another in endothelial cells, and 4) inhibit
TGF-.beta. signalling (which is considered to be beneficial to
atherosclerosis), rchd534 and fchd540 proteins are attractive
targets for therapeutic intervention in cardiovascular disease. In
particular, treatment regimens that inhibit the interaction or
activity of the rchd534 and fchd540 proteins can be beneficial for
the treatment cardiovascular disease.
[0379] Further analyses demonstrated that the rchd534 protein
interacts with itself to form a homodimer. Thus, treatment regimens
that inhibit the interaction of the rchd534 protein with itself can
be beneficial for the treatment cardiovascular disease.
[0380] In addition, the analyses described below demonstrated novel
interactions of both the rchd534 and fchd540 proteins with other
proteins known to be involved in the TGF-.beta. signalling pathway.
The protein members of the TGF-.beta. signalling pathway tested
included MADR1 (Hoodless et al., 1996, Cell 85:489-500), MADR2
(Eppert et al., 1996, Cell 86: 543-552), DPC4 (Raftery et al.,
1988, Genetics 139: 241-254), T.beta.RI, TSR1, ActRIb, ALK3, and
ALK6 (Wieser et al., 1995, EMBO J. 14: 2199-2208). For example, the
rchd534 protein interacts strongly in endothelial cells with MADR1,
MADR2, DPC4, and weakly in 293 (human embryonic kidney) cells with
activated forms of receptors T.beta.RI and ActRI. The fchd540
protein interacts strongly in 293 cells with activated forms of
receptors T.beta.RI and ALK6.
[0381] In the absence of transfected rchd543 and fchd540 genes,
transfected MADR1 or transfected MADR2 mediated a 20-fold induction
of a TGF-.beta. inducible promoter in BAECs. Co-expression of
either transfected rchd534 or transfected rchd540 in this system
eliminated the induction, and also prevented the localization of
MADR2 in the nucleus in response to TGF-.beta. signalling.
Therefore, treatment regimens that inhibit the interaction of the
rchd534 and fchd540 proteins with other proteins involved in the
TGF-.beta. pathway also can be beneficial for the treatment
cardiovascular of disease. As described above, the expression of
rchd534 and fchd540 is specific, within arterial tissue, to
endothelial cells. Accordingly, the rchd534 and rchd540 genes may
be targets for intervention in a variety of inflammatory and
fibroproliferative disorders that involve endothelial cells,
including, but not limited to, cancer, angiogenesis, inflammation,
and fibrosis.
11.1. MATERIALS AND METHODS
11.1.1. YEAST STRAINS, MEDIA, AND MICROBIOLOGICAL TECHNIQUES
[0382] Standard yeast media including synthetic complete medium
lacking L-leucine, L-tryptophan, and L-histidine were prepared and
yeast genetic manipulations were performed as described (Sherman,
1991, Meth. Enzymol., 194:3-21). Yeast transformations were
performed using standard protocols (Gietz et al., 1992, Nucleic
Acids Res., 20:1425. Ito et al., 1983, J. Bacteriol., 153:163-168).
Plasmid DNAs were isolated from yeast strains by a standard method
(Hoffman and Winston, 1987, Gene, 57:267-272).
11.1.2. PLASMID AND YEAST STRAIN CONSTRUCTION
[0383] The coding region of human fchd540 was amplified by PCR and
cloned in frame into pGBT9 (Bartel et al., 1993, Cellular
Interactions in Development. pp. 153-159) resulting in plasmid
pGBT9-fchd540. pGBT9-fchd540 was transformed into two-hybrid
screening strain HF7c and one resulting transformant was designated
TB35.
11.1.3. TWO-HYBRID SCREENING
[0384] Two-hybrid screening was carried out essentially as
described (Bartel et al., 1993, supra) using TB35 as the recipient
strain and a human breast two-hybrid library.
11.1.4. PAPER FILTER BETA-GALACTOSIDASE ASSAYS
[0385] The paper filter beta-galactosidase (beta-gal) assay was
performed essentially as previously described (Brill et al., 1994,
Mol. Biol. Cell 5: 297-312).
11.2. RESULTS
11.2.1. STRONG PHYSICAL INTERACTION OF RCHD534 AND FCHD540 MEASURED
BY TWO-HYBRID ASSAY
[0386] The fchd540 coding sequence was amplified by PCR and cloned
into pGBT9 creating a GAL4 DNA-binding domain-fchd540 fusion gene.
The screening strain HF7c was transformed with this construct. The
rchd534 coding sequence was cloned into pGAD424 (Bartel et al.,
1993, supra) creating a GAL4 transcriptional activation
domain-rchd534 fusion gene, which was then used to transform strain
Y187.
[0387] Yeast expression plasmids encoding the GAL4 DNA-binding
domain either alone or fused in frame to fchd540, rchd534,
Drosophilia MAD, DPC4, or p53 were transformed into MATa two-hybrid
screening strain HF7c. Yeast expression plasmids encoding the GAL4
transcriptional activation domain alone and GAL4 activation domain
fusions to rchd534 and SV40 were transformed into MATA two-hybrid
screening strain Y187. p53 and SV40 interact with each other and
should not interact with the experimental proteins. The HF7c
transformants were propagated as stripes on semisolid synthetic
complete medium lacking L-tryptophan and the Y187 transformants
were grown as stripes on semisolid synthetic complete medium
lacking L-leucine. Both sets of stripes were replica plated in the
form of a grid onto a single rich YPAD plate and the haploid
strains of opposite mating types were allowed to mate overnight at
30.degree. C. The yeast strains on the mating plate were then
replica plated to a synthetic complete plate lacking L-leucine and
L-tryptophan to select for diploids and incubated at 30.degree. C.
overnight. Diploid strains on the synthetic complete plate lacking
L-leucine and L-tryptophan were replica plated to a synthetic
complete plate lacking L-leucine, L-tryptophan, and L-histidine to
assay HIS3 expression and a paper filter on a synthetic complete
plate lacking L-leucine and L-tryptophan. The next day the paper
filter was subjected to the paper filter beta-galactosidase assay
to measure expression of the lacZ reporter gene. HIS3 expression
was scored after 3 days of growth at 30.degree. C. The results are
shown in Table 3.
[0388] The rchd534 fish protein was found to interact strongly with
the fchd540 bait protein and not to interact with the rchd534, MAD,
DPC4, p53, and GAL4 DNA binding domain bait proteins. This result
demonstrated that rchd534 and fchd540 strongly physically interact
with each other with significant specificity.
11.2.2. IDENTIFICATION OF PROTEINS THAT PHYSICALLY INTERACT WITH
FCHD540
[0389] The fchd540 coding sequence was amplified by PCR and cloned
into pGBT9 (Bartel et al., 1993, supra) creating a GAL4 DNA-binding
domain-fchd540 fusion gene. HF7c was transformed with this
construct resulting in strain TB35. TB35 grew on synthetic complete
medium lacking L-tryptophan but not on synthetic complete medium
lacking L-tryptophan and L-histidine demonstrating that the GAL4
DNA-binding domain-fchd540 fusion does not have intrinsic
transcriptional activation activity.
[0390] TB35 was transformed with the human breast two-hybrid
library and 5 million transformants were obtained. The
transformants were plated on synthetic complete medium lacking
L-leucine, L-tryptophan, and L-histidine and yeast colonies that
both grew on synthetic complete medium lacking L-leucine,
L-tryptophan, and L-histidine and expressed the beta-galactosidase
reporter gene were identified. The 30 strains with the strongest
beta-galactosidase induction were characterized. Library plasmids
were isolated from these strains, and the 5' ends of all of the
cDNA inserts were sequenced.
11.2.3. RETRANSFORMATION AND SPECIFICITY TESTING OF TCHV03A AND
TCHVR4A
[0391] Two of the plasmids that encoded the strongest interactors
were found to contain rchd534 cDNAs. Plasmid tchv03A was found to
encode amino acids 17-235 of rchd534 and plasmid tchvR4A was found
to encode amino acids 25-235 of rchd534.
[0392] It was confirmed that these rchd534 cDNAs encode proteins
that physically interact specifically with fchd540. Yeast
expression plasmids encoding the GAL4 DNA-binding domain either
alone or fused in frame to fchd540, rchd534, Drosophila MAD, DPC4,
and p53 were transformed into MATa two-hybrid screening strain
HF7c. Yeast expression plasmids encoding the GAL4 transcriptional
activation domain (GAL4 AD) alone and GAL4 activation domain
fusions to tchv03a, tchvR4A and SV40 were transformed into MATA
two-hybrid screening strain Y187. p53 and SV40 interact with each
other and should not interact with the experimental proteins. The
HF7c transformants were propagated as stripes on semi-solid
synthetic complete medium lacking L-leucine. Both sets of stripes
were replica plated in the form of a grid onto a single rich YPAD
plate and the haploid strains of opposite mating types were allowed
to mate overnight at 30.degree. C. The yeast strains on the mating
plate were then replica plated to a synthetic complete plate
lacking L-leucine and L-tryptophan to select for diploids and
incubated at 30.degree. C. overnight. Diploid strains on the
synthetic complete plate lacking L-leucine and L-tryptophan were
replica plated to a synthetic complete plate lacking L-leucine,
L-tryptophan, and L-histidine to assay HIS3 expression and a paper
filter on a synthetic complete plate lacking L-leucine and
L-tryptophan. The next day the paper filter was subjected to the
paper filter beta-galactosidase assay to measure expression of the
lacZ reporter gene. HIS3 expression was scored after 3 days of
growth at 30.degree. C. The results are shown in the table below.
The strength or absence of physical interaction between each
combination of test proteins is listed. Strong interactions are
defined as interactions that cause the activation of both the HIS3
and lacZ reporter genes.
9TABLE 3 GAL4 DNA- Binding cDNA-GAL4 Activation Domain Fusion
Tested Domain GAL4 AD Fusions rchd534 tchv03A tchvR4A SV40 alone
fchd540 Strong Strong Strong None None rchd534 None None None None
None Dros. MAD None None None None None DPC4 None None None None
None p53 None None None Strong None GAL4 DNA- None None None None
None Binding Domain alone
[0393] The tchv03A and tchvR4A fish proteins were found to interact
strongly with the fchd540 bait protein and to not interact with the
rchd534, MAD, DPC4, p53, and GAL4 DNA binding domain bait proteins.
These results confirm the result that the rchd534 and fchd540
proteins interact strongly with each other.
11.3. FURTHER ANALYSIS OF RCHD534 AND FCHD540 FUNCTION
[0394] The significance of the rchd534/fchd540 protein interaction
was confirmed by examination of their expression and activity in
human cells and animal models.
11.3.1. CHROMOSOMAL LOCALIZATION
[0395] The rchd534 gene was localized to chromosome 15 and the
fchd540 gene was localized to chromosome 18, regions of the human
genome that contain other MAD homologues. These regions of the
human genome have also been implicated in the pathogenesis of
several human malignancies.
11.3.2. TISSUE EXPRESSION PATTERNS
[0396] The expression patterns were examined using in situ
hybridization techniques. Fluorescently labeled DNA probes of both
the rchd534 and fchd540 genes were used to probe human carotid
endartectomy samples. The expression of rchd534 and fchd540 was
specific to endothelial cells lining the luminal surface of the
carotid artery. In addition, a rabbit polyclonal antiserum
generated against the rchd534 gene product prominently and
selectively stained the endothelium present in large vessels such
as human coronary arteries as well as smaller vessels present
within human myocardium. Neither gene showed expression in any
other cell type present in the arterial tissue sample, including
smooth muscle cells and macrophages.
[0397] Expression patterns of both genes were also examined in
response to certain stimulus. Both genes are selectively
upregulated under the steady laminar shear stress (LSS) paradigm,
but not under the turbulent shear stress paradigm or in response to
stimulus by the cytokines rhIL-1.beta., TNF.alpha., IFN.gamma. or
active TGF.beta. as measured in HUVEC cells. Thus, the rchd534 and
the fchd540 genes appear to be selectively responsive to a LSS
stimulus, manifesting no response to a non-laminar fluid mechanical
stimulus, nor any other humoral stimuli tested. Thus, given that
these two genes are: (1) localized to a region of the human genome
that has been implicated in the pathogenesis of several human
malignancies; (2) specifically expressed in a cell-type that is
found only in vascular tissue, including atherosclerotic plaques;
(3) up-regulated under the steady laminar shear stress
cardiovascular disease paradigm; and (4) specifically inhibit
TGF-.beta. signalling indicate that rchd534 and fchd540 are
excellent and specific targets for therapeutic intervention in the
treatment of fibroproliferative and oncogenic disorders including
tumor growth and vascularization.
11.3.3. CELLULAR LOCALIZATION
[0398] The cellular localization of the rchd534 and fchd540
proteins in bovine aortic endothelial cells (BAECs) was examined in
relationship to other proteins involved in the TGF-.beta.
signalling pathway. In all experiments, the rchd534 and fchd540
proteins were located in the cytoplasm. MADR2 was located in the
cytoplasm when transfected alone and in the nucleus when
co-transfected with activated T.beta.RI or when TGF-.beta. was
added to the culture medium. Co-transfection of rchd534 or fchd540
with MADR2 prevented the localization of MADR2 in the nucleus in
response to TGF-.beta. signalling.
11.3.4. PROTEIN INTERACTIONS IN HUMAN CELLS
[0399] The interaction of the rchd534 and fchd540 proteins,
observed in yeast cells as described above, was tested in mammalian
endothelial cell tissue culture. Either bovine aortic endothelial
cells (BAECs) or 293 cells (human embryonic kidney cells, ATCC
Accession No. CRL-1573) were transfected with constructs encoding
both the rchd534 and fchd540 proteins, each fused to a different
flag peptide allowing for specific immunoprecipitation. The rchd534
and fchd540 proteins were found to co-immunoprecipitate as
heterodimers in extracts produced from both 293 cells and BAECs.
The co-immunoprecipitation of rchd534 and fchd540 further supports
that these proteins interact in human cells that are
physiologically relevant to cardiovascular disease.
[0400] The ability of the rchd534 and fchd540 proteins to interact
with themselves and with other protein members of the TGF-.beta.
signalling pathway (MADR1, MADR2, DPC4, TbR1, TSR1, ActR1b, ALK3,
ALK6), was tested using this co-immunoprecipitation method. Each
gene was transfected alone and in various combinations with other
TGF-.beta. pathway genes in either 293 cells or BAECs. The rchd534
protein formed homodimers in 293 cells and BAECs. The fchd540
protein did not form homodimers in 293 cells or BAECs. As mentioned
above, the rchd534 and fchd540 proteins formed heterodimers in 293
cells and BAECs. This interaction is about 50 fold stronger in
BAECs than 293 cells based on equal amounts of protein. However,
the rchd534-fchd540 protein interaction was significantly less avid
than the rchd534 protein's interaction with itself.
[0401] The rchd534 protein interacted with MADR1, MADR2, and DPC4
in 293 cells and BAECs. The strength of MADR1 and MADR2
interactions was about the same between 293 cells and BAECs and
much greater in BAECs for DPC4. The fchd540 protein interacted very
weakly with MADR1, MADR2, and DPC4 in 293 cells. The rchd534
protein interacted strongly with activated forms of T.beta.RI and
ActRI and weakly with activated ALK6 in 293 cells. The fchd540
protein interacted strongly with activated T.beta.RI and ALK6
receptors, and weakly with activated forms of TSRI, ALK3, and
ActRIb in 293 cells. Thus, in addition to the interaction of the
rchd534 and fchd540 proteins, the interaction of the rchd534
protein with itself, as well as the interaction of the rchd534
protein and the fchd540 protein with the other proteins in the
TGF-.beta. pathway described above are excellent targets for
therapeutic intervention.
11.3.5. EFFECT OF EXPRESSION ON TGF-B SIGNALLING
[0402] The effect of both rchd534 and fchd540 on the TGF-.beta.
signalling pathway was tested in vitro. Primary BAECs were
transfected with a construct called p3TP-Lux, containing a
TGF-.beta. responsive promoter fused to a reporter gene (Wrana et
al., 19944, Nature 370: 341-347). The rchd534 gene or the fchd540
gene in pCI expression vectors (Promega) was transfected with and
without MADR1 (pCMV5MADR1-Flag, Hoodless et al. 1996 Cell 85:
489-500) or MADR2 (pCMV5MADR2-Flag, Eppert et al. 1996 Cell 86:
543-552). The TGF-.beta. response was induced 20-fold by either
MADR1 or MADR2. Co-expression of either rchd534 or fchd540
completely eliminated this induction. Thus, the rchd534 and fchd540
proteins inhibited MADR1- and MADR2-mediated TGF-.beta. signalling
in endothelial cells. To confirm the specificity of this inhibitory
effect, site specific mutants of both rchd534 or fchd540 were
constructed, based on known mutations identified in Drosophila
homologues, that would be predicted to disrupt MAD-like signaling
functions (Sekelsky et al., 1995, Genetics 139:1347-58; Raftery,
1995, Genetics 139:241-54; Newfeld et al., 1996, Development
122:2099-108; Wiersdorff et al., 1996, Development 122:2153-62).
Unlike wild type rchd534 and fchd540, these mutant proteins were
unable to inhibit the activation of the p3TP promoter in response
to TGF-.beta.. The expression levels of the mutant and wild-type
proteins were comparable indicating the loss of function was not
due to secondary instability.
[0403] Interestingly, Smad3, the C. elegans homolog to MAD3 which
also functions in TGF.beta. signalling is over 90% identical to
Smad2, the C. elegans MAD2 homolog, in the MH2 domain. Although
this has not yet been directly investigated, it is likely that
Smad7, the C. elegans homolog of the fchd540 gene, may function
similarly to its inhibition to prevent association and activation
of Smad3 by the TGF.beta. receptor, that is, to inhibit the
phosphorylation of Smad3 and its association with protein
components of the TGF-.beta. signalling pathway.
[0404] These results further demonstrate that the interactions of
either the rchd534 protein or the fchd540 protein with MADR2 or
with activated T.beta.R1 are excellent targets for therapeutic
intervention. As described above, the expression of rchd534 and
fchd540 is specific, within arterial tissue, to endothelial cells.
Accordingly, the rchd534 and rchd540 genes may be targets for
intervention in a variety of inflammatory and fibroproliferative
disorders that involve endothelial cells, including, but not
limited to, cancer angiogenesis, inflammation, and fibrosis.
12. EXAMPLE: ANTISENSE AND RIBOZYME MOLECULES FOR INHIBITION OF
RCHD534 AND FCHD540 EXPRESSION
[0405] The principles presented in Section 5.6.1.1, above, can be
used to design oligonucleotides for use in inhibiting the
expression of target genes, such as the rchd534 or fchd540
genes.
[0406] The following antisense molecules can be used to inhibit the
expression of the rchd534 gene:
[0407] Antisense:
[0408] a) 5'-CATTTCATTTCATACAA-3' which is complementary to
nucleotides -14 to +3 of rchd534 in FIG. 6.
[0409] b) 5'-CATTTCATTTCATACAATATATG-3' which is complementary to
nucleotides -20 to +3 of rchd534 in FIG. 6.
[0410] c) 5'-CATTTCATTTCATACAATATATGGCCTTT-3' which is
complementary to nucleotides -26 to +3 of rchd534 in FIG. 6.
[0411] d) 5'-CATTTCATTTCATACAATATATGGCCTTTTGTGGC-3' which is
complementary to nucleotides -32 to +3 of rchd534 in FIG. 6.
[0412] e) 5'-GGACATTTCATTTCATACAATATATGGCCTTTTGT-3' which is
complementary to nucleotides -29 to +6 of rchd534 in FIG. 6.
[0413] f) 5'-TTCATTTCATACAATATATGGCCTTTTGT-3' which is
complementary to nucleotides -29 to -1 of rchd534 in FIG. 6.
[0414] f) 5'-TCATACAATATATGGCCTTTTGT-3' which is complementary to
nucleotides -29 to -7 of rchd534 in FIG. 6.
[0415] h) 5'-AATATATGGCCTTTTGT-3' which is complementary to
nucleotides -29 to -13 of rchd534 in FIG. 6.
[0416] The following antisense molecules can be used to inhibit the
expression of the fchd540 gene:
[0417] a) 5'-CATGCGGGGCGAGGAGG-3' which is complementary to
nucleotides -14 to +3 of fchd540 in FIG. 2.
[0418] b) 5'-CATGCGGGGCGAGGAGGCGAGGA-3' which is complementary to
nucleotides -20 to +3 of fchd540 in FIG. 2.
[0419] c) 5'-CATGCGGGGCGAGGAGGCGAGGAGAAAAG-3' which is
complementary to nucleotides -26 to +3 of fchd540 in FIG. 2.
[0420] d) 5'-CATGCGGGGCGAGGAGGCGAGGAGAAAAGTCGTTT-3' which is
complementary to nucleotides -32 to +3 of fchd540 in FIG. 2.
[0421] e) 5'-GAACATGCGGGGCGAGGAGGCGAGGAGAAAAGTCG-3' which is
complementary to nucleotides -29 to +6 of fchd540 in FIG. 2.
[0422] f) 5'-GCGGGGCGAGGAGGCGAGGAGAAAAGTCG-3' which is
complementary to nucleotides -29 to -1 of fchd540 in FIG. 2.
[0423] g) 5'-CGAGGAGGCGAGGAGAAAAGTCG-3' which is complementary to
nucleotides -29 to -7 of fchd540 in FIG. 2.
[0424] h) 5'-GGCGAGGAGAAAAGTCG-3' which is complementary to
nucleotides -29 to -13 of fchd540 in FIG. 2.
[0425] Ribozymes:
[0426] The central, catalytic portion of a hammerhead ribozyme
molecule consist of the following sequence:
5'-CAAAGCNGNXXXXNCNGAGNAGUC-3';
[0427] wherein the 5'-proximal CA bases hybridize to a
complementary 5'-UG-3' in the target mRNA. The first four
underlined bases form a stem by base pairing with the second set of
underlined bases, with the intervening bases, shown as X's, forming
a non-pairing loop. In order to hybridize to a target mRNA, a
hammerhead ribozyme contains additional bases flanking each end of
the central segment shown above. The 5' ribozyme flanking segment
is complementary to the respective flanking sequences immediately
3' to the target UG; and the 3' flanking segment is complementary
to the respective flanking sequence beginning two bases upstream of
the target U, and extending 5'-ward (in effect, skipping the first
base upstream of the target U). Cleavage occurs between first and
second bases upstream of (i.e., 5' to) the U in the target 5'-UG-3'
site.
[0428] The following ribozyme molecules can be used to inhibit the
expression of the rchd534 gene:
[0429] a) 5'-GGUGGAGCCCCAGGGCAUUACCUCAAAGCNGNXXXXNCNGAGNAGUCGUGG
GCAAGGUGGGCACUCAGGUGGG-3' which will cleave the rchd534 mRNA
between nucleotides 716 and 717 in FIG. 6.
[0430] b) 5'-GUGUCUCUAUGGGUUUGCCCAAAGCNGNXXXXNCNGAGNAGUCUCUGGACA
UUUCAUUUCAUAC-3' which will cleave the rchd534 mRNA between
nucleotides 1040 and 1041 in FIG. 6.
[0431] c) 5'-GGCCCUCUCGCCGUCGGGCUCCUUGCUGAGCAAAGCNGNXXXXNCNGAGNA
GUCGAUGCCGAAGCCGAUCUUGCUGCGCG-3' which will cleave between
nucleotides 1421 and 1422 in FIG. 6.
[0432] The following ribozYme molecules can be used to inhibit the
expression of the fchd540 gene:
[0433] a) 5'-CGUUUGCCUGCUAAGGAGCGAACAAAGCNGNXXXXNCNGAGNAGUCGAUGU
UUCUUUGUGAGUCGGGCGCCG-3', which will cleave the fchd540 mRNA
between nucleotides -53 and -52 in FIG. 2.
[0434] b) 5'-CGCCGGACGAGCGCAGAUCGUUUGGUCCUGAACAAAGCNGNXXXXNCNGAG
NAGUCCGGGGCGAGGAGGCGAGGAGAAAAGUCG-3', which will cleave the fchd540
mRNA between nucleotides -1 and +1 in FIG. 2.
[0435] c) 5'-GGAGUAAGGAGGGGGGGGAGACUCUAGUUCGCAAAGCNGNXXXXNCNGAGN
AGUCAGUCGGCUAAGGUGAUGGGGGUUGCAGCACACC-3' which will cleave the
fchd540 mRNA between nucleotides +602 and +603 in FIG. 2.
13. DEPOSIT OF MICROORGANISMS
[0436] The following microorganisms were deposited with the
American Type Culture Collection (ATCC), Rockville, Md., on the
indicated dates and assigned the indicated accession numbers:
10 Microorganism ATCC Accession No. Date of Deposit pFCHD531 69983
February 7, 1996 pFCHD540 69984 February 7, 1996 fchd545 69974
January 5, 1996
[0437] The following microorganism was deposited with the
Agricultural Research Service Culture Collection (NRRL), Peoria,
Ill., on the indicated date and assigned the indicated accession
number:
11 Microorganism NRRL Accession No. Date of Deposit FCHD534 B-21459
June 6, 1995
[0438] 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.
* * * * *