U.S. patent application number 11/981488 was filed with the patent office on 2008-05-15 for novel 27875, 22025, 27420, 17906, 16319, 55092 and 10218 molecules and uses therefor.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Joseph M. Carroll, Miyoung Chun, William James Cook, Rosana Kapeller-Libermann, Kyle J. MacBeth, Rachel E. Meyers, Keith E. Robison, David White, Mark J. Williamson.
Application Number | 20080113399 11/981488 |
Document ID | / |
Family ID | 30004156 |
Filed Date | 2008-05-15 |
United States Patent
Application |
20080113399 |
Kind Code |
A1 |
Kapeller-Libermann; Rosana ;
et al. |
May 15, 2008 |
Novel 27875, 22025, 27420, 17906, 16319, 55092 and 10218 molecules
and uses therefor
Abstract
The invention provides isolated nucleic acids molecules,
designated 27875, 22025, 27420, 16319, 55092 and 10218 nucleic acid
molecules. The invention also provides antisense nucleic acid
molecules, recombinant expression vectors containing 27875, 22025,
27420, 16319, 55092 and 10218 nucleic acid molecules, host cells
into which the expression vectors have been introduced, and
nonhuman transgenic animals in which a 27875, 22025, 27420, 16319,
55092 and 10218 gene has been introduced or disrupted. The
invention still further provides isolated 27875, 22025, 27420,
17906, 16319, 55092 or 10218 proteins, fusion proteins, antigenic
peptides and anti-27875, 22025, 27420, 17906, 16319, 55092 or 10218
antibodies. Diagnostic and therapeutic methods utilizing
compositions of the invention are also provided.
Inventors: |
Kapeller-Libermann; Rosana;
(Chestnut Hill, MA) ; White; David; (Braintree,
MA) ; Robison; Keith E.; (Wilmington, MA) ;
MacBeth; Kyle J.; (Boston, MA) ; Carroll; Joseph
M.; (Cambridge, MA) ; Cook; William James;
(Hanover, NH) ; Meyers; Rachel E.; (Newton,
MA) ; Chun; Miyoung; (Belmont, MA) ;
Williamson; Mark J.; (Saugus, MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
30004156 |
Appl. No.: |
11/981488 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11226701 |
Sep 14, 2005 |
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11981488 |
Oct 31, 2007 |
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10386414 |
Mar 11, 2003 |
7098015 |
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11226701 |
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09426282 |
Oct 25, 1999 |
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10386414 |
Mar 11, 2003 |
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09668266 |
Sep 22, 2000 |
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10386414 |
Mar 11, 2003 |
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09330970 |
Jun 11, 1999 |
6146876 |
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09668266 |
Sep 22, 2000 |
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09724599 |
Nov 28, 2000 |
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10386414 |
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09860193 |
May 16, 2001 |
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10386414 |
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09571689 |
May 16, 2000 |
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09860193 |
May 16, 2001 |
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10283023 |
Oct 29, 2002 |
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10386414 |
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10010943 |
Dec 6, 2001 |
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10386414 |
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09833082 |
Apr 10, 2001 |
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10386414 |
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60335044 |
Oct 31, 2001 |
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60254037 |
Dec 7, 2000 |
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Current U.S.
Class: |
435/24 |
Current CPC
Class: |
G01N 33/502 20130101;
G01N 33/56994 20130101; G01N 2500/04 20130101; G01N 33/5011
20130101; G01N 33/68 20130101; G01N 2333/96486 20130101; C12Q
2600/158 20130101; G01N 33/5767 20130101; C12Q 1/6883 20130101;
A61P 19/08 20180101; C12Q 1/37 20130101; G01N 33/56983 20130101;
G01N 2333/916 20130101; A61K 38/00 20130101; C12Y 301/04017
20130101; C12N 9/1007 20130101; A61P 35/00 20180101; G01N 33/57426
20130101; G01N 33/5008 20130101; A61P 7/00 20180101; G01N 33/5761
20130101; A61P 9/00 20180101; C12N 9/16 20130101; A61P 31/12
20180101; C12N 9/6489 20130101 |
Class at
Publication: |
435/024 |
International
Class: |
C12Q 1/37 20060101
C12Q001/37 |
Claims
1. A method for identifying a compound capable of treating a
bone-associated disorder, comprising: (a) contacting a polypeptide
which comprises the amino acid sequence of SEQ ID NO:11 with a test
compound; (b) assaying the ability of the test compound to modulate
the carboxypeptidase activity of the polypeptide, thereby
identifying a compound capable of treating a bone-associated
disorder.
2. A method for identifying a compound capable of modulating a bone
cell activity comprising: (a) contacting a bone cell with a test
compound; (b) assaying the ability of the test compound to modulate
the expression or carboxypeptidase activity of a polypeptide
comprising the amino acid sequence of SEQ ID NO:11, thereby
identifying a compound capable of modulating a bone cell activity.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 11/226,701, filed Sep. 14, 2005 (pending),
which is a divisional of U.S. patent application Ser. No.
10/386,414, filed Mar. 11, 2003, now U.S. Pat. No. 7,098,015, which
is a continuation-in-part of U.S. patent application Ser. No.
09/426,282, filed Oct. 25, 1999 (abandoned). U.S. patent
application Ser. No. 10/386,414 is also a continuation-in-part of
U.S. patent application Ser. No. 09/668,266, filed Sep. 22, 2000
(abandoned), which is a divisional of U.S. application Ser. No.
09/330,970, filed Jun. 11, 1999, now U.S. Pat. No. 6,146,876. U.S.
patent application Ser. No. 10/386,414 is also a
continuation-in-part of U.S. patent application Ser. No.
09/724,599, filed Nov. 28, 2000 (abandoned). U.S. patent
application Ser. No. 10/386,414 is also a continuation-in-part of
U.S. patent application Ser. No. 09/860,193, filed May 16, 2001
(abandoned), which is a divisional of U.S. application Ser. No.
09/571,689, filed May 16, 2000 (abandoned). U.S. patent application
Ser. No. 10/386,414 is also a continuation-in-part of U.S. patent
application Ser. No. 10/283,023, filed Oct. 29, 2002 (abandoned),
which claims the benefit of U.S. Provisional Application Ser. No.
60/335,044, filed Oct. 31, 2001 (abandoned). U.S. patent
application Ser. No. 10/386,414 is also a continuation-in-part of
U.S. patent application Ser. No. 10/010,943, filed Dec. 6, 2001
(abandoned), which claims the benefit of U.S. Provisional
Application Ser. No. 60/254,037, filed Dec. 7, 2000 (abandoned).
U.S. patent application Ser. No. 10/386,414 is also a
continuation-in-part of U.S. patent application Ser. No.
09/833,082, filed Apr. 10, 2001 (abandoned). The entire contents of
each of the above-referenced patent applications are incorporated
herein by this reference. TABLE-US-00001 INDEX Chapter Page Title
I. 2 27875, A NOVEL HUMAN ADAMS-TS HOMOLOG II. 78 22025, A NOVEL
HUMAN CYCLIC NUCLEOTIDE PHOSPHODIESTERASE III. 143 METHODS AND
COMPOSITIONS FOR DIAGNOSIS AND TREATMENT OF CANCER USING 27420 IV.
228 METHOD OF TREATING BONE DISEASE USING 17906 V. 318 METHODS AND
COMPOSITIONS FOR THE DIAGNOSIS AND TREATMENT OF HEMATOLOGICAL
DISORDERS USING 16319 VI. 375 METHODS AND COMPOSITIONS FOR THE
DIAGNOSIS AND TREATMENT OF VIRAL DISEASE USING 55092 VII. 451
METHODS AND COMPOSITIONS FOR TREATING CARDIOVASCULAR DISEASE USING
10218
I. 27875, A NOVEL HUMAN ADAMS-TS HOMOLOG
Background of the Invention
[0002] Metalloproteinases are a group of widely distributed
proteolytic enzymes that depend on bound Ca.sup.2+ or Zn.sup.2+ for
activity; however, certain metalloproteinases can readily utilize
Mn.sup.2+ and Mg.sup.2+. Biological functions of metalloproteinases
include protein maturation, degradation of proteins, such as
extracellular matrix proteins, tumor growth, metastasis and
angiogenesis.
[0003] Disintegrins are integrin ligands that disrupt cell/cell
(aggregation) and cell-matrix (adhesion) interactions by inhibiting
the binding of other physiological ligands to integrins.
Disintegrins have a conserved spacing of cysteine residues that is
required for their direct binding to integrin metalloproteinases
(Niewiarowski et al. (1994) Semin Hematol 31:289).
[0004] TSP I motifs are conserved domains in Thrombospondin 1 and
2, multifunctional secretory glycoproteins involved in blood
clotting, inhibiting angiogenesis and regulating the proliferation,
adhesion and migration of normal and tumor cells. The biological
activities of thrombospondin 1 and 2 are mediated by the binding of
the TSP type I motifs to extracellular matrix molecules, such as
heparan sulfate, proteoglycans, fibronectin, laminin and collagen.
Thrombospondin-1 is a platelet-derived glycoprotein that is
released from platelet alpha granules in response to thrombin
stimulation. It is involved in cell adhesion and modulates cell
movement, cell proliferation, neurite outgrowth and
angiogenesis.
[0005] ADAMs comprise a broad family of multifunctional proteins,
characterized as having a disintegrin and metalloproteinase domain
(Wolfsberg et al. (1995) Developmental Biol 169:378-383; Wolfsberg
et al. (1995) J Cell Biol 131:275-278). Approximately 20 ADAMs have
been identified to date. The prototypical ADAM is a
membrane-anchored glycoprotein with pro-, metalloproteinase,
disintegrin, cystine-rich, epidermal growth factor-like,
transmembrane and cytoplasmic domains. Members of the ADAM family
of proteins include MDC (ADAM1), fertilin .beta. (ADAM2),
cryitestin (ADAM3), epididymal apical protein I, meltrin, MS2,
TNF-.alpha. converting enzyme, Kuzbanian and metargidin.
[0006] ADAMs participate in a variety of roles, including cell-cell
and cell-matrix interactions and polypeptide processing. Examples
of ADAM functions include tumor cell adhesion (Iba et al. (1999) Am
J Pathol 154:1489-1501), tumor suppression (Emi et al. (1993)
Nature Genet 5:151-157), spermatogenesis and mediation of fusion of
gamete membranes (Evans et al. (1999) Biol Reprod 59:145-152),
blastocyst implantation (Olson et al. (1998) Cell Tissue Res
293:489-498), myotube formation and myoblast fusion (Gilpin et al.
(1998) J Biol Chem 273:157-166), immunity (Higuchi et al. (1999)
Immunol Today 20:278-284), proteolytic processing of ligands that
activate epidermal growth factor metalloproteinase (Dong et al.
(1999) Proc Natl Acad Sci USA 96:6235-6240), proteolytic cleavage
of Alzheimer's amyloid precursor protein (Lammich et al (1999) Proc
Natl Acad Sci USA 96:3922-3927; Buxbaum et al. (1998) J Biol Chem
273:27765-27767), processing of Notch ligands (Qi et al. (1999)
Science 283:91-94), neurogenesis (Rooke et al. (1996) Science
273:1227-1231), cleavage of murine mannose metalloproteinase to
produce a soluble mannose metalloproteinase (Martinez-Pomares et
al. (1998) J Biol Chem 273:23376-23380), and maturation of
TNF-.alpha. (Lunn et al. (1997) FEBS Lett 400:333-335). The
cell-cell interactions are thought to be mediated by the
disintegrin domain.
[0007] The cloning of ADAM-TS-1, a novel murine ADAM, was reported
(Kuno et al. (1997) J Biol Chem 272:556-562). ADAM-TS-1 is
selectively expressed in the cachexigenic colon 26 adenocarcinoma
cell line and is believed to be associated with acute inflammation
and cancer cachexia. ADAM-TS-1 is a 951 amino acid polypeptide
comprising a signal peptide, a prodomain, a catalytically active
zinc-dependent metalloproteinase domain, a disintegrin domain, and
three thrombospondin (TSP) type 1 domains, which are responsible
for anchoring ADAM-TS-1 to the extracellular matrix. In contrast to
other ADAMs, ADAM-TS-1 does not possess a transmembrane domain or
an epidermal growth factor-like domain. Rather, ADAM-TS-1 is
secreted and is associated with the extracellular matrix.
[0008] More recent reports from this group (Kuno et al. (1999) J.
Biol. Chem. 274:18821-18826; Kuno et al. (1998) J. Biol. Chem.
273:13912-13917) also showed ADAM-TS-1 to be a unique ADAM family
protein with respect to the presence of thrombospondin type 1
motifs and the capacity to bind to the extracellular matrix. Like
the other members of the ADAM family, the amino terminal half
region of ADAM-TS-1 consists of a proprotein and a
metalloproteinase domain and a disintegrin-like domain that share
sequence similarity to snake venom metalloproteinases. In contrast,
the domain organization of the carboxy terminal half is completely
different from other ADAMs. Instead of the transmembrane region,
ADAM-TS-1 has three thrombospondin-type 1 motifs found in
thrombospondins 1 and 2. These motifs are functional for binding
two molecules of heparin. The ADAM-TS-1 is secreted and
incorporated into the extracellular matrix. The three
thrombospondin-type 1 motifs are responsible for anchoring to the
extracellular matrix. The ADAM-TS-1 was shown to have a
zinc-binding motif in the metalloproteinase domain providing the
capacity to bind to .alpha..sub.2-macroglobulin. Accordingly,
soluble ADAM-TS-1 was shown to be able to form a covalent binding
complex with .alpha..sub.2-macroglobulin. A point mutation in this
motif was shown to eliminate the capacity to bind to the
.alpha..sub.2-macroglobulin. In addition, the studies reported that
the removal of the prodomain from the ADAM-TS-1 precursor was
impaired in a furin-deficient cell line and that the processing
ability of the cells was restored by coexpression of the furin
cDNA. These results provided evidence that the ADAM-TS-1 precursor
is processed in vivo by furin endopeptidase in the secretory
pathway. It was accordingly proposed that ADAM-TS-1 plays a role in
the inflammatory process through its protease activity.
[0009] Expression of the gene was shown to be induced in kidney and
in heart by in vivo administration of lipopolysaccharide,
suggesting a possible role in the inflammatory reaction. (Kuno et
al. (1998)).
[0010] Using a transient expression system, it was shown that both
precursor and processed forms of ADAM-TS-1 are secreted from cells.
The majority was associated with the extracellular matrix. When
cells were cultured in the presence of heparin, the mature form of
ADAM-TS-1 was detected in cell culture medium, suggesting that the
binding of the protein to the extracellular matrix is mediated
through a sulfated glycosaminoglycan. Deletion mutation analysis
showed that the spacer region and the three thrombospondin-type 1
motifs in the carboxy terminal region are important for interaction
with the extracellular matrix (Kuno et al. (1998)).
[0011] The thrombospondin-type 1 motif is conserved in
thrombospondins 1 and 2 which are multifunctional extracellular
matrix proteins that influence cell adhesion, motility, and growth
(Kuno et al. (1998)). Thrombospondin-type 1 motifs and
thrombospondins have two conserved heparin-binding segments:
W(S/G)XWSXW and CSVTCG). ADAM-TS-1 contains a middle thrombospondin
1 motif with sequences similar to the following heparin-binding
segments in thrombospondins: WGPWGPW and CS(R/K)TCG. The carboxy
terminal submotifs have only the latter sequence. Kuno et al.
(1998) show that the middle and carboxy terminal TSP submotifs of
the ADAM-TS-1 protein are able to bind heparin. The report
concluded that the data demonstrate that the interaction between
the three motifs and sulfated glycosaminoglycans in the
extracellular matrix, such as heparan sulfate, plays a role in the
extracellular matrix binding of the ADAM-TS protein. However, the
report also showed that truncation of the spacer region intervening
between the middle and carboxyl terminal TSP-type 1 motifs
significantly reduced the extracellular matrix binding of the
protein. Accordingly, it was concluded that, in addition to the
three TSP Type 1 motifs, the carboxy terminal spacer domain is
important for tight binding to the extracellular matrix. Finally,
the report showed that the protein is associated with the
extracellular matrix through multiple independent extracellular
matrix attachment sites in the carboxy terminal region.
[0012] Within the proprotein domain, there are two cleavage sites
(RRRR, 178-182) (RKKR, 233-236) for the furin-like protease. Furin
cleaves a wide variety of precursor proteins at the consensus
sequence RX(K/R)R. Furin cleavage sites are found in a number of
precursor proteins that are transported to the cell surface. (Kuno
et al. (1998)). The ADAM-TS-1 protein has a zinc-binding motif
(HEXXH) in its metalloproteinase domain. Accordingly, it was
suggested that this protein is secreted from cells as a
proteolytically active form by cleavage with a furin-like
enzyme.
[0013] Tortorella et al. ((1999) Science 284:1664-1666) purified
the metalloproteinase aggrecanase-1 (ADAM-TS-4) from
IL-1-stimulated bovine nasal cartilage conditioned medium and then
cloned and expressed the human ortholog. This protease represents a
cartilage aggrecanase that cleaves aggrecan at the
Glu.sup.373-Ala.sup.374 bond to produce fragments similar to those
found in the sinovial fluid of patients with various types of
arthritis. This recombinant molecule provides a target for
development of therapeutics to prevent the loss of articular
cartilage in arthritis. Aggrecan degradation is an important factor
in the erosion of articular cartilage in arthritic diseases. The
degradation involves proteolysis in the core protein near the amino
terminus where two major cleavage sites have been identified. One
of these is the Glu.sup.373-Ala.sup.374 cleavage site. Aggrecan
fragments cleaved from this site have been identified in cultures
undergoing cartilage matrix degradation and in arthritic sinovial
fluids. Incubation of purified aggrecanase-1 with bovine aggrecan
produced fragments generated by cleavage at this site. The
fragments were identified by an assay using the neoepitope
antibody, BC-3, to detect products formed by specific cleavage at
this bond. Further, including SF775, a potent aggrecanase
inhibitor, blocked binding of the aggrecanase to a specific
inhibitor resin.
[0014] The amino terminal and two internal sequences of bovine
aggrecanase 1 were found to be 50 to 60% identical to the
inflammation-associated murine protein ADAM-TS-1. The aggrecanase 1
contains a signal sequence followed by a propeptide domain with a
potential cysteine switch at Cys.sup.194 and a potential furin
cleavage site that precedes the catalytic domain. The catalytic
domain has a zinc-binding motif similar to the HEXXHXXGXXH motif
found in matrix metalloproteinases and ADAMs. The enzyme also
contains a disintegrin-like domain and lacks the transmembrane
domain and cytoplasmic tail present in many ADAMs. It ends with a
carboxy terminal domain that contains a thrombospondin-type 1 motif
similar to those present in ADAM-TS-1. It is likely synthesized as
a zymogen that is cleaved to remove the propeptide domain to
generate the mature active enzyme. A compound that interferes with
the normal pro-MMP activation through a cysteine switch mechanism
inhibits cleavage of aggrecan in cartilage organ cultures. The
enzyme was shown to be ineffective in cleaving several substrates
that are cleaved by matrix metalloproteinases including the
extracellular matrix molecules type II collagen, thrombospondin,
and fibronectin, as well as more general protease substrates,
casein and gelatin. The activity was inhibited by several
hydroxamates that are effective in blocking the cleavage of
aggrecan at the Glu-Ala bond by native bovine aggrecanase. These
researchers also identified a second aggrecanase designated
aggrecanase-2 with a similar specificity for the cleavage of
aggrecan at the Glu-Ala bond. Preliminary data from this group
indicated that ADAM-TS-1 does not cleave aggrecan at the Glu-Ala
bond.
[0015] Vazquez et al. ((1999) J. Biol. Chem. 274:R23349-23357)
reported studies of two ADAM proteins that were designated METH-1
AND METH-2. Both proteins suppressed fibroblast growth factor
2-induced vascularization in the cornea pocket assay and inhibited
vascular endothelial growth factor-induced angiogenesis in the
chorioallantoic membrane assay. The suppression was reported to be
considerably greater than that mediated by either thrombospondin 1
or endostatin on a molar basis. Both proteins were also shown to
inhibit endothelial cell proliferation but not fibroblast or smooth
muscle growth. Accordingly, the proteins show an
endothelial-specific response. Although not designated as ADAM-TS
proteins, the proteins are clearly members of the ADAM-TS family,
containing metalloproteinase, disintegrin, and thrombospondin
domains. In fact, the reference indicates that the mouse homolog of
one of the cloned genes is the ADAM-TS-1. The report also refers to
pNP-1 (procollagenase 1 N-proteinase) having a structural
resemblance and high sequence similarity to both of the cloned METH
proteins. The reference cites Colige et al. (Proc. Natl. Acad. Sci.
USA 94:2374-2379 (1997)) for the identification of this new
protein. The authors discussed the two proteins as novel inhibitors
of angiogenesis. They cited four additional members of the family
represented as partial ESTs. The authors also pointed out that
despite the identical structure and the high levels of amino acid
similarities in the two proteins, the pattern of expression differs
significantly. It was suggested that the differences are most
likely the result of specific cis-acting elements in the non-coding
regulatory sequences. It was proposed that proteins with similar or
identical function, but different tissue specificity, may
participate as specific angiogenic inhibitors regulating vascular
networks in different organs or in specific physiological
responses. Alternatively, it was proposed that small differences in
sequence might confer significant differences in tissue
specificity. Further, whereas ADAM-TS-1 was identified in a screen
of genes associated with the induction of cachexia and appears to
be regulated by inflammatory cytokines, the METH-2 is not reported
to have these features. Finally, the authors discussed the
disintegrin motif present in both proteins. The disintegrin motif
can contain an RGD (or RGX) motif with a negatively charged residue
at the X-position. This sequence binds two integrins and serves as
ligand or an antagonist of ligand binding. The authors pointed out
that inactivation of integrins with antibodies has been shown to
inhibit neovascularization during development and in
tumorigenesis.
[0016] Abbaszade et al. ((1999) J. Biol. Chem. 274:23443-23450))
report the cloning and characterization of a second aggrecanase,
designated ADAM-TS-11. It was shown to have extensive homology to
ADAM-TS-4 (aggrecanase-1) and to ADAM-TS-1. The recombinant human
ADAM-TS-11 was expressed in insect cells and shown to cleave
aggrecan at the Glu-Ala site. Aggrecan is the major proteoglycan of
cartilage and is responsible for its compressibility and stiffness.
Results from several studies cited by the authors suggest that the
cleavage at the Glu-Ala site is responsible for increased aggrecan
degredation observed in inflammatory joint disease. Gene expression
of both the ADAM-TS-4 and ADAM-TS-1 were examined in a variety of
normal and arthritic human tissues. ADAM-TS-1 was shown to be
highly expressed in arthritic fibrous tissues and arthritic joint
capsule. The ADAM-TS-4 and ADAM-TS-11 both showed moderate
expression in arthritic fibrous tissue and arthritic joint capsule.
However, expression was not limited to these tissues alone. The
ADAM-TS-11 appears to be synthesized in an inactive pro form. The
N-terminal peptide sequence of the enzyme purified from
bovine-cartilage-conditioned medium starts immediately C terminal
of the consensus furin cleavage site. Accordingly, the inhibition
of furin can block aggrecan cleavage.
[0017] Accordingly, ADAMs and ADAM-TSs are a major target for drug
action and development. Therefore, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown ADAMs and ADAM-TSs. The present invention advances the
state of the art by providing a previously unidentified human
ADAM-TS having 39% sequence identity and 67% sequence similarity
with murine ADAM-TS-1 and a second human metalloproteinase with
homology to the ADAM-TS family, and especially high homology to the
above novel ADAM-TS.
Summary of the Invention
[0018] A novel ADAM-TS cDNA, 27875 metalloproteinase, and the
deduced 27875 metalloproteinase polypeptide are described herein.
The human 27875 sequence (SEQ ID NO:1), is approximately 5366
nucleotides long including untranslated regions. The coding
sequence, located at about nucleic acid 46 to 5106 of SEQ ID NO:1,
encodes a 1687 amino acid protein (SEQ ID NO: 2).
[0019] It is also an object of the invention to provide nucleic
acid molecules encoding the 27875 metalloproteinase polypeptide,
and variants and fragments thereof. Such nucleic acid molecules are
useful as targets and reagents in 27875 metalloproteinase
expression assays, are applicable to treatment and diagnosis of
27875 metalloproteinase-related disorders and are useful for
producing novel 27875 metalloproteinase polypeptides by recombinant
methods.
[0020] The invention also provides a partial cDNA and deduced amino
acid sequence for a second human metalloproteinase with homology to
the ADAM-TS family, and particularly high homology to the 27875
metalloproteinase. This protein has been designated 42812. Further,
where appropriate, although the disclosure herein and all
embodiments are explicitly directed to the 27875 metalloproteinase,
these embodiments apply as well to the 42812 metalloproteinase
protein. An alignment between these two proteins is shown
herein.
[0021] The invention thus further provides nucleic acid constructs
comprising the nucleic acid molecules described herein. In a
preferred embodiment, the nucleic acid molecules of the invention
are operatively linked to a regulatory sequence. The invention also
provides vectors and host cells for expressing the 27875
metalloproteinase nucleic acid molecules and polypeptides, and
particularly recombinant vectors and host cells.
[0022] In another aspect, it is an object of the invention to
provide isolated 27875 metalloproteinase polypeptides and fragments
and variants thereof, including a polypeptide having the amino acid
sequence shown in SEQ ID NO:2 or the amino acid sequence encoded by
the deposited cDNA. The disclosed 27875 metalloproteinase
polypeptides are useful as reagents or targets in 27875
metalloproteinase assays and are applicable to treatment and
diagnosis of 27875 metalloproteinase-related disorders.
[0023] The invention also provides assays for determining the
activity of or the presence or absence of the 27875
metalloproteinase polypeptides or nucleic acid molecules in a
biological sample, including for disease diagnosis. In addition,
the invention provides assays for determining the presence of a
mutation in the polypeptides or nucleic acid molecules, including
for disease diagnosis.
[0024] A further object of the invention is to provide compounds
that modulate expression of the 27875 metalloproteinase for
treatment and diagnosis of 27875 metalloproteinase-related
disorders. Such compounds may be used to treat conditions related
to aberrant activity or expression of the 27875 metalloproteinase
polypeptides or nucleic acids.
[0025] The disclosed invention further relates to methods and
compositions for the study, modulation, diagnosis and treatment of
27875 metalloproteinase related disorders. The compositions include
27875 metalloproteinase polypeptides, nucleic acids, vectors,
transformed cells and related variants thereof. In particular, the
invention relates to the diagnosis and treatment of 27875
metalloproteinase-related disorders of bone, lung, heart, skeletal
muscle, aorta, testis, and kidney, and more specifically of bone.
Since the gene is highly expressed in undifferentiated osteoblasts,
the invention even more specifically relates to disorders involving
osteoblast function, growth, and differentiation, and to modulation
of gene expression in osteoblasts. Accordingly, specific disorders
include, but are not limited to, osteoporosis and
osteopetrosis.
[0026] In yet another aspect, the invention provides antibodies or
antigen-binding fragments thereof that selectively bind the 27875
metalloproteinase polypeptides and fragments. Such antibodies and
antigen binding fragments have use in the detection of the 27875
metalloproteinase polypeptide, and in the prevention, diagnosis and
treatment of 27875 metalloproteinase related disorders.
Detailed Description of the Invention
[0027] The growth, development and maintenance of bone is a highly
regulated process. Bone mass reflects the balance of bone formation
and resorption which at the cellular level involves the coordinate
regulation of bone forming (osteoblast) and bone resorbing
(osteoclast) cells. Each of these cell types is influenced by a
wide variety of hormones, inflammatory mediators and growth
factors. Importantly, osteoblast-derived secreted factors are known
regulators of osteoclast formation and/or activity in vivo.
Accordingly, it would be beneficial to identify these
osteoblast-secreted factors. Such factors may function to regulate
osteoblast activity including both cytokine and hormone processing
as well as extracellular matrix homeostasis. Modulation of the
activity of such factors (for example, via the use of small
molecule inhibitors) may prove beneficial for blocking activities
of osteoblasts that are associated with accelerated osteoclast
formation/activities and subsequent bone resorptive function.
[0028] The invention is based on the identification of the novel
human ADAM-TS 27875 metalloproteinase, which is expressed at high
levels in undifferentiated osteoblast, fetal heart and fetal
kidney. The 27875 metalloproteinase cDNA was identified based on
consensus motifs or protein domains characteristic of the ADAM-TS
family of metalloproteases. Specifically, a novel human gene,
termed the 27875 metalloproteinase, is provided. This sequence, and
other nucleotide sequences encoding the 27875 metalloproteinase
protein or fragments and variants thereof, are referred to as
"27875 metalloproteinase sequences".
[0029] The 27875 metalloproteinase cDNA was identified in a human
bone cell cDNA library. Specifically, an expressed sequence tag
(EST) found in a human bone library was selected based on homology
to known ADAM-TS sequences. Based on this EST sequence, primers
were designed to identify a full length clone from a human bone
cDNA library. Positive clones were sequenced and the overlapping
fragments were assembled.
[0030] Analysis of the assembled sequence revealed that the cloned
cDNA molecule encodes an ADAM-TS-like polypeptide. BLAST analysis
indicated that the 27875 metalloproteinase protein displays closest
similarity to the murine ADAM-TS-1 protein, with approximately 39%
identity and 67% overall similarity, indicating that the 27875
metalloproteinase is the human ortholog of this murine protein.
[0031] The 27875 metalloproteinase sequence of the invention
belongs to the ADAM-TS family of molecules having conserved
functional features. The term "family" when referring to the
proteins and nucleic acid molecules of the invention is intended to
mean two or more proteins or nucleic acid molecules having
sufficient amino acid or nucleotide sequence identity as defined
herein to provide a specific function. Such family members can be
naturally occurring and can be from either the same or different
species. For example, a family can contain a first protein of
murine origin and an ortholog of that protein of human origin, as
well as a second, distinct protein of human origin and a murine
ortholog homolog of that protein.
[0032] The 27875 metalloproteinase nucleotide sequence (SEQ ID
NO:1), is approximately 5366 nucleotides long including
untranslated regions. The coding sequence, located at about nucleic
acid 46 to 5106 of SEQ ID NO:1, encodes a 1687 amino acid protein
(SEQ ID NO:2). The 27875 metalloproteinase contains a
metalloproteinase domain at residues 244-259 of SEQ ID NO:2 and a
disintegrin domain at residues 541-592 of SEQ ID NO:2. A
zinc-binding domain (active site) is found at approximately amino
acids 385-394 of SEQ ID NO:2. The protein also contains 5
thrombospondin domains located from about amino acid 542-592,
825-868, 949-988, 1415-1463, and 1466-1521 of SEQ ID NO:2. SignalP
(eukaryote) analysis of the amino terminal 70 amino acids of the
polypeptide predicts a 30 amino acid signal peptide, which is
cleaved to produce the mature 27875 metalloproteinase polypeptide
(residues 31-1687 of SEQ ID NO:2).
[0033] Prosite program analysis was used to predict various sites
within the 27875 metalloproteinase protein. N-glycosylation sites
were predicted at about amino acid residues 94-97, 693-696,
778-781, 950-953, 971-974, 1412-1415, 1419-1422 and 1470 to 1473 of
SEQ ID NO:2. A glycosaminoglycan attachment site was predicted at
about amino acid residues 1006-1009 of SEQ ID NO:2. cAMP- and
cGMP-dependent protein kinase phosphorylation sites were predicted
at amino acid residues 872-875 and 1606-1609 of SEQ ID NO:2.
Protein kinase C phosphorylation sites were predicted at amino acid
residues 6-8, 73-75, 110-112, 214-216, 313-315, 342-344, 569-571,
598-600, 901-903, 962-964, 1035-1037, 1370-1372, 1385-1387,
1440-1442, 1483-1485, 1528-1530, 1599-1601, 1620-1622, 1649-1651
and 1660-1662 of SEQ ID NO:2. Casein kinase II phosphorylation
sites were predicted at amino acid residues 147-150, 159-162,
214-217, 342-345, 373-376, 401-404, 505-508, 605-608, 703-706,
917-920, 957-960, 1011-1014, 1192-1195, 1308-1311, 1397-1400,
1440-1443, 1483-1486, 1528-1531 and 1546-1549 of SEQ ID NO:2. A
tyrosine kinase phosphorylation site was predicted at amino acid
residues 740-747 of SEQ ID NO:2. N-myristoylation sites were
predicted at amino acid residues 55-60, 115-120, 141-146, 379-384,
479-484, 513-518, 539-544, 557-562, 614-619, 667-672, 688-693,
716-721, 765-770, 774-779, 1005-1010, 1039-1044, 1263-1252,
1263-1268, 1358-1363, 1517-1522, 1592-1597 and 1625-1630 of SEQ ID
NO:2. An amidation site was predicted at amino acid residues
408-411 of SEQ ID NO:2. A cell attachment sequence was predicted at
amino acid residues 195-197 of SEQ ID NO:2. A zinc binding domain
is predicted at residues 385 to 394 of SEQ ID NO:2. A Cytochrome C
family heme-binding site was predicted at amino acid residues
687-692 of SEQ ID NO:2. A crystallins beta and gamma Greek key
motif is predicted at amino acid residues 78-93 of SEQ ID NO:2. A
growth factor and cytokine metalloproteinase family signature 2
domain was predicted at amino acid residues 539-545 of SEQ ID NO:2.
Thrombospondin domains were predicted by HMMer, Version 2, at amino
acid residues 488-567, 542-592, 825-879, 949-994, 1415-1463 and
1466-1521 of SEQ ID NO:2.
[0034] Northern blot analysis of 27875 metalloproteinase expression
in human tissues shows high level expression in cells of osteoblast
lineage. A transcript of approximately 4 kb was detected in
osteoblast-derived polyA.sup.+ RNA (not shown). In situ
hybridization with human fetal bone also showed significant levels
of expression in mature and stromal osteoblast progenitors. High
27875 metalloproteinase expression was also detected in human fetal
kidney and fetal heart. The gene is also significantly expressed in
human adult skeletal muscle, heart, lung, aorta, testes, and lymph
node as well as in thymus and normal foreskin melanocytes (not
shown).
[0035] Expression of 27875 metalloproteinase mRNA in the above
cells and tissues indicates that the 27875 metalloproteinase is
likely to be involved in the proper function and in disorders of
these tissues, especially the bone, where the gene is expressed in
osteoblasts. Accordingly, the disclosed invention further relates
to methods and compositions for the study, modulation, diagnosis
and treatment of 27875 metalloproteinase related disorders,
especially disorders of the bone that include, but are not limited
to, osteoporosis and osteopetrosis. Since the gene is expressed in
undifferentiated osteoblasts, disorders related to osteoblast
production, function, and differentiation are particularly relevant
to the invention. The compositions include 27875 metalloproteinase
polypeptides, nucleic acids, vectors, transformed cells and related
variants and fragments thereof, as well as agents that modulate
expression of the polypeptides and polynucleotides. In particular,
the invention relates to the modulation, diagnosis and treatment of
27875 metalloproteinase related disorders as described herein.
[0036] Disorders involving the lung include, but are not limited
to, congenital anomalies; atelectasis; diseases of vascular origin,
such as pulmonary congestion and edema, including hemodynamic
pulmonary edema and edema caused by microvascular injury, adult
respiratory distress syndrome (diffuse alveolar damage), pulmonary
embolism, hemorrhage, and infarction, and pulmonary hypertension
and vascular sclerosis; chronic obstructive pulmonary disease, such
as emphysema, chronic bronchitis, bronchial asthma, and
bronchiectasis; diffuse interstitial (infiltrative, restrictive)
diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary
fibrosis, desquamative interstitial pneumonitis, hypersensitivity
pneumonitis, pulmonary eosinophilia (pulmonary infiltration with
eosinophilia), Bronchiolitis obliterans-organizing pneumonia,
diffuse pulmonary hemorrhage syndromes, including Goodpasture
syndrome, idiopathic pulmonary hemosiderosis and other hemorrhagic
syndromes, pulmonary involvement in collagen vascular disorders,
and pulmonary alveolar proteinosis; complications of therapies,
such as drug-induced lung disease, radiation-induced lung disease,
and lung transplantation; tumors, such as bronchogenic carcinoma,
including paraneoplastic syndromes, bronchioloalveolar carcinoma,
neuroendocrine tumors, such as bronchial carcinoid, miscellaneous
tumors, and metastatic tumors; pathologies of the pleura, including
inflammatory pleural effusions, noninflammatory pleural effusions,
pneumothorax, and pleural tumors, including solitary fibrous tumors
(pleural fibroma) and malignant mesothelioma.
[0037] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[0038] Disorders involving the skeletal muscle include tumors such
as rhabdomyosarcoma.
[0039] Disorders involving blood vessels include, but are not
limited to, responses of vascular cell walls to injury, such as
endothelial dysfunction and endothelial activation and intimal
thickening; vascular diseases including, but not limited to,
congenital anomalies, such as arteriovenous fistula,
atherosclerosis, and hypertensive vascular disease, such as
hypertension; inflammatory disease--the vasculitides, such as giant
cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa
(classic), Kawasaki syndrome (mucocutaneous lymph node syndrome),
microscopic polyanglitis (microscopic polyarteritis,
hypersensitivity or leukocytoclastic anglitis), Wegener
granulomatosis, thromboanglitis obliterans (Buerger disease),
vasculitis associated with other disorders, and infectious
arteritis; Raynaud disease; aneurysms and dissection, such as
abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and
aortic dissection (dissecting hematoma); disorders of veins and
lymphatics, such as varicose veins, thrombophlebitis and
phlebothrombosis, obstruction of superior vena cava (superior vena
cava syndrome), obstruction of inferior vena cava (inferior vena
cava syndrome), and lymphangitis and lymphedema; tumors, including
benign tumors and tumor-like conditions, such as hemangioma,
lymphangioma, glomus tumor (glomangioma), vascular ectasias, and
bacillary angiomatosis, and intermediate-grade (borderline
low-grade malignant) tumors, such as Kaposi sarcoma and
hemangloendothelioma, and malignant tumors, such as angiosarcoma
and hemangiopericytoma; and pathology of therapeutic interventions
in vascular disease, such as balloon angioplasty and related
techniques and vascular replacement, such as coronary artery bypass
graft surgery.
[0040] Disorders involving the testis and epididymis include, but
are not limited to, congenital anomalies such as cryptorchidism,
regressive changes such as atrophy, inflammations such as
nonspecific epididymitis and orchitis, granulomatous (autoimmune)
orchitis, and specific inflammations including, but not limited to,
gonorrhea, mumps, tuberculosis, and syphilis, vascular disturbances
including torsion, testicular tumors including germ cell tumors
that include, but are not limited to, seminoma, spermatocytic
seminoma, embryonal carcinoma, yolk sac tumor choriocarcinoma,
teratoma, and mixed tumors, tumore of sex cord-gonadal stroma
including, but not limited to, leydig (interstitial) cell tumors
and sertoli cell tumors (androblastoma), and testicular lymphoma,
and miscellaneous lesions of tunica vaginalis.
[0041] Disorders involving the kidney include, but are not limited
to, congenital anomalies including, but not limited to, cystic
diseases of the kidney, that include but are not limited to, cystic
renal dysplasia, autosomal dominant (adult) polycystic kidney
disease, autosomal recessive (childhood) polycystic kidney disease,
and cystic diseases of renal medulla, which include, but are not
limited to, medullary sponge kidney, and nephronophthisis-uremic
medullary cystic disease complex, acquired (dialysis-associated)
cystic disease, such as simple cysts; glomerular diseases including
pathologies of glomerular injury that include, but are not limited
to, in situ immune complex deposition, that includes, but is not
limited to, anti-GBM nephritis, Heymann nephritis, and antibodies
against planted antigens, circulating immune complex nephritis,
antibodies to glomerular cells, cell-mediated immunity in
glomerulonephritis, activation of alternative complement pathway,
epithelial cell injury, and pathologies involving mediators of
glomerular injury including cellular and soluble mediators, acute
glomerulonephritis, such as acute proliferative (poststreptococcal,
postinfectious) glomerulonephritis, including but not limited to,
poststreptococcal glomerulonephritis and nonstreptococcal acute
glomerulonephritis, rapidly progressive (crescentic)
glomerulonephritis, nephrotic syndrome, membranous
glomerulonephritis (membranous nephropathy), minimal change disease
(lipoid nephrosis), focal segmental glomerulosclerosis,
membranoproliferative glomerulonephritis, IgA nephropathy (Berger
disease), focal proliferative and necrotizing glomerulonephritis
(focal glomerulonephritis), hereditary nephritis, including but not
limited to, Alport syndrome and thin membrane disease (benign
familial hematuria), chronic glomerulonephritis, glomerular lesions
associated with systemic disease, including but not limited to,
systemic lupus erythematosus, Henoch-Schonlein purpura, bacterial
endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary
and immunotactoid glomerulonephritis, and other systemic disorders;
diseases affecting tubules and interstitium, including acute
tubular necrosis and tubulointerstitial nephritis, including but
not limited to, pyelonephritis and urinary tract infection, acute
pyelonephritis, chronic pyelonephritis and reflux nephropathy, and
tubulointerstitial nephritis induced by drugs and toxins, including
but not limited to, acute drug-induced interstitial nephritis,
analgesic abuse nephropathy, nephropathy associated with
nonsteroidal anti-inflammatory drugs, and other tubulointerstitial
diseases including, but not limited to, urate nephropathy,
hypercalcemia and nephrocalcinosis, and multiple myeloma; diseases
of blood vessels including benign nephrosclerosis, malignant
hypertension and accelerated nephrosclerosis, renal artery
stenosis, and thrombotic microangiopathies including, but not
limited to, classic (childhood) hemolytic-uremic syndrome, adult
hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura,
idiopathic HUS/TTP, and other vascular disorders including, but not
limited to, atherosclerotic ischemic renal disease, atheroembolic
renal disease, sickle cell disease nephropathy, diffuse cortical
necrosis, and renal infarcts; urinary tract obstruction
(obstructive uropathy); urolithiasis (renal calculi, stones); and
tumors of the kidney including, but not limited to, benign tumors,
such as renal papillary adenoma, renal fibroma or hamartoma
(renomedullary interstitial cell tumor), angiomyolipoma, and
oncocytoma, and malignant tumors, including renal cell carcinoma
(hypemephroma, adenocarcinoma of kidney), which includes urothelial
carcinomas of renal pelvis.
[0042] Bone-forming cells include the osteoprogenitor cells,
osteoblasts, and osteocytes. The disorders of the bone are complex
because they may have an impact on the skeleton during any of its
stages of development. Hence, the disorders may have variable
manifestations and may involve one, multiple or all bones of the
body. Such disorders include, congenital malformations,
achondroplasia and thanatophoric dwarfism, diseases associated with
abnormal matix such as type 1 collagen disease, osteoporosis, Paget
disease, rickets, osteomalacia, high-turnover osteodystrophy,
low-turnover aplastic disease, osteonecrosis, pyogenic
osteomyelitis, tuberculous osteomyelitism, osteoma, osteoid
osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondromas,
chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous
cortical defects, fibrous dysplasia, fibrosarcoma, malignant
fibrous histiocytoma, Ewing sarcoma, primitive neuroectodermal
tumor, giant cell tumor, and metastatic tumors.
[0043] Disorders involving the thymus include developmental
disorders, such as DiGeorge syndrome with thymic hypoplasia or
aplasia; thymic cysts; thymic hypoplasia, which involves the
appearance of lymphoid follicles within the thymus, creating thymic
follicular hyperplasia; and thymomas, including germ cell tumors,
lynphomas, Hodgkin disease, and carcinoids. Thymomas can include
benign or encapsulated thymoma, and malignant thymoma Type I
(invasive thymoma) or Type II, designated thymic carcinoma.
[0044] The sequences of the invention find use in diagnosis of
disorders involving an increase or decrease in 27875
metalloproteinase expression relative to normal expression, such as
a proliferative disorder, a differentiative disorder, or a
developmental disorder. The sequences also find use in modulating
27875 metalloproteinase-related responses. By "modulating" is
intended the upregulating or downregulating of a response. That is,
the compositions of the invention affect the targeted activity in
either a positive or negative fashion.
27875 Metalloproteinase Polypeptides
[0045] The invention relates to the novel 27875 metalloproteinase,
having the deduced amino acid sequence (SEQ ID NO:2).
[0046] Thus, present invention provides an isolated or purified
27875 metalloproteinase polypeptide and variants and fragments
thereof. "27875 metalloproteinase polypeptide" or "27875
metalloproteinase protein" refers to the polypeptide in SEQ ID NO:2
or encoded by the deposited cDNA. The term "27875 metalloproteinase
protein" or "27875 metalloproteinase polypeptide", however, further
includes the numerous variants described herein, as well as
fragments derived from the full-length 27875 metalloproteinase and
variants.
[0047] 27875 metalloproteinase polypeptides can be purified to
homogeneity. It is understood, however, that preparations in which
the polypeptide is not purified to homogeneity are useful and
considered to contain an isolated form of the polypeptide. The
critical feature is that the preparation allows for the desired
function of the polypeptide, even in the presence of considerable
amounts of other components. Thus, the invention encompasses
various degrees of purity.
[0048] As used herein, a polypeptide is said to be "isolated" or
"purified" when it is substantially free of cellular material when
it is isolated from recombinant and non-recombinant cells, or free
of chemical precursors or other chemicals when it is chemically
synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell and
still be considered "isolated" or "purified."
[0049] In one embodiment, the language "substantially free of
cellular material" includes preparations of 27875 metalloproteinase
having less than about 30% (by dry weight) other proteins (i.e.,
contaminating protein), less than about 20% other proteins, less
than about 10% other proteins, or less than about 5% other
proteins. When the polypeptide is recombinantly produced, it can
also be substantially free of culture medium, i.e., culture medium
represents less than about 20%, less than about 10%, or less than
about 5% of the volume of the protein preparation.
[0050] The 27875 metalloproteinase polypeptide is also considered
to be isolated when it is part of a membrane preparation or is
purified and then reconstituted with membrane vesicles or
liposomes.
[0051] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the 27875
metalloproteinase polypeptide in which it is separated from
chemical precursors or other chemicals that are involved in its
synthesis. The language "substantially free of chemical precursors
or other chemicals" includes, but is not limited to, preparations
of the polypeptide having less than about 30% (by dry weight)
chemical precursors or other chemicals, less than about 20%
chemical precursors or other chemicals, less than about 10%
chemical precursors or other chemicals, or less than about 5%
chemical precursors or other chemicals.
[0052] In one embodiment, the 27875 metalloproteinase polypeptide
comprises the amino acid sequence shown in SEQ ID NO:2. However,
the invention also encompasses sequence variants. Variants include
a substantially homologous protein encoded by the same genetic
locus in an organism, i.e., an allelic variant. Variants also
encompass proteins derived from other genetic loci in an organism,
but having substantial homology to 27875 metalloproteinase of SEQ
ID NO:1. Variants also include proteins substantially homologous to
27875 metalloproteinase but derived from another organism, i.e., an
ortholog. Variants also include proteins that are substantially
homologous to 27875 metalloproteinase that are produced by chemical
synthesis. Variants also include proteins that are substantially
homologous to 27875 metalloproteinase that are produced by
recombinant methods. It is understood, however, that variants
exclude any amino acid sequences disclosed prior to the
invention.
[0053] Preferred 27875 metalloproteinase polypeptides of the
present invention have an amino acid sequence sufficiently
identical to the amino acid sequence of SEQ ID NO:2. The term
"sufficiently identical" is used herein to refer to a first amino
acid or nucleotide sequence that contains a sufficient or minimum
number of identical or equivalent (e.g., with a similar side chain)
amino acid residues or nucleotides to a second amino acid or
nucleotide sequence such that the first and second amino acid or
nucleotide sequences have a common structural domain and/or common
functional activity. For example, amino acid or nucleotide
sequences that contain a common structural domain having at least
about 45%, 55%, or 65% identity, preferably 75% identity, more
preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity are defined herein as sufficiently identical.
[0054] The determination of percent identity between two sequences
using the algorithms of Karlin and Altschul (1990) Proc. Natl.
Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993)
Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul et al.
(1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be
performed with the NBLAST program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to 27875 metalloproteinase
nucleic acid molecules of the invention. BLAST protein searches can
be performed with the XBLAST program, score=50, wordlength=3, to
obtain amino acid sequences homologous to 27875 metalloproteinase
protein molecules of the invention. When utilizing BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used.
[0055] As used herein, two proteins (or a region of the proteins)
are substantially homologous when the amino acid sequences are at
least about 60-65%, 65-70%, 70-75%, typically at least about
80-85%, and most typically at least about 90-95% or more
homologous. A substantially homologous amino acid sequence,
according to the present invention, will be encoded by a nucleic
acid sequence hybridizing to the nucleic acid sequence, or portion
thereof, of the sequence shown in SEQ ID NO:1 under stringent
conditions as more fully described below.
[0056] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence (e.g., when aligning a second sequence to
the amino acid sequences herein having 502 amino acid residues, at
least 165, preferably at least 200, more preferably at least 250,
even more preferably at least 300, and even more preferably at
least 350, 400, 450, and 500 amino acid residues are aligned). The
amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position (as
used herein amino acid or nucleic acid "identity" is equivalent to
amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[0057] The invention also encompasses polypeptides having a lower
degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by 27875
metalloproteinase. Similarity is determined by conservative amino
acid substitution, as shown in Table 1. Such substitutions are
those that substitute a given amino acid in a polypeptide by
another amino acid of like characteristics. Conservative
substitutions are likely to be phenotypically silent. Typically
seen as conservative substitutions are the replacements, one for
another, among the aliphatic amino acids Ala, Val, Leu, and Ile;
interchange of the hydroxyl residues Ser and Thr, exchange of the
acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr. Guidance
concerning which amino acid changes are likely to be phenotypically
silent are found in Bowie et al., Science 247:1306-1310 (1990).
TABLE-US-00002 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0058] A variant polypeptide can differ in amino acid sequence by
one or more substitutions, deletions, insertions, inversions,
fusions, and truncations or a combination of any of these. Variant
polypeptides can be fully functional or can lack function in one or
more activities. Thus, in the present case, variations can affect
the function, for example, of one or more of regions including any
of the five thrombospondin domains, the disintegrin domain,
zinc-binding domain, metalloproteinase domain, the region
containing the propeptide, regulatory regions, other substrate
binding regions, regions involved in membrane association, regions
involved in post-translational modification, for example, by
phosphorylation, and regions that are important for effector
function (i.e., agents that act upon the protein, such as
pro-peptide cleavage).
[0059] Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. Functional variants can also contain
substitution of similar amino acids, which results in no change or
an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree.
[0060] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0061] As indicated, variants can be naturally-occurring or can be
made by recombinant means or chemical synthesis to provide useful
and novel characteristics for 27875 metalloproteinase polypeptide.
This includes preventing immunogenicity from pharmaceutical
formulations by preventing protein aggregation.
[0062] Useful variations further include alteration of functional
activity. For example, one embodiment involves a variation at the
substrate peptide binding site that results in binding but not
hydrolysis or slower hydrolysis of the peptide substrate. A further
useful variation at the same site can result in altered affinity
for the peptide substrate. Useful variations also include changes
that provide for affinity for another peptide substrate. Useful
variations further include the ability to bind integrin with
greater or lesser affinity, such as not to bind integrin or to bind
integrin but not release it. Further useful variations include
alteration in the ability of the propeptide to be cleaved by a
cleavage protein, for example, by furin, including alteration in
the binding or recognition site. Further, the cleavage site can
also be modified so that recognition and cleavage are by a
different protease. A useful variation includes binding, but not
cleavage, by such a protease. Further useful variations involve
variations in the TSP domain, such as in the ability to bind
heparin or other sulfated glycosaminoglycan, such as greater or
lesser affinity, or a change in specificity. A further useful
variation involves a variation in the ability to be bound by zinc,
including a greater or lesser affinity for the metal. Further
variation could include a variation in the specificity of metal
binding, in other words, the ability to be bound by a different
metal ion.
[0063] Another useful variation provides a fusion protein in which
one or more domains or subregions are operationally fused to one or
more domains, subregions, or motifs from another ADAMs-TS or ADAM.
For example, the transmembrane domain from an ADAM protein can be
introduced into the 27875 ADAM-TS such that the protein is anchored
in the cell surface. Other permutations include the number of
thrombospondin domains, mixing of thrombospondin domains from
different ADAM-TS families, spacer regions (between thrombospondin
domains), from different ADAM-TS families, the metalloproteinase
domain, the propeptide domain, and the disintegrin domain. Mixing
these various domains can allow the formation of novel ADAM-TS
molecules with different host cell, substrate, and effector
molecule (one that acts on the ADAM-TS) specificity.
[0064] The term "substrate" is intended to refer not only to the
peptide substrate that is cleaved by the metalloproteinase domain,
but to refer to any component with which the 27875 polypeptide
interacts in order to produce an effect on that component or a
subsequent biological effect that is a result of interacting with
that component. This includes, but is not limited to, for example,
interaction with extracellular matrix components and integrin.
However, it is understood that a substrate also includes peptides
that are cleaved as a result of catalysis in the metalloproteinase
domain.
[0065] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.
(1985) Science 244:1081-1085). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity,
such as peptide bond hydrolysis in vitro or related biological
activity, such as proliferative activity. Sites that are critical
for binding can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith et al. (1992) J. Mol. Biol. 224:899-904; de Vos et
al. (1992) Science 255:306-312).
[0066] The invention thus also includes polypeptide fragments of
27875 metalloproteinase. Fragments can be derived from the amino
acid sequence shown in SEQ ID NO:1. However, the invention also
encompasses fragments of the variants of the 27875
metalloproteinase polypeptide as described herein. The fragments to
which the invention pertains, however, are not to be construed as
encompassing fragments that may be disclosed prior to the present
invention.
[0067] The longest contiguous stretch of amino acid homology
between the 27875 metalloproteinase and ADAM-TS-1 is 9 contiguous
amino acids. Accordingly, a fragment can comprise at least about
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,
35, 40, 45, 50 or more contiguous amino acids. Fragments can retain
one or more of the biological activities of the protein, for
example as discussed above, as well as fragments that can be used
as an immunogen to generate 27875 metalloproteinase antibodies.
[0068] Biologically active fragments (peptides which are, for
example, 5, 7, 10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100
or more amino acids in length) can comprise a functional site. Such
sites include but are not limited to those discussed above, such as
a catalytic site, regulatory site, site important for substrate
recognition or binding, zinc binding region, regions containing a
metalloproteinase, disintegrin or TSP motif, phosphorylation sites,
glycosylation sites, and other functional sites disclosed herein.
Such sites or motifs can be identified by means of routine
computerized homology searching procedures, such as those disclosed
herein.
[0069] Fragments, for example, can extend in one or both directions
from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or
up to 100 amino acids. Further, fragments can include sub-fragments
of the specific sites or regions disclosed herein, which
sub-fragments retain the function of the site or region from which
they are derived.
[0070] The invention also provides fragments with immunogenic
properties. These contain an epitope-bearing portion of the 27875
metalloproteinase polypeptide and variants. These epitope-bearing
peptides are useful to raise antibodies that bind specifically to
an 27875 metalloproteinase polypeptide or region or fragment. These
peptides can contain at least 10, 12, at least 14, or between at
least about 15 to about 30 amino acids. The epitope-bearing 27875
metalloproteinase polypeptides may be produced by any conventional
means (Houghten, R. A. (1985) Proc. Natl. Acad. Sci. USA
82:5131-5135). Simultaneous multiple peptide synthesis is described
in U.S. Pat. No. 4,631,211.
[0071] Non-limiting examples of antigenic polypeptides that can be
used to generate antibodies include but are not limited to peptides
derived from extracellular regions. However, intracellularly-made
antibodies ("intrabodies") are also encompassed, which would
recognize intracellular peptide regions.
[0072] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the 27875 metalloproteinase
polypeptide fragment and an additional region fused to the carboxyl
terminus of the fragment.
[0073] The invention thus provides chimeric or fusion proteins.
These comprise an 27875 metalloproteinase peptide sequence
operatively linked to a heterologous peptide having an amino acid
sequence not substantially homologous to the 27875
metalloproteinase polypeptide. "Operatively linked" indicates that
the 27875 metalloproteinase polypeptide and the heterologous
peptide are fused in-frame. The heterologous peptide can be fused
to the N-terminus or C-terminus of the 27875 metalloproteinase
polypeptide or can be internally located.
[0074] In one embodiment the fusion protein does not affect 27875
metalloproteinase function per se. For example, the fusion protein
can be a GST-fusion protein in which 27875 metalloproteinase
sequences are fused to the N- or C-terminus of the GST sequences.
Other types of fusion proteins include, but are not limited to,
enzymatic fusion proteins, for example beta-galactosidase fusions,
yeast two-hybrid GAL4 fusions, poly-His fusions and Ig fusions.
Such fusion proteins, particularly poly-His fusions, can facilitate
the purification of recombinant 27875 metalloproteinase
polypeptide. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of a protein can be increased by using
a heterologous signal sequence. Therefore, in another embodiment,
the fusion protein contains a heterologous signal sequence at its
C- or N-terminus.
[0075] EP-A-O 464 533 discloses fusion proteins comprising various
portions of immunoglobulin constant regions. The Fc is useful in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). In drug discovery, for
example, human proteins have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
(Bennett et al. (1995) J. Mol. Recog. 8:52-58 (1995) and Johanson
et al. J. Biol. Chem. 270:9459-9471). Thus, this invention also
encompasses soluble fusion proteins containing an 27875
metalloproteinase polypeptide and various portions of the constant
regions of heavy or light chains of immunoglobulins of various
subclass (IgG, IgM, IgA, IgE). Preferred as immunoglobulin is the
constant part of the heavy chain of human IgG, particularly IgG1,
where fusion takes place at the hinge region. For some uses it is
desirable to remove the Fc after the fusion protein has been used
for its intended purpose, for example when the fusion protein is to
be used as antigen for immunizations. In a particular embodiment,
the Fc part can be removed in a simple way by a cleavage sequence,
which is also incorporated and can be cleaved with factor Xa.
[0076] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see Ausubel et al. (1992)
Current Protocols in Molecular Biology). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). An 27875 metalloproteinase-encoding
nucleic acid can be cloned into such an expression vector such that
the fusion moiety is linked in-frame to 27875
metalloproteinase.
[0077] Another form of fusion protein is one that directly affects
27875 metalloproteinase functions. Accordingly, an 27875
metalloproteinase polypeptide is encompassed by the present
invention in which one or more of the 27875 metalloproteinase
regions (or parts thereof) has been replaced by heterologous or
homologous regions (or parts thereof) from another ADAM-TS or an
ADAM. Accordingly, various permutations are possible, for example,
as discussed above. Thus, chimeric 27875 metalloproteinases can be
formed in which one or more of the native domains or subregions has
been replaced by another. This includes metalloproteinase,
disintegrin or thrombospondin domains.
[0078] It is understood however that such regions could be derived
from an ADAM-TS, ADAM, metalloprotein, disintegrin or
thrombospondin that has not yet been characterized. Moreover,
disintegrin, metalloprotein, and thrombospondin function can be
derived from peptides that contain these functions but are not
found in either an ADAM or ADAM-TS family. Accordingly, these
domains could be provided from other metalloproteins, disintegrins
or thrombospondins.
[0079] The isolated 27875 metalloproteinase protein can be purified
from cells that naturally express it, such as cells of osteoblast,
lung, heart or kidney lineage, especially purified from cells that
have been altered to express it (recombinant), or synthesized using
known protein synthesis methods.
[0080] In one embodiment, the protein is produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding the
27875 metalloproteinase polypeptide is cloned into an expression
vector, the expression vector introduced into a host cell and the
protein expressed in the host cell. The protein can then be
isolated from the cells by an appropriate purification scheme using
standard protein purification techniques. Polypeptides often
contain amino acids other than the 20 amino acids commonly referred
to as the 20 naturally-occurring amino acids. Further, many amino
acids, including the terminal amino acids, may be modified by
natural processes, such as processing and other post-translational
modifications, or by chemical modification techniques well known in
the art. Common modifications that occur naturally in polypeptides
are described in basic texts, detailed monographs, and the research
literature, and they are well known to those of skill in the
art.
[0081] Accordingly, the polypeptides also encompass derivatives or
analogs in which a substituted amino acid residue is not one
encoded by the genetic code, in which a substituent group is
included, in which the mature polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence for purification of
the mature polypeptide or a pro-protein sequence.
[0082] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0083] Such modifications are well-known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd ed., T. E.
Creighton, W.H. Freeman and Company, New York (1993). Many detailed
reviews are available on this subject, such as by Wold, F.,
Posttranslational Covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York 1-12 (1983); Seifter et al. (1990)
Meth. Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. N.Y.
Acad. Sci. 663:48-62).
[0084] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquitination, and they may be circular, with or without
branching, generally as a result of post-translation events,
including natural processing events and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translational natural processes and by synthetic methods.
[0085] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. Blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, is common in
naturally-occurring and synthetic polypeptides. For instance, the
aminoterminal residue of polypeptides made in E. coli, prior to
proteolytic processing, almost invariably will be
N-formylmethionine.
[0086] The modifications can be a function of how the protein is
made. For recombinant polypeptides, for example, the modifications
will be determined by the host cell posttranslational modification
capacity and the modification signals in the polypeptide amino acid
sequence. Accordingly, when glycosylation is desired, a polypeptide
should be expressed in a glycosylating host, generally a eukaryotic
cell. Insect cells often carry out the same posttranslational
glycosylations as mammalian cells and, for this reason, insect cell
expression systems have been developed to efficiently express
mammalian proteins having native patterns of glycosylation. Similar
considerations apply to other modifications.
[0087] The same type of modification may be present in the same or
varying degree at several sites in a given polypeptide. Also, a
given polypeptide may contain more than one type of
modification.
Polypeptide Uses
[0088] The protein sequences of the present invention can be used
as a "query sequence" to perform a search against public databases
to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to the nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the proteins of the invention. To obtain gapped
alignments for comparison purposes, Gapped BLAST can be utilized as
described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used.
[0089] 27875 metalloproteinase polypeptides are useful for
producing antibodies specific for 27875 metalloproteinase, regions,
or fragments.
[0090] 27875 metalloproteinase polypeptides are useful for
biological assays related to metalloproteinases, disintegrins or
thrombospondins, particularly those functions found in ADAMs and
ADAM-TSs. Such assays involve any of the known ADAM, ADAM-TS,
metalloproteinase, disintegrin or thrombospondin functions or
activities or properties useful for diagnosis and treatment of
27875 metalloproteinase-related conditions.
[0091] These assays include, but are not limited to, binding
extracellular matrix, binding integrin, binding zinc or other
metals, binding .alpha..sub.2-macroglobulin, cleaving specific
peptide substrates to produce fragments, affecting cell adhesion,
binding heparin or other sulfated glycosaminoglycan, such as
heparan sulfate, suppressing vascularization, suppressing vascular
endothelial growth, breaking down cartilage, inducing apoptosis of
endothelial cells, suppressing tumor growth, inhibiting
angiogenesis, affecting cellular chemotaxis, affecting cell-cell
interaction or cell-matrix interaction, binding integrin, and any
of the other biological or functional properties of these proteins,
including, but not limited to, those disclosed herein, and in the
references cited herein which are incorporated herein by reference
for the disclosure of these properties and for the assays based on
these properties. Further, assays may relate to changes in the
protein, per se, and on the effects of these changes, for example,
cleavage of the propeptide by furin or other specific proteinase,
activation of the protein following cleavage, induction of
expression of the protein in vivo by LPS, inhibition of function by
such agents as SF775, as well as any other effects on the protein
mentioned herein or cited in the references herein, which are
incorporated herein by reference for these effects and for the
subsequent biological consequences of these effects.
[0092] Such assays include, but are not limited to, those disclosed
in Tang et al. (FEBS Letters 445:223-225 (1999)) (for example,
induction by interleukin I in vitro and by intravenous
administration of lipopolysaccharide in vivo, as well as effects on
cell adhesion, motility, and growth); Abbaszade et al., above (for
example, products resulting from cleavage at the Glu-Ala site in
cartilage explants and chondrocyte cultures treated with
interleukin I and retinoic acid, determination of aggrecan cleaving
activity with and without hydroxamate inhibitors); Kuno et al.
(1998), above (binding to the extracellular matrix, binding to
sulfated glycosaminoglycans, binding to heparan sulfate); Kuno et
al. (1999) proteinase trapping of .alpha..sub.2-macroglobulin,
furin processing); Tortorella et al. (1999), above (detection of
aggrecan fragments, especially by neoepitope antibodies, inhibition
of cleavage by ADAM-TS inhibitors, inhibition of pro-MMP
activation); Vasquez et al., above (suppression of fibroblast
growth factor-2-induced vascularization in the cornea pocket assay
and inhibition of vascular endothelial growth factor-induced
angiogenesis in the chorioallantoic membrane assay, inhibition of
endothelial cell proliferation, competitive inhibition with
endostatin, proliferation of human dermal endothelial cells, use of
the antiangiogenic region of the TSP-1 motif as bait); Kuno et al.
(1997), above (heparin binding, induction of expression in vitro by
interleukin I, induction of expression in vivo by LPS); Wolfsberg
et al., above (degradation of basement membrane, binding of
integrin, and fusogenic activity); Guilpin et al. (1988) J. Biol.
Chem. 273:157-166 (.alpha..sub.2-macroglobulin trapping, cleavage
of prodomain at the furin site to generate active
metalloproteinase); Rosendahl et al., above (J. Biol. Chem.
272:24588-24593 (1997)) (TNF .alpha. processing); Wolfsberg et al.,
Developmental Biology 169:378-383 (1995) (adhesion by integrin
binding in the disintegrin domain, antiadhesive function by
zinc-dependent metalloproteinase domain). These references are
incorporated herein by reference for these specific assays.
[0093] Recombinant assay systems include, but are not limited to,
those shown in Abbaszade et al., above; Kuno et al. (1998), above;
Kuno et al. (1999), above; Tortorella et al., above; Vasquez et
al., above, Kuno et al. (1997), above; Wolfsberg et al.
(Developmental Biology), above. These references are also
incorporated herein by reference for the cloning and expression
systems disclosed therein.
[0094] 27875 metalloproteinase polypeptides are also useful in drug
screening assays, in cell-based or cell-free systems. Cell-based
systems can be native, i.e., cells that normally express 27875
metalloproteinase, such as lung, fetal kidney, fetal heart, adult
lung and osteoblasts, as a biopsy, or expanded in cell culture. In
one embodiment, however, cell-based assays involve recombinant host
cells expressing 27875 metalloproteinase. Accordingly, these
drug-screening assays can be based on effects on protein function
as described above for biological assays useful for diagnosis and
treatment.
[0095] Determining the ability of the test compound to interact
with 27875 metalloproteinase can also comprise determining the
ability of the test compound to preferentially bind to the
polypeptide as compared to the ability of a known binding molecule
to bind to the polypeptide.
[0096] The polypeptides can be used to identify compounds that
modulate 27875 metalloproteinase activity. Such compounds, for
example, can increase or decrease affinity or rate of binding to
substrate, compete with substrate for binding to 27875
metalloproteinase, or displace substrate bound to 27875
metalloproteinase. Both 27875 metalloproteinase and appropriate
variants and fragments can be used in high-throughput screens to
assay candidate compounds for the ability to bind to 27875
metalloproteinase. These compounds can be further screened against
a functional 27875 metalloproteinase to determine the effect of the
compound on 27875 metalloproteinase activity. Compounds can be
identified that activate (agonist) or inactivate (antagonist) 27875
metalloproteinase to a desired degree. Modulatory methods can be
performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g., by administering the agent to a
subject).
[0097] 27875 metalloproteinase polypeptides can be used to screen a
compound for the ability to stimulate or inhibit interaction
between 27875 metalloproteinase protein and a target molecule that
normally interacts with 27875 metalloproteinase, for example,
furin, zinc or other metal, substrate peptide of the
metalloproteinase module, substrate of the disintegrin module, for
example, integrin, or substrate of the thrombospondin module, i.e.,
sulfated glycosaminoglycan, such as heparin and heparan sulfate,
and accordingly, extracellular matrix. The assay includes the steps
of combining 27875 metalloproteinase protein with a candidate
compound under conditions that allow the 27875 metalloproteinase
protein or fragment to interact with the target molecule, and to
detect the formation of a complex between the 27875
metalloproteinase protein and the target or to detect the
biochemical consequence of the interaction with 27875
metalloproteinase and the target.
[0098] Determining the ability of 27875 metalloproteinase to bind
to a target molecule can also be accomplished using a technology
such as real-time Bimolecular Interaction Analysis (BIA). Sjolander
et al. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)
Curr. Opin. Struct. Biol. 5:699-705. As used herein, "BIA" is a
technology for studying biospecific interactions in real time,
without labeling any of the interactants (e.g., BIAcore.TM.).
Changes in the optical phenomenon surface plasmon resonance (SPR)
can be used as an indication of real-time reactions between
biological molecules.
[0099] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to polypeptide libraries, while the
other four approaches are applicable to polypeptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, K. S.
(1997) Anticancer Drug Des. 12:145).
[0100] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in DeWitt et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.
37:1233. Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 97:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra).
[0101] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al. (1991)
Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al. (1993) Cell 72:767-778); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0102] One candidate compound is a soluble full-length 27875
metalloproteinase or fragment that competes for peptide, integrin,
metal, or glycan binding. Other candidate compounds include mutant
27875 metalloproteinases or appropriate fragments containing
mutations that affect 27875 metalloproteinase function and compete
for peptide, integrin, metal, or glycan substrate. Accordingly, a
fragment that competes for substrate, for example with a higher
affinity, or a fragment that binds substrate but does not process
or otherwise affect it, is encompassed by the invention.
[0103] The invention provides other end points to identify
compounds that modulate (stimulate or inhibit) 27875
metalloproteinase activity. The assays typically involve an assay
of cellular events that indicate 27875 metalloproteinase activity.
Thus, the expression of genes that are up- or down-regulated in
response to 27875 metalloproteinase activity can be assayed. In one
embodiment, the regulatory region of such genes can be operably
linked to a marker that is easily detectable, such as luciferase.
Alternatively, modification of 27875 metalloproteinase could also
be measured.
[0104] Any of the biological or biochemical functions mediated by
the 27875 metalloproteinase can be used as an endpoint assay. These
include all of the biochemical or biochemical/biological events
described herein, in the references cited herein, incorporated by
reference for these endpoint assay targets, and other functions
known to those of ordinary skill in the art. In the case of the
27875 metalloproteinase, specific end points can include, but are
not limited to, the events resulting from expression (or lack
thereof) of metalloproteinase, disintegrin or thrombospondin
activity. With respect to disorders, this would include, but not be
limited to, cartilage breakdown, effects on angiogenesis, such as
inhibition, induction of apoptosis of endothelial cells, cell-cell
adhesion, as well as cell-matrix interaction stimulation of cell
surface receptors by cleavage of extracellular ligand, and
resulting clinical effects, such as arthritis and tumor growth. In
addition, osteoblast function, differentiation, and proliferation
can be assayed as well as the biological effects of osteoblast
function such as osteoporosis and osteopetrosis and other disorders
and pathology, such as that disclosed above, for bone-forming
cells.
[0105] Binding and/or activating compounds can also be screened by
using chimeric 27875 metalloproteinase proteins in which one or
more regions, segments, sites, and the like, as disclosed herein,
or parts thereof, can be replaced by heterologous and homologous
counterparts derived from other ADAM-TSs, ADAMs,
metalloproteinases, disintegrins or thrombospondins. For example, a
catalytic region can be used that interacts with a different
peptide or glycan specificity and/or affinity than the native 27875
metalloproteinase. Accordingly, a different set of components is
available as an end-point assay for activation. As a further
alternative, the site of modification by an effector protein, for
example phosphorylation, can be replaced with the site for a
different effector protein. Activation can also be detected by a
reporter gene containing an easily detectable coding region
operably linked to a transcriptional regulatory sequence that is
part of the native pathway in which 27875 metalloproteinase is
involved.
[0106] 27875 metalloproteinase polypeptides are also useful in
competition binding assays in methods designed to discover
compounds that interact with 27875 metalloproteinase. Thus, a
compound is exposed to an 27875 metalloproteinase polypeptide under
conditions that allow the compound to bind or to otherwise interact
with the polypeptide. Soluble 27875 metalloproteinase polypeptide
is also added to the mixture. If the test compound interacts with
the soluble 27875 metalloproteinase polypeptide, it decreases the
amount of complex formed or activity from 27875 metalloproteinase
target. This type of assay is particularly useful in cases in which
compounds are sought that interact with specific regions of 27875
metalloproteinase. Thus, the soluble polypeptide that competes with
the target 27875 metalloproteinase region is designed to contain
peptide sequences corresponding to the region of interest.
[0107] Another type of competition-binding assay can be used to
discover compounds that interact with specific functional sites. As
an example, bindable zinc and a candidate compound can be added to
a sample of 27875 metalloproteinase. Compounds that interact with
27875 metalloproteinase at the same site as the zinc will reduce
the amount of complex formed between 27875 metalloproteinase and
the zinc. Accordingly, it is possible to discover a compound that
specifically prevents interaction between 27875 metalloproteinase
and the zinc component. Another example involves adding a candidate
compound to a sample of 27875 metalloproteinase and substrate
peptide. A compound that competes with the peptide will reduce the
amount of hydrolysis or binding of the peptide to 27875
metalloproteinase. Accordingly, compounds can be discovered that
directly interact with 27875 metalloproteinase and compete with the
peptide. Such assays can involve any other component that interacts
with 27875 metalloproteinase, such as integrin or sulfated
glycosaminoglycan.
[0108] To perform cell free drug screening assays, it is desirable
to immobilize either 27875 metalloproteinase, or fragment, or its
target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[0109] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase/27875
metalloproteinase fusion proteins can be adsorbed onto glutathione
sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione
derivatized microtitre plates, which are then combined with the
cell lysates (e.g., .sup.35S-labeled) and the candidate compound,
and the mixture incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and pH).
Following incubation, the beads are washed to remove any unbound
label, and the matrix immobilized and radiolabel determined
directly, or in the supernatant after the complexes is dissociated.
Alternatively, the complexes can be dissociated from the matrix,
separated by SDS-PAGE, and the level of 27875
metalloproteinase-binding protein found in the bead fraction
quantitated from the gel using standard electrophoretic techniques.
For example, either the polypeptide or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin using
techniques well known in the art. Alternatively, antibodies
reactive with the protein but which do not interfere with binding
of the protein to its target molecule can be derivatized to the
wells of the plate, and the protein trapped in the wells by
antibody conjugation. Preparations of an 27875
metalloproteinase-binding target component, such as a peptide or
zinc component, and a candidate compound are incubated in 27875
metalloproteinase-presenting wells and the amount of complex
trapped in the well can be quantitated. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with 27875 metalloproteinase target
molecule, or which are reactive with 27875 metalloproteinase and
compete with the target molecule; as well as enzyme-linked assays
which rely on detecting an enzymatic activity associated with the
target molecule.
[0110] Modulators of 27875 metalloproteinase activity identified
according to these drug screening assays can be used to treat a
subject with a disorder related to 27875 metalloproteinase, by
treating cells that express the 27875 metalloproteinase. These
methods of treatment include the steps of administering the
modulators of 27875 metalloproteinase activity in a pharmaceutical
composition as described herein, to a subject in need of such
treatment.
[0111] 27875 metalloproteinase is highly expressed in fetal kidney,
fetal heart, and undifferentiated osteoblasts. As such it is
specifically involved in disorders relating to these tissues.
Examples include, but are not limited to, osteoporosis and
osteopetrosis, as well as other disorders involving osteoblast
differentiation, function, and growth. Furthermore, expression is
also relevant to disorders of several other tissues have been
described. Disorders of these tissues are disclosed hereinabove.
27875 metalloproteinase polypeptides are thus useful for treating
an 27875 metalloproteinase-associated disorder characterized by
aberrant expression or activity of an 27875 metalloproteinase. In
one embodiment, the method involves administering an agent (e.g.,
an agent identified by a screening assay described herein), or
combination of agents that modulates (e.g., upregulates or
downregulates) expression or activity of the protein. In another
embodiment, the method involves administering 27875
metalloproteinase as therapy to compensate for reduced or aberrant
expression or activity of the protein.
[0112] Methods for treatment include but are not limited to the use
of soluble 27875 metalloproteinase or fragments of 27875
metalloproteinase protein that compete for substrate or any other
component that directly interacts with 27875 metalloproteinase,
such as integrin, glycan, zinc, or any of the enzymes that modify
27875 metalloproteinase. These 27875 metalloproteinases or
fragments can have a higher affinity for the target so as to
provide effective competition.
[0113] Stimulation of activity is desirable in situations in which
the protein is abnormally downregulated and/or in which increased
activity is likely to have a beneficial effect. Likewise,
inhibition of activity is desirable in situations in which the
protein is abnormally upregulated and/or in which decreased
activity is likely to have a beneficial effect. In one example of
such a situation, a subject has a disorder characterized by
aberrant development or cellular differentiation. In another
example, the subject has a disorder characterized by an aberrant
hematopoietic response. In another example, it is desirable to
achieve tissue regeneration in a subject (e.g., where a subject has
undergone bone trauma or osteoporosis).
[0114] In yet another aspect of the invention, the proteins of the
invention can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO
94/10300), to identify other proteins (captured proteins) which
bind to or interact with the proteins of the invention and modulate
their activity.
[0115] 27875 metalloproteinase polypeptides also are useful to
provide a target for diagnosing a disease or predisposition to
disease mediated by 27875 metalloproteinase, including, but not
limited to, those diseases discussed herein, and particularly
bone-related disorders, as disclosed above. Targets are useful for
diagnosing a disease or predisposition to disease mediated by 27875
metalloproteinase. Accordingly, methods are provided for detecting
the presence, or levels of, 27875 metalloproteinase in a cell,
tissue, or organism. The method involves contacting a biological
sample with a compound capable of interacting with 27875
metalloproteinase such that the interaction can be detected. One
agent for detecting 27875 metalloproteinase is an antibody capable
of selectively binding to 27875 metalloproteinase. A biological
sample includes tissues, cells and biological fluids isolated from
a subject, as well as tissues, cells and fluids present within a
subject.
[0116] The 27875 metalloproteinase also provides a target for
diagnosing active disease, or predisposition to disease, in a
patient having a variant 27875 metalloproteinase. Thus, 27875
metalloproteinase can be isolated from a biological sample and
assayed for the presence of a genetic mutation that results in an
aberrant protein. This includes amino acid substitution, deletion,
insertion, rearrangement, (as the result of aberrant splicing
events), and inappropriate post-translational modification.
Analytic methods include altered electrophoretic mobility, altered
tryptic peptide digest, altered 27875 metalloproteinase activity in
cell-based or cell-free assay, alteration in peptide binding or
degradation, integrin binding, glycan binding, zinc binding or
antibody-binding pattern, altered isoelectric point, direct amino
acid sequencing, and any other of the known assay techniques useful
for detecting mutations in a protein in general or in an 27875
metalloproteinase specifically, such as are disclosed herein.
[0117] In vitro techniques for detection of 27875 metalloproteinase
include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. Alternatively, the
protein can be detected in vivo in a subject by introducing into
the subject a labeled anti-27875 metalloproteinase antibody. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques. Particularly useful are methods, which
detect the allelic variant of 27875 metalloproteinase expressed in
a subject, and methods, which detect fragments of 27875
metalloproteinase in a sample.
[0118] 27875 metalloproteinase polypeptides are also useful in
pharmacogenomic analysis. Pharmacogenomics deal with clinically
significant hereditary variations in the response to drugs due to
altered drug disposition and abnormal action in affected persons.
See, e.g., Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol.
23(10-11):983-985, and Linder, M. W. (1997) Clin. Chem.
43(2):254-266. The clinical outcomes of these variations result in
severe toxicity of therapeutic drugs in certain individuals or
therapeutic failure of drugs in certain individuals as a result of
individual variation in metabolism. Thus, the genotype of the
individual can determine the way a therapeutic compound acts on the
body or the way the body metabolizes the compound. Further, the
activity of drug metabolizing enzymes affects both the intensity
and duration of drug action. Thus, the pharmacogenomics of the
individual permit the selection of effective compounds and
effective dosages of such compounds for prophylactic or therapeutic
treatment based on the individual's genotype. The discovery of
genetic polymorphisms in some drug metabolizing enzymes has
explained why some patients do not obtain the expected drug
effects, show an exaggerated drug effect, or experience serious
toxicity from standard drug dosages. Polymorphisms can be expressed
in the phenotype of the extensive metabolizer and the phenotype of
the poor metabolizer. Accordingly, genetic polymorphism may lead to
allelic protein variants of 27875 metalloproteinase in which one or
more of 27875 metalloproteinase functions in one population is
different from those in another population. The polypeptides thus
allow a target to ascertain a genetic predisposition that can
affect treatment modality. Thus, in a peptide-based treatment,
polymorphism may give rise to catalytic regions that are more or
less active. Accordingly, dosage would necessarily be modified to
maximize the therapeutic effect within a given population
containing the polymorphism. As an alternative to genotyping,
specific polymorphic polypeptides could be identified.
[0119] 27875 metalloproteinase polypeptides are also useful for
monitoring therapeutic effects during clinical trials and other
treatment. Thus, the therapeutic effectiveness of an agent that is
designed to increase or decrease gene expression, protein levels or
27875 metalloproteinase activity can be monitored over the course
of treatment using 27875 metalloproteinase polypeptides as an
end-point target. The monitoring can be, for example, as follows:
(i) obtaining a pre-administration sample from a subject prior to
administration of the agent; (ii) detecting the level of expression
or activity of the protein in the pre-administration sample; (iii)
obtaining one or more post-administration samples from the subject;
(iv) detecting the level of expression or activity of the protein
in the post-administration samples; (v) comparing the level of
expression or activity of the protein in the pre-administration
sample with the protein in the post-administration sample or
samples; and (vi) increasing or decreasing the administration of
the agent to the subject accordingly.
Antibodies
[0120] The invention also provides antibodies that selectively bind
to 27875 metalloproteinase and its variants and fragments. An
antibody is considered to selectively bind, even if it also binds
to other proteins that are not substantially homologous with 27875
metalloproteinase. These other proteins share homology with a
fragment or domain of 27875 metalloproteinase. This conservation in
specific regions gives rise to antibodies that bind to both
proteins by virtue of the homologous sequence. In this case, it
would be understood that antibody binding to 27875
metalloproteinase is still selective.
[0121] Antibodies can be polyclonal or monoclonal. An intact
antibody, or a fragment thereof (e.g. Fab or F(ab').sub.2) can be
used. An appropriate immunogenic preparation can be derived from
native, recombinantly expressed, or chemically synthesized
peptides.
[0122] To generate antibodies, an isolated 27875 metalloproteinase
polypeptide is used as an immunogen to generate antibodies using
standard techniques for polyclonal and monoclonal antibody
preparation. Either the full-length protein or antigenic peptide
fragment can be used. Regions having a high antigenicity index are
described.
[0123] Antibodies are preferably prepared from these regions or
from discrete fragments in these regions. However, antibodies can
be prepared from any region of the peptide as described herein. A
preferred fragment produces an antibody that diminishes or
completely prevents peptide hydrolysis or binding. Antibodies can
be developed against the entire 27875 metalloproteinase or domains
of 27875 metalloproteinase as described herein, for example, the
zinc binding region, metalloproteinase motif, the disintegrin
domain, the TSP motif, or subregions thereof. Antibodies can also
be developed against specific functional sites as disclosed
herein.
[0124] The antigenic peptide can comprise a contiguous sequence of
at least 12, 14, 15, or 30 amino acid residues. In one embodiment,
fragments correspond to regions that are located on the surface of
the protein, e.g., hydrophilic regions. These fragments are not to
be construed, however, as encompassing any fragments, which may be
disclosed prior to the invention.
[0125] Detection can be facilitated by coupling (i.e., physically
linking) the antibody to a detectable substance. Examples of
detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
Antibody Uses
[0126] The antibodies can be used to isolate a 27875
metalloproteinase by standard techniques, such as affinity
chromatography or immunoprecipitation. The antibodies can
facilitate the purification of the natural 27875 metalloproteinase
from cells and recombinantly produced 27875 metalloproteinase
expressed in host cells.
[0127] The antibodies are useful to detect the presence of 27875
metalloproteinase in cells or tissues to determine the pattern of
expression of 27875 metalloproteinase among various tissues in an
organism and over the course of normal development. The antibodies
can be used to detect 27875 metalloproteinase in situ, in vitro, or
in a cell lysate or supernatant in order to evaluate the abundance
and pattern of expression. Antibody detection of circulating
fragments of the full length 27875 metalloproteinase can be used to
identify 27875 metalloproteinase turnover. In addition, the
antibodies can be used to assess abnormal tissue distribution or
abnormal expression during development.
[0128] Further, the antibodies can be used to assess 27875
metalloproteinase expression in disease states such as in active
stages of the disease or in an individual with a predisposition
toward disease related to 27875 metalloproteinase function. When a
disorder is caused by an inappropriate tissue distribution,
developmental expression, or level of expression of 27875
metalloproteinase protein, the antibody can be prepared against the
normal 27875 metalloproteinase protein. If a disorder is
characterized by a specific mutation in 27875 metalloproteinase,
antibodies specific for this mutant protein can be used to assay
for the presence of the specific mutant 27875 metalloproteinase.
However, intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular 27875
metalloproteinase peptide regions.
[0129] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Antibodies can be developed against the whole 27875
metalloproteinase or portions of 27875 metalloproteinase.
[0130] The diagnostic uses can be applied, not only in genetic
testing, but also in monitoring a treatment modality. Accordingly,
where treatment is ultimately aimed at correcting 27875
metalloproteinase expression level or the presence of aberrant
27875 metalloproteinases and aberrant tissue distribution or
developmental expression, antibodies directed against 27875
metalloproteinase or relevant fragments can be used to monitor
therapeutic efficacy.
[0131] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic 27875
metalloproteinase can be used to identify individuals that require
modified treatment modalities.
[0132] The antibodies are also useful as diagnostic tools as an
immunological marker for aberrant 27875 metalloproteinase analyzed
by electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0133] The antibodies are also useful for tissue typing. Thus,
where a specific 27875 metalloproteinase has been correlated with
expression in a specific tissue, antibodies that are specific for
this 27875 metalloproteinase can be used to identify a tissue
type.
[0134] The antibodies are also useful in forensic identification.
Accordingly, where an individual has been correlated with a
specific genetic polymorphism resulting in a specific polymorphic
protein, an antibody specific for the polymorphic protein can be
used as an aid in identification.
[0135] The antibodies are also useful for inhibiting 27875
metalloproteinase function, for example, zinc binding,
metalloproteinase activity, disintegrin activity or TSP activity.
For example, metalloproteinase activity may be measured by the
ability to form a covalent binding complex with
.alpha..sub.2-macroglobulin (Kuno et al. (1999) J Biol Chem
274:18821-18826).
[0136] These uses can also be applied in a therapeutic context in
which treatment involves inhibiting 27875 metalloproteinase
function. An antibody can be used, for example, to block peptide
binding. Antibodies can be prepared against specific fragments
containing sites required for function or against intact 27875
metalloproteinase associated with a cell.
[0137] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. For an overview of this
technology for producing human antibodies, see Lonberg et al.
(1995) Int. Rev. Immunol. 13:65-93. For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, e.g., U.S.
Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No.
5,545,806.
[0138] The invention also encompasses kits for using antibodies to
detect the presence of an 27875 metalloproteinase protein in a
biological sample. The kit can comprise antibodies such as a
labeled or labelable antibody and a compound or agent for detecting
27875 metalloproteinase in a biological sample; means for
determining the amount of 27875 metalloproteinase in the sample;
and means for comparing the amount of 27875 metalloproteinase in
the sample with a standard. The compound or agent can be packaged
in a suitable container. The kit can further comprise instructions
for using the kit to detect 27875 metalloproteinase.
Polynucleotides
[0139] The nucleotide sequence in SEQ ID NO:1 was obtained by
sequencing the deposited human cDNA. Accordingly, the sequence of
the deposited clone is controlling as to any discrepancies between
the two and any reference to the sequence of SEQ ID NO:1 includes
reference to the sequence of the deposited cDNA.
[0140] The specifically disclosed cDNA comprises the coding region
and 5' and 3' untranslated sequences in SEQ ID NO:1.
[0141] The invention provides isolated polynucleotides encoding the
novel 27875 metalloproteinase. The term "27875 metalloproteinase
polynucleotide" or "27875 metalloproteinase nucleic acid" refers to
the sequence shown in SEQ ID NO:1 or in the deposited cDNA. The
term "27875 metalloproteinase polynucleotide" or "27875
metalloproteinase nucleic acid" further includes variants and
fragments of 27875 metalloproteinase polynucleotides.
[0142] An "isolated" 27875 metalloproteinase nucleic acid is one
that is separated from other nucleic acid present in the natural
source of 27875 metalloproteinase nucleic acid. Preferably, an
"isolated" nucleic acid is free of sequences which naturally flank
27875 metalloproteinase nucleic acid (i.e., sequences located at
the 5' and 3' ends of the nucleic acid) in the genomic DNA of the
organism from which the nucleic acid is derived. However, there can
be some flanking nucleotide sequences, for example up to about 5
KB. The important point is that the 27875 metalloproteinase nucleic
acid is isolated from flanking sequences such that it can be
subjected to the specific manipulations described herein, such as
recombinant expression, preparation of probes and primers, and
other uses specific to the 27875 metalloproteinase nucleic acid
sequences. In one embodiment, the 27875 metalloproteinase nucleic
acid comprises only the coding region.
[0143] Moreover, an "isolated" nucleic acid molecule, such as a
cDNA or RNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0144] In some instances, the isolated material will form part of a
composition (for example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0145] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0146] In some instances, the isolated material will form part of a
composition (or example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0147] 27875 metalloproteinase polynucleotides can encode the
mature protein plus additional amino or carboxyterminal amino
acids, or amino acids interior to the mature polypeptide (when the
mature form has more than one polypeptide chain, for instance).
Such sequences may play a role in processing of a protein from
precursor to a mature form, facilitate protein trafficking, prolong
or shorten protein half-life or facilitate manipulation of a
protein for assay or production, among other things. As generally
is the case in situ, the additional amino acids may be processed
away from the mature protein by cellular enzymes.
[0148] 27875 metalloproteinase polynucleotides include, but are not
limited to, the sequence encoding the mature polypeptide alone, the
sequence encoding the mature polypeptide and additional coding
sequences, such as a leader or secretory sequence (e.g., a pre-pro
or pro-protein sequence), the sequence encoding the mature
polypeptide, with or without the additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but non-translated
sequences that play a role in transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding
and stability of mRNA. In addition, the polynucleotide may be fused
to a marker sequence encoding, for example, a peptide that
facilitates purification.
[0149] 27875 metalloproteinase polynucleotides can be in the form
of RNA, such as mRNA, or in the form DNA, including cDNA and
genomic DNA obtained by cloning or produced by chemical synthetic
techniques or by a combination thereof. The nucleic acid,
especially DNA, can be double-stranded or single-stranded.
Single-stranded nucleic acid can be the coding strand (sense
strand) or the non-coding strand (anti-sense strand).
[0150] The invention further provides variant 27875
metalloproteinase polynucleotides, and fragments thereof, that
differ from the nucleotide sequence shown in SEQ ID NO:1 due to
degeneracy of the genetic code and thus encode the same protein as
that encoded by the nucleotide sequence shown in SEQ ID NO:1.
[0151] The invention also provides 27875 metalloproteinase nucleic
acid molecules encoding the variant polypeptides described herein.
Such polynucleotides may be naturally occurring, such as allelic
variants (same locus), homologs (different locus), and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Such non-naturally occurring
variants may be made by mutagenesis techniques, including those
applied to polynucleotides, cells, or organisms. Accordingly, as
discussed above, the variants can contain nucleotide substitutions,
deletions, inversions and insertions.
[0152] Typically, variants have a substantial identity with a
nucleic acid molecules of SEQ ID NO:1 and the complements thereof.
Variation can occur in either or both the coding and non-coding
regions. The variations can produce both conservative and
non-conservative amino acid substitutions.
[0153] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. These variants comprise a
nucleotide sequence encoding a 27875 metalloproteinase that is
typically at least about 60-65%, 65-70%, 70-75%, more typically at
least about 80-85%, and most typically at least about 90-95% or
more homologous to the nucleotide sequence shown in SEQ ID NO:1 or
a fragment of this sequence. Such nucleic acid molecules can
readily be identified as being able to hybridize under stringent
conditions, to the nucleotide sequence shown in SEQ ID NO:1 or a
fragment of the sequence. It is understood that stringent
hybridization does not indicate substantial homology where it is
due to general homology, such as polyA.sup.+ sequences, or
sequences common to all or most proteins, metalloproteinases, zinc
binding proteins, thrombospondins, disintegrins, ADAMs, proteins in
the ADAM-TS family, or even all proteins in specific ADAM-TS
subfamilies, such as ADAM-TS-1, 3, etc. Moreover, it is understood
that variants do not include any of the nucleic acid sequences that
may have been disclosed prior to the invention.
[0154] As used herein, the term "stringent conditions" is intended
to describe conditions comprising hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more washes in 0.2.times.SSC/0.1% SDS at 65.degree. C.
Methods of hybridization are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1998), incorporated by reference. In one
embodiment, an isolated nucleic acid molecule that hybridizes under
stringent conditions to the sequence of SEQ ID NO:1 corresponds to
a naturally-occurring nucleic acid molecule. As used herein, a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA
molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein).
[0155] The present invention also provides isolated nucleic acids
that contain a single or double stranded fragment or portion that
hybridizes under stringent conditions to the nucleotide sequence of
SEQ ID NO:1 or the complement of SEQ ID NO:1. In one embodiment,
the nucleic acid consists of a portion of the nucleotide sequence
of SEQ ID NO:1 and the complement of SEQ ID NO:1. The nucleic acid
fragments of the invention are at least about 15, preferably at
least about 16, 17, 18, 19, 20, 23 or 25 contiguous nucleotides,
and can be 30, 33, 35, 40, 50, 60, 70, 75, 80, 90, 100, 200, 500 or
more nucleotides in length. Longer fragments, for example, 600 or
more nucleotides in length, which encode antigenic proteins or
polypeptides described herein are also useful.
[0156] Furthermore, the invention provides polynucleotides that
comprise a fragment of the full-length 27875 metalloproteinase
polynucleotides. The fragment can be single or double-stranded and
can comprise DNA or RNA. The fragment can be derived from either
the coding or the non-coding sequence.
[0157] In one embodiment, the nucleic acid sequence is selected
from the group consisting of:
[0158] (a) a nucleotide sequence encoding a fragment of the amino
acid sequence shown in SEQ ID NO:2, wherein the fragment comprises
at least 26 contiguous amino acids;
[0159] (b) a nucleotide sequence comprising at least 75 consecutive
nucleotides of the sequence shown in SEQ ID NO:1;
[0160] (c) a nucleotide sequence comprising at least 33 consecutive
nucleotides of residues 1-4800 of SEQ ID NO:1;
[0161] (d) a nucleotide sequence encoding residues 31-1687 of the
amino acid shown in SEQ ID NO:2;
[0162] (e) a nucleotide sequence encoding residues 244-259 of SEQ
ID NO:2;
[0163] (f) a nucleotide sequence encoding residues 385-394 of SEQ
ID NO:2;
[0164] (g) a nucleotide sequence encoding residues 541-592 of SEQ
ID NO:2;
[0165] (h) a nucleotide sequence encoding residues 542-592 of SEQ
ID NO:2;
[0166] (i) a nucleotide sequence encoding residues 825-868 of SEQ
ID NO:2;
[0167] (j) a nucleotide sequence encoding residues 949-988 of SEQ
ID NO:2;
[0168] (k) a nucleotide sequence encoding residues 1415-1463 of SEQ
ID NO:2; and
[0169] (l) a nucleotide sequence complementary to a nucleotide
sequences of (a)-(l).
[0170] In another embodiment an isolated 27875 metalloproteinase
nucleic acid encodes the entire coding region. In another
embodiment the isolated 27875 metalloproteinase nucleic acid
encodes a sequence corresponding to the mature protein that may be
from about amino acid 6 to the last amino acid. Other fragments
include nucleotide sequences encoding the amino acid fragments
described herein.
[0171] Thus, 27875 metalloproteinase nucleic acid fragments further
include sequences corresponding to the regions described herein,
subregions also described, and specific functional sites. 27875
metalloproteinase nucleic acid fragments also include combinations
of the regions, segments, motifs, and other functional sites
described above. It is understood that a 27875 metalloproteinase
fragment includes any nucleic acid sequence that does not include
the entire gene. A person of ordinary skill in the art would be
aware of the many permutations that are possible. Nucleic acid
fragments, according to the present invention, are not to be
construed as encompassing those fragments that may have been
disclosed prior to the invention.
[0172] Where the location of the regions or sites have been
predicted by computer analysis, one of ordinary sill would
appreciate that the amino acid residues constituting these regions
can vary depending on the criteria used to define the regions.
Polynucleotide Uses
[0173] The nucleotide sequences of the present invention can be
used as a "query sequence" to perform a search against public
databases, for example, to identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol.
Biol. 215:403-10. BLAST protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to the proteins of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al. (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used.
[0174] The nucleic acid fragments of the invention provide probes
or primers in assays such as those described below. "Probes" are
oligonucleotides that hybridize in a base-specific manner to a
complementary strand of nucleic acid. Such probes include
polypeptide nucleic acids, as described in Nielsen et al. (1991)
Science 254:1497-1500. Typically, a probe comprises a region of
nucleotide sequence that hybridizes under highly stringent
conditions to at least about 15, typically about 20-25, and more
typically about 40, 50 or 75 consecutive nucleotides of the nucleic
acid sequence shown in SEQ ID NO:1 and the complements thereof.
More typically, the probe further comprises a label, e.g.,
radioisotope, fluorescent compound, enzyme, or enzyme
co-factor.
[0175] As used herein, the term "primer" refers to a
single-stranded oligonucleotide which acts as a point of initiation
of template-directed DNA synthesis using well-known methods (e.g.,
PCR, LCR) including, but not limited to those described herein. The
appropriate length of the primer depends on the particular use, but
typically ranges from about 15 to 30 nucleotides. The term "primer
site" refers to the area of the target DNA to which a primer
hybridizes. The term "primer pair" refers to a set of primers
including a 5' (upstream) primer that hybridizes with the 5' end of
the nucleic acid sequence to be amplified and a 3' (downstream)
primer that hybridizes with the complement of the sequence to be
amplified.
[0176] 27875 metalloproteinase polynucleotides are thus useful for
probes, primers, and in biological assays. Where the
polynucleotides are used to assess 27875 metalloproteinase
properties or functions, such as in the assays described herein,
all or less than all of the entire cDNA can be useful. Assays
specifically directed to 27875 metalloproteinase functions, such as
assessing agonist or antagonist activity, encompass the use of
known fragments. Further, diagnostic methods for assessing 27875
metalloproteinase function can also be practiced with any fragment,
including those fragments that may have been known prior to the
invention. Similarly, in methods involving treatment of 27875
metalloproteinase dysfunction, all fragments are encompassed
including those, which may have been known in the art.
[0177] 27875 metalloproteinase polynucleotides are useful as a
hybridization probe for cDNA and genomic DNA to isolate a
full-length cDNA and genomic clones encoding the polypeptides
described in SEQ ID NO:1 and to isolate cDNA and genomic clones
that correspond to variants producing the same polypeptides shown
in SEQ ID NO:2 or the other variants described herein. Variants can
be isolated from the same tissue and organism from which the
polypeptides shown in SEQ ID NO:2 were isolated, different tissues
from the same organism, or from different organisms. This method is
useful for isolating genes and cDNA that are
developmentally-controlled and therefore may be expressed in the
same tissue or different tissues at different points in the
development of an organism.
[0178] The probe can correspond to any sequence along the entire
length of the gene encoding the 27875 metalloproteinase
polypeptide. Accordingly, it could be derived from 5' noncoding
regions, the coding region, and 3' noncoding regions.
[0179] The nucleic acid probe can be, for example, the full-length
cDNA of SEQ ID NO:1, or a fragment thereof, such as an
oligonucleotide of at least 12, 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to mRNA or DNA.
[0180] Fragments of the polynucleotides described herein are also
useful to synthesize larger fragments or full-length
polynucleotides described herein, ribozymes or antisense molecules.
For example, a fragment can be hybridized to any portion of an mRNA
and a larger or full-length cDNA can be produced.
[0181] Antisense nucleic acids of the invention can be designed
using the nucleotide sequences of SEQ ID NO:1, and constructed
using chemical synthesis and enzymatic ligation reactions using
procedures known in the art. For example, an antisense nucleic acid
(e.g., an antisense oligonucleotide) can be chemically synthesized
using naturally occurring nucleotides or variously modified
nucleotides designed to increase the biological stability of the
molecules or to increase the physical stability of the duplex
formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate derivatives and acridine substituted nucleotides
can be used. Examples of modified nucleotides which can be used to
generate the antisense nucleic acid include 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, 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-isopentenyladenine,
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. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest).
[0182] Additionally, the nucleic acid molecules of the invention
can be modified at the base moiety, sugar moiety or phosphate
backbone to improve, e.g., the stability, hybridization, or
solubility of the molecule. For example, the deoxyribose phosphate
backbone of the nucleic acids can be modified to generate peptide
nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4:5). As used herein, the terms "peptide nucleic acids"
or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which
the deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93:14670. PNAs can be further modified, e.g., to
enhance their stability, specificity or cellular uptake, by
attaching lipophilic or other helper groups to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other
techniques of drug delivery known in the art. The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63, Mag et
al. (1989) Nucleic Acids Res. 17:5973, and Peterser et al. (1975)
Bioorganic Med. Chem. Lett. 5:1119.
[0183] The nucleic acid molecules and fragments of the invention
can also include other appended groups such as peptides (e.g., for
targeting host cell 27875 metalloproteinases in vivo), or agents
facilitating transport across the cell membrane (see, e.g.,
Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;
Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT
Publication No. WO 88/0918) or the blood brain barrier (see, e.g.,
PCT Publication No. WO 89/10134). In addition, oligonucleotides can
be modified with hybridization-triggered cleavage agents (see,
e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating
agents (see, e.g., Zon (1988) Pharm Res. 5:539-549).
[0184] 27875 metalloproteinase polynucleotides are also useful as
primers for PCR to amplify any given region of an 27875
metalloproteinase polynucleotide.
[0185] 27875 metalloproteinase polynucleotides are also useful for
constructing recombinant vectors. Such vectors include expression
vectors that express a portion of, or all of, the 27875
metalloproteinase polypeptides. Vectors also include insertion
vectors, used to integrate into another polynucleotide sequence,
such as into the cellular genome, to alter in situ expression of
27875 metalloproteinase genes and gene products. For example, an
endogenous 27875 metalloproteinase coding sequence can be replaced
via homologous recombination with all or part of the coding region
containing one or more specifically introduced mutations.
[0186] 27875 metalloproteinase polynucleotides are also useful for
expressing antigenic portions of 27875 metalloproteinase
proteins.
[0187] 27875 metalloproteinase polynucleotides are also useful as
probes for determining the chromosomal positions of 27875
metalloproteinase polynucleotides by means of in situ hybridization
methods, such as FISH. (For a review of this technique, see Verma
et al. (1988) Human Chromosomes: A Manual of Basic Techniques
(Pergamon Press, New York), and PCR mapping of somatic cell
hybrids. The mapping of the sequences to chromosomes is an
important first step in correlating these sequences with genes
associated with disease.
[0188] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0189] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland et al. ((1987) Nature 325:783-787).
[0190] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
a specified gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or
translocations, that are visible from chromosome spreads, or
detectable using PCR based on that DNA sequence. Ultimately,
complete sequencing of genes from several individuals can be
performed to confirm the presence of a mutation and to distinguish
mutations from polymorphisms.
[0191] 27875 metalloproteinase polynucleotide probes are also
useful to determine patterns of the presence of the gene encoding
27875 metalloproteinases and their variants with respect to tissue
distribution, for example, whether gene duplication has occurred
and whether the duplication occurs in all or only a subset of
tissues. The genes can be naturally occurring or can have been
introduced into a cell, tissue, or organism exogenously.
[0192] 27875 metalloproteinase polynucleotides are also useful for
designing ribozymes corresponding to all, or a part, of the mRNA
produced from genes encoding the polynucleotides described
herein.
[0193] 27875 metalloproteinase polynucleotides are also useful for
constructing host cells expressing a part, or all, of 27875
metalloproteinase polynucleotides and polypeptides.
[0194] 27875 metalloproteinase polynucleotides are also useful for
constructing transgenic animals expressing all, or a part, of 27875
metalloproteinase polynucleotides and polypeptides.
[0195] 27875 metalloproteinase polynucleotides are also useful for
making vectors that express part, or all, of 27875
metalloproteinase polypeptides.
[0196] 27875 metalloproteinase polynucleotides are also useful as
hybridization probes for determining the level of 27875
metalloproteinase nucleic acid expression. Accordingly, the probes
can be used to detect the presence of, or to determine levels of,
27875 metalloproteinase nucleic acid in cells, tissues, and in
organisms. The nucleic acid whose level is determined can be DNA or
RNA. Accordingly, probes corresponding to the polypeptides
described herein can be used to assess gene copy number in a given
cell, tissue, or organism. This is particularly relevant in cases
in which there has been an amplification of 27875 metalloproteinase
genes.
[0197] Alternatively, the probe can be used in an in situ
hybridization context to assess the position of extra copies of
27875 metalloproteinase genes, as on extrachromosomal elements or
as integrated into chromosomes in which the 27875 metalloproteinase
gene is not normally found, for example as a homogeneously staining
region.
[0198] These uses are relevant for diagnosis of disorders involving
an increase or decrease in 27875 metalloproteinase expression
relative to normal, such as a proliferative disorder, a
differentiative or developmental disorder, or a hematopoietic
disorder. Disorders in which 27875 metalloproteinase expression is
relevant include, but are not limited to, those of bone, such as
osteoporosis and osteopetrosis.
[0199] Thus, the present invention provides a method for
identifying a disease or disorder associated with aberrant
expression or activity of 27875 metalloproteinase nucleic acid, in
which a test sample is obtained from a subject and nucleic acid
(e.g., mRNA, genomic DNA) is detected, wherein the presence of the
nucleic acid is diagnostic for a subject having or at risk of
developing a disease or disorder associated with aberrant
expression or activity of the nucleic acid.
[0200] One aspect of the invention relates to diagnostic assays for
determining nucleic acid expression as well as activity in the
context of a biological sample (e.g., blood, serum, cells, tissue)
to determine whether an individual has a disease or disorder, or is
at risk of developing a disease or disorder, associated with
aberrant nucleic acid expression or activity. Such assays can be
used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with expression or activity
of the nucleic acid molecules.
[0201] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[0202] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express 27875 metalloproteinase,
such as by measuring the level of an 27875
metalloproteinase-encoding nucleic acid in a sample of cells from a
subject e.g., mRNA or genomic DNA, or determining if the 27875
metalloproteinase gene has been mutated.
[0203] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate 27875 metalloproteinase nucleic
acid expression (e.g., antisense, polypeptides, peptidomimetics,
small molecules or other drugs). A cell is contacted with a
candidate compound and the expression of mRNA determined. The level
of expression of the mRNA in the presence of the candidate compound
is compared to the level of expression of the mRNA in the absence
of the candidate compound. The candidate compound can then be
identified as a modulator of nucleic acid expression based on this
comparison and be used, for example to treat a disorder
characterized by aberrant nucleic acid expression. The modulator
can bind to the nucleic acid or indirectly modulate expression,
such as by interacting with other cellular components that affect
nucleic acid expression.
[0204] Modulatory methods can be performed in vitro (e.g., by
culturing the cell with the agent) or, alternatively, in vivo
(e.g., by administering the gent to a subject) in patients or in
transgenic animals. The invention thus provides a method for
identifying a compound that can be used to treat a disorder
associated with nucleic acid expression of the 27875
metalloproteinase gene. The method typically includes assaying the
ability of the compound to modulate the expression of the 27875
metalloproteinase nucleic acid and thus identifying a compound that
can be used to treat a disorder characterized by undesired 27875
metalloproteinase nucleic acid expression.
[0205] The assays can be performed in cell-based and cell-free
systems. Cell-based assays include cells naturally expressing the
27875 metalloproteinase nucleic acid or recombinant cells
genetically engineered to express specific nucleic acid sequences.
Alternatively, candidate compounds can be assayed in vivo in
patients or in transgenic animals.
[0206] The assay for 27875 metalloproteinase nucleic acid
expression can involve direct assay of nucleic acid levels, such as
mRNA levels, or on collateral compounds (such as peptide
hydrolysis). Further, the expression of genes that are up- or
down-regulated in response to 27875 metalloproteinase activity can
also be assayed. In this embodiment the regulatory regions of these
genes can be operably linked to a reporter gene such as
luciferase.
[0207] Thus, modulators of 27875 metalloproteinase gene expression
can be identified in a method wherein a cell is contacted with a
candidate compound and the expression of mRNA determined. The level
of expression of 27875 metalloproteinase mRNA in the presence of
the candidate compound is compared to the level of expression of
27875 metalloproteinase mRNA in the absence of the candidate
compound. The candidate compound can then be identified as a
modulator of nucleic acid expression based on this comparison and
be used, for example to treat a disorder characterized by aberrant
nucleic acid expression. When expression of mRNA is statistically
significantly greater in the presence of the candidate compound
than in its absence, the candidate compound is identified as a
stimulator of nucleic acid expression. When nucleic acid expression
is statistically significantly less in the presence of the
candidate compound than in its absence, the candidate compound is
identified as an inhibitor of nucleic acid expression.
[0208] Accordingly, the invention provides methods of treatment,
with the nucleic acid as a target, using a compound identified
through drug screening as a gene modulator to modulate 27875
metalloproteinase nucleic acid expression. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or effects on nucleic acid activity
(e.g. when nucleic acid is mutated or improperly modified).
Treatment is of disorders characterized by aberrant expression or
activity of the nucleic acid.
[0209] Alternatively, a modulator for 27875 metalloproteinase
nucleic acid expression can be a small molecule or drug identified
using the screening assays described herein as long as the drug or
small molecule inhibits 27875 metalloproteinase nucleic acid
expression.
[0210] 27875 metalloproteinase polynucleotides are also useful for
monitoring the effectiveness of modulating compounds on the
expression or activity of the 27875 metalloproteinase gene in
clinical trials or in a treatment regimen. Thus, the gene
expression pattern can serve as a barometer for the continuing
effectiveness of treatment with the compound, particularly with
compounds to which a patient can develop resistance. The gene
expression pattern can also serve as a marker indicative of a
physiological response of the affected cells to the compound.
Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative
compounds to which the patient has not become resistant. Similarly,
if the level of nucleic acid expression falls below a desirable
level, administration of the compound could be commensurately
decreased.
[0211] Monitoring can be, for example, as follows: (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of a specified
mRNA or genomic DNA of the invention in the pre-administration
sample; (iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression or
activity of the mRNA or genomic DNA in the post-administration
samples; (v) comparing the level of expression or activity of the
mRNA or genomic DNA in the pre-administration sample with the mRNA
or genomic DNA in the post-administration sample or samples; and
(vi) increasing or decreasing the administration of the agent to
the subject accordingly.
[0212] 27875 metalloproteinase polynucleotides are also useful in
diagnostic assays for qualitative changes in 27875
metalloproteinase nucleic acid, and particularly in qualitative
changes that lead to pathology. The polynucleotides can be used to
detect mutations in 27875 metalloproteinase genes and gene
expression products such as mRNA. The polynucleotides can be used
as hybridization probes to detect naturally-occurring genetic
mutations in the 27875 metalloproteinase gene and thereby to
determine whether a subject with the mutation is at risk for a
disorder caused by the mutation. Mutations include deletion,
addition, or substitution of one or more nucleotides in the gene,
chromosomal rearrangement, such as inversion or transposition,
modification of genomic DNA, such as aberrant methylation patterns
or changes in gene copy number, such as amplification. Detection of
a mutated form of the 27875 metalloproteinase gene associated with
a dysfunction provides a diagnostic tool for an active disease or
susceptibility to disease when the disease results from
overexpression, underexpression, or altered expression of an 27875
metalloproteinase.
[0213] Mutations in the 27875 metalloproteinase gene can be
detected at the nucleic acid level by a variety of techniques.
Genomic DNA can be analyzed directly or can be amplified by using
PCR prior to analysis. RNA or cDNA can be used in the same way.
[0214] In certain embodiments, detection of the mutation involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) PNAS 91:360-364), the latter of which
can be particularly useful for detecting point mutations in the
gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682).
This method can include the steps of collecting a sample of cells
from a patient, isolating nucleic acid (e.g., genomic, mRNA or
both) from the cells of the sample, contacting the nucleic acid
sample with one or more primers which specifically hybridize to a
gene under conditions such that hybridization and amplification of
the gene (if present) occurs, and detecting the presence or absence
of an amplification product, or detecting the size of the
amplification product and comparing the length to a control sample.
Deletions and insertions can be detected by a change in size of the
amplified product compared to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to normal RNA or
antisense DNA sequences.
[0215] It is anticipated that PCR and/or LCR may be desirable to
use as a preliminary amplification step in conjunction with any of
the techniques used for detecting mutations described herein.
[0216] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi 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.
[0217] Alternatively, mutations in an 27875 metalloproteinase gene
can be directly identified, for example, by alterations in
restriction enzyme digestion patterns determined by gel
electrophoresis.
[0218] Further, sequence-specific ribozymes (U.S. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
[0219] Perfectly matched sequences can be distinguished from
mismatched sequences by nuclease cleavage digestion assays or by
differences in melting temperature.
[0220] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method.
[0221] Furthermore, sequence differences between a mutant 27875
metalloproteinase gene and a wild-type gene can be determined by
direct DNA sequencing. A variety of automated sequencing procedures
can be utilized when performing the diagnostic assays ((1995)
Biotechniques 19:448), including sequencing by mass spectrometry
(see, e.g., PCT International Publication No. WO 94/16101; Cohen et
al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993)
Appl. Biochem. Biotechnol. 38:147-159).
[0222] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.
(1985) Science 230:1242); Cotton et al. (1988) PNAS 85:4397;
Saleeba et al. (1992) Meth. Enzymol. 217:286-295), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al. (1989) PNAS 86:2766; Cotton et al. (1993) Mutat. Res.
285:125-144; and Hayashi et al. (1992) Genet. Anal. Tech. Appl.
9:73-79), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al. (1985)
Nature 313:495). The sensitivity of the assay may be enhanced by
using RNA (rather than DNA), in which the secondary structure is
more sensitive to a change in sequence. In one embodiment, the
subject method utilizes heteroduplex analysis to separate double
stranded heteroduplex molecules on the basis of changes in
electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
Examples of other techniques for detecting point mutations include,
selective oligonucleotide hybridization, selective amplification,
and selective primer extension.
[0223] In other embodiments, genetic mutations can be identified by
hybridizing a sample and control nucleic acids, e.g., DNA or RNA,
to high density arrays containing hundreds or thousands of
oligonucleotide probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two dimensional
arrays containing light-generated DNA probes as described in Cronin
et al. supra. Briefly, a first hybridization array of probes can be
used to scan through long stretches of DNA in a sample and control
to identify base changes between the sequences by making linear
arrays of sequential overlapping probes. This step allows the
identification of point mutations. This step is followed by a
second hybridization array that allows the characterization of
specific mutations by using smaller, specialized probe arrays
complementary to all variants or mutations detected. Each mutation
array is composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant gene.
[0224] 27875 metalloproteinase polynucleotides are also useful for
testing an individual for a genotype that while not necessarily
causing the disease, nevertheless affects the treatment modality.
Thus, the polynucleotides can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship). In the
present case, for example, a mutation in the 27875
metalloproteinase gene that results in altered affinity for zinc
could result in an excessive or decreased drug effect with standard
concentrations of zinc. Accordingly, the 27875 metalloproteinase
polynucleotides described herein can be used to assess the mutation
content of the gene in an individual in order to select an
appropriate compound or dosage regimen for treatment.
[0225] Thus polynucleotides displaying genetic variations that
affect treatment provide a diagnostic target that can be used to
tailor treatment in an individual. Accordingly, the production of
recombinant cells and animals containing these polymorphisms allow
effective clinical design of treatment compounds and dosage
regimens.
[0226] The methods can involve obtaining a control biological
sample from a control subject, contacting the control sample with a
compound or agent capable of detecting mRNA, or genomic DNA, such
that the presence of mRNA or genomic DNA is detected in the
biological sample, and comparing the presence of mRNA or genomic
DNA in the control sample with the presence of mRNA or genomic DNA
in the test sample.
[0227] 27875 metalloproteinase polynucleotides are also useful for
chromosome identification when the sequence is identified with an
individual chromosome and to a particular location on the
chromosome. First, the DNA sequence is matched to the chromosome by
in situ or other chromosome-specific hybridization. Sequences can
also be correlated to specific chromosomes by preparing PCR primers
that can be used for PCR screening of somatic cell hybrids
containing individual chromosomes from the desired species. Only
hybrids containing the chromosome containing the gene homologous to
the primer will yield an amplified fragment. Sublocalization can be
achieved using chromosomal fragments. Other strategies include
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to chromosome-specific libraries. Further mapping
strategies include fluorescence in situ hybridization, which allows
hybridization with probes shorter than those traditionally used.
Reagents for chromosome mapping can be used individually to mark a
single chromosome or a single site on the chromosome, or panels of
reagents can be used for marking multiple sites and/or multiple
chromosomes. Reagents corresponding to noncoding regions of the
genes actually are preferred for mapping purposes. Coding sequences
are more likely to be conserved within gene families, thus
increasing the chance of cross hybridizations during chromosomal
mapping.
[0228] 27875 metalloproteinase polynucleotides can also be used to
identify individuals from small biological samples. This can be
done for example using restriction fragment-length polymorphism
(RFLP) to identify an individual. Thus, the polynucleotides
described herein are useful as DNA markers for RFLP (See U.S. Pat.
No. 5,272,057).
[0229] Furthermore, the 27875 metalloproteinase sequence can be
used to provide an alternative technique, which determines the
actual DNA sequence of selected fragments in the genome of an
individual. Thus, the 27875 metalloproteinase sequences described
herein can be used to prepare two PCR primers from the 5' and 3'
ends of the sequences. These primers can then be used to amplify
DNA from an individual for subsequent sequencing.
[0230] Panels of corresponding DNA sequences from individuals
prepared in this manner can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences. It is estimated that allelic variation in humans
occurs with a frequency of about once per each 500 bases. Allelic
variation occurs to some degree in the coding regions of these
sequences, and to a greater degree in the noncoding regions. 27875
metalloproteinase sequences can be used to obtain such
identification sequences from individuals and from tissue. The
sequences represent unique fragments of the human genome. Each of
the sequences described herein can, to some degree, be used as a
standard against which DNA from an individual can be compared for
identification purposes.
[0231] If a panel of reagents from the sequences is used to
generate a unique identification database for an individual, those
same reagents can later be used to identify tissue from that
individual. Using the unique identification database, positive
identification of the individual, living or dead, can be made from
extremely small tissue samples.
[0232] 27875 metalloproteinase polynucleotides can also be used in
forensic identification procedures. PCR technology can be used to
amplify DNA sequences taken from very small biological samples,
such as a single hair follicle, body fluids (e.g. blood, saliva, or
semen). The amplified sequence can then be compared to a standard
allowing identification of the origin of the sample.
[0233] 27875 metalloproteinase polynucleotides can thus be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As described
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to the
noncoding region are particularly useful since greater polymorphism
occurs in the noncoding regions, making it easier to differentiate
individuals using this technique.
[0234] 27875 metalloproteinase polynucleotides can further be used
to provide polynucleotide reagents, e.g., labeled or labelable
probes which can be used in, for example, an in situ hybridization
technique, to identify a specific tissue. This is useful in cases
in which a forensic pathologist is presented with a tissue of
unknown origin. Panels of 27875 metalloproteinase probes can be
used to identify tissue by species and/or by organ type.
[0235] In a similar fashion, these primers and probes can be used
to screen tissue culture for contamination (i.e. screen for the
presence of a mixture of different types of cells in a
culture).
[0236] Alternatively, 27875 metalloproteinase polynucleotides can
be used directly to block transcription or translation of 27875
metalloproteinase gene sequences by means of antisense or ribozyme
constructs. Thus, in a disorder characterized by abnormally high or
undesirable 27875 metalloproteinase gene expression, nucleic acids
can be directly used for treatment.
[0237] 27875 metalloproteinase polynucleotides are thus useful as
antisense constructs to control 27875 metalloproteinase gene
expression in cells, tissues, and organisms. A DNA antisense
polynucleotide is designed to be complementary to a region of the
gene involved in transcription, preventing transcription and hence
production of 27875 metalloproteinase protein. An antisense RNA or
DNA polynucleotide would hybridize to the mRNA and thus block
translation of mRNA into 27875 metalloproteinase protein.
[0238] Examples of antisense molecules useful to inhibit nucleic
acid expression include antisense molecules complementary to a
fragment of the 5' untranslated region of SEQ ID NO:2 which also
includes the start codon and antisense molecules which are
complementary to a fragment of the 3' untranslated region of SEQ ID
NO:1.
[0239] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of 27875
metalloproteinase nucleic acid. Accordingly, these molecules can
treat a disorder characterized by abnormal or undesired 27875
metalloproteinase nucleic acid expression. This technique involves
cleavage by means of ribozymes containing nucleotide sequences
complementary to one or more regions in the mRNA that attenuate the
ability of the mRNA to be translated. Possible regions include
coding regions and particularly coding regions corresponding to the
catalytic and other functional activities of the 27875
metalloproteinase protein.
[0240] 27875 metalloproteinase polynucleotides also provide vectors
for gene therapy in patients containing cells that are aberrant in
27875 metalloproteinase gene expression. Thus, recombinant cells,
which include the patient's cells that have been engineered ex vivo
and returned to the patient, are introduced into an individual
where the cells produce the desired 27875 metalloproteinase protein
to treat the individual.
[0241] The invention also encompasses kits for detecting the
presence of an 27875 metalloproteinase nucleic acid in a biological
sample. For example, the kit can comprise reagents such as a
labeled or labelable nucleic acid or agent capable of detecting
27875 metalloproteinase nucleic acid in a biological sample; means
for determining the amount of 27875 metalloproteinase nucleic acid
in the sample; and means for comparing the amount of 27875
metalloproteinase nucleic acid in the sample with a standard. The
compound or agent can be packaged in a suitable container. The kit
can further comprise instructions for using the kit to detect 27875
metalloproteinase mRNA or DNA.
Computer Readable Means
[0242] The nucleotide or amino acid sequences of the invention are
also provided in a variety of mediums to facilitate use thereof. As
used herein, "provided" refers to a manufacture, other than an
isolated nucleic acid or amino acid molecule, which contains a
nucleotide or amino acid sequence of the present invention. Such a
manufacture provides the nucleotide or amino acid sequences, or a
subset thereof (e.g., a subset of open reading frames (ORFs)) in a
form which allows a skilled artisan to examine the manufacture
using means not directly applicable to examining the nucleotide or
amino acid sequences, or a subset thereof, as they exists in nature
or in purified form.
[0243] In one application of this embodiment, a nucleotide or amino
acid sequence of the present invention can be recorded on computer
readable media. As used herein, "computer readable media" refers to
any medium that can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media,
such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. The skilled artisan will readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide or amino acid sequence
of the present invention.
[0244] As used herein, "recorded" refers to a process for storing
information on computer readable medium. The skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide or amino acid sequence information of the
present invention.
[0245] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of data processor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0246] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[0247] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[0248] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[0249] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software includes, but is not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[0250] For example, software which implements the BLAST (Altschul
et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al.
(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system
can be used to identify open reading frames (ORFs) of the sequences
of the invention which contain homology to ORFs or proteins from
other libraries. Such ORFs are protein encoding fragments and are
useful in producing commercially important proteins such as enzymes
used in various reactions and in the production of commercially
useful metabolites.
Vectors/Host Cells
[0251] The invention also provides vectors containing 27875
metalloproteinase polynucleotides. The term "vector" refers to a
vehicle, preferably a nucleic acid molecule that can transport
27875 metalloproteinase polynucleotides. When the vector is a
nucleic acid molecule, the 27875 metalloproteinase polynucleotides
are covalently linked to the vector nucleic acid. With this aspect
of the invention, the vector includes a plasmid, single or double
stranded phage, a single or double stranded RNA or DNA viral
vector, or artificial chromosome, such as a BAC, PAC, YAC, OR
MAC.
[0252] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of 27875 metalloproteinase polynucleotides.
Alternatively, the vector may integrate into the host cell genome
and produce additional copies of 27875 metalloproteinase
polynucleotides when the host cell replicates.
[0253] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of 27875
metalloproteinase polynucleotides. The vectors can function in
procaryotic or eukaryotic cells or in both (shuttle vectors).
[0254] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to 27875 metalloproteinase
polynucleotides such that transcription of the polynucleotides is
allowed in a host cell. The polynucleotides can be introduced into
the host cell with a separate polynucleotide capable of affecting
transcription. Thus, the second polynucleotide may provide a
trans-acting factor interacting with the cis-regulatory control
region to allow transcription of 27875 metalloproteinase
polynucleotides from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself.
[0255] It is understood, however, that in some embodiments,
transcription and/or translation of 27875' metalloproteinase
polynucleotides can occur in a cell-free system.
[0256] The regulatory sequence to which the polynucleotides
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lamda., the lac, TRP, and
TAC promoters from E. coli, the early and late promoters from SV40,
the CMV immediate early promoter, the adenovirus early and late
promoters, and retrovirus long-terminal repeats.
[0257] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0258] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[0259] A variety of expression vectors can be used to express an
27875 metalloproteinase polynucleotide. Such vectors include
chromosomal, episomal, and virus-derived vectors, for example
vectors derived from bacterial plasmids, from bacteriophage, from
yeast episomes, from yeast chromosomal elements, including yeast
artificial chromosomes, from viruses such as baculoviruses,
papovaviruses such as SV40, Vaccinia viruses, adenoviruses,
poxviruses, pseudorabies viruses, and retroviruses. Vectors may
also be derived from combinations of these sources such as those
derived from plasmid and bacteriophage genetic elements, e.g.
cosmids and phagemids. Appropriate cloning and expression vectors
for prokaryotic and eukaryotic hosts are described in Sambrook et
al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0260] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0261] 27875 metalloproteinase polynucleotides can be inserted into
the vector nucleic acid by well-known methodology. Generally, the
DNA sequence that will ultimately be expressed is joined to an
expression vector by cleaving the DNA sequence and the expression
vector with one or more restriction enzymes and then ligating the
fragments together. Procedures for restriction enzyme digestion and
ligation are well known to those of ordinary skill in the art.
[0262] The vector containing the appropriate polynucleotide can be
introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0263] As described herein, it may be desirable to express the
polypeptide as a fusion protein. Accordingly, the invention
provides fusion vectors that allow for the production of 27875
metalloproteinase polypeptides. Fusion vectors can increase the
expression of a recombinant protein, increase the solubility of the
recombinant protein, and aid in the purification of the protein by
acting for example as a ligand for affinity purification. A
proteolytic cleavage site may be introduced at the junction of the
fusion moiety so that the desired polypeptide can ultimately be
separated from the fusion moiety. Proteolytic enzymes include, but
are not limited to, factor Xa, thrombin, and enterokinase. Typical
fusion expression vectors include pGEX (Smith et al. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein. Examples of suitable inducible
non-fusion E. coli expression vectors include pTrc (Amann et al.
(1988) Gene 69:301-315) and pET 11d (Studier et al. (1990) Gene
Expression Technology: Methods in Enzymology 185:60-89).
[0264] Recombinant protein expression can be maximized in a host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S. (1990) Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. 119-128).
Alternatively, the sequence of the polynucleotide of interest can
be altered to provide preferential codon usage for a specific host
cell, for example E. coli. (Wada et al. (1992) Nucleic Acids Res.
20:2111-2118).
[0265] 27875 metalloproteinase polynucleotides can also be
expressed by expression vectors that are operative in yeast.
Examples of vectors for expression in yeast e.g., S. cerevisiae
include pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa
(Kurjan et al. (1982) Cell 30:933-943), pJRY88 (Schultz et al.
(1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San
Diego, Calif.).
[0266] 27875 metalloproteinase polynucleotides can also be
expressed in insect cells using, for example, baculovirus
expression vectors. Baculovirus vectors available for expression of
proteins in cultured insect cells (e.g., Sf 9 cells) include the
pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and
the pVL series (Lucklow et al. (1989) Virology 170:31-39).
[0267] In certain embodiments of the invention, the polynucleotides
described herein are expressed in mammalian cells using mammalian
expression vectors. Examples of mammalian expression vectors
include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman
et al. (1987) EMBO J. 6:187-195).
[0268] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express 27875
metalloproteinase polynucleotides. The person of ordinary skill in
the art would be aware of other vectors suitable for maintenance
propagation or expression of the polynucleotides described herein.
These are found for example in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[0269] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the
polynucleotide sequences described herein, including both coding
and non-coding regions. Expression of this antisense RNA is subject
to each of the parameters described above in relation to expression
of the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0270] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0271] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).
[0272] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, 27875 metalloproteinase polynucleotides can
be introduced either alone or with other polynucleotides that are
not related to 27875 metalloproteinase polynucleotides such as
those providing trans-acting factors for expression vectors. When
more than one vector is introduced into a cell, the vectors can be
introduced independently, co-introduced or joined to the 27875
metalloproteinase polynucleotide vector.
[0273] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0274] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the polynucleotides described herein or
may be on a separate vector. Markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0275] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0276] Where secretion of the polypeptide is desired, appropriate
secretion signals are incorporated into the vector. The signal
sequence can be endogenous to the 27875 metalloproteinase
polypeptides or heterologous to these polypeptides.
[0277] Where the polypeptide is not secreted into the medium, the
protein can be isolated from the host cell by standard disruption
procedures, including freeze thaw, sonication, mechanical
disruption, use of lysing agents and the like. The polypeptide can
then be recovered and purified by well-known purification methods
including ammonium sulfate precipitation, acid extraction, anion or
cationic exchange chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0278] It is also understood that depending upon the host cell in
recombinant production of the polypeptides described herein, the
polypeptides can have various glycosylation patterns, depending
upon the cell, or maybe non-glycosylated as when produced in
bacteria. In addition, the polypeptides may include an initial
modified methionine in some cases as a result of a host-mediated
process.
Uses of Vectors and Host Cells
[0279] It is understood that "host cells" and "recombinant host
cells" refer not only to the particular subject cell but also to
the progeny or potential progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term as used herein.
[0280] The host cells expressing the polypeptides described herein,
and particularly recombinant host cells, have a variety of uses.
First, the cells are useful for producing 27875 metalloproteinase
proteins or polypeptides that can be further purified to produce
desired amounts of 27875 metalloproteinase protein or fragments.
Thus, host cells containing expression vectors are useful for
polypeptide production.
[0281] Host cells are also useful for conducting cell-based assays
involving 27875 metalloproteinase or 27875 metalloproteinase
fragments. Thus, a recombinant host cell expressing a native 27875
metalloproteinase is useful to assay for compounds that stimulate
or inhibit 27875 metalloproteinase function. This includes zinc or
peptide binding, gene expression at the level of transcription or
translation, and interaction with other cellular components.
[0282] Host cells are also useful for identifying 27875
metalloproteinase mutants in which these functions are affected. If
the mutants naturally occur and give rise to a pathology, host
cells containing the mutations are useful to assay compounds that
have a desired effect on the mutant 27875 metalloproteinase (for
example, stimulating or inhibiting function) which may not be
indicated by their effect on the native 27875
metalloproteinase.
[0283] Recombinant host cells are also useful for expressing the
chimeric polypeptides described herein to assess compounds that
activate or suppress activation by means of a heterologous domain,
segment, site, and the like, as disclosed herein.
[0284] Further, mutant 27875 metalloproteinases can be designed in
which one or more of the various functions is engineered to be
increased or decreased and used to augment or replace 27875
metalloproteinase proteins in an individual. Thus, host cells can
provide a therapeutic benefit by replacing an aberrant 27875
metalloproteinase or providing an aberrant 27875 metalloproteinase
that provides a therapeutic result. In one embodiment, the cells
provide 27875 metalloproteinases that are abnormally active.
[0285] In another embodiment, the cells provide 27875
metalloproteinases that are abnormally inactive. These 27875
metalloproteinases can compete with endogenous 27875
metalloproteinases in the individual.
[0286] In another embodiment, cells expressing 27875
metalloproteinases that cannot be activated, are introduced into an
individual in order to compete with endogenous 27875
metalloproteinases for zinc, glycan, or peptide. For example, in
the case in which excessive zinc is part of a treatment modality,
it may be necessary to effectively inactivate zinc at a specific
point in treatment. Providing cells that compete for the molecule,
but which cannot be affected by 27875 metalloproteinase activation
would be beneficial.
[0287] Homologously recombinant host cells can also be produced
that allow the in situ alteration of endogenous metalloproteinase
polynucleotide sequences in a host cell genome. The host cell
includes, but is not limited to, a stable cell line, cell in vivo,
or cloned microorganism. This technology is more fully described in
WO 93/09222, WO 91/12650, WO 91/06667, U.S. Pat. No. 5,272,071, and
U.S. Pat. No. 5,641,670. Briefly, specific polynucleotide sequences
corresponding to the metalloproteinase polynucleotides or sequences
proximal or distal to a metalloproteinase gene are allowed to
integrate into a host cell genome by homologous recombination where
expression of the gene can be affected. In one embodiment,
regulatory sequences are introduced that either increase or
decrease expression of an endogenous sequence. Accordingly, a
metalloproteinase protein can be produced in a cell not normally
producing it. Alternatively, increased expression of
metalloproteinase protein can be effected in a cell normally
producing the protein at a specific level. Further, expression can
be decreased or eliminated by introducing a specific regulatory
sequence. The regulatory sequence can be heterologous to the
metalloproteinase protein sequence or can be a homologous sequence
with a desired mutation that affects expression. Alternatively, the
entire gene can be deleted. The regulatory sequence can be specific
to the host cell or capable of functioning in more than one cell
type. Still further, specific mutations can be introduced into any
desired region of the gene to produce mutant metalloproteinase
proteins. Such mutations could be introduced, for example, into the
specific functional regions such as the peptide substrate-binding
site.
[0288] In one embodiment, the host cell can be a fertilized oocyte
or embryonic stem cell that can be used to produce a transgenic
animal containing the altered 27875 metalloproteinase gene.
Alternatively, the host cell can be a stem cell or other early
tissue precursor that gives rise to a specific subset of cells and
can be used to produce transgenic tissues in an animal. See also
Thomas et al., Cell 51:503 (1987) for a description of homologous
recombination vectors. The vector is introduced into an embryonic
stem cell line (e.g., by electroporation) and cells in which the
introduced gene has homologously recombined with the endogenous
27875 metalloproteinase gene is selected (see e.g., Li, E. et al.
(1992) Cell 69:915). The selected cells are then injected into a
blastocyst of an animal (e.g., a mouse) to form aggregation
chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,
Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted
into a suitable pseudopregnant female foster animal and the embryo
brought to term. Progeny harboring the homologously recombined DNA
in their germ cells can be used to breed animals in which all cells
of the animal contain the homologously recombined DNA by germline
transmission of the transgene. Methods for constructing homologous
recombination vectors and homologous recombinant animals are
described further in Bradley, A. (1991) Current Opinion in
Biotechnology 2:823-829 and in PCT International Publication Nos.
WO 90/11354; WO 91/01140; and WO 93/04169.
[0289] The genetically engineered host cells can be used to produce
non-human transgenic animals. A transgenic animal is preferably a
mammal, for example a rodent, such as a rat or mouse, in which one
or more of the cells of the animal include a transgene. A transgene
is exogenous DNA which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal in one or more cell types or tissues of the
transgenic animal. These animals are useful for studying the
function of an 27875 metalloproteinase protein and identifying and
evaluating modulators of 27875 metalloproteinase protein
activity.
[0290] Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[0291] In one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which 27875 metalloproteinase
polynucleotide sequences have been introduced.
[0292] A transgenic animal can be produced by introducing nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. Any of the 27875
metalloproteinase nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[0293] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the 27875
metalloproteinase protein to particular cells.
[0294] Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0295] In another embodiment, transgenic non-human animals can be
produced which contain selected systems, which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
PNAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. (1991)
Science 251:1351-1355. If a cre/loxP recombinase system is used to
regulate expression of the transgene, animals containing transgenes
encoding both the Cre recombinase and a selected protein is
required. Such animals can be provided through the construction of
"double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0296] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al. (1997) Nature 385:810-813 and PCT International Publication
Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic
cell, from the transgenic animal can be isolated and induced to
exit the growth cycle and enter G.sub.o phase. The quiescent cell
can then be fused, e.g., through the use of electrical pulses, to
an enucleated oocyte from an animal of the same species from which
the quiescent cell is isolated. The reconstructed oocyte is then
cultured such that it develops to morula or blastocyst and then
transferred to a pseudopregnant female foster animal. The offspring
born of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[0297] Transgenic animals containing recombinant cells that express
the polypeptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
affect binding or activation, may not be evident from in vitro
cell-free or cell-based assays. Accordingly, it is useful to
provide non-human transgenic animals to assay in vivo 27875
metalloproteinase function, including peptide interaction, the
effect of specific mutant 27875 metalloproteinases on 27875
metalloproteinase function and peptide interaction, and the effect
of chimeric 27875 metalloproteinases. It is also possible to assess
the effect of null mutations, that is mutations that substantially
or completely eliminate one or more 27875 metalloproteinase
functions.
[0298] In general, methods for producing transgenic animals include
introducing a nucleic acid sequence according to the present
invention, the nucleic acid sequence capable of expressing the
protein in a transgenic animal, into a cell in culture or in vivo.
When introduced in vivo, the nucleic acid is introduced into an
intact organism such that one or more cell types and, accordingly,
one or more tissue types, express the nucleic acid encoding the
protein. Alternatively, the nucleic acid can be introduced into
virtually all cells in an organism by transfecting a cell in
culture, such as an embryonic stem cell, as described herein for
the production of transgenic animals, and this cell can be used to
produce an entire transgenic organism. As described, in a further
embodiment, the host cell can be a fertilized oocyte. Such cells
are then allowed to develop in a female foster animal to produce
the transgenic organism.
Pharmaceutical Compositions
[0299] 27875 metalloproteinase nucleic acid molecules, proteins,
modulators of the protein, and antibodies (also referred to herein
as "active compounds") can be incorporated into pharmaceutical
compositions suitable for administration to a subject, e.g., a
human. Such compositions typically comprise the nucleic acid
molecule, protein, modulator, or antibody and a pharmaceutically
acceptable carrier.
[0300] The term "administer" is used in its broadest sense and
includes any method of introducing the compositions of the present
invention into a subject. This includes producing polypeptides or
polynucleotides in vivo by in vivo transcription or translation of
polynucleotides that have been exogenously introduced into a
subject. Thus, polypeptides or nucleic acids produced in the
subject from the exogenous compositions are encompassed in the term
"administer."
[0301] As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, such media can be used in the compositions of the
invention. Supplementary active compounds can also be incorporated
into the compositions. A pharmaceutical composition of the
invention is formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral
(e.g., inhalation), transdermal (topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampules, disposable
syringes or multiple dose vials made of glass or plastic.
[0302] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0303] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., an 27875 metalloproteinase
protein or anti-27875 metalloproteinase antibody) in the required
amount in an appropriate solvent with one or a combination of
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0304] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For oral administration, the agent can be
contained in enteric forms to survive the stomach or further coated
or mixed to be released in a particular region of the GI tract by
known methods. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules. Oral compositions can
also be prepared using a fluid carrier for use as a mouthwash,
wherein the compound in the fluid carrier is applied orally and
swished and expectorated or swallowed. Pharmaceutically compatible
binding agents, and/or adjuvant materials can be included as part
of the composition. The tablets, pills, capsules, troches and the
like can contain any of the following ingredients, or compounds of
a similar nature: a binder such as microcrystalline cellulose, gum
tragacanth or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0305] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser, which contains a suitable propellant, e.g., a gas
such as carbon dioxide, or a nebulizer.
[0306] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0307] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0308] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0309] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. "Dosage unit form" as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0310] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) PNAS
91:3054-3057). The pharmaceutical preparation of the gene therapy
vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. Alternatively, where the complete
gene delivery vector can be produced intact from recombinant cells,
e.g. retroviral vectors, the pharmaceutical preparation can include
one or more cells which produce the gene delivery system.
[0311] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
[0312] The skilled artisan will appreciate that certain factors may
influence the dosage required to effectively treat a subject,
including but not limited to the severity of the disease or
disorder, previous treatments, the general health and/or age of the
subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a protein,
polypeptide, or antibody can include a single treatment or,
preferably, can include a series of treatments. In a preferred
example, a subject is treated with antibody, protein, or
polypeptide in the range of between about 0.1 to 20 mg/kg body
weight, one time per week for between about 1 to 10 weeks,
preferably between 2 to 8 weeks, more preferably between about 3 to
7 weeks, and even more preferably for about 4, 5, or 6 weeks. It
will also be appreciated that the effective dosage of antibody,
protein, or polypeptide used for treatment may increase or decrease
over the course of a particular treatment. Changes in dosage may
result and become apparent from the results of diagnostic assays as
described herein.
[0313] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds.
[0314] It is understood that appropriate doses of small molecule
agents depends upon a number of factors within the ken of the
ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention. Exemplary doses
include milligram or microgram amounts of the small molecule per
kilogram of subject or sample weight (e.g., about 1 microgram per
kilogram to about 500 milligrams per kilogram, about 100 micrograms
per kilogram to about 5 milligrams per kilogram, or about 1
microgram per kilogram to about 50 micrograms per kilogram. It is
furthermore understood that appropriate doses of a small molecule
depend upon the potency of the small molecule with respect to the
expression or activity to be modulated. Such appropriate doses may
be determined using the assays described herein. When one or more
of these small molecules is to be administered to an animal (e.g.,
a human) in order to modulate expression or activity of a
polypeptide or nucleic acid of the invention, a physician,
veterinarian, or researcher may, for example, prescribe a
relatively low dose at first, subsequently increasing the dose
until an appropriate response is obtained. In addition, it is
understood that the specific dose level for any particular animal
subject will depend upon a variety of factors including the
activity of the specific compound employed, the age, body weight,
general health, gender, and diet of the subject, the time of
administration, the route of administration, the rate of excretion,
any drug combination, and the degree of expression or activity to
be modulated.
[0315] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0316] This invention may be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will fully convey the invention to those skilled in the
art. Many modifications and other embodiments of the invention will
come to mind in one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Although specific terms are employed, they
are used as in the art unless otherwise indicated.
[0317] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein.
II. 22025, A NOVEL HUMAN CYCLIC NUCLEOTIDE PHOSPHODIESTERASE
Background of the Invention
[0318] Cyclic nucleotide phosphodiesterases show specificity for
purine cyclic nucleotide substrates and catalyze cyclic AMP (cAMP)
and cyclic GMP (cGMP) hydrolysis (Thompson W. J. (1991) Pharma.
Ther. 51:13-33). Cyclic nucleotide phosphodiesterases regulate the
steady-state levels of cAMP and cGMP and modulate both the
amplitude and duration of cyclic nucleotide signal. At least eight
different but homologous gene families are currently known to exist
in mammalian tissues. Most families contain distinct genes, many of
which are expressed in different tissues as functionally unique
alternative splice variants. (Beavo (1995) Physiological Reviews
75:725-748 and U.S. Pat. No. 5,798,246).
[0319] All cyclic nucleotide phosphodiesterases contain a core of
about 270 conserved amino acids in the COOH-terminal half of the
protein thought to be the catalytic domain of the enzyme. The
cyclic nucleotide phosphodiesterases within each family display
about 65% amino acid homology and the similarity drops to less than
40% when compared between different families with most of the
similarity occurring in the catalytic domains.
[0320] Most cyclic nucleotide phosphodiesterase genes have more
than one alternatively spliced mRNA transcribed from them and in
many cases the alternative splicing appears to be highly tissue
specific, providing a mechanism for selective expression of
different cyclic nucleotide phosphodiesterases (Beavo supra).
Cell-type-specific expression suggests that the different isozymes
are likely to have different cell-type-specific properties.
[0321] Type 1 cyclic nucleotide phosphodiesterases are
Ca.sup.2+/calmodulin dependent, are reported to contain three
different genes, each of which appears to have at least two
different splice variants, and have been found in the lung, heart
and brain. Some of the calmodulin-dependent phosphodiesterases are
regulated in vitro by phosphorylation/dephosphorylation events. The
effect of phosphorylation is to decrease the affinity of the enzyme
for calmodulin, which decreases phosphodiesterase activity, thereby
increasing the steady state level of cAMP. Type 2 cyclic nucleotide
phosphodiesterases are cGMP stimulated, are localized in the brain
and are thought to mediate the effects of cAMP on catecholamine
secretion. Type 3 cyclic nucleotide phosphodiesterases are
cGMP-inhibited, have a high specificity for cAMP as a substrate,
and are one of the major phosphodiesterase isozymes present in
vascular smooth muscle and play a role in cardiac function. One
isozyme of type 3 is regulated by one or more insulin-dependent
kinases. Type 4 cyclic nucleotide phosphodiesterases are the
predominant isoenzyme in most inflammatory cells, with some of the
members being activated by cAMP-dependent phosphorylation. Type 5
cyclic nucleotide phosphodiesterases have traditionally been
thought of as regulators of cGMP function but may also affect cAMP
function. High levels of type 5 cyclic nucleotide
phosphodiesterases are found in most smooth muscle preparations,
platelets and kidney. Type 6 cyclic nucleotide phosphodiesterase
family members play a role in vision and are regulated by light and
cGMP. A Type 7 cyclic nucleotide phosphodiesterase family member is
found in high concentrations in skeletal muscle. A listing of
cyclic nucleotide phosphodiesterase families 1-7, their
localization and physiological role is given in Beavo supra. A Type
8 family is reported in U.S. Pat. No. 5,798,246.
[0322] Many functions of the immune and inflammatory responses are
inhibited by agents that increase intracellular levels of cAMP
(Verghese (1995) Mol. Pharmacol. 47:1164-1171) while the metabolism
of cGMP is involved in smooth muscle, lung and brain cell function
(Thompson W. (1991) Pharma. Ther. 51:13-33). A variety of diseases
have been attributed to increased cyclic nucleotide
phosphodiesterase activity which results in decreased levels of
cyclic nucleotides. For example, one form of diabetes insipidus in
the mouse has been associated with increased phosphodiesterase
Family 4 activity and an increase in low-K.sub.m cAMP
phosphodiesterase activity has been reported in leukocytes of
atopic patients. Defects in cyclic nucleotide phosphodiesterases
have also been associated with retinal disease. Retinal
degeneration in the rd mouse, human autosomal recessive retinitis
pigmentosa, and rod/cone dysplasia 1 in Irish setter dogs has been
attributed to mutations in the Family 6 phosphodiesterase, gene B.
Family 3 phosphodiesterase has been associated with cardiac
disease.
[0323] Many inhibitors of different cyclic nucleotide
phosphodiesterases have been identified and some have undergone
clinical evaluation. For example, Family 3 phosphodiesterase
inhibitors are being developed as antithrombotic agents, as
antihypertensive agents and as cardiotonic agents useful in the
treatment of congestive heart failure. Rolipram, a Family 4
phosphodiesterase inhibitor, has been used in the treatment of
depression and other inhibitors of Family 4 phosphodiesterase are
undergoing evaluation as anti-inflammatory agents. Rolipram has
also been shown to inhibit lipopolysaccharide (LPS) induced
TNF-alpha which has been shown to enhance HIV-1 replication in
vitro. Therefore, rolipram may inhibit HIV-1 replication (Angel et
al. (1995) AIDS 9:1137-44). Additionally, based on its ability to
suppress the production of TNF alpha and beta and interferon gamma,
rolipram has been shown to be effective in the treatment of
encephalomyelitis, the experimental animal model for multiple
sclerosis (Sommer et al. (1995) Nat. Med. 1:244-248) and may be
effective in the treatment of tardive dyskinesia (Sasaki et al.
(1995) Eur. J. Pharmacol. 282:72-76).
[0324] There are also nonspecific phosphodiesterase inhibitors such
as theophylline, used in the treatment of bronchial asthma and
other respiratory diseases, and pentoxifylline, used in the
treatment of intermittent claudication and diabetes-induced
peripheral vascular disease. Theophylline is thought to act on
airway smooth muscle function as well as in an anti-inflammatory or
immunomodulatory capacity in the treatment of respiratory diseases
(Banner et al. (1995) Eur. Respir. J 8:996-1000) where it is
thought to act by inhibiting both cyclic nucleotide
phosphodiesterase cAMP and cGMP hydrolysis (Banner et al. (1995)
Monaldi Arch. Chest Dis. 50:286-292). Pentoxifylline, also known to
block TNF-alpha production, may inhibit HIV-1 replication (Angel et
al. supra). A list of cyclic nucleotide phosphodiesterase
inhibitors is given in Beavo supra.
[0325] Cyclic nucleotide phosphodiesterases have also been reported
to affect cellular proliferation of a variety of cell types and
have been implicated in the treatment of various cancers. (Bang et
al. (1994) Proc. Natl. Acad. Sci. USA 91:5330-5334) reported that
the prostate carcinoma cell lines DU 145 and LNCaP were
growth-inhibited by delivery of cAMP derivatives and
phosphodiesterase inhibitors and observed a permanent conversion in
phenotype from epithelial to neuronal morphology; Matousovic et al.
((1995) J. Clin. Invest. 96:401-410) suggest that cyclic nucleotide
phosphodiesterase isozyme inhibitors have the potential to regulate
mesangial cell proliferation; Joulain et al. ((1995) J. Mediat.
Cell Signal 11:63-79) reports that cyclic nucleotide
phosphodiesterase has been shown to be an important target involved
in the control of lymphocyte proliferation; and Deonarain et al.
((1994) Brit. J. Cancer 70:786-94) suggest a tumor targeting
approach to cancer treatment that involves intracellular delivery
of phosphodiesterases to particular cellular compartments,
resulting in cell death.
[0326] Accordingly, cyclic nucleotide phosphodiesterases are a
major target for drug action and development. Accordingly, it is
valuable to the field of pharmaceutical development to identify and
characterize previously unknown phosphodiesterases. The present
invention advances the state of the art by providing a previously
unidentified human cyclic nucleotide phosphodiesterase.
Summary of Invention
[0327] It is an object of the invention to identify novel cyclic
nucleotide phosphodiesterases. It is a further object of the
invention to provide novel cyclic nucleotide phosphodiesterase
polypeptides that are useful as reagents or targets in
phosphodiesterase assays applicable to treatment and diagnosis of
cyclic nucleotide phosphodiesterase-mediated or -related
disorders.
[0328] It is a further object of the invention to provide
polynucleotides corresponding to the novel phosphodiesterase
polypeptides that are useful as targets and reagents in
phosphodiesterase assays applicable to treatment and diagnosis of
phosphodiesterase-mediated or -related disorders and useful for
producing novel phosphodiesterase polypeptides by recombinant
methods.
[0329] A specific object of the invention is to identify compounds
that act as agonists and antagonists and modulate the expression of
the novel phosphodiesterase.
[0330] A further specific object of the invention is to provide
compounds that modulate expression of the phosphodiesterase for
treatment and diagnosis of phosphodiesterase-related disorders.
[0331] The invention is thus based on the identification of a novel
human cyclic nucleotide phosphodiesterase. The invention
encompasses a long and short form of the phosphodiesterase. The
amino acid sequence of the longer form is shown in SEQ ID NO:4 and
the amino acid sequence of the shorter form is shown as SEQ ID
NO:6. The nucleotide sequence of the longer form is shown as SEQ ID
NO:3 and the nucleotide sequence of the shorter form is shown as
SEQ ID NO:5.
[0332] The invention also provides variant polypeptides having an
amino acid sequence that is substantially homologous to the amino
acid sequence shown in SEQ ID NO:4 or SEQ ID NO:6 or encoded by the
deposited cDNA.
[0333] The invention also provides variant nucleic acid sequences
that are substantially homologous to the nucleotide sequence shown
in SEQ ID NO:3 or SEQ ID NO:5 or in the deposited cDNA.
[0334] The invention also provides fragments of the polypeptide
shown in SEQ ID NO:4 or SEQ ID NO:6 and nucleotide sequence shown
in SEQ ID NO:3 or SEQ ID NO:5, as well as substantially homologous
fragments of the polypeptide or nucleic acid.
[0335] The invention further provides nucleic acid constructs
comprising the nucleic acid molecules described herein. In a
preferred embodiment, the nucleic acid molecules of the invention
are operatively linked to a regulatory sequence.
[0336] The invention also provides vectors and host cells for
expressing the phosphodiesterase nucleic acid molecules and
polypeptides, and particularly recombinant vectors and host
cells.
[0337] The invention also provides methods of making the vectors
and host cells and methods for using them to produce the
phosphodiesterase nucleic acid molecules and polypeptides.
[0338] The invention also provides antibodies or antigen-binding
fragments thereof that selectively bind the phosphodiesterase
polypeptides and fragments.
[0339] The invention also provides methods of screening for
compounds that modulate expression or activity of the
phosphodiesterase polypeptides or nucleic acid (RNA or DNA).
[0340] The invention also provides a process for modulating
phosphodiesterase polypeptide or nucleic acid expression or
activity, especially using the screened compounds. Modulation may
be used to treat conditions related to aberrant activity or
expression of the phosphodiesterase polypeptides or nucleic
acids.
[0341] The invention also provides assays for determining the
activity of or the presence or absence of the phosphodiesterase
polypeptides or nucleic acid molecules in a biological sample,
including for disease diagnosis.
[0342] The invention also provides assays for determining the
presence of a mutation in the polypeptides or nucleic acid
molecules, including for disease diagnosis.
[0343] In still a further embodiment, the invention provides a
computer readable means containing the nucleotide and/or amino acid
sequences of the nucleic acids and polypeptides of the invention,
respectively.
Detailed Description of the Invention
[0344] As used herein, a "signaling pathway" refers to the
modulation (e.g., stimulation or inhibition) of a cellular
function/activity upon the binding of a ligand to a receptor.
Examples of such functions include mobilization of intracellular
molecules that participate in a signal transduction pathway, e.g.,
phosphatidylinositol 4,5-bisphosphate (PIP.sub.2), inositol
1,4,5-triphosphate (IP.sub.3) and adenylate cyclase; polarization
of the plasma membrane; production or secretion of molecules;
alteration in the structure of a cellular component; cell
proliferation, e.g., synthesis of DNA; cell migration; cell
differentiation; and cell survival.
[0345] The response depends on the type of cell. In some cells,
binding of a ligand to the receptor may stimulate an activity such
as release of compounds, gating of a channel, cellular adhesion,
migration, differentiation, etc., through phosphatidylinositol or
cyclic AMP metabolism and turnover while in other cells, binding
will produce a different result.
[0346] A signaling pathway is the cAMP turnover pathway. As used
herein, "cyclic AMP turnover and metabolism" refers to the
molecules involved in the turnover and metabolism of cAMP as well
as to the activities of these molecules. Cyclic AMP is a second
messenger produced in response to ligand-induced stimulation of
certain receptors. In the cAMP signaling pathway, binding of a
ligand can lead to the activation of the enzyme adenyl cyclase,
which catalyzes the synthesis of cAMP. The newly synthesized cAMP
can in turn activate a cAMP-dependent protein kinase. This
activated kinase can phosphorylate a voltage-gated potassium
channel protein, or an associated protein, and lead to the
inability of the potassium channel to open during an action
potential. The inability of the potassium channel to open results
in a decrease in the outward flow of potassium, which normally
repolarizes the membrane of a neuron, leading to prolonged membrane
depolarization.
Polypeptides
[0347] The invention is based on the discovery of a novel human
cyclic nucleotide phosphodiesterase. Specifically, an expressed
sequence tag (EST) was selected based on homology to
phosphodiesterase sequences. This EST was used to design primers
based on sequences that it contains and used to identify a cDNA
from a kidney and adrenal gland cDNA library. Positive clones were
sequenced and the overlapping fragments were assembled. Analysis of
the assembled sequence revealed that the cloned cDNA molecule
encodes a cyclic nucleotide phosphodiesterase. Nucleic acid
encoding a truncated form of the enzyme was also isolated from an
osteoblast cDNA library.
[0348] Novel phosphodiesterase nucleotides and the deduced
polypeptides are described herein. The human 22025 (long) sequence
(SEQ ID NO:3), is approximately 2662 nucleotides long including
untranslated regions, which encodes a 508 amino acid protein (SEQ
ID NO:4). The human 22025 (short) sequence (SEQ ID NO:5), is
approximately 3336 nucleotides long including untranslated regions
which encodes a 320 amino acid protein (SEQ ID NO:6)
[0349] "Phosphodiesterase polypeptide" or "phosphodiesterase
protein" refers to the polypeptides in SEQ ID NO:4 or SEQ ID NO:6
or encoded by the deposited cDNAs. The term "phosphodiesterase
protein" or "phosphodiesterase polypeptide", however, further
includes the numerous variants described herein, as well as
fragments derived from the full-length phosphodiesterases and
variants.
[0350] Tissues and/or cells in which the phosphodiesterases are
found include, but are not limited to heart (including fetal),
ovary, brain, pancreas, kidneys, breast, liver, testis, prostate,
skeletal muscle, and osteoblasts. In addition, the
phosphodiesterases are expressed in diseased tissues, including but
limited to, those involved in congestive heart failure and breast
cancer. Expression has been confirmed by Northern blot analysis and
addition, in osteoblasts, by in situ hybridization.
[0351] The present invention thus provides an isolated or purified
phosphodiesterase polypeptide and variants and fragments
thereof.
[0352] The phosphodiesterases include a catalytic signature,
HDVDHPG, at residues 265-271.
[0353] Based on a BLAST search, highest homology was shown to
Family 7. The long form is designated B2 and the short form B1.
[0354] As used herein, a polypeptide is said to be "isolated" or
"purified" when it is substantially free of cellular material when
it is isolated from recombinant and non-recombinant cells, or free
of chemical precursors or other chemicals when it is chemically
synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell and
still be considered "isolated" or "purified."
[0355] The phosphodiesterase polypeptides can be purified to
homogeneity. It is understood, however, that preparations in which
the polypeptide is not purified to homogeneity are useful and
considered to contain an isolated form of the polypeptide. The
critical feature is that the preparation allows for the desired
function of the polypeptide, even in the presence of considerable
amounts of other components. Thus, the invention encompasses
various degrees of purity.
[0356] In one embodiment, the language "substantially free of
cellular material" includes preparations of the phosphodiesterase
having less than about 30% (by dry weight) other proteins (i.e.,
contaminating protein), less than about 20% other proteins, less
than about 10% other proteins, or less than about 5% other
proteins. When the polypeptide is recombinantly produced, it can
also be substantially free of culture medium, i.e., culture medium
represents less than about 20%, less than about 10%, or less than
about 5% of the volume of the protein preparation.
[0357] A phosphodiesterase polypeptide is also considered to be
isolated when it is part of a membrane preparation or is purified
and then reconstituted with membrane vesicles or liposomes.
[0358] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the phosphodiesterase
polypeptide in which it is separated from chemical precursors or
other chemicals that are involved in its synthesis. In one
embodiment, the language "substantially free of chemical precursors
or other chemicals" includes preparations of the polypeptide having
less than about 30% (by dry weight) chemical precursors or other
chemicals, less than about 20% chemical precursors or other
chemicals, less than about 10% chemical precursors or other
chemicals, or less than about 5% chemical precursors or other
chemicals.
[0359] In one embodiment, the phosphodiesterase polypeptide
comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID
NO:6. However, the invention also encompasses sequence variants.
Variants include a substantially homologous protein encoded by the
same genetic locus in an organism, i.e., an allelic variant. The
phosphodiesterase has been mapped to human chromosome 6
(6q21-q23.2), with flanking markers AFMA074ZG9 (2.6cR) and
AFM214ZF6 (7.9cR). Mutations near this locus include, but are not
limited to, the following: PPAC, arthropathy, progressive
pseudorheumatoid, of childhood; ODDD, oculodentodigital dysplasia;
heterocellular hereditary persistence of fetal hemoglobin; DFNA10,
deafness, autosomal dominant nonsyndromic sensorineural 10; CMD1F,
cardiomyopathy, dilated, 1F; and diabetes mellitus, transient
neonatal. In the mouse this locus is associated with the following:
gl, grey-lethal; dl, downless; Cat5, dominant cataract 5; Lwq3,
liver weight QTL 3; mshi, male sterility and histoincompatibility;
Mop2, morphine preference 2; H60, histocompatibility 60; Daq4,
directional asymmetry QTL 4; Daq5, directional asymmetry QTL 5; and
kd/kidney disease. Genes near this locus include PDNP1
(phosphodiesterase 1/nucleotide pyrophosphatase 1 (homologous to
mouseLy-41 antigen)), MACS, PTPRK, ARG1, PCMT1, DFNA10, MEKK5,
CTGF, SGK, HIVEP2, CMD1F, EPB41L2, HPFH, UTRN, IFNGR1, and
ESR1.
[0360] Variants also encompass proteins derived from other genetic
loci in an organism, but having substantial homology to the
phosphodiesterase of SEQ ID NO:4 or SEQ ID NO:6. Variants also
include proteins substantially homologous to the phosphodiesterase
but derived from another organism, i.e., an ortholog. Variants also
include proteins that are substantially homologous to the
phosphodiesterase that are produced by chemical synthesis. Variants
also include proteins that are substantially homologous to the
phosphodiesterase that are produced by recombinant methods. It is
understood, however, that variants exclude any amino acid sequences
disclosed prior to the invention.
[0361] As used herein, two proteins (or a region of the proteins)
are substantially homologous when the amino acid sequences are at
least about 70-75%, typically at least about 80-85%, and most
typically at least about 90-95% or more homologous. A substantially
homologous amino acid sequence, according to the present invention,
will be encoded by a nucleic acid sequence hybridizing to the
nucleic acid sequence, or portion thereof, of the sequence shown in
SEQ ID NO:3 or SEQ ID NO:5 under stringent conditions as more fully
described below.
[0362] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence (e.g., when aligning a second sequence to
the amino acid sequences herein having 502 amino acid residues, at
least 165, preferably at least 200, more preferably at least 250,
even more preferably at least 300, and even more preferably at
least 350, 400, 450, and 500 amino acid residues are aligned). The
amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position (as
used herein amino acid or nucleic acid "identity" is equivalent to
amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[0363] The invention also encompasses polypeptides having a lower
degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by the
phosphodiesterase. Similarity is determined by conserved amino acid
substitution. Such substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics. Conservative substitutions are likely to be
phenotypically silent. Typically seen as conservative substitutions
are the replacements, one for another, among the aliphatic amino
acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues
Ser and Thr, exchange of the acidic residues Asp and Glu,
substitution between the amide residues Asn and Gln, exchange of
the basic residues Lys and Arg and replacements among the aromatic
residues Phe, Tyr. Guidance concerning which amino acid changes are
likely to be phenotypically silent are found in Bowie et al.,
Science 247:1306-1310 (1990). TABLE-US-00003 TABLE 2 Conservative
Amino Acid Substitutions. Aromatic Phenylalanine Tryptophan
Tyrosine Hydrophobic Leucine Isoleucine Valine Polar Glutamine
Asparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid
Glutamic Acid Small Alanine Serine Threonine Methionine Glycine
[0364] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991).
[0365] A preferred, non-limiting example of such a mathematical
algorithm is described in Karlin et al. (1993) Proc. Natl. Acad.
Sci. USA 90:5873-5877. Such an algorithm is incorporated into the
NBLAST and XBLAST programs (version 2.0) as described in Altschul
et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., NBLAST) can be used. In one embodiment, parameters
for sequence comparison can be set at score=100, wordlength=12, or
can be varied (e.g., W=5 or W=20).
[0366] In a preferred embodiment, the percent identity between two
amino acid sequences is determined using the Needleman et al.
(1970) (J. Mol. Biol. 48:444-453) algorithm which has been
incorporated into the GAP program in the GCG software package,
using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap
weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,
3, 4, 5, or 6. In yet another preferred embodiment, the percent
identity between two nucleotide sequences is determined using the
GAP program in the GCG software package (Devereux et al. (1984)
Nucleic Acids Res. 12(1):387), using a NWSgapdna.CMP matrix and a
gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3,
4, 5, or 6.
[0367] Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the CGC sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis et
al. (1994) Comput. Appl. Biosci. 10:3-5; and FASTA described in
Pearson et al. (1988) PNAS 85:2444-8.
[0368] A variant polypeptide can differ in amino acid sequence by
one or more substitutions, deletions, insertions, inversions,
fusions, and truncations or a combination of any of these.
[0369] Variant polypeptides can be fully functional or can lack
function in one or more activities. Thus, in the present case,
variations can affect the function, for example, of one or more of
the regions corresponding to the conserved catalytic region,
carboxyterminal regulatory regions, aminoterminal regulatory
regions, aminoterminal targeting regions, regions involved in
membrane association, regions involved in enzyme activation, for
example, by phosphorylation, and regions involved in interaction
with components of other cyclic nucleotide (e.g., AMP,
GMP)-dependent signal transduction pathways.
[0370] Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. Functional variants can also contain
substitution of similar amino acids, which results in no change or
an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree.
[0371] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0372] As indicated, variants can be naturally-occurring or can be
made by recombinant means or chemical synthesis to provide useful
and novel characteristics for the phosphodiesterase polypeptide.
This includes preventing immunogenicity from pharmaceutical
formulations by preventing protein aggregation.
[0373] Useful variations further include alteration of catalytic
activity. For example, one embodiment involves a variation at the
binding site that results in binding but not hydrolysis, or slower
hydrolysis, of cAMP. A further useful variation at the same site
can result in altered affinity for cAMP. Useful variations also
include changes that provide for affinity for another cyclic
nucleotide. Another useful variation includes one that prevents
activation by protein kinase A. Another useful variation provides a
fusion protein in which one or more domains or subregions are
operationally fused to one or more domains or subregions from
another phosphodiesterase isoform or family.
[0374] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.
(1985) Science 244:1081-1085). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity,
such as cAMP hydrolysis in vitro or cAMP-dependent in vitro
activity, such as proliferative activity. Sites that are critical
for cAMP or protein kinase A binding can also be determined by
structural analysis such as crystallization, nuclear magnetic
resonance or photoaffinity labeling (Smith et al. (1992) J. Mol.
Biol. 224:899-904; de Vos et al. (1992) Science 255:306-312).
[0375] Substantial homology can be to the entire nucleic acid or
amino acid sequence or to fragments of these sequences.
[0376] The invention thus also includes polypeptide fragments of
the phosphodiesterase. Fragments can be derived from the amino acid
sequence shown in SEQ ID NO:4 or SEQ ID NO:6. However, the
invention also encompasses fragments of the variants of the
phosphodiesterases as described herein.
[0377] The fragments to which the invention pertains, however, are
not to be construed as encompassing fragments that may be disclosed
prior to the present invention.
[0378] Accordingly, a fragment can comprise at least about 10, 15,
20, 25, 30, 35, 40, 45, 50 or more contiguous amino acids.
Fragments can retain one or more of the biological activities of
the protein, for example the ability to bind to or hydrolyze cAMP,
as well as fragments that can be used as an immunogen to generate
phosphodiesterase antibodies.
[0379] Biologically active fragments (peptides which are, for
example, 5, 7, 10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100
or more amino acids in length) can comprise a domain or motif,
e.g., catalytic site, phosphodiesterase signature, and sites for
glycosylation, cAMP and cGMP-dependent protein kinase
phosphorylation, protein kinase C phosphorylation, casein kinase II
phosphorylation, tyrosine kinase phosphorylation, N-myristoylation,
amidation, and glycosaminoglycan attachment. Further possible
fragments include the catalytic site, an allosteric binding site,
sites important for cellular and subcellular targeting, sites
functional for interacting with components of other cAMP-dependent
signal transduction pathways, and aminoterminal and carboxyterminal
regulatory sites.
[0380] Such domains or motifs can be identified by means of routine
computerized homology searching procedures.
[0381] Fragments, for example, can extend in one or both directions
from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or
up to 100 amino acids. Further, fragments can include sub-fragments
of the specific domains mentioned above, which sub-fragments retain
the function of the domain from which they are derived.
[0382] These regions can be identified by well-known methods
involving computerized homology analysis.
[0383] The invention also provides fragments with immunogenic
properties. These contain an epitope-bearing portion of the
phosphodiesterase and variants. These epitope-bearing peptides are
useful to raise antibodies that bind specifically to a
phosphodiesterase polypeptide or region or fragment. These peptides
can contain at least 10, 12, at least 14, or between at least about
15 to about 30 amino acids.
[0384] Non-limiting examples of antigenic polypeptides that can be
used to generate antibodies include but are not limited to peptides
derived from an extracellular site. However, intracellularly-made
antibodies ("intrabodies") are also encompassed, which would
recognize intracellular peptide regions.
[0385] The epitope-bearing phosphodiesterase polypeptides may be
produced by any conventional means (Houghten, R. A. (1985) Proc.
Natl. Acad. Sci. USA 82:5131-5135). Simultaneous multiple peptide
synthesis is described in U.S. Pat. No. 4,631,211.
[0386] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the phosphodiesterase fragment and
an additional region fused to the carboxyl terminus of the
fragment.
[0387] The invention thus provides chimeric or fusion proteins.
These comprise a phosphodiesterase peptide sequence operatively
linked to a heterologous peptide having an amino acid sequence not
substantially homologous to the phosphodiesterase. "Operatively
linked" indicates that the phosphodiesterase peptide and the
heterologous peptide are fused in-frame. The heterologous peptide
can be fused to the N-terminus or C-terminus of the
phosphodiesterase or can be internally located.
[0388] In one embodiment the fusion protein does not affect
phosphodiesterase function per se. For example, the fusion protein
can be a GST-fusion protein in which the phosphodiesterase
sequences are fused to the C-terminus of the GST sequences. Other
types of fusion proteins include, but are not limited to, enzymatic
fusion proteins, for example beta-galactosidase fusions, yeast
two-hybrid GAL-4 fusions, poly-His fusions and Ig fusions. Such
fusion proteins, particularly poly-His fusions, can facilitate the
purification of recombinant phosphodiesterase. In certain host
cells (e.g., mammalian host cells), expression and/or secretion of
a protein can be increased by using a heterologous signal sequence.
Therefore, in another embodiment, the fusion protein contains a
heterologous signal sequence at its N-terminus.
[0389] EP-A-O 464 533 discloses fusion proteins comprising various
portions of immunoglobulin constant regions. The Fc is useful in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). In drug discovery, for
example, human proteins have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
(Bennett et al. (1995) J. Mol. Recog. 8:52-58 (1995) and Johanson
et al. J. Biol. Chem. 270:9459-9471). Thus, this invention also
encompasses soluble fusion proteins containing a phosphodiesterase
polypeptide and various portions of the constant regions of heavy
or light chains of immunoglobulins of various subclass (IgG, IgM,
IgA, IgE). Preferred as immunoglobulin is the constant part of the
heavy chain of human IgG, particularly IgG1, where fusion takes
place at the hinge region. For some uses it is desirable to remove
the Fc after the fusion protein has been used for its intended
purpose, for example when the fusion protein is to be used as
antigen for immunizations. In a particular embodiment, the Fc part
can be removed in a simple way by a cleavage sequence, which is
also incorporated and can be cleaved with factor Xa.
[0390] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see Ausubel et al. (1992)
Current Protocols in Molecular Biology). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A phosphodiesterase-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the phosphodiesterase.
[0391] Another form of fusion protein is one that directly affects
phosphodiesterase functions. Accordingly, a phosphodiesterase
polypeptide is encompassed by the present invention in which one or
more of the phosphodiesterase domains (or parts thereof) has been
replaced by homologous domains (or parts thereof) from another
Family 7 phosphodiesterase or other phosphodiesterase family.
Accordingly, various permutations are possible. For example, the
aminoterminal regulatory domain, or subregion thereof, can be
replaced with the domain or subregion from another Family 7 isoform
or phosphodiesterase family. As a further example, the catalytic
domain or parts thereof, can be replaced; the carboxyterminal
domain or subregion can be replaced. Thus, chimeric
phosphodiesterases can be formed in which one or more of the native
domains or subregions has been replaced by another.
[0392] Additionally, chimeric phosphodiesterase proteins can be
produced in which one or more functional sites is derived from a
different Family 7 isoform, or from another phosphodiesterase
family, such as 1-6 and 8. It is understood however that sites
could be derived from phosphodiesterase families that occur in the
mammalian genome but which have not yet been discovered or
characterized. Such sites include but are not limited to the
catalytic site, aminoterminal regulatory site, carboxyterminal
regulatory site, sites important for targeting to subcellular and
cellular locations, sites functional for interaction with
components of other cyclic AMP dependent signal transduction
pathways, protein kinase A phosphorylation sites, glycosylation
sites, and other functional sites disclosed herein.
[0393] The isolated phosphodiesterases can be purified from cells
that naturally express it, such as from heart (including fetal),
ovary, brain, pancreas, kidneys, breast, liver, testis, prostate,
skeletal muscle, and osteoblasts, among others, especially purified
from cells that have been altered to express it (recombinant), or
synthesized using known protein synthesis methods.
[0394] In one embodiment, the protein is produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding the
phosphodiesterase polypeptide is cloned into an expression vector,
the expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques. Polypeptides often contain amino
acids other than the 20 amino acids commonly referred to as the 20
naturally-occurring amino acids. Further, many amino acids,
including the terminal amino acids, may be modified by natural
processes, such as processing and other post-translational
modifications, or by chemical modification techniques well known in
the art. Common modifications that occur naturally in polypeptides
are described in basic texts, detailed monographs, and the research
literature, and they are well known to those of skill in the
art.
[0395] Accordingly, the polypeptides also encompass derivatives or
analogs in which a substituted amino acid residue is not one
encoded by the genetic code, in which a substituent group is
included, in which the mature polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence for purification of
the mature polypeptide or a pro-protein sequence.
[0396] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0397] Such modifications are well-known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd ed., T. E.
Creighton, W.H. Freeman and Company, New York (1993). Many detailed
reviews are available on this subject, such as by Wold, F.,
Posttranslational Covalent Modification of Proteins, B. C. Johnson,
Ed., Academic Press, New York 1-12 (1983); Seifter et al. (1990)
Meth. Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. N.Y.
Acad. Sci. 663:48-62).
[0398] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquitination, and they may be circular, with or without
branching, generally as a result of post-translation events,
including natural processing events and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translational natural processes and by synthetic methods.
[0399] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. Blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, is common in
naturally-occurring and synthetic polypeptides. For instance, the
aminoterminal residue of polypeptides made in E. coli, prior to
proteolytic processing, almost invariably will be
N-formylmethionine.
[0400] The modifications can be a function of how the protein is
made. For recombinant polypeptides, for example, the modifications
will be determined by the host cell posttranslational modification
capacity and the modification signals in the polypeptide amino acid
sequence. Accordingly, when glycosylation is desired, a polypeptide
should be expressed in a glycosylating host, generally a eukaryotic
cell. Insect cells often carry out the same posttranslational
glycosylations as mammalian cells and, for this reason, insect cell
expression systems have been developed to efficiently express
mammalian proteins having native patterns of glycosylation. Similar
considerations apply to other modifications.
[0401] The same type of modification may be present in the same or
varying degree at several sites in a given polypeptide. Also, a
given polypeptide may contain more than one type of
modification.
Polypeptide Uses
[0402] The protein sequences of the present invention can be used
as a "query sequence" to perform a search against public databases
to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to the nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the proteins of the invention. To obtain gapped
alignments for comparison purposes, Gapped BLAST can be utilized as
described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used.
[0403] The phosphodiesterase polypeptides are useful for producing
antibodies specific for the phosphodiesterase, regions, or
fragments. Regions having a high antigenicity index score are
described herein.
[0404] The phosphodiesterase polypeptides are useful for biological
assays related to phosphodiesterases, especially from Family 7.
Such assays involve any of the known phosphodiesterase functions or
activities or properties useful for diagnosis and treatment of
phosphodiesterase-related conditions.
[0405] The phosphodiesterase polypeptides are also useful in drug
screening assays, in cell-based or cell-free systems. Cell-based
systems can be native, i.e., cells that normally express the
phosphodiesterase, as a biopsy or expanded in cell culture. In one
embodiment, however, cell-based assays involve recombinant host
cells expressing the phosphodiesterase.
[0406] Determining the ability of the test compound to interact
with the phosphodiesterase can also comprise determining the
ability of the test compound to preferentially bind to the
polypeptide as compared to the ability of a known binding molecule
(e.g. cAMP) to bind to the polypeptide.
[0407] The polypeptides can be used to identify compounds that
modulate phosphodiesterase activity. Such compounds, for example,
can increase or decrease affinity or rate of binding to cAMP,
compete with cAMP for binding to the phosphodiesterase, or displace
cAMP bound to the phosphodiesterase. Both phosphodiesterase and
appropriate variants and fragments can be used in high-throughput
screens to assay candidate compounds for the ability to bind to the
phosphodiesterase. These compounds can be further screened against
a functional phosphodiesterase to determine the effect of the
compound on the phosphodiesterase activity. Compounds can be
identified that activate (agonist) or inactivate (antagonist) the
phosphodiesterase to a desired degree. Modulatory methods can be
performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g., by administering the agent to a
subject.
[0408] The phosphodiesterase polypeptides can be used to screen a
compound for the ability to stimulate or inhibit interaction
between the phosphodiesterase protein and a target molecule that
normally interacts with the phosphodiesterase protein. The target
can be a cyclic nucleotide or another component of the signal
pathway with which the phosphodiesterase protein normally interacts
(for example, protein kinase A or other interactor involved in cAMP
turnover). The assay includes the steps of combining the
phosphodiesterase protein with a candidate compound under
conditions that allow the phosphodiesterase protein or fragment to
interact with the target molecule, and to detect the formation of a
complex between the phosphodiesterase protein and the target or to
detect the biochemical consequence of the interaction with the
phosphodiesterase and the target, such as any of the associated
effects of signal transduction such as protein kinase A
phosphorylation, cAMP turnover, and biological endpoints of the
pathway.
[0409] Determining the ability of the phosphodiesterase to bind to
a target molecule can also be accomplished using a technology such
as real-time Bimolecular Interaction Analysis (BIA). Sjolander et
al. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr.
Opin. Struct. Biol. 5:699-705. As used herein, "BIA" is a
technology for studying biospecific interactions in real time,
without labeling any of the interactants (e.g., BIAcore.TM.).
Changes in the optical phenomenon surface plasmon resonance (SPR)
can be used as an indication of real-time reactions between
biological molecules.
[0410] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to polypeptide libraries, while the
other four approaches are applicable to polypeptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, K. S.
(1997) Anticancer Drug Des. 12:145).
[0411] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in DeWitt et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.
37:1233. Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 97:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra).
[0412] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al. (1991)
Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al. (1993) Cell 72:767-778); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0413] One candidate compound is a soluble full-length
phosphodiesterase or fragment that competes for cAMP binding. Other
candidate compounds include mutant phosphodiesterases or
appropriate fragments containing mutations that affect
phosphodiesterase function and thus compete for cAMP. Accordingly,
a fragment that competes for cAMP, for example with a higher
affinity, or a fragment that binds cAMP but does not degrade it, is
encompassed by the invention.
[0414] The invention provides other end points to identify
compounds that modulate (stimulate or inhibit) phosphodiesterase
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate phosphodiesterase
activity. Thus, the expression of genes that are up- or
down-regulated in response to the phosphodiesterase dependent
signal cascade can be assayed. In one embodiment, the regulatory
region of such genes can be operably linked to a marker that is
easily detectable, such as luciferase. Alternatively,
phosphorylation of the phosphodiesterase, or a phosphodiesterase
target, could also be measured.
[0415] Any of the biological or biochemical functions mediated by
the phosphodiesterase can be used as an endpoint assay. These
include all of the biochemical or biochemical/biological events
described herein, in the references cited herein, incorporated by
reference for these endpoint assay targets, and other functions
known to those of ordinary skill in the art.
[0416] In the case of the phosphodiesterase, specific end points
can include cAMP hydrolysis and a decrease in protein kinase A
activation.
[0417] Binding and/or activating compounds can also be screened by
using chimeric phosphodiesterase proteins in which one or more
domains, sites, and the like, as disclosed herein, or parts
thereof, can be replaced by their heterologous counterparts derived
from other Family 7 phosphodiesterases or from phosphodiesterase
isoforms of any other phosphodiesterase family. For example, a
catalytic region can be used that interacts with a different cyclic
nucleotide specificity and/or affinity than the native
phosphodiesterase. Accordingly, a different set of signal
transduction components is available as an end-point assay for
activation. Alternatively, a heterologous targeting sequence can
replace the native targeting sequence. This will result in
different subcellular or cellular localization and accordingly can
result in having an effect on a different signal transduction
pathway. Accordingly, a different set of signal transduction
components is available as an endpoint assay for activation. As a
further alternative, the site of modification by an effector
protein, for example phosphorylation by protein kinase A, can be
replaced with the site from a different effector protein. This
could also provide the use of a different signal transduction
pathway for endpoint determination. Activation can also be detected
by a reporter gene containing an easily detectable coding region
operably linked to a transcriptional regulatory sequence that is
part of the native signal transduction pathway.
[0418] The phosphodiesterase polypeptides are also useful in
competition binding assays in methods designed to discover
compounds that interact with the phosphodiesterase. Thus, a
compound is exposed to a phosphodiesterase polypeptide under
conditions that allow the compound to bind or to otherwise interact
with the polypeptide. Soluble phosphodiesterase polypeptide is also
added to the mixture. If the test compound interacts with the
soluble phosphodiesterase polypeptide, it decreases the amount of
complex formed or activity from the phosphodiesterase target. This
type of assay is particularly useful in cases in which compounds
are sought that interact with specific regions of the
phosphodiesterase. Thus, the soluble polypeptide that competes with
the target phosphodiesterase region is designed to contain peptide
sequences corresponding to the region of interest.
[0419] Another type of competition-binding assay can be used to
discover compounds that interact with specific functional sites. As
an example, protein kinase A and a candidate compound can be added
to a sample of the phosphodiesterase. Compounds that interact with
the phosphodiesterase at the same site as the protein kinase A will
reduce the amount of complex formed between the phosphodiesterase
and protein kinase A. Accordingly, it is possible to discover a
compound that specifically prevents interaction between the
phosphodiesterase and protein kinase A. Another example involves
adding a candidate compound to a sample of phosphodiesterase and
cAMP. A compound that competes with cAMP will reduce the amount of
hydrolysis or binding of the cAMP to the phosphodiesterase.
Accordingly, compounds can be discovered that directly interact
with the phosphodiesterase and compete with cAMP. Such assays can
involve any other component that interacts with the
phosphodiesterase.
[0420] To perform cell free drug screening assays, it is desirable
to immobilize either the phosphodiesterase, or fragment, or its
target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[0421] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example,
glutathione-S-transferase/phosphodiesterase fusion proteins can be
adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
Louis, Mo.) or glutathione derivatized microtitre plates, which are
then combined with the cell lysates (e.g., .sup.35S-labeled) and
the candidate compound, and the mixture incubated under conditions
conducive to complex formation (e.g., at physiological conditions
for salt and pH). Following incubation, the beads are washed to
remove any unbound label, and the matrix immobilized and radiolabel
determined directly, or in the supernatant after the complexes is
dissociated. Alternatively, the complexes can be dissociated from
the matrix, separated by SDS-PAGE, and the level of
phosphodiesterase-binding protein found in the bead fraction
quantitated from the gel using standard electrophoretic techniques.
For example, either the polypeptide or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin using
techniques well known in the art. Alternatively, antibodies
reactive with the protein but which do not interfere with binding
of the protein to its target molecule can be derivatized to the
wells of the plate, and the protein trapped in the wells by
antibody conjugation. Preparations of a phosphodiesterase-binding
target component, such as cAMP or protein kinase A, and a candidate
compound are incubated in the phosphodiesterase-presenting wells
and the amount of complex trapped in the well can be quantitated.
Methods for detecting such complexes, in addition to those
described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the
phosphodiesterase target molecule, or which are reactive with
phosphodiesterase and compete with the target molecule; as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the target molecule.
[0422] Modulators of phosphodiesterase activity identified
according to these drug screening assays can be used to treat a
subject with a disorder mediated by the phosphodiesterase pathway,
by treating cells that express the phosphodiesterase, such as
heart, ovary, brain, pancreas, kidneys, breast, liver, testis,
prostate, skeletal muscle, and osteoblast-containing tissue, such
as bone. These methods of treatment include the steps of
administering the modulators of phosphodiesterase activity in a
pharmaceutical composition as described herein, to a subject in
need of such treatment.
[0423] The phosphodiesterase is expressed in osteoblasts and is
involved in osteoblast differentiation. Accordingly, it is involved
in bone matrix deposition and thus, bone formation. As such, the
gene is particularly relevant for the treatment of disorders
involving bone tissue and particularly in osteoporosis.
[0424] Disorders in which the phosphodiesterase expression is
relevant include, but are not limited to, dementia, memory loss,
congestive heart failure, thrombosis, pulmonary hypertension,
glomerulonephritis, bipolar depression, bronchial asthma, atopic
diseases, autoimmune encephalomyelitis, organ transplantation, salt
retention in nephrotic syndrome, and erectile dysfunction.
[0425] The phosphodiesterases are also specifically involved in
heart disease, such as in congestive heart failure and breast
cancer.
[0426] The phosphodiesterase polypeptides are thus useful for
treating a phosphodiesterase-associated disorder characterized by
aberrant expression or activity of a phosphodiesterase. In one
embodiment, the method involves administering an agent (e.g., an
agent identified by a screening assay described herein), or
combination of agents that modulates (e.g., upregulates or
down-regulates) expression or activity of the protein. In another
embodiment, the method involves administering the phosphodiesterase
as therapy to compensate for reduced or aberrant expression or
activity of the protein.
[0427] Methods for treatment include but are not limited to the use
of soluble phosphodiesterase or fragments of the phosphodiesterase
protein that compete for cAMP or protein kinase A. These
phosphodiesterases or fragments can have a higher affinity for the
target so as to provide effective competition.
[0428] Stimulation of activity is desirable in situations in which
the protein is abnormally downregulated and/or in which increased
activity is likely to have a beneficial effect. Likewise,
inhibition of activity is desirable in situations in which the
protein is abnormally upregulated and/or in which decreased
activity is likely to have a beneficial effect. In one example of
such a situation, a subject has a disorder characterized by
aberrant development or cellular differentiation. In another
example, the subject has a proliferative disease (e.g., cancer) or
a disorder characterized by an aberrant hematopoietic response. In
another example, it is desirable to achieve tissue regeneration in
a subject (e.g., where a subject has undergone brain or spinal cord
injury and it is desirable to regenerate neuronal tissue in a
regulated manner).
[0429] In yet another aspect of the invention, the proteins of the
invention can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO
94/10300), to identify other proteins (captured proteins) which
bind to or interact with the proteins of the invention and modulate
their activity.
[0430] The phosphodiesterase polypeptides also are useful to
provide a target for diagnosing a disease or predisposition to
disease mediated by the phosphodiesterase, including, but not
limited to, diseases involving tissues in which the
phosphodiesterases are expressed as disclosed herein, and
particularly in osteoporosis, breast cancer, and congestive heart
failure. Accordingly, methods are provided for detecting the
presence, or levels of, the phosphodiesterase in a cell, tissue, or
organism. The method involves contacting a biological sample with a
compound capable of interacting with the phosphodiesterase such
that the interaction can be detected.
[0431] One agent for detecting phosphodiesterase is an antibody
capable of selectively binding to phosphodiesterase. A biological
sample includes tissues, cells and biological fluids isolated from
a subject, as well as tissues, cells and fluids present within a
subject.
[0432] The phosphodiesterase also provides a target for diagnosing
active disease, or predisposition to disease, in a patient having a
variant phosphodiesterase. Thus, phosphodiesterase can be isolated
from a biological sample and assayed for the presence of a genetic
mutation that results in an aberrant protein. This includes amino
acid substitution, deletion, insertion, rearrangement, (as the
result of aberrant splicing events), and inappropriate
post-translational modification. Analytic methods include altered
electrophoretic mobility, altered tryptic peptide digest, altered
phosphodiesterase activity in cell-based or cell-free assay,
alteration in cAMP binding or degradation, protein kinase A binding
or phosphorylation, or antibody-binding pattern, altered
isoelectric point, direct amino acid sequencing, and any other of
the known assay techniques useful for detecting mutations in a
protein in general or in a phosphodiesterase specifically.
[0433] In vitro techniques for detection of phosphodiesterase
include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. Alternatively, the
protein can be detected in vivo in a subject by introducing into
the subject a labeled anti-phosphodiesterase antibody. For example,
the antibody can be labeled with a radioactive marker whose
presence and location in a subject can be detected by standard
imaging techniques. Particularly useful are methods, which detect
the allelic variant of the phosphodiesterase expressed in a
subject, and methods, which detect fragments of the
phosphodiesterase in a sample.
[0434] The phosphodiesterase polypeptides are also useful in
pharmacogenomic analysis. Pharmacogenomics deal with clinically
significant hereditary variations in the response to drugs due to
altered drug disposition and abnormal action in affected persons.
See, e.g., Eichelbaum, M. (1996) Clin. Exp. Pharmacol. Physiol.
23(10-11):983-985, and Linder, M. W. (1997) Clin. Chem.
43(2):254-266. The clinical outcomes of these variations result in
severe toxicity of therapeutic drugs in certain individuals or
therapeutic failure of drugs in certain individuals as a result of
individual variation in metabolism. Thus, the genotype of the
individual can determine the way a therapeutic compound acts on the
body or the way the body metabolizes the compound. Further, the
activity of drug metabolizing enzymes affects both the intensity
and duration of drug action. Thus, the pharmacogenomics of the
individual permit the selection of effective compounds and
effective dosages of such compounds for prophylactic or therapeutic
treatment based on the individual's genotype. The discovery of
genetic polymorphisms in some drug metabolizing enzymes has
explained why some patients do not obtain the expected drug
effects, show an exaggerated drug effect, or experience serious
toxicity from standard drug dosages. Polymorphisms can be expressed
in the phenotype of the extensive metabolizer and the phenotype of
the poor metabolizer. Accordingly, genetic polymorphism may lead to
allelic protein variants of the phosphodiesterase in which one or
more of the phosphodiesterase functions in one population is
different from those in another population. The polypeptides thus
allow a target to ascertain a genetic predisposition that can
affect treatment modality. Thus, in a cAMP-based treatment,
polymorphism may give rise to catalytic regions that are more or
less active. Accordingly, dosage would necessarily be modified to
maximize the therapeutic effect within a given population
containing the polymorphism. As an alternative to genotyping,
specific polymorphic polypeptides could be identified.
[0435] The phosphodiesterase polypeptides are also useful for
monitoring therapeutic effects during clinical trials and other
treatment. Thus, the therapeutic effectiveness of an agent that is
designed to increase or decrease gene expression, protein levels or
phosphodiesterase activity can be monitored over the course of
treatment using the phosphodiesterase polypeptides as an end-point
target. The monitoring can be, for example, as follows: (i)
obtaining a pre-administration sample from a subject prior to
administration of the agent; (ii) detecting the level of expression
or activity of the protein in the pre-administration sample; (iii)
obtaining one or more post-administration samples from the subject;
(iv) detecting the level of expression or activity of the protein
in the post-administration samples; (v) comparing the level of
expression or activity of the protein in the pre-administration
sample with the protein in the post-administration sample or
samples; and (vi) increasing or decreasing the administration of
the agent to the subject accordingly.
Antibodies
[0436] The invention also provides antibodies that selectively bind
to the phosphodiesterase and its variants and fragments. An
antibody is considered to selectively bind, even if it also binds
to other proteins that are not substantially homologous with the
phosphodiesterase. These other proteins share homology with a
fragment or domain of the phosphodiesterase. This conservation in
specific regions gives rise to antibodies that bind to both
proteins by virtue of the homologous sequence. In this case, it
would be understood that antibody binding to the phosphodiesterase
is still selective.
[0437] To generate antibodies, an isolated phosphodiesterase
polypeptide is used as an immunogen to generate antibodies using
standard techniques for polyclonal and monoclonal antibody
preparation. Either the full-length protein or antigenic peptide
fragment can be used.
[0438] Antibodies are preferably prepared from these regions or
from discrete fragments in these regions. However, antibodies can
be prepared from any region of the peptide as described herein. A
preferred fragment produces an antibody that diminishes or
completely prevents cAMP hydrolysis or binding. Antibodies can be
developed against the entire phosphodiesterase or domains of the
phosphodiesterase as described herein. Antibodies can also be
developed against specific functional sites as disclosed
herein.
[0439] The antigenic peptide can comprise a contiguous sequence of
at least 12, 14, 15, or 30 amino acid residues. In one embodiment,
fragments correspond to regions that are located on the surface of
the protein, e.g., hydrophilic regions. These fragments are not to
be construed, however, as encompassing any fragments, which may be
disclosed prior to the invention.
[0440] Antibodies can be polyclonal or monoclonal. An intact
antibody, or a fragment thereof (e.g. Fab or F(ab').sub.2) can be
used.
[0441] Detection can be facilitated by coupling (i.e., physically
linking) the antibody to a detectable substance. Examples of
detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0442] An appropriate immunogenic preparation can be derived from
native, recombinantly expressed, or chemically synthesized
peptides.
Antibody Uses
[0443] The antibodies can be used to isolate a phosphodiesterase by
standard techniques, such as affinity chromatography or
immunoprecipitation. The antibodies can facilitate the purification
of the natural phosphodiesterase from cells and recombinantly
produced phosphodiesterase expressed in host cells.
[0444] The antibodies are useful to detect the presence of
phosphodiesterase in cells or tissues to determine the pattern of
expression of the phosphodiesterase among various tissues in an
organism and over the course of normal development.
[0445] The antibodies can be used to detect phosphodiesterase in
situ, in vitro, or in a cell lysate or supernatant in order to
evaluate the abundance and pattern of expression.
[0446] The antibodies can be used to assess abnormal tissue
distribution or abnormal expression during development.
[0447] Antibody detection of circulating fragments of the full
length phosphodiesterase can be used to identify phosphodiesterase
turnover.
[0448] Further, the antibodies can be used to assess
phosphodiesterase expression in disease states such as in active
stages of the disease or in an individual with a predisposition
toward disease related to phosphodiesterase function. When a
disorder is caused by an inappropriate tissue distribution,
developmental expression, or level of expression of the
phosphodiesterase protein, the antibody can be prepared against the
normal phosphodiesterase protein. If a disorder is characterized by
a specific mutation in the phosphodiesterase, antibodies specific
for this mutant protein can be used to assay for the presence of
the specific mutant phosphodiesterase. However,
intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular phosphodiesterase
peptide regions.
[0449] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Antibodies can be developed against the whole
phosphodiesterase or portions of the phosphodiesterase.
[0450] The diagnostic uses can be applied, not only in genetic
testing, but also in monitoring a treatment modality. Accordingly,
where treatment is ultimately aimed at correcting phosphodiesterase
expression level or the presence of aberrant phosphodiesterases and
aberrant tissue distribution or developmental expression,
antibodies directed against the phosphodiesterase or relevant
fragments can be used to monitor therapeutic efficacy.
[0451] Antibodies accordingly can be used diagnostically to monitor
protein levels in tissue as part of a clinical testing procedure,
e.g., to, for example, determine the efficacy of a given treatment
regimen.
[0452] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic
phosphodiesterase can be used to identify individuals that require
modified treatment modalities.
[0453] The antibodies are also useful as diagnostic tools as an
immunological marker for aberrant phosphodiesterase analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0454] The antibodies are also useful for tissue typing. Thus,
where a specific phosphodiesterase has been correlated with
expression in a specific tissue, antibodies that are specific for
this phosphodiesterase can be used to identify a tissue type.
[0455] The antibodies are also useful in forensic identification.
Accordingly, where an individual has been correlated with a
specific genetic polymorphism resulting in a specific polymorphic
protein, an antibody specific for the polymorphic protein can be
used as an aid in identification.
[0456] The antibodies are also useful for inhibiting
phosphodiesterase function, for example, blocking cAMP, protein
kinase A, or the catalytic site.
[0457] These uses can also be applied in a therapeutic context in
which treatment involves inhibiting phosphodiesterase function. An
antibody can be used, for example, to block cAMP binding.
Antibodies can be prepared against specific fragments containing
sites required for function or against intact phosphodiesterase
associated with a cell.
[0458] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. For an overview of this
technology for producing human antibodies, see Lonberg et al.
(1995) Int. Rev. Immunol. 13:65-93. For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, e.g., U.S.
Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No.
5,545,806.
[0459] The invention also encompasses kits for using antibodies to
detect the presence of a phosphodiesterase protein in a biological
sample. The kit can comprise antibodies such as a labeled or
labelable antibody and a compound or agent for detecting
phosphodiesterase in a biological sample; means for determining the
amount of phosphodiesterase in the sample; and means for comparing
the amount of phosphodiesterase in the sample with a standard. The
compound or agent can be packaged in a suitable container. The kit
can further comprise instructions for using the kit to detect
phosphodiesterase.
Polynucleotides
[0460] The nucleotide sequences in SEQ ID NO:3 or SEQ ID NO:5 were
obtained by sequencing the deposited human cDNA. Accordingly, the
sequence of the deposited clone is controlling as to any
discrepancies between the two and any reference to the sequences of
SEQ ID NO:3 or SEQ ID NO:5 includes reference to the sequences of
the deposited cDNA.
[0461] The specifically disclosed cDNAs comprise the coding region
and 5' and 3' untranslated sequences in SEQ ID NO:3 or SEQ ID
NO:5.
[0462] The invention provides isolated polynucleotides encoding the
novel phosphodiesterases. The term "phosphodiesterase
polynucleotide" or "phosphodiesterase nucleic acid" refers to the
sequences shown in SEQ ID NO:3 or SEQ ID NO:5 or in the deposited
cDNAs. The term "phosphodiesterase polynucleotide" or
"phosphodiesterase nucleic acid" further includes variants and
fragments of the phosphodiesterase polynucleotides.
[0463] An "isolated" phosphodiesterase nucleic acid is one that is
separated from other nucleic acid present in the natural source of
the phosphodiesterase nucleic acid. Preferably, an "isolated"
nucleic acid is free of sequences which naturally flank the
phosphodiesterase nucleic acid (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. However, there can be some
flanking nucleotide sequences, for example up to about 5 KB. The
important point is that the phosphodiesterase nucleic acid is
isolated from flanking sequences such that it can be subjected to
the specific manipulations described herein, such as recombinant
expression, preparation of probes and primers, and other uses
specific to the phosphodiesterase nucleic acid sequences.
[0464] Moreover, an "isolated" nucleic acid molecule, such as a
cDNA or RNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0465] In some instances, the isolated material will form part of a
composition (for example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0466] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0467] In some instances, the isolated material will form part of a
composition (or example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0468] The phosphodiesterase polynucleotides can encode the mature
protein plus additional amino or carboxyterminal amino acids, or
amino acids interior to the mature polypeptide (when the mature
form has more than one polypeptide chain, for instance). Such
sequences may play a role in processing of a protein from precursor
to a mature form, facilitate protein trafficking, prolong or
shorten protein half-life or facilitate manipulation of a protein
for assay or production, among other things. As generally is the
case in situ, the additional amino acids may be processed away from
the mature protein by cellular enzymes.
[0469] The phosphodiesterase polynucleotides include, but are not
limited to, the sequence encoding the mature polypeptide alone, the
sequence encoding the mature polypeptide and additional coding
sequences, such as a leader or secretory sequence (e.g., a pre-pro
or pro-protein sequence), the sequence encoding the mature
polypeptide, with or without the additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but non-translated
sequences that play a role in transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding
and stability of mRNA. In addition, the polynucleotide may be fused
to a marker sequence encoding, for example, a peptide that
facilitates purification.
[0470] Phosphodiesterase polynucleotides can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0471] Phosphodiesterase nucleic acid can comprise the nucleotide
sequences shown in SEQ ID NO:3 or SEQ ID NO:5, corresponding to
human osteoblast (short form) and kidney and adrenal gland (long
form) cDNA.
[0472] In one embodiment, the phosphodiesterase nucleic acid
comprises only the coding region.
[0473] The invention further provides variant phosphodiesterase
polynucleotides, and fragments thereof, that differ from the
nucleotide sequences shown in SEQ ID NO:3 or SEQ ID NO:5 due to
degeneracy of the genetic code and thus encode the same protein as
that encoded by the nucleotide sequences shown in SEQ ID NO:3 or
SEQ ID NO:5.
[0474] The invention also provides phosphodiesterase nucleic acid
molecules encoding the variant polypeptides described herein. Such
polynucleotides may be naturally occurring, such as allelic
variants (same locus), homologs (different locus), and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Such non-naturally occurring
variants may be made by mutagenesis techniques, including those
applied to polynucleotides, cells, or organisms. Accordingly, as
discussed above, the variants can contain nucleotide substitutions,
deletions, inversions and insertions.
[0475] Typically, variants have a substantial identity with a
nucleic acid molecules of SEQ ID NO:3 or SEQ ID NO:5 and the
complements thereof. Variation can occur in either or both the
coding and non-coding regions. The variations can produce both
conservative and non-conservative amino acid substitutions.
[0476] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. These variants comprise a
nucleotide sequence encoding a phosphodiesterase that is at least
about 60-65%, 65-70%, typically at least about 70-75%, more
typically at least about 80-85%, and most typically at least about
90-95% or more homologous to the nucleotide sequence shown in SEQ
ID NO:3 or SEQ ID NO:5 or a fragment of this sequence. Such nucleic
acid molecules can readily be identified as being able to hybridize
under stringent conditions, to the nucleotide sequence shown in SEQ
ID NO:3 or SEQ ID NO:5 or a fragment of the sequence. It is
understood that stringent hybridization does not indicate
substantial homology where it is due to general homology, such as
poly A sequences, or sequences common to all or most proteins, all
cyclic nucleotide phosphodiesterases, or all Family 7
phosphodiesterases. Moreover, it is understood that variants do not
include any of the nucleic acid sequences that may have been
disclosed prior to the invention.
[0477] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a polypeptide
at least about 60-65% homologous to each other typically remain
hybridized to each other. The conditions can be such that sequences
at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 90%, at least about 95% or more
identical to each other remain hybridized to one another. Such
stringent conditions are known to those skilled in the art and can
be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6, incorporated by reference.
One example of stringent hybridization conditions are hybridization
in 6.times. sodium chloride/sodium citrate (SSC) at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 50-65.degree. C. In another non-limiting example,
nucleic acid molecules are allowed to hybridize in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more low stringency washes in 0.2.times.SSC/0.1% SDS at room
temperature, or by one or more moderate stringency washes in
0.2.times.SSC/0.1% SDS at 42.degree. C., or washed in
0.2.times.SSC/0.1% SDS at 65.degree. C. for high stringency. In one
embodiment, an isolated nucleic acid molecule that hybridizes under
stringent conditions to the sequence of SEQ ID NO:3 or SEQ ID NO:5
corresponds to a naturally-occurring nucleic acid molecule. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein).
[0478] As understood by those of ordinary skill, the exact
conditions can be determined empirically and depend on ionic
strength, temperature and the concentration of destabilizing agents
such as formamide or denaturing agents such as SDS. Other factors
considered in determining the desired hybridization conditions
include the length of the nucleic acid sequences, base composition,
percent mismatch between the hybridizing sequences and the
frequency of occurrence of subsets of the sequences within other
non-identical sequences. Thus, equivalent conditions can be
determined by varying one or more of these parameters while
maintaining a similar degree of identity or similarity between the
two nucleic acid molecules.
[0479] The present invention also provides isolated nucleic acids
that contain a single or double stranded fragment or portion that
hybridizes under stringent conditions to the nucleotide sequence of
SEQ ID NO:3 or SEQ ID NO:5 or the complement of SEQ ID NO:3 or SEQ
ID NO:5. In one embodiment, the nucleic acid consists of a portion
of the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:5 and the
complement of SEQ ID NO:3 or SEQ ID NO:5. The nucleic acid
fragments of the invention are at least about 15, preferably at
least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50,
100, 200, 500 or more nucleotides in length. Longer fragments, for
example, 30 or more nucleotides in length, which encode antigenic
proteins or polypeptides described herein are useful.
[0480] Furthermore, the invention provides polynucleotides that
comprise a fragment of the full-length phosphodiesterase
polynucleotides. The fragment can be single or double-stranded and
can comprise DNA or RNA. The fragment can be derived from either
the coding or the non-coding sequence.
[0481] In another embodiment an isolated phosphodiesterase nucleic
acid encodes the entire coding region. In another embodiment the
isolated phosphodiesterase nucleic acid encodes a sequence
corresponding to the mature protein that may be from about amino
acid 6 to the last amino acid. Other fragments include nucleotide
sequences encoding the amino acid fragments described herein.
[0482] Thus, phosphodiesterase nucleic acid fragments further
include sequences corresponding to the domains described herein,
subregions also described, and specific functional sites.
Phosphodiesterase nucleic acid fragments also include combinations
of the domains, segments, and other functional sites described
above. A person of ordinary skill in the art would be aware of the
many permutations that are possible.
[0483] Where the location of the domains or sites have been
predicted by computer analysis, one of ordinary sill would
appreciate that the amino acid residues constituting these domains
can vary depending on the criteria used to define the domains.
[0484] However, it is understood that a phosphodiesterase fragment
includes any nucleic acid sequence that does not include the entire
gene.
[0485] The invention also provides phosphodiesterase nucleic acid
fragments that encode epitope bearing regions of the
phosphodiesterase proteins described herein.
[0486] Nucleic acid fragments, according to the present invention,
are not to be construed as encompassing those fragments that may
have been disclosed prior to the invention.
Polynucleotide Uses
[0487] The nucleotide sequences of the present invention can be
used as a "query sequence" to perform a search against public
databases, for example, to identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol.
Biol. 215:403-10. BLAST protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to the proteins of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al. (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used.
[0488] The nucleic acid fragments of the invention provide probes
or primers in assays such as those described below. "Probes" are
oligonucleotides that hybridize in a base-specific manner to a
complementary strand of nucleic acid. Such probes include
polypeptide nucleic acids, as described in Nielsen et al. (1991)
Science 254:1497-1500. Typically, a probe comprises a region of
nucleotide sequence that hybridizes under highly stringent
conditions to at least about 15, typically about 20-25, and more
typically about 40, 50 or 75 consecutive nucleotides of the nucleic
acid sequence shown in SEQ ID NO:3 or SEQ ID NO:5 and the
complements thereof. More typically, the probe further comprises a
label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme
co-factor.
[0489] As used herein, the term "primer" refers to a
single-stranded oligonucleotide which acts as a point of initiation
of template-directed DNA synthesis using well-known methods (e.g.,
PCR, LCR) including, but not limited to those described herein. The
appropriate length of the primer depends on the particular use, but
typically ranges from about 15 to 30 nucleotides. The term "primer
site" refers to the area of the target DNA to which a primer
hybridizes. The term "primer pair" refers to a set of primers
including a 5' (upstream) primer that hybridizes with the 5' end of
the nucleic acid sequence to be amplified and a 3' (downstream)
primer that hybridizes with the complement of the sequence to be
amplified.
[0490] The phosphodiesterase polynucleotides are thus useful for
probes, primers, and in biological assays.
[0491] Where the polynucleotides are used to assess
phosphodiesterase properties or functions, such as in the assays
described herein, all or less than all of the entire cDNA can be
useful. Assays specifically directed to phosphodiesterase
functions, such as assessing agonist or antagonist activity,
encompass the use of known fragments. Further, diagnostic methods
for assessing phosphodiesterase function can also be practiced with
any fragment, including those fragments that may have been known
prior to the invention. Similarly, in methods involving treatment
of phosphodiesterase dysfunction, all fragments are encompassed
including those, which may have been known in the art.
[0492] The phosphodiesterase polynucleotides are useful as a
hybridization probe for cDNA and genomic DNA to isolate a
full-length cDNA and genomic clones encoding the polypeptides
described in SEQ ID NO:4 or SEQ ID NO:6 and to isolate cDNA and
genomic clones that correspond to variants producing the same
polypeptides shown in SEQ ID NO:3 or SEQ ID NO:5 or the other
variants described herein. Variants can be isolated from the same
tissue and organism from which the polypeptides shown in SEQ ID
NO:4 or SEQ ID NO:6 were isolated, different tissues from the same
organism, or from different organisms. This method is useful for
isolating genes and cDNA that are developmentally-controlled and
therefore may be expressed in the same tissue or different tissues
at different points in the development of an organism.
[0493] The probe can correspond to any sequence along the entire
length of the gene encoding the phosphodiesterase. Accordingly, it
could be derived from 5' noncoding regions, the coding region, and
3' noncoding regions.
[0494] The nucleic acid probe can be, for example, the full-length
cDNA of SEQ ID NO:3 or SEQ ID NO:5, or a fragment thereof, such as
an oligonucleotide of at least 12, 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to mRNA or DNA.
[0495] Fragments of the polynucleotides described herein are also
useful to synthesize larger fragments or full-length
polynucleotides described herein. For example, a fragment can be
hybridized to any portion of an mRNA and a larger or full-length
cDNA can be produced.
[0496] The fragments are also useful to synthesize antisense
molecules of desired length and sequence.
[0497] Antisense nucleic acids of the invention can be designed
using the nucleotide sequences of SEQ ID NO:3 or SEQ ID NO:5, and
constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. Examples of modified nucleotides which can
be used to generate the antisense nucleic acid include
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, 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-isopentenyladenine,
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. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest).
[0498] Additionally, the nucleic acid molecules of the invention
can be modified at the base moiety, sugar moiety or phosphate
backbone to improve, e.g., the stability, hybridization, or
solubility of the molecule. For example, the deoxyribose phosphate
backbone of the nucleic acids can be modified to generate peptide
nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4:5). As used herein, the terms "peptide nucleic acids"
or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which
the deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93:14670. PNAs can be further modified, e.g., to
enhance their stability, specificity or cellular uptake, by
attaching lipophilic or other helper groups to PNA, by the
formation of PNA-DNA chimeras, or by the use of liposomes or other
techniques of drug delivery known in the art. The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63, Mag et
al. (1989) Nucleic Acids Res. 17:5973, and Peterser et al. (1975)
Bioorganic Med. Chem. Lett. 5:1119.
[0499] The nucleic acid molecules and fragments of the invention
can also include other appended groups such as peptides (e.g., for
targeting host cell phosphodiesterases in vivo), or agents
facilitating transport across the cell membrane (see, e.g.,
Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;
Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT
Publication No. WO 88/0918) or the blood brain barrier (see, e.g.,
PCT Publication No. WO 89/10134). In addition, oligonucleotides can
be modified with hybridization-triggered cleavage agents (see,
e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating
agents (see, e.g., Zon (1988) Pharm Res. 5:539-549).
[0500] The phosphodiesterase polynucleotides are also useful as
primers for PCR to amplify any given region of a phosphodiesterase
polynucleotide.
[0501] The phosphodiesterase polynucleotides are also useful for
constructing recombinant vectors. Such vectors include expression
vectors that express a portion of, or all of, the phosphodiesterase
polypeptides. Vectors also include insertion vectors, used to
integrate into another polynucleotide sequence, such as into the
cellular genome, to alter in situ expression of phosphodiesterase
genes and gene products. For example, an endogenous
phosphodiesterase coding sequence can be replaced via homologous
recombination with all or part of the coding region containing one
or more specifically introduced mutations.
[0502] The phosphodiesterase polynucleotides are also useful for
expressing antigenic portions of the phosphodiesterase
proteins.
[0503] The phosphodiesterase polynucleotides are also useful as
probes for determining the chromosomal positions of the
phosphodiesterase polynucleotides by means of in situ hybridization
methods, such as FISH. (For a review of this technique, see Verma
et al. (1988) Human Chromosomes: A Manual of Basic
Techniques(Pergamon Press, New York), and PCR mapping of somatic
cell hybrids. The mapping of the sequences to chromosomes is an
important first step in correlating these sequences with genes
associated with disease.
[0504] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0505] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland et al. ((1987) Nature 325:783-787).
[0506] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
a specified gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or
translocations, that are visible from chromosome spreads, or
detectable using PCR based on that DNA sequence. Ultimately,
complete sequencing of genes from several individuals can be
performed to confirm the presence of a mutation and to distinguish
mutations from polymorphisms.
[0507] The phosphodiesterase polynucleotide probes are also useful
to determine patterns of the presence of the gene encoding the
phosphodiesterases and their variants with respect to tissue
distribution, for example, whether gene duplication has occurred
and whether the duplication occurs in all or only a subset of
tissues. The genes can be naturally occurring or can have been
introduced into a cell, tissue, or organism exogenously.
[0508] The phosphodiesterase polynucleotides are also useful for
designing ribozymes corresponding to all, or a part, of the mRNA
produced from genes encoding the polynucleotides described
herein.
[0509] The phosphodiesterase polynucleotides are also useful for
constructing host cells expressing a part, or all, of the
phosphodiesterase polynucleotides and polypeptides.
[0510] The phosphodiesterase polynucleotides are also useful for
constructing transgenic animals expressing all, or a part, of the
phosphodiesterase polynucleotides and polypeptides.
[0511] The phosphodiesterase polynucleotides are also useful for
making vectors that express part, or all, of the phosphodiesterase
polypeptides.
[0512] The phosphodiesterase polynucleotides are also useful as
hybridization probes for determining the level of phosphodiesterase
nucleic acid expression. Accordingly, the probes can be used to
detect the presence of, or to determine levels of,
phosphodiesterase nucleic acid in cells, tissues, and in organisms.
The nucleic acid whose level is determined can be DNA or RNA.
Accordingly, probes corresponding to the polypeptides described
herein can be used to assess gene copy number in a given cell,
tissue, or organism. This is particularly relevant in cases in
which there has been an amplification of the phosphodiesterase
genes.
[0513] Alternatively, the probe can be used in an in situ
hybridization context to assess the position of extra copies of the
phosphodiesterase genes, as on extrachromosomal elements or as
integrated into chromosomes in which the phosphodiesterase gene is
not normally found, for example as a homogeneously staining
region.
[0514] These uses are relevant for diagnosis of disorders involving
an increase or decrease in phosphodiesterase expression relative to
normal, such as a proliferative disorder, a differentiative or
developmental disorder, or a hematopoietic disorder.
[0515] The phosphodiesterases are expressed in osteoblasts and are
involved in osteoblast differentiation. Accordingly, they are
involved in bone matrix deposition and thus, bone formation. As
such, the gene is particularly relevant for the treatment of
disorders involving bone tissue and particularly in
osteoporosis.
[0516] The phosphodiesterases are also specifically involved in
heart disease, such as congestive heart failure, and in breast
cancer.
[0517] Disorders in which phosphodiesterase expression is relevant
also include, but are not limited to, dementia, memory loss,
congestive heart failure, thrombosis, pulmonary hypertesion,
glomerutonephritis, bipolar depression, bronchial asthma, atopic
diseases, autoimmune enceptholomyelitis, organ transplantation,
salt retention in nephrotic syndrome, and erectile dysfunction.
[0518] Thus, the present invention provides a method for
identifying a disease or disorder associated with aberrant
expression or activity of phosphodiesterase nucleic acid, in which
a test sample is obtained from a subject and nucleic acid (e.g.,
mRNA, genomic DNA) is detected, wherein the presence of the nucleic
acid is diagnostic for a subject having or at risk of developing a
disease or disorder associated with aberrant expression or activity
of the nucleic acid.
[0519] One aspect of the invention relates to diagnostic assays for
determining nucleic acid expression as well as activity in the
context of a biological sample (e.g., blood, serum, cells, tissue)
to determine whether an individual has a disease or disorder, or is
at risk of developing a disease or disorder, associated with
aberrant nucleic acid expression or activity. Such assays can be
used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with expression or activity
of the nucleic acid molecules.
[0520] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[0521] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express the phosphodiesterase,
such as by measuring the level of a phosphodiesterase-encoding
nucleic acid in a sample of cells from a subject e.g., mRNA or
genomic DNA, or determining if the phosphodiesterase gene has been
mutated.
[0522] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate phosphodiesterase nucleic acid
expression (e.g., antisense, polypeptides, peptidomimetics, small
molecules or other drugs). A cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of the mRNA in the presence of the candidate compound is
compared to the level of expression of the mRNA in the absence of
the candidate compound. The candidate compound can then be
identified as a modulator of nucleic acid expression based on this
comparison and be used, for example to treat a disorder
characterized by aberrant nucleic acid expression. The modulator
can bind to the nucleic acid or indirectly modulate expression,
such as by interacting with other cellular components that affect
nucleic acid expression.
[0523] Modulatory methods can be performed in vitro (e.g., by
culturing the cell with the agent) or, alternatively, in vivo
(e.g., by administering the gent to a subject) in patients or in
transgenic animals.
[0524] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the phosphodiesterase gene. The method
typically includes assaying the ability of the compound to modulate
the expression of the phosphodiesterase nucleic acid and thus
identifying a compound that can be used to treat a disorder
characterized by undesired phosphodiesterase nucleic acid
expression.
[0525] The assays can be performed in cell-based and cell-free
systems. Cell-based assays include cells naturally expressing the
phosphodiesterase nucleic acid or recombinant cells genetically
engineered to express specific nucleic acid sequences.
[0526] Alternatively, candidate compounds can be assayed in vivo in
patients or in transgenic animals.
[0527] The assay for phosphodiesterase nucleic acid expression can
involve direct assay of nucleic acid levels, such as mRNA levels,
or on collateral compounds involved in the signal pathway (such as
cyclic AMP turnover). Further, the expression of genes that are up-
or down-regulated in response to the phosphodiesterase signal
pathway can also be assayed. In this embodiment the regulatory
regions of these genes can be operably linked to a reporter gene
such as luciferase.
[0528] Thus, modulators of phosphodiesterase gene expression can be
identified in a method wherein a cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of phosphodiesterase mRNA in the presence of the
candidate compound is compared to the level of expression of
phosphodiesterase mRNA in the absence of the candidate compound.
The candidate compound can then be identified as a modulator of
nucleic acid expression based on this comparison and be used, for
example to treat a disorder characterized by aberrant nucleic acid
expression. When expression of mRNA is statistically significantly
greater in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of
nucleic acid expression. When nucleic acid expression is
statistically significantly less in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of nucleic acid expression.
[0529] Accordingly, the invention provides methods of treatment,
with the nucleic acid as a target, using a compound identified
through drug screening as a gene modulator to modulate
phosphodiesterase nucleic acid expression. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or effects on nucleic acid activity
(e.g. when nucleic acid is mutated or improperly modified).
Treatment is of disorders characterized by aberrant expression or
activity of the nucleic acid.
[0530] The gene is particularly relevant for the treatment of
disorders involving bone tissue and particularly in osteoporosis.
The gene is also involved in heart disease, such as congestive
heart failure, and in breast cancer. Further disorders in which
expression is relevant include, but are not limited to, dementia,
memory loss, congestive heart failure, thrombosis, pulmonary
hypertesion, glomerulonephritis, bipolar depression, bronchial
asthma, atopic diseases, autoimmune encephalomyelitis, organ
transplantation, salt retention in nephrotic syndrome, and erectile
dysfunction.
[0531] Alternatively, a modulator for phosphodiesterase nucleic
acid expression can be a small molecule or drug identified using
the screening assays described herein as long as the drug or small
molecule inhibits the phosphodiesterase nucleic acid
expression.
[0532] The phosphodiesterase polynucleotides are also useful for
monitoring the effectiveness of modulating compounds on the
expression or activity of the phosphodiesterase gene in clinical
trials or in a treatment regimen. Thus, the gene expression pattern
can serve as a barometer for the continuing effectiveness of
treatment with the compound, particularly with compounds to which a
patient can develop resistance. The gene expression pattern can
also serve as a marker indicative of a physiological response of
the affected cells to the compound. Accordingly, such monitoring
would allow either increased administration of the compound or the
administration of alternative compounds to which the patient has
not become resistant. Similarly, if the level of nucleic acid
expression falls below a desirable level, administration of the
compound could be commensurately decreased.
[0533] Monitoring can be, for example, as follows: (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of a specified
mRNA or genomic DNA of the invention in the pre-administration
sample; (iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression or
activity of the mRNA or genomic DNA in the post-administration
samples; (v) comparing the level of expression or activity of the
mRNA or genomic DNA in the pre-administration sample with the mRNA
or genomic DNA in the post-administration sample or samples; and
(vi) increasing or decreasing the administration of the agent to
the subject accordingly.
[0534] The phosphodiesterase polynucleotides are also useful in
diagnostic assays for qualitative changes in phosphodiesterase
nucleic acid, and particularly in qualitative changes that lead to
pathology. The polynucleotides can be used to detect mutations in
phosphodiesterase genes and gene expression products such as mRNA.
The polynucleotides can be used as hybridization probes to detect
naturally-occurring genetic mutations in the phosphodiesterase gene
and thereby to determine whether a subject with the mutation is at
risk for a disorder caused by the mutation. Mutations include
deletion, addition, or substitution of one or more nucleotides in
the gene, chromosomal rearrangement, such as inversion or
transposition, modification of genomic DNA, such as aberrant
methylation patterns or changes in gene copy number, such as
amplification. Detection of a mutated form of the phosphodiesterase
gene associated with a dysfunction provides a diagnostic tool for
an active disease or susceptibility to disease when the disease
results from overexpression, underexpression, or altered expression
of a phosphodiesterase.
[0535] Mutations in the phosphodiesterase gene can be detected at
the nucleic acid level by a variety of techniques. Genomic DNA can
be analyzed directly or can be amplified by using PCR prior to
analysis. RNA or cDNA can be used in the same way.
[0536] In certain embodiments, detection of the mutation involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) PNAS 91:360-364), the latter of which
can be particularly useful for detecting point mutations in the
gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682).
This method can include the steps of collecting a sample of cells
from a patient, isolating nucleic acid (e.g., genomic, mRNA or
both) from the cells of the sample, contacting the nucleic acid
sample with one or more primers which specifically hybridize to a
gene under conditions such that hybridization and amplification of
the gene (if present) occurs, and detecting the presence or absence
of an amplification product, or detecting the size of the
amplification product and comparing the length to a control sample.
Deletions and insertions can be detected by a change in size of the
amplified product compared to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to normal RNA or
antisense DNA sequences.
[0537] It is anticipated that PCR and/or LCR may be desirable to
use as a preliminary amplification step in conjunction with any of
the techniques used for detecting mutations described herein.
[0538] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi 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.
[0539] Alternatively, mutations in a phosphodiesterase gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[0540] Further, sequence-specific ribozymes (U.S. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
[0541] Perfectly matched sequences can be distinguished from
mismatched sequences by nuclease cleavage digestion assays or by
differences in melting temperature.
[0542] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method.
[0543] Furthermore, sequence differences between a mutant
phosphodiesterase gene and a wild-type gene can be determined by
direct DNA sequencing. A variety of automated sequencing procedures
can be utilized when performing the diagnostic assays ((1995)
Biotechniques 19:448), including sequencing by mass spectrometry
(see, e.g., PCT International Publication No. WO 94/16101; Cohen et
al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993)
Appl. Biochem. Biotechnol. 38:147-159).
[0544] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.
(1985) Science 230:1242); Cotton et al. (1988) PNAS 85:4397;
Saleeba et al. (1992) Meth. Enzymol. 217:286-295), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al. (1989) PNAS 86:2766; Cotton et al. (1993) Mutat. Res.
285:125-144; and Hayashi et al. (1992) Genet. Anal. Tech. Appl.
9:73-79), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al. (1985)
Nature 313:495). The sensitivity of the assay may be enhanced by
using RNA (rather than DNA), in which the secondary structure is
more sensitive to a change in sequence. In one embodiment, the
subject method utilizes heteroduplex analysis to separate double
stranded heteroduplex molecules on the basis of changes in
electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
Examples of other techniques for detecting point mutations include,
selective oligonucleotide hybridization, selective amplification,
and selective primer extension.
[0545] In other embodiments, genetic mutations can be identified by
hybridizing a sample and control nucleic acids, e.g., DNA or RNA,
to high density arrays containing hundreds or thousands of
oligonucleotide probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two dimensional
arrays containing light-generated DNA probes as described in Cronin
et al. supra. Briefly, a first hybridization array of probes can be
used to scan through long stretches of DNA in a sample and control
to identify base changes between the sequences by making linear
arrays of sequential overlapping probes. This step allows the
identification of point mutations. This step is followed by a
second hybridization array that allows the characterization of
specific mutations by using smaller, specialized probe arrays
complementary to all variants or mutations detected. Each mutation
array is composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant gene.
[0546] The phosphodiesterase polynucleotides are also useful for
testing an individual for a genotype that while not necessarily
causing the disease, nevertheless affects the treatment modality.
Thus, the polynucleotides can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship). In the
present case, for example, a mutation in the phosphodiesterase gene
that results in altered affinity for cAMP could result in an
excessive or decreased drug effect with standard concentrations of
cAMP that activates the phosphodiesterase. Accordingly, the
phosphodiesterase polynucleotides described herein can be used to
assess the mutation content of the gene in an individual in order
to select an appropriate compound or dosage regimen for
treatment.
[0547] Thus polynucleotides displaying genetic variations that
affect treatment provide a diagnostic target that can be used to
tailor treatment in an individual. Accordingly, the production of
recombinant cells and animals containing these polymorphisms allow
effective clinical design of treatment compounds and dosage
regimens.
[0548] The methods can involve obtaining a control biological
sample from a control subject, contacting the control sample with a
compound or agent capable of detecting mRNA, or genomic DNA, such
that the presence of mRNA or genomic DNA is detected in the
biological sample, and comparing the presence of mRNA or genomic
DNA in the control sample with the presence of mRNA or genomic DNA
in the test sample.
[0549] The phosphodiesterase polynucleotides are also useful for
chromosome identification when the sequence is identified with an
individual chromosome and to a particular location on the
chromosome. First, the DNA sequence is matched to the chromosome by
in situ or other chromosome-specific hybridization. Sequences can
also be correlated to specific chromosomes by preparing PCR primers
that can be used for PCR screening of somatic cell hybrids
containing individual chromosomes from the desired species. Only
hybrids containing the chromosome containing the gene homologous to
the primer will yield an amplified fragment. Sublocalization can be
achieved using chromosomal fragments. Other strategies include
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to chromosome-specific libraries. Further mapping
strategies include fluorescence in situ hybridization, which allows
hybridization with probes shorter than those traditionally used.
Reagents for chromosome mapping can be used individually to mark a
single chromosome or a single site on the chromosome, or panels of
reagents can be used for marking multiple sites and/or multiple
chromosomes. Reagents corresponding to noncoding regions of the
genes actually are preferred for mapping purposes. Coding sequences
are more likely to be conserved within gene families, thus
increasing the chance of cross hybridizations during chromosomal
mapping.
[0550] The phosphodiesterase polynucleotides can also be used to
identify individuals from small biological samples. This can be
done for example using restriction fragment-length polymorphism
(RFLP) to identify an individual. Thus, the polynucleotides
described herein are useful as DNA markers for RFLP (See U.S. Pat.
No. 5,272,057).
[0551] Furthermore, the phosphodiesterase sequence can be used to
provide an alternative technique, which determines the actual DNA
sequence of selected fragments in the genome of an individual.
Thus, the phosphodiesterase sequences described herein can be used
to prepare two PCR primers from the 5' and 3' ends of the
sequences. These primers can then be used to amplify DNA from an
individual for subsequent sequencing.
[0552] Panels of corresponding DNA sequences from individuals
prepared in this manner can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences. It is estimated that allelic variation in humans
occurs with a frequency of about once per each 500 bases. Allelic
variation occurs to some degree in the coding regions of these
sequences, and to a greater degree in the noncoding regions. The
phosphodiesterase sequences can be used to obtain such
identification sequences from individuals and from tissue. The
sequences represent unique fragments of the human genome. Each of
the sequences described herein can, to some degree, be used as a
standard against which DNA from an individual can be compared for
identification purposes.
[0553] If a panel of reagents from the sequences is used to
generate a unique identification database for an individual, those
same reagents can later be used to identify tissue from that
individual. Using the unique identification database, positive
identification of the individual, living or dead, can be made from
extremely small tissue samples.
[0554] The phosphodiesterase polynucleotides can also be used in
forensic identification procedures. PCR technology can be used to
amplify DNA sequences taken from very small biological samples,
such as a single hair follicle, body fluids (e.g. blood, saliva, or
semen). The amplified sequence can then be compared to a standard
allowing identification of the origin of the sample.
[0555] The phosphodiesterase polynucleotides can thus be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As described
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to the
noncoding region are particularly useful since greater polymorphism
occurs in the noncoding regions, making it easier to differentiate
individuals using this technique.
[0556] The phosphodiesterase polynucleotides can further be used to
provide polynucleotide reagents, e.g., labeled or labelable probes
which can be used in, for example, an in situ hybridization
technique, to identify a specific tissue. This is useful in cases
in which a forensic pathologist is presented with a tissue of
unknown origin. Panels of phosphodiesterase probes can be used to
identify tissue by species and/or by organ type.
[0557] In a similar fashion, these primers and probes can be used
to screen tissue culture for contamination (i.e. screen for the
presence of a mixture of different types of cells in a
culture).
[0558] Alternatively, the phosphodiesterase polynucleotides can be
used directly to block transcription or translation of
phosphodiesterase gene sequences by means of antisense or ribozyme
constructs. Thus, in a disorder characterized by abnormally high or
undesirable phosphodiesterase gene expression, nucleic acids can be
directly used for treatment.
[0559] The phosphodiesterase polynucleotides are thus useful as
antisense constructs to control phosphodiesterase gene expression
in cells, tissues, and organisms. A DNA antisense polynucleotide is
designed to be complementary to a region of the gene involved in
transcription, preventing transcription and hence production of
phosphodiesterase protein. An antisense RNA or DNA polynucleotide
would hybridize to the mRNA and thus block translation of mRNA into
phosphodiesterase protein.
[0560] Examples of antisense molecules useful to inhibit nucleic
acid expression include antisense molecules complementary to a
fragment of the 5' untranslated region of SEQ ID NO:3 or SEQ ID
NO:5 which also includes the start codon and antisense molecules
which are complementary to a fragment of the 3' untranslated region
of SEQ ID NO:3 or SEQ ID NO:5.
[0561] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of
phosphodiesterase nucleic acid. Accordingly, these molecules can
treat a disorder characterized by abnormal or undesired
phosphodiesterase nucleic acid expression. This technique involves
cleavage by means of ribozymes containing nucleotide sequences
complementary to one or more regions in the mRNA that attenuate the
ability of the mRNA to be translated. Possible regions include
coding regions and particularly coding regions corresponding to the
catalytic and other functional activities of the phosphodiesterase
protein.
[0562] The phosphodiesterase polynucleotides also provide vectors
for gene therapy in patients containing cells that are aberrant in
phosphodiesterase gene expression. Thus, recombinant cells, which
include the patient's cells that have been engineered ex vivo and
returned to the patient, are introduced into an individual where
the cells produce the desired phosphodiesterase protein to treat
the individual.
[0563] The invention also encompasses kits for detecting the
presence of a phosphodiesterase nucleic acid in a biological
sample. For example, the kit can comprise reagents such as a
labeled or labelable nucleic acid or agent capable of detecting
phosphodiesterase nucleic acid in a biological sample; means for
determining the amount of phosphodiesterase nucleic acid in the
sample; and means for comparing the amount of phosphodiesterase
nucleic acid in the sample with a standard. The compound or agent
can be packaged in a suitable container. The kit can further
comprise instructions for using the kit to detect phosphodiesterase
mRNA or DNA.
Computer Readable Means
[0564] The nucleotide or amino acid sequences of the invention are
also provided in a variety of mediums to facilitate use thereof. As
used herein, "provided" refers to a manufacture, other than an
isolated nucleic acid or amino acid molecule, which contains a
nucleotide or amino acid sequence of the present invention. Such a
manufacture provides the nucleotide or amino acid sequences, or a
subset thereof (e.g., a subset of open reading frames (ORFs)) in a
form which allows a skilled artisan to examine the manufacture
using means not directly applicable to examining the nucleotide or
amino acid sequences, or a subset thereof, as they exists in nature
or in purified form.
[0565] In one application of this embodiment, a nucleotide or amino
acid sequence of the present invention can be recorded on computer
readable media. As used herein, "computer readable media" refers to
any medium that can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media,
such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. The skilled artisan will readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide or amino acid sequence
of the present invention.
[0566] As used herein, "recorded" refers to a process for storing
information on computer readable medium. The skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide or amino acid sequence information of the
present invention.
[0567] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of data processor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0568] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[0569] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[0570] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[0571] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software includes, but is not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[0572] For example, software which implements the BLAST (Altschul
et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al.
(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system
can be used to identify open reading frames (ORFs) of the sequences
of the invention which contain homology to ORFs or proteins from
other libraries. Such ORFs are protein encoding fragments and are
useful in producing commercially important proteins such as enzymes
used in various reactions and in the production of commercially
useful metabolites.
Vectors/Host Cells
[0573] The invention also provides vectors containing the
phosphodiesterase polynucleotides. The term "vector" refers to a
vehicle, preferably a nucleic acid molecule that can transport the
phosphodiesterase polynucleotides. When the vector is a nucleic
acid molecule, the phosphodiesterase polynucleotides are covalently
linked to the vector nucleic acid. With this aspect of the
invention, the vector includes a plasmid, single or double stranded
phage, a single or double stranded RNA or DNA viral vector, or
artificial chromosome, such as a BAC, PAC, YAC, OR MAC.
[0574] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the phosphodiesterase polynucleotides.
Alternatively, the vector may integrate into the host cell genome
and produce additional copies of the phosphodiesterase
polynucleotides when the host cell replicates.
[0575] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
phosphodiesterase polynucleotides. The vectors can function in
procaryotic or eukaryotic cells or in both (shuttle vectors).
[0576] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the phosphodiesterase
polynucleotides such that transcription of the polynucleotides is
allowed in a host cell. The polynucleotides can be introduced into
the host cell with a separate polynucleotide capable of affecting
transcription. Thus, the second polynucleotide may provide a
trans-acting factor interacting with the cis-regulatory control
region to allow transcription of the phosphodiesterase
polynucleotides from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself.
[0577] It is understood, however, that in some embodiments,
transcription and/or translation of the phosphodiesterase
polynucleotides can occur in a cell-free system.
[0578] The regulatory sequence to which the polynucleotides
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lamda., the lac, TRP, and
TAC promoters from E. coli, the early and late promoters from SV40,
the CMV immediate early promoter, the adenovirus early and late
promoters, and retrovirus long-terminal repeats.
[0579] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0580] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[0581] A variety of expression vectors can be used to express a
phosphodiesterase polynucleotide. Such vectors include chromosomal,
episomal, and virus-derived vectors, for example vectors derived
from bacterial plasmids, from bacteriophage, from yeast episomes,
from yeast chromosomal elements, including yeast artificial
chromosomes, from viruses such as baculoviruses, papovaviruses such
as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies
viruses, and retroviruses. Vectors may also be derived from
combinations of these sources such as those derived from plasmid
and bacteriophage genetic elements, e.g. cosmids and phagemids.
Appropriate cloning and expression vectors for prokaryotic and
eukaryotic hosts are described in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.
[0582] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0583] The phosphodiesterase polynucleotides can be inserted into
the vector nucleic acid by well-known methodology. Generally, the
DNA sequence that will ultimately be expressed is joined to an
expression vector by cleaving the DNA sequence and the expression
vector with one or more restriction enzymes and then ligating the
fragments together. Procedures for restriction enzyme digestion and
ligation are well known to those of ordinary skill in the art.
[0584] The vector containing the appropriate polynucleotide can be
introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0585] As described herein, it may be desirable to express the
polypeptide as a fusion protein. Accordingly, the invention
provides fusion vectors that allow for the production of the
phosphodiesterase polypeptides. Fusion vectors can increase the
expression of a recombinant protein, increase the solubility of the
recombinant protein, and aid in the purification of the protein by
acting for example as a ligand for affinity purification. A
proteolytic cleavage site may be introduced at the junction of the
fusion moiety so that the desired polypeptide can ultimately be
separated from the fusion moiety. Proteolytic enzymes include, but
are not limited to, factor Xa, thrombin, and enterokinase. Typical
fusion expression vectors include pGEX (Smith et al. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein. Examples of suitable inducible
non-fusion E. coli expression vectors include pTrc (Amann et al.
(1988) Gene 69:301-315) and pET 11d (Studier et al. (1990) Gene
Expression Technology: Methods in Enzymology 185:60-89).
[0586] Recombinant protein expression can be maximized in a host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S. (1990) Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. 119-128).
Alternatively, the sequence of the polynucleotide of interest can
be altered to provide preferential codon usage for a specific host
cell, for example E. coli. (Wada et al. (1992) Nucleic Acids Res.
20:2111-2118).
[0587] The phosphodiesterase polynucleotides can also be expressed
by expression vectors that are operative in yeast. Examples of
vectors for expression in yeast e.g., S. cerevisiae include
pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan et
al. (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene
54:113-123), and pYES2 (Invitrogen Corporation, San Diego,
Calif.).
[0588] The phosphodiesterase polynucleotides can also be expressed
in insect cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL
series (Lucklow et al. (1989) Virology 170:31-39).
[0589] In certain embodiments of the invention, the polynucleotides
described herein are expressed in mammalian cells using mammalian
expression vectors. Examples of mammalian expression vectors
include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman
et al. (1987) EMBO J. 6:187-195).
[0590] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
phosphodiesterase polynucleotides. The person of ordinary skill in
the art would be aware of other vectors suitable for maintenance
propagation or expression of the polynucleotides described herein.
These are found for example in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[0591] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the
polynucleotide sequences described herein, including both coding
and non-coding regions. Expression of this antisense RNA is subject
to each of the parameters described above in relation to expression
of the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0592] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0593] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).
[0594] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the phosphodiesterase polynucleotides can be
introduced either alone or with other polynucleotides that are not
related to the phosphodiesterase polynucleotides such as those
providing trans-acting factors for expression vectors. When more
than one vector is introduced into a cell, the vectors can be
introduced independently, co-introduced or joined to the
phosphodiesterase polynucleotide vector.
[0595] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0596] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the polynucleotides described herein or
may be on a separate vector. Markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0597] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0598] Where secretion of the polypeptide is desired, appropriate
secretion signals are incorporated into the vector. The signal
sequence can be endogenous to the phosphodiesterase polypeptides or
heterologous to these polypeptides.
[0599] Where the polypeptide is not secreted into the medium, the
protein can be isolated from the host cell by standard disruption
procedures, including freeze thaw, sonication, mechanical
disruption, use of lysing agents and the like. The polypeptide can
then be recovered and purified by well-known purification methods
including ammonium sulfate precipitation, acid extraction, anion or
cationic exchange chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0600] It is also understood that depending upon the host cell in
recombinant production of the polypeptides described herein, the
polypeptides can have various glycosylation patterns, depending
upon the cell, or maybe non-glycosylated as when produced in
bacteria. In addition, the polypeptides may include an initial
modified methionine in some cases as a result of a host-mediated
process.
Uses of Vectors and Host Cells
[0601] It is understood that "host cells" and "recombinant host
cells" refer not only to the particular subject cell but also to
the progeny or potential progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term as used herein.
[0602] The host cells expressing the polypeptides described herein,
and particularly recombinant host cells, have a variety of uses.
First, the cells are useful for producing phosphodiesterase
proteins or polypeptides that can be further purified to produce
desired amounts of phosphodiesterase protein or fragments. Thus,
host cells containing expression vectors are useful for polypeptide
production.
[0603] Host cells are also useful for conducting cell-based assays
involving the phosphodiesterase or phosphodiesterase fragments.
Thus, a recombinant host cell expressing a native phosphodiesterase
is useful to assay for compounds that stimulate or inhibit
phosphodiesterase function. This includes cAMP binding, gene
expression at the level of transcription or translation, protein
kinase A interaction, and components of the signal transduction
pathway.
[0604] Host cells are also useful for identifying phosphodiesterase
mutants in which these functions are affected. If the mutants
naturally occur and give rise to a pathology, host cells containing
the mutations are useful to assay compounds that have a desired
effect on the mutant phosphodiesterase (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native phosphodiesterase.
[0605] Recombinant host cells are also useful for expressing the
chimeric polypeptides described herein to assess compounds that
activate or suppress activation by means of a heterologous domain,
segment, site, and the like, as disclosed herein.
[0606] Further, mutant phosphodiesterases can be designed in which
one or more of the various functions is engineered to be increased
or decreased (e.g., cAMP binding or kinase A binding) and used to
augment or replace phosphodiesterase proteins in an individual.
Thus, host cells can provide a therapeutic benefit by replacing an
aberrant phosphodiesterase or providing an aberrant
phosphodiesterase that provides a therapeutic result. In one
embodiment, the cells provide phosphodiesterases that are
abnormally active.
[0607] In another embodiment, the cells provide phosphodiesterases
that are abnormally inactive. These phosphodiesterases can compete
with endogenous phosphodiesterases in the individual.
[0608] In another embodiment, cells expressing phosphodiesterases
that cannot be activated, are introduced into an individual in
order to compete with endogenous phosphodiesterases for cAMP. For
example, in the case in which excessive cAMP is part of a treatment
modality, it may be necessary to inactivate this molecule at a
specific point in treatment. Providing cells that compete for the
molecule, but which cannot be affected by phosphodiesterase
activation would be beneficial.
[0609] Homologously recombinant host cells can also be produced
that allow the in situ alteration of endogenous phosphodiesterase
polynucleotide sequences in a host cell genome. This technology is
more fully described in WO 93/09222, WO 91/12650 and U.S. Pat. No.
5,641,670. Briefly, specific polynucleotide sequences corresponding
to the phosphodiesterase polynucleotides or sequences proximal or
distal to a phosphodiesterase gene are allowed to integrate into a
host cell genome by homologous recombination where expression of
the gene can be affected. In one embodiment, regulatory sequences
are introduced that either increase or decrease expression of an
endogenous sequence. Accordingly, a phosphodiesterase protein can
be produced in a cell not normally producing it, or increased
expression of phosphodiesterase protein can result in a cell
normally producing the protein at a specific level. Alternatively,
the entire gene can be deleted. Still further, specific mutations
can be introduced into any desired region of the gene to produce
mutant phosphodiesterase proteins. Such mutations could be
introduced, for example, into the specific regions disclosed
herein.
[0610] In one embodiment, the host cell can be a fertilized oocyte
or embryonic stem cell that can be used to produce a transgenic
animal containing the altered phosphodiesterase gene.
Alternatively, the host cell can be a stem cell or other early
tissue precursor that gives rise to a specific subset of cells and
can be used to produce transgenic tissues in an animal. See also
Thomas et al., Cell 51:503 (1987) for a description of homologous
recombination vectors. The vector is introduced into an embryonic
stem cell line (e.g., by electroporation) and cells in which the
introduced gene has homologously recombined with the endogenous
phosphodiesterase gene is selected (see e.g., Li, E. et al. (1992)
Cell 69:915). The selected cells are then injected into a
blastocyst of an animal (e.g., a mouse) to form aggregation
chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,
Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted
into a suitable pseudopregnant female foster animal and the embryo
brought to term. Progeny harboring the homologously recombined DNA
in their germ cells can be used to breed animals in which all cells
of the animal contain the homologously recombined DNA by germline
transmission of the transgene. Methods for constructing homologous
recombination vectors and homologous recombinant animals are
described further in Bradley, A. (1991) Current Opinion in
Biotechnology 2:823-829 and in PCT International Publication Nos.
WO 90/11354; WO 91/01140; and WO 93/04169.
[0611] The genetically engineered host cells can be used to produce
non-human transgenic animals. A transgenic animal is preferably a
mammal, for example a rodent, such as a rat or mouse, in which one
or more of the cells of the animal include a transgene. A transgene
is exogenous DNA which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal in one or more cell types or tissues of the
transgenic animal. These animals are useful for studying the
function of a phosphodiesterase protein and identifying and
evaluating modulators of phosphodiesterase protein activity.
[0612] Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[0613] In one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which phosphodiesterase polynucleotide
sequences have been introduced.
[0614] A transgenic animal can be produced by introducing nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. Any of the
phosphodiesterase nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[0615] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
phosphodiesterase protein to particular cells.
[0616] Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0617] In another embodiment, transgenic non-human animals can be
produced which contain selected systems, which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
PNAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. (1991)
Science 251:1351-1355. If a cre/loxP recombinase system is used to
regulate expression of the transgene, animals containing transgenes
encoding both the Cre recombinase and a selected protein is
required. Such animals can be provided through the construction of
"double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0618] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al. (1997) Nature 385:810-813 and PCT International Publication
Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic
cell, from the transgenic animal can be isolated and induced to
exit the growth cycle and enter G.sub.o phase. The quiescent cell
can then be fused, e.g., through the use of electrical pulses, to
an enucleated oocyte from an animal of the same species from which
the quiescent cell is isolated. The reconstructed oocyte is then
cultured such that it develops to morula or blastocyst and then
transferred to a pseudopregnant female foster animal. The offspring
born of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[0619] Transgenic animals containing recombinant cells that express
the polypeptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
affect cAMP binding, phosphodiesterase activation, and signal
transduction, may not be evident from in vitro cell-free or
cell-based assays. Accordingly, it is useful to provide non-human
transgenic animals to assay in vivo phosphodiesterase function,
including cAMP interaction, the effect of specific mutant
phosphodiesterases on phosphodiesterase function and cAMP
interaction, and the effect of chimeric phosphodiesterases. It is
also possible to assess the effect of null mutations, that is
mutations that substantially or completely eliminate one or more
phosphodiesterase functions.
Pharmaceutical Compositions
[0620] The phosphodiesterase nucleic acid molecules, protein (such
as an extracellular loop), modulators of the protein, and
antibodies (also referred to herein as "active compounds") can be
incorporated into pharmaceutical compositions suitable for
administration to a subject, e.g., a human. Such compositions
typically comprise the nucleic acid molecule, protein, modulator,
or antibody and a pharmaceutically acceptable carrier.
[0621] The term "administer" is used in its broadest sense and
includes any method of introducing the compositions of the present
invention into a subject. This includes producing polypeptides or
polynucleotides in vivo as by transcription or translation, in
vivo, of polynucleotides that have been exogenously introduced into
a subject. Thus, polypeptides or nucleic acids produced in the
subject from the exogenous compositions are encompassed in the term
"administer."
[0622] As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, such media can be used in the compositions of the
invention. Supplementary active compounds can also be incorporated
into the compositions. A pharmaceutical composition of the
invention is formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral
(e.g., inhalation), transdermal (topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampules, disposable
syringes or multiple dose vials made of glass or plastic.
[0623] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0624] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a phosphodiesterase
protein or anti-phosphodiesterase antibody) in the required amount
in an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0625] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For oral administration, the agent can be
contained in enteric forms to survive the stomach or further coated
or mixed to be released in a particular region of the GI tract by
known methods. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules. Oral compositions can
also be prepared using a fluid carrier for use as a mouthwash,
wherein the compound in the fluid carrier is applied orally and
swished and expectorated or swallowed. Pharmaceutically compatible
binding agents, and/or adjuvant materials can be included as part
of the composition. The tablets, pills, capsules, troches and the
like can contain any of the following ingredients, or compounds of
a similar nature: a binder such as microcrystalline cellulose, gum
tragacanth or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0626] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser, which contains a suitable propellant, e.g., a gas
such as carbon dioxide, or a nebulizer.
[0627] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0628] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0629] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0630] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. "Dosage unit form" as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0631] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) PNAS
91:3054-3057). The pharmaceutical preparation of the gene therapy
vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. Alternatively, where the complete
gene delivery vector can be produced intact from recombinant cells,
e.g. retroviral vectors, the pharmaceutical preparation can include
one or more cells which produce the gene delivery system.
[0632] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0633] This invention may be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will fully convey the invention to those skilled in the
art. Many modifications and other embodiments of the invention will
come to mind in one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Although specific terms are employed, they
are used as in the art unless otherwise indicated.
III. METHODS AND COMPOSITIONS FOR DIAGNOSIS AND TREATMENT OF CANCER
USING 27420
Background of the Invention
[0634] The methyltransferase family is a large superfamily of
enzymes that regulate biological processes by catalyzing the
transfer of methyl groups to a wide variety of endogenous and
exogenous compounds, including DNA, RNA, proteins, hormones,
neurotransmitters, drugs, and xenobiotics (Weinshilboum, R. M. et
al. (1999) Annu. Rev. Pharmacol. Toxicol. 39:19-52)
[0635] Methylation of DNA can play an important role in the control
of gene expression in mammalian cells. The enzyme involved in DNA
methylation is DNA methyltransferase, which catalyzes the transfer
of a methyl group from S-adenosylmethionine to cytosine residues to
form 5-methylcytosine, a modified base that is found mostly at CpG
sites in the genome. The presence of methylated CpG islands in the
promoter region of genes can suppress their expression. This
process may be due to the presence of 5-methylcytosine, which
apparently interferes with the binding of transcription factors or
other DNA-binding proteins, and thus, blocks transcription. In
different types of tumors, aberrant or accidental methylation of
CpG islands in the promoter region has been observed for many tumor
suppressor genes, genes that suppress metastasis, and genes that
repair DNA, silencing their expression (Momparler, R. L. and
Bovenzi, V. (2000) J. Cell Physiol. 183:145-54).
[0636] Methylation of proteins is a post-translational modification
which can regulate the activity and subcellular localization of
numerous proteins. Methylation of proteins can play an important
role in protein repair and reversal of protein aging. Proteins
undergo a variety of spontaneous degradation processes, including
oxidation, glycation, deamidation, isomerization, and racemization
(Finch, C. E. (1990) Longevity, Senescence, and the Genome (Univ.
of Chicago Press, Chicago); Harding, J. J. et al. (1989) Mech.
Aging Dev. 50:7-16; Stadtman, E. R. (1990) Biochemistry
29:6323-6331; Stadtman, E. R. (1992) Science 257:1220-1224; Geiger,
T. and Clarke, S. (1987) J. Biol. Chem. 262:785-794; Yuan, P. M. et
al. (1981) Mech. Agin. Dev. 17:151-172; Wright, H. T. (1991) Crit.
Rev. Biochem. Mol. Biol. 26:1-52; Visick, J. E. and Clarke, S.
(1995) Mol. Microbiol. 16:835-845). These non-enzymatic
modifications can produce functionally damaged species that reflect
the action of aging at the molecular level (Stadtman (1992) supra;
Martin, G. M. et al. (1996) Nat. Genet. 13:25-34), and methylation
of these damaged proteins can play a part in the repair
pathway.
[0637] Protein methylation, which uses S-adenosylmethionine as the
methyl donor (Kim and Paik (1965) J. Biol. Chem. 240:4629-4634;
Paik and Kim (1980) in Biochemistry: A Series of Monographs
(Meister, A. ed.), vol 1, pp. 112-141, John Wiley & Sons, New
York), can be classified into three major categories (Paik and Kim
(1980) in Biochemistry: A Series of Monographs (Meister, A. ed.),
vol 1, pp. 112-141, John Wiley & Sons, New York; Paik and Kim
(1985) in Enzymology of Post-translational Modification of Proteins
(Freedman, R. B. and Hawkins, H. C., eds.), vol. 2, pp. 187-228,
Academic Press, London; Clarke (1985) Annu. Rev. Biochem.
54:479-506; Clarke et al. (1987) Proc. Natl. Acad. Sci. USA
85:4643-4647; Kim et al. (1990) in Protein Methylation (Paik, W. K.
and Kim, S. eds.), pp. 97-123, CRC Press, Boca Raton, Fla.);
N-methylation involving methylation of arginine, lysine, and
histidine side chains; O-methylation of either the internal carboxy
group of glutamate and isoaspartate residues or the C-terminal
cysteine residue; and S-methylation of either cysteine or
methionine residues.
[0638] Protein methylation is also known to be important in
cellular stress responses (Desrosiers, R. and Tanguay, R. (1988) J.
Biol. Chem. 263:4686-4692). Moreover, protein methyltransferases
have recently been demonstrated to be important in cellular
signaling events, for example, in receptor-mediated and/or
differentiation-dependent signaling (Lin, W. et al. (1996) J. Biol.
Chem. 271:15034-15044; Abramovich, C. et al. (1997) EMBO J.
16:260-266).
[0639] One type of protein methylation is mediated by arginine
methyltransferases. A subtype of arginine methyltransferase, the
type I arginine methyltransferases, catalyze the formation of
monomethylarginine and asymmetric NG,NG-dimethylarginine in a
variety of substrates (Tang, J. et al. (2000) J. Biol. Chem.
275:19866-19876), including many RNA-binding proteins (Najbauer, J.
et al. (1993) J. Biol. Chem. 268:10501-10509), RNA-transporting
proteins (Najbauer et al. (1993) supra), transcription factors
(Gary, J. D. and Clarke, S. (1998) Prog. Nucleic Acids Res. Mol.
Biol. 61:65-131; Chen, D. et al. (1999) Science 284:2174-2177),
nuclear matrix proteins (Gary and Clarke (1998) supra), and
cytokines (Sommer, A. et al. (1989) Biochem. Biophys. Res. Commun.
160:1267-1274). Methylation by type I arginine methyltransferases
modifies the activities of transcription factors (Gary and Clarke
(1998) supra), modulates the affinity of nucleic acid binding
proteins for nucleic acids (Gary and Clarke (1998) supra),
regulates interferon signaling pathways (Abramovich, C. et al.
(1997) EMBO J. 16:260-266), and alters targeting of nuclear
proteins (Pintucci, G. et al. (1996) Mol. Biol. Cell
7:1249-1258).
[0640] Given the important role of methyltransferases in a variety
of distinct cellular functions, there exists a need to identify
novel methyltransferases, as well as modulators of such
methyltransferases, for use in regulating diverse biological
processes, including biological processes which have a role in
human diseases or disorders, such as cancer.
[0641] Cancer is the second leading cause of death in the United
States, after heart disease (Boring, et al., (1993) CA Cancer J.
Clin. 43:7). Cancer is characterized primarily by an increase in
the number of abnormal, or neoplastic, cells derived from a normal
tissue which proliferate to form a tumor mass, the invasion of
adjacent tissues by these neoplastic tumor cells, and the
generation of malignant cells which spread via the blood or
lymphatic system to regional lymph nodes and to distant sites. The
latter progression to malignancy is referred to as metastasis.
[0642] Colorectal cancer is among the most common cancers affecting
the western world. An estimated 129,400 new cases of colorectal
cancer occurred in the United States in 1999 (Rudy, et al. (2000)
Am Fam Physician 61(6):1759-70, 1773-4). By the age of 70 years, at
least 50% of the Western population will develop some form of
colorectal tumor, including early benign polyps and invasive
adenocarcinomas. It is estimated that approximately 10% of the
benign polypoid lesions will progress to invasive carcinoma (Fahy
et al. (1998) Surg Oncol 7(3-4):115-23). Colorectal cancer arises
from a precursor lesion, the adenomatous polyp, which forms in a
field of epithelial cell hyperproliferation and crypt dysplasia.
Progression from this precursor lesion to colorectal cancer is a
multistep process (Winawer (1999) Am J Med 106(1A):3S-6S).
Summary of the Invention
[0643] The present invention provides methods and compositions for
the diagnosis and treatment of cellular growth or proliferation
disorders, e.g., cancer, including, but not limited to colon
cancer. The present invention is based, at least in part, on the
discovery of novel human arginine methyltransferase family members,
referred to herein as "arginine methyltransferase-3" or "MTR-3"
nucleic acid and protein molecules. The present invention is also
based, at least in part, on the discovery that the novel MTR-3
molecules of the present invention are differentially expressed in
tumor cells, e.g., colon tumor cells, as compared to normal cells,
e.g., normal colon cells, and are useful in the diagnosis and
treatment of cellular growth and proliferation disorders, e.g.,
cancer, including, but not limited to, colon cancer.
[0644] The novel MTR-3 nucleic acid and protein molecules of the
present invention are useful as modulating agents in regulating a
variety of cellular processes, e.g., transcriptional activation and
cellular growth and proliferation. Accordingly, in one aspect, this
invention provides isolated nucleic acid molecules encoding MTR-3
proteins or biologically active portions thereof, as well as
nucleic acid fragments suitable as primers or hybridization probes
for the detection of MTR-3-encoding nucleic acids.
[0645] In one embodiment, the invention features an isolated
nucleic acid molecule that includes the nucleotide sequence set
forth in SEQ ID NO:7 or SEQ ID NO:9. In another embodiment, the
invention features an isolated nucleic acid molecule that encodes a
polypeptide including the amino acid sequence set forth in SEQ ID
NO:8.
[0646] In still other embodiments, the invention features isolated
nucleic acid molecules including nucleotide sequences that are
substantially identical (e.g., 77% identical) to the entire length
of the nucleotide sequence set forth as SEQ ID NO:7 or SEQ ID NO:9.
The invention further features isolated nucleic acid molecules
including at least 1123 contiguous nucleotides of the nucleotide
sequence set forth as SEQ ID NO:7 or SEQ ID NO:9. In another
embodiment, the invention features isolated nucleic acid molecules
which encode a polypeptide including an amino acid sequence that is
substantially identical (e.g., 98% identical) to the entire length
of the amino acid sequence set forth as SEQ ID NO:8. The present
invention also features nucleic acid molecules which encode allelic
variants of the polypeptide having the amino acid sequence set
forth as SEQ ID NO:8. In addition to isolated nucleic acid
molecules encoding full-length polypeptides, the present invention
also features nucleic acid molecules which encode fragments, for
example, biologically active or antigenic fragments, of the
full-length polypeptides of the present invention (e.g., fragments
including at least 433 or 448 contiguous amino acid residues of the
amino acid sequence of SEQ ID NO:8). In still other embodiments,
the invention features nucleic acid molecules that are
complementary to, antisense to, or hybridize under stringent
conditions to the isolated nucleic acid molecules described
herein.
[0647] In another aspect, the invention provides vectors including
the isolated nucleic acid molecules described herein (e.g.,
MTR-3-encoding nucleic acid molecules). Such vectors can optionally
include nucleotide sequences encoding heterologous polypeptides.
Also featured are host cells including such vectors (e.g., host
cells including vectors suitable for producing MTR-3 nucleic acid
molecules and polypeptides).
[0648] In another aspect, the invention features isolated MTR-3
polypeptides and/or biologically active or antigenic fragments
thereof. Exemplary embodiments feature a polypeptide including the
amino acid sequence set forth as SEQ ID NO:8, a polypeptide
including an amino acid sequence at least 98% identical to the
entire length of the amino acid sequence set forth as SEQ ID NO:8,
a polypeptide encoded by a nucleic acid molecule including a
nucleotide sequence at least 77% identical to the entire length of
the nucleotide sequence set forth as SEQ ID NO:7 or SEQ ID NO:9.
Also featured are fragments of the full-length polypeptides
described herein (e.g., fragments including at least 443 or 448
contiguous amino acid residues of the sequence set forth as SEQ ID
NO:8) as well as allelic variants of the polypeptide having the
amino acid sequence set forth as SEQ ID NO:8.
[0649] The MTR-3 polypeptides and/or biologically active or
antigenic fragments thereof, are useful, for example, as reagents
or targets in assays applicable to treatment and/or diagnosis of
cellular growth or proliferation disorders, such as cancer, e.g.,
colon cancer. In one embodiment, an MTR-3 polypeptide or fragment
thereof, has an MTR-3 activity. In another embodiment, an MTR-3
polypeptide or fragment thereof, includes at least one of the
following domains: a VLD binding domain, a transmembrane domain,
and optionally, has an MTR-3 activity. In a related aspect, the
invention features antibodies (e.g., antibodies which specifically
bind to any one of the polypeptides described herein) as well as
fusion polypeptides including all or a fragment of a polypeptide
described herein.
[0650] The present invention further features methods for detecting
MTR-3 polypeptides and/or MTR-3 nucleic acid molecules, such
methods featuring, for example, a probe, primer or antibody
described herein. Also featured are kits, e.g., kits for the
detection of MTR-3 polypeptides and/or MTR-3 nucleic acid
molecules.
[0651] The present invention also provides diagnostic assays for
identifying the presence or absence of a genetic alteration
characterized by at least one of (i) aberrant modification or
mutation of a gene encoding an MTR-3 protein; (ii) mis-regulation
of the gene; and (iii) aberrant post-translational modification of
an MTR-3 protein, wherein a wild-type form of the gene encodes a
protein with an MTR-3 activity.
[0652] In another aspect, the invention provides a method for
identifying a compound which binds to an MTR-3 polypeptide by
contacting the polypeptide, or a cell expressing the polypeptide
with a test compound, and determining whether the polypeptide binds
to the test compound. In yet another aspect, the invention provides
a method for identifying a compound which modulates the activity of
an MTR-3 polypeptide comprising contacting an MTR-3 polypeptide
with a test compound and determining the effect of the test
compound on the activity of the polypeptide.
[0653] In another aspect, the invention provides a method for
identifying the presence of a nucleic acid molecule associated with
a cellular growth or proliferation disorder, in a sample, by
contacting a sample comprising nucleic acid molecules with a
hybridization probe comprising at least 25 contiguous nucleotides
of SEQ ID NO:7 or 9, and detecting the presence of a nucleic acid
molecule associated with a cellular growth or proliferation
disorder, when the sample contains a nucleic acid molecule that
hybridizes to the nucleic acid probe. In one embodiment, the
hybridization probe is detectably labeled. In another embodiment
the sample comprising nucleic acid molecules is subjected to
agarose gel electrophoresis and southern blotting prior to
contacting with the hybridization probe. In a further embodiment,
the sample comprising nucleic acid molecules is subjected to
agarose gel electrophoresis and northern blotting prior to
contacting with the hybridization probe. In yet another embodiment,
the detecting is by in situ hybridization. In other embodiments,
the method is used to detect mRNA or genomic DNA in the sample.
[0654] The invention also provides a method for identifying a
nucleic acid molecule associated with a cellular growth or
proliferation disorder, in a sample, e.g., a colon tissue sample,
by contacting a sample comprising nucleic acid molecules with a
first and a second amplification primer, the first primer
comprising at least 25 contiguous nucleotides of SEQ ID NO:7 or 9
and the second primer comprising at least 25 contiguous nucleotides
from the complement of SEQ ID NO:7 or 9, incubating the sample
under conditions that allow for nucleic acid amplification, and
detecting the presence of a nucleic acid molecule associated with a
cellular growth or proliferation disorder, when the sample contains
a nucleic acid molecule that is amplified. In one embodiment, the
sample comprising nucleic acid molecules is subjected to agarose
gel electrophoresis after the incubation step.
[0655] In addition, the invention provides a method for identifying
a polypeptide associated with a cellular growth or proliferation
disorder, in a sample, by contacting a sample comprising
polypeptide molecules with a binding substance specific for an
MTR-3 polypeptide, and detecting the presence of a polypeptide
associated with a cellular growth or proliferation disorder, when
the sample contains a polypeptide molecule that binds to the
binding substance. The binding substance may be an antibody or an
MTR-3 ligand, and may be detectably labeled.
[0656] In another aspect, the invention provides a method of
identifying a subject at risk for a cellular growth or
proliferation disorder. The method includes contacting a sample
obtained from the subject comprising nucleic acid molecules with a
hybridization probe comprising at least 25 contiguous nucleotides
of SEQ ID NO:7 or 9, and detecting the presence of a nucleic acid
molecule which identifies a subject a risk for a cellular growth or
proliferation disorder, when the sample contains a nucleic acid
molecule that hybridizes to the nucleic acid probe.
[0657] In a further aspect, the invention provides a method for
identifying a subject at risk for a cellular growth or
proliferation disorder, by contacting a sample obtained from a
subject comprising nucleic acid molecules with a first and a second
amplification primer, the first primer comprising at least 25
contiguous nucleotides of SEQ ID NO:7 or 9 and the second primer
comprising at least 25 contiguous nucleotides from the complement
of SEQ ID NO:7 or 9, incubating the sample under conditions that
allow for nucleic acid amplification, and detecting a nucleic acid
molecule which identifies a subject at risk for a cellular growth
or proliferation disorder, when the sample contains a nucleic acid
molecule that is amplified.
[0658] In yet another aspect, the invention provides a method of
identifying a subject at risk for a cellular growth or
proliferation disorder by contacting a sample obtained from the
subject comprising polypeptide molecules with a binding substance
specific for an MTR-3 polypeptide, and detecting the presence of a
polypeptide molecule in the sample that binds to the binding
substance.
[0659] In another aspect, the invention provides a method for
identifying a compound capable of treating a cellular growth or
proliferation disorder such as cancer, e.g., colon cancer,
characterized by aberrant MTR-3 nucleic acid expression or MTR-3
protein activity. The method includes assaying the ability of the
compound to modulate the expression of an MTR-3 nucleic acid or the
activity of an MTR-3 protein.
[0660] In addition, the invention provides a method for treating a
subject having a cellular growth or proliferation disorder, such as
cancer e.g., colon cancer, that is characterized by aberrant MTR-3
protein activity or aberrant MTR-3 nucleic acid expression by
administering to the subject an MTR-3 modulator. The MTR-3
modulator may be administered in a pharmaceutically acceptable
formulation or may be administered using a gene therapy vector.
[0661] In one embodiment, an MTR-3 modulator is capable of
modulating MTR-3 polypeptide activity. For example, the MTR-3
modulator may be a small molecule, an anti-MTR-3 antibody, an MTR-3
polypeptide comprising the amino acid sequence of SEQ ID NO:8, or a
fragment thereof, an MTR-3 polypeptide comprising an amino acid
sequence which is at least 96 percent identical to the entire
length of the amino acid sequence of SEQ ID NO:8, or an isolated
naturally occurring allelic variant of a polypeptide consisting of
the amino acid sequence of SEQ ID NO:8.
[0662] In another embodiment, the MTR-3 modulator is capable of
modulating MTR-3 nucleic acid expression. For example, the MTR-3
modulator may be a small molecule, an antisense MTR-3 nucleic acid
molecule, a ribozyme, a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:7 or 9, or a fragment thereof, a
nucleic acid molecule that is 77% identical to the entire length of
the nucleotide sequence of SEQ ID NO:7 or 9, or a nucleic acid
molecule encoding a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:8.
[0663] Also featured are methods of regulating metastasis in an
individual or inhibiting tumor progression in an individual which
include administering to the individual an MTR-3 modulator (e.g.,
an MTR-3 inhibitor).
[0664] Furthermore, the invention provides a method for modulating
cellular growth or proliferation comprising contacting a cell with
an MTR-3 modulator.
[0665] In another embodiment, the invention provides a method for
modulating transcriptional activation comprising contacting a cell
with an MTR-3 modulator.
[0666] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
Detailed Description of the Invention
[0667] The present invention is based, at least in part, on the
discovery of novel human arginine methyltransferase family members,
referred to herein as "arginine methyltransferase-3" or "MTR-3"
nucleic acid and protein molecules. These novel molecules are
capable of catalyzing the transfer of a methyl group to or from
biological molecules (e.g., polypeptides or amino acids such as
arginine residues and/or S-adenosylmethionine) and, thus, play a
role in or function in a variety of cellular processes, e.g.,
protein methylation, arginine methylation, indirect or direct
modulation (e.g., activation or inactivation) of gene
transcription, and/or cellular proliferation, growth, and/or
differentiation.
[0668] The present invention further provides methods and
compositions for the diagnosis and treatment of a cellular growth
or proliferation disorder, e.g., cancer, including, but not limited
to, colon cancer. The novel MTR-3 molecules of the present
invention may be involved in the modulation (e.g., activation or
inactivation) of transcription, e.g., nuclear hormone receptor
(e.g., androgen receptor, progesterone receptor, or estrogen
receptor) mediated transcription. "Treatment", as used herein, is
defined as the application or administration of a therapeutic agent
to a patient, or application or administration of a therapeutic
agent to an isolated tissue or cell line from a patient, who has a
disease or disorder, a symptom of a disease or disorder, or a
predisposition toward a disease or disorder, with the purpose to
cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve
or affect the disease or disorder, the symptoms of the disease or
disorder, or the predisposition toward a disease or disorder. A
therapeutic agent includes, but is not limited to, small molecules,
peptides, antibodies, ribozymes and antisense oligonucleotides.
[0669] The novel MTR-3 molecules of the present invention are
differentially expressed in tumor cells, e.g., colon tumor cells
and colon cells which have metastasized to the liver, as compared
to normal cells, e.g., normal colon cells and normal liver cells.
Increased expression of MTR-3 in tumor cells results in an increase
in transcriptional activation by nuclear hormone receptors (e.g.,
nuclear hormone receptors present in tumors, such as, for example,
estrogen and/or progesterone receptors). Increased transcriptional
activation by nuclear receptors, e.g., hormone receptors,
contributes to cellular growth and proliferation, thereby
increasing tumorigenesis and metastasis of tumor cells, e.g., colon
tumor cells or colon cells which have metastasized to the liver
(colon metastases to the liver). In addition, methylation, e.g.,
arginine methylation, has been associated with cellular
proliferation in cancer cells (Kim, et al. (1999) Life Sci.
65(8):737-45). Therefore, methylation by the MTR-3 molecules of the
present invention may be involved in cellular growth,
proliferation, and tumorigenesis. Accordingly, the MTR-3 molecules
of the present invention provide novel diagnostic targets and
therapeutic agents to control cellular growth or proliferation
disorders.
[0670] As used herein, a "cellular growth or proliferation
disorder" includes a disease or disorder that affects a cell growth
or proliferation process. As used herein, a "cellular growth or
proliferation process" is a process by which a cell increases in
number, size or content, by which a cell develops a specialized set
of characteristics which differ from that of other cells, or by
which a cell moves closer to or further from a particular location
or stimulus. A cellular growth or proliferation process includes
the metabolic processes of the cell and cellular transcriptional
activation mechanisms. A cellular growth or proliferation disorder
may be characterized by aberrantly regulated cell growth,
proliferation, differentiation, or migration. Cellular growth or
proliferation disorders include tumorigenic disease or disorders.
As used herein, a "tumorigenic disease or disorder" includes a
disease or disorder characterized by aberrantly regulated cell
growth, proliferation, differentiation, adhesion, or migration,
resulting in the production of or tendency to produce tumors. As
used herein, a "tumor" includes a normal benign or malignant mass
of tissue. Examples of cellular growth or proliferation disorders
include, but are not limited to, cancer, e.g., carcinoma, sarcoma,
or leukemia, examples of which include, but are not limited to,
colon, ovarian, lung, breast, endometrial, uterine, hepatic,
gastrointestinal, prostate, and brain cancer; tumorigenesis and
metastasis; skeletal dysplasia; and hematopoietic and/or
myeloproliferative disorders.
[0671] "Differential expression", as used herein, includes both
quantitative as well as qualitative differences in the temporal
and/or tissue expression pattern of a gene. Thus, a differentially
expressed gene may have its expression activated or inactivated in
normal versus cellular growth or proliferation disease states. The
degree to which expression differs in normal versus cellular growth
or proliferation disease states or control versus experimental
states need only be large enough to be visualized via standard
characterization techniques, e.g., quantitative PCR, Northern
analysis, or subtractive hybridization. The expression pattern of a
differentially expressed gene may be used as part of a prognostic
or diagnostic cellular growth or proliferation disorder evaluation,
or may be used in methods for identifying compounds useful for the
treatment of cellular growth or proliferation disorder. In
addition, a differentially expressed gene involved in tumorigenic
disorders may represent a target gene such that modulation of the
expression level of this gene or the activity of the gene product
may act to ameliorate a cellular growth or proliferation disorder.
Compounds that modulate target gene expression or activity of the
target gene product can be used in the treatment of cellular growth
or proliferation disorders. Although the MTR-3 genes described
herein may be differentially expressed with respect to cellular
growth or proliferation disorders, and/or their products may
interact with gene products important to cellular growth or
proliferation disorders, the genes may also be involved in
mechanisms important to additional tumor cell processes.
[0672] The MTR-3 molecules of the present invention are involved in
the modulation of transcriptional activation and function to
modulate cell proliferation, differentiation, and motility. Thus,
the MTR-3 molecules of the present invention may play a role in the
modulation of cellular transcriptional activation mechanisms, such
as the regulation of the activation of transcription (e.g., by
nuclear receptors, such as nuclear hormone receptors), the
recruitment of a transcription initiation complex to the promoter
of genes, and/or cell transversal through the cell cycle.
[0673] The term "family" when referring to the protein and nucleic
acid molecules of the invention is intended to mean two or more
proteins or nucleic acid molecules having a common structural
domain or motif and having sufficient amino acid or nucleotide
sequence homology as defined herein. Such family members can be
naturally or non-naturally occurring and can be from either the
same or different species. For example, a family can contain a
first protein of human origin as well as other distinct proteins of
human origin or alternatively, can contain homologues of non-human
origin, e.g., rat proteins. Members of a family can also have
common functional characteristics.
[0674] For example, the family of MTR-3 polypeptides comprise at
least one "transmembrane domain" and preferably four transmembrane
domains. As used herein, the term "transmembrane domain" includes
an amino acid sequence of about 15-25 amino acid residues in length
which spans the plasma membrane. More preferably, a transmembrane
domain includes about at least 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 amino acid residues and spans the plasma membrane.
Transmembrane domains are rich in hydrophobic residues, and
typically have an alpha-helical structure. In a preferred
embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the
amino acids of a transmembrane domain are hydrophobic, e.g.,
leucines, isoleucines, alanines, valines, phenylalanines, prolines
or methionines. Transmembrane domains are described in, for
example, Zagotta W. N. et al, (1996) Annual Rev. Neurosci. 19:
235-263, the contents of which are incorporated herein by
reference. A MEMSAT analysis and a structural, hydrophobicity, and
antigenicity analysis resulted in the identification of four
transmembrane domains in the amino acid sequence of human MTR-3
(SEQ ID NO:8) at about residues 18-41, 97-113, 187-203, and 382404.
Accordingly, MTR-3 polypeptides having at least 50-60% homology,
preferably about 60-70%, more preferably about 70-80%, or about
80-90% homology with a transmembrane domain of human MTR-3 are
within the scope of the invention.
[0675] In another embodiment, members of the MTR-3 family of
proteins include at least one "VLD binding domain" in the protein
or corresponding nucleic acid molecule. As used herein, the term
"VLD binding domain" includes a protein domain having at least
about 3 amino acid residues with the amino acid consensus sequence
Valine-Leucine-Aspartic Acid (V-L-D). The amino acid residues of
the VLD binding domain have been shown to be important for
methyltransferase activity and for transcriptional activation
(Chen, et al. (1999) Science 284:2174-2177). A VLD binding domain
in the proteins of the present invention has at least 3 amino acid
residues matching the VLD binding domain consensus sequence, and
may also have additional amino acid residues. A VLD binding domain
motif was identified in the amino acid sequence of human MTR-3 at
about residues 188-190 of SEQ ID NO:8.
[0676] Preferably a VLD binding domain comprises at least about
3-10 amino acid residues and has a "VLD binding activity," for
example, the ability to interact with an MTR-3 substrate or target
molecule (e.g., a non-MTR-3 protein); to convert an MTR-3 substrate
or target molecule to a product (e.g., transfer a methyl group to
or from the substrate or target molecule); to interact with and/or
transfer a methyl group to a second non-MTR-3 protein; to transfer
a methyl group to an arginine residue; to modulate intra- or
inter-cellular signaling; to modulate transcriptional activation
(e.g., either directly or indirectly); to modulate cellular
targeting and/or transport of proteins; and/or to modulate cellular
proliferation, growth, or differentiation. Accordingly, identifying
the presence of a VLD binding domain can include isolating a
fragment of an MTR-3 molecule (e.g., an MTR-3 polypeptide) and
assaying for the ability of the fragment to exhibit one of the
aforementioned VLD binding domain activities.
[0677] Isolated MTR-3 proteins of the present invention, have an
amino acid sequence sufficiently homologous to the amino acid
sequence of SEQ ID NO:8, or are encoded by a nucleotide sequence
sufficiently identical to SEQ ID NO:7 or 9. As used herein, the
term "sufficiently identical" refers to a first amino acid or
nucleotide sequence which contains a sufficient or minimum number
of identical or equivalent (e.g., an amino acid residue which has a
similar side chain) amino acid residues or nucleotides to a second
amino acid or nucleotide sequence such that the first and second
amino acid or nucleotide sequences share common structural domains
or motifs and/or a common functional activity. For example, amino
acid or nucleotide sequences which share common structural domains
having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology or
identity across the amino acid sequences of the domains and contain
at least one structural domains or motifs, are defined herein as
sufficiently homologous. Furthermore, amino acid or nucleotide
sequences which share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
homology or identity and share a common functional activity are
defined herein as sufficiently homologous or identical.
[0678] In a preferred embodiment, an MTR-3 protein, preferably a
human MTR-3 protein, includes a VLD binding domain, a transmembrane
domain, and has an amino acid sequence at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the
amino acid sequence of SEQ ID NO:8. In yet another preferred
embodiment, an MTR-3 protein, preferably a human MTR-3 protein,
includes a VLD binding domain, a transmembrane domain, and is
encoded by a nucleic acid molecule having a nucleotide sequence
which hybridizes under stringent hybridization conditions to a
complement of a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:7 or 9. In another preferred embodiment, an
MTR-3 protein, preferably a human MTR-3 protein, includes a VLD
binding domain, a transmembrane domain, and has an MTR-3
activity.
[0679] As used interchangeably herein, an "MTR-3 activity",
"biological activity of MTR-3" or "functional activity of MTR-3",
includes an activity exerted or mediated by an MTR-3 protein,
polypeptide or nucleic acid molecule on an MTR-3 responsive cell or
on an MTR-3 substrate, as determined in vivo or in vitro, according
to standard techniques. In one embodiment, an MTR-3 activity is a
direct activity, such as an association with an MTR-3 target
molecule. As used herein, a "target molecule" or "binding partner"
is a molecule which an MTR-3 protein binds or interacts with in
nature, such that MTR-3-mediated function is achieved. An MTR-3
target molecule can be a non-MTR-3 molecule or an MTR-3 protein or
polypeptide of the present invention. In an exemplary embodiment,
an MTR-3 target molecule is an MTR-3 substrate (e.g., a polypeptide
substrate, an arginine residue, or S-adenosylmethionine). An MTR-3
activity can also be an indirect activity, such as a cellular
transcription modulating activity mediated by interaction of the
MTR-3 protein with an MTR-3 substrate.
[0680] In a preferred embodiment, an MTR-3 activity is at least one
of the following activities: (i) modulation of transcriptional
activation (e.g., either directly or indirectly); (ii) modulation
of (directly or indirectly) chromatin structure to, for example,
regulate the recruitment of an RNA polymerase II transcription
initiation complex to a gene promoter; (iii) modulation of the
methylation state of proteins in the transcription machinery; (iv)
interaction with an MTR-3 substrate or target molecule (e.g., a
non-MTR-3 protein); (v) conversion of an MTR-3 substrate or target
molecule to a product (e.g., transfer of a methyl group to or from
the substrate or target molecule); (vi) interaction with and/or
methyl transfer to a second non-MTR-3 protein; (vii) transfer of a
methyl group to an arginine residue; (viii) modulation of
protein-protein interaction (e.g., MTR-3-MTR-3 and/or
MTR-3-non-MTR-3 interaction); (ix) modulation and/or coordination
of protein complex formation (e.g., MTR-3-containing complex
formation); (x) regulation of substrate or target molecule
activity; (xi) modulation of intra- or inter-cellular signaling,
(xii) modulation of cellular targeting and/or transport of
proteins; and/or (xiii) modulation of cellular proliferation,
growth, or differentiation.
[0681] The nucleotide sequence of the isolated human MTR-3 cDNA and
the predicted amino acid sequence encoded by the MTR-3 cDNA in SEQ
ID NO:7 and 8, respectively.
[0682] The human MTR-3 gene, which is approximately 2898
nucleotides in length, encodes a polypeptide which is approximately
608 amino acid residues in length.
Screening Assays
[0683] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules (organic or inorganic) or other drugs) which bind to
MTR-3 proteins, have a stimulatory or inhibitory effect on, for
example, MTR-3 expression or MTR-3 activity, or have a stimulatory
or inhibitory effect on, for example, the expression or activity of
an MTR-3 substrate.
[0684] These assays are designed to identify compounds that bind to
an MTR-3 protein, bind to other inter- or extra-cellular proteins
that interact with an MTR-3 protein, and interfere with the
interaction of the MTR-3 protein with other inter- or
extra-cellular proteins. For example, in the case of the MTR-3
protein, which is a protein that is capable of binding to a
substrate and thereby modulating transcriptional activation, methyl
transfer to a second non-MTR-3 protein, transfer of a methyl group
to an arginine residue, and modulation and/or coordination of
protein complex formation, such techniques can be used to identify
compounds that stimulate or inhibit any or all of these activities.
Such compounds may include, but are not limited to MTR-3 peptides,
anti-MTR-3 antibodies, or small organic or inorganic compounds.
Such compounds may also include other cellular proteins or
peptides.
[0685] Compounds identified via assays such as those described
herein may be useful, for example, for ameliorating cellular growth
and proliferation disorders, e.g., cancer. In instances whereby a
cellular growth or proliferation disorder results from an overall
lower level of MTR-3 gene expression and/or MTR-3 protein activity
in a cell or tissue, MTR-3 modulators may include compounds which
accentuate or amplify the activity of the MTR-3 protein such as
MTR-3 agonists. Such compounds would bring about an effective
increase in MTR-3 protein activity, thus ameliorating symptoms.
[0686] In other instances, mutations within the MTR-3 gene may
cause aberrant types or excessive amounts of MTR-3 proteins to be
made which have a deleterious effect that leads to a cellular
growth or proliferation disorder. Similarly, physiological
conditions may cause an excessive increase in MTR-3 gene expression
leading to a cellular growth or proliferation disease or disorder.
In such cases, compounds, e.g., compounds that bind to an MTR-3
protein, may be identified that inhibit the activity of the MTR-3
protein. Assays for testing the effectiveness of compounds
identified by techniques such as those described in this section
are discussed herein.
[0687] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of an
MTR-3 protein or polypeptide or biologically active portion
thereof. In another embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of an MTR-3 protein or polypeptide or biologically active
portion thereof. The test compounds of the present invention can be
obtained using any of the numerous approaches in combinatorial
library methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K. S. (1997) Anticancer
Drug Des. 12:145).
[0688] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0689] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra.).
[0690] In one embodiment, an assay is a cell-based assay in which a
cell which expresses an MTR-3 protein or biologically active
portion thereof is contacted with a test compound and the ability
of the test compound to modulate MTR-3 activity is determined.
Determining the ability of the test compound to modulate MTR-3
activity can be accomplished by monitoring, for example: (i)
modulate transcriptional activation (e.g., either directly or
indirectly); (ii) modulate (directly or indirectly) chromatin
structure to, for example, regulate the recruitment of an RNA
polymerase II transcription initiation complex to a gene promoter;
(iii) modulate the methylation state of proteins in the
transcription machinery; (iv) interaction with an MTR-3 substrate
or target molecule (e.g., a non-MTR-3 protein); (v) conversion of
an MTR-3 substrate or target molecule to a product (e.g., transfer
of a methyl group to or from the substrate or target molecule);
(vi) interaction with and/or methyl transfer to a second non-MTR-3
protein; (vii) transfer of a methyl group to an arginine residue;
(viii) modulation of protein-protein interaction (e.g., MTR-3-MTR-3
and/or MTR-3-non-MTR-3 interaction); (ix) modulation and/or
coordination of protein complex formation (e.g., MTR-3-containing
complex formation); (x) regulation of substrate or target molecule
activity; (xi) modulation of intra- or intercellular signaling,
(xii) modulation of cellular targeting and/or transport of
proteins; and/or (xiii) modulation of cellular proliferation,
growth, or differentiation. The cell, for example, can be of
mammalian origin, e.g., an epithelial cell, for example a colon
epithelial cell, or a tumor cell. The ability of the test compound
to modulate MTR-3 binding to a substrate or to bind to MTR-3 can
also be determined.
[0691] Determining the ability of the test compound to modulate
MTR-3 binding to a substrate can be accomplished, for example, by
coupling the MTR-3 substrate with a radioisotope or enzymatic label
such that binding of the MTR-3 substrate to MTR-3 can be determined
by detecting the labeled MTR-3 substrate in a complex.
Alternatively, MTR-3 could be coupled with a radioisotope or
enzymatic label to monitor the ability of a test compound to
modulate MTR-3 binding to an MTR-3 substrate in a complex.
Determining the ability of the test compound to bind MTR-3 can be
accomplished, for example, by coupling the compound with a
radioisotope or enzymatic label such that binding of the compound
to MTR-3 can be determined by detecting the labeled MTR-3 compound
in a complex. For example, compounds (e.g., MTR-3 substrates) can
be labeled with .sup.125I, .sup.35S, .sup.14C, or .sup.3H, either
directly or indirectly, and the radioisotope detected by direct
counting of radioemmission or by scintillation counting.
Alternatively, compounds can be enzymatically labeled with, for
example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0692] It is also within the scope of this invention to determine
the ability of a compound (e.g., an MTR-3 substrate) to interact
with MTR-3 without the labeling of any of the interactants. For
example, a microphysiometer can be used to detect the interaction
of a compound with MTR-3 without the labeling of either the
compound or the MTR-3. McConnell, H. M. et al. (1992) Science
257:1906-1912. As used herein, a "microphysiometer" (e.g.,
Cytosensor) is an analytical instrument that measures the rate at
which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate
can be used as an indicator of the interaction between a compound
and MTR-3.
[0693] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing an MTR-3 target molecule
(e.g., an MTR-3 substrate) with a test compound and determining the
ability of the test compound to modulate (e.g., stimulate or
inhibit) the activity of the MTR-3 target molecule. Determining the
ability of the test compound to modulate the activity of an MTR-3
target molecule can be accomplished, for example, by determining
the ability of the MTR-3 protein to bind to or interact with the
MTR-3 target molecule.
[0694] Determining the ability of the MTR-3 protein, or a
biologically active fragment thereof, to bind to or interact with
an MTR-3 target molecule can be accomplished by one of the methods
described above for determining direct binding. In a preferred
embodiment, determining the ability of the MTR-3 protein to bind to
or interact with an MTR-3 target molecule can be accomplished by
determining the activity of the target molecule. For example, the
activity of the target molecule can be determined by detecting
induction of a cellular response, detecting catalytic/enzymatic
activity of the target on an appropriate substrate, detecting the
induction of a reporter gene (comprising a target-responsive
regulatory element operatively linked to a nucleic acid encoding a
detectable marker, e.g., luciferase), or detecting a
target-regulated cellular response (e.g., cell growth or
proliferation).
[0695] In yet another embodiment, an assay of the present invention
is a cell-free assay in which an MTR-3 protein or biologically
active portion thereof is contacted with a test compound and the
ability of the test compound to bind to the MTR-3 protein or
biologically active portion thereof is determined. Preferred
biologically active portions of the MTR-3 proteins to be used in
assays of the present invention include fragments which participate
in interactions with non-MTR-3 molecules, e.g., fragments with high
surface probability scores. Binding of the test compound to the
MTR-3 protein can be determined either directly or indirectly as
described above. In a preferred embodiment, the assay includes
contacting the MTR-3 protein or biologically active portion thereof
with a known compound which binds MTR-3 to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with an MTR-3 protein,
wherein determining the ability of the test compound to interact
with an MTR-3 protein comprises determining the ability of the test
compound to preferentially bind to MTR-3 or biologically active
portion thereof as compared to the known compound.
[0696] In another embodiment, the assay is a cell-free assay in
which an MTR-3 protein or biologically active portion thereof is
contacted with a test compound and the ability of the test compound
to modulate (e.g., stimulate or inhibit) the activity of the MTR-3
protein or biologically active portion thereof is determined.
Determining the ability of the test compound to modulate the
activity of an MTR-3 protein can be accomplished, for example, by
determining the ability of the MTR-3 protein to bind to an MTR-3
target molecule by one of the methods described above for
determining direct binding. Determining the ability of the MTR-3
protein to bind to an MTR-3 target molecule can also be
accomplished using a technology such as real-time Biomolecular
Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991)
Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin.
Struct. Biol. 5:699-705. As used herein, "BIA" is a technology for
studying biospecific interactions in real time, without labeling
any of the interactants (e.g., BIAcore). Changes in the optical
phenomenon of surface plasmon resonance (SPR) can be used as an
indication of real-time reactions between biological molecules.
[0697] In an alternative embodiment, determining the ability of the
test compound to modulate the activity of an MTR-3 protein can be
accomplished by determining the ability of the MTR-3 protein to
interact with and/or convert an MTR-3 substrate (e.g., to methylate
arginine residues of specific proteins, e.g., histones, hnRNPA1,
fibrillarin, or nucleolin) or to regulate transcription (e.g.,
transcriptional activation by nuclear receptors). For example, to
determine the ability of an MTR-3 protein to methylate a substrate,
assays for methylation such as those described in Chen, et al.
(1999) Science 284:2174) and Gu, et al. (1999) Life Sciences
65:737-745 may be carried out. In another embodiment, determining
the ability of the test compound to modulate the activity of an
MTR-3 protein can be accomplished by determining the ability of the
MTR-3 protein to further modulate the activity of a downstream
effector of an MTR-3 target molecule. For example, the activity of
the effector molecule on an appropriate target can be determined or
the binding of the effector to an appropriate target can be
determined as previously described.
[0698] In yet another embodiment, the cell-free assay involves
contacting an MTR-3 protein or biologically active portion thereof
with a known compound which binds the MTR-3 protein to form an
assay mixture, contacting the assay mixture with a test compound,
and determining the ability of the test compound to interact with
the MTR-3 protein, wherein determining the ability of the test
compound to interact with the MTR-3 protein comprises determining
the ability of the MTR-3 protein to preferentially bind to or
methylate the target substrate.
[0699] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either
MTR-3 or its target molecule to facilitate separation of complexed
from uncomplexed forms of one or both of the proteins, as well as
to accommodate automation of the assay. Binding of a test compound
to an MTR-3 protein, or interaction of an MTR-3 protein with a
target molecule in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtitre plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/MTR-3 fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or MTR-3 protein, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtitre plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of MTR-3 binding or activity
determined using standard techniques.
[0700] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either an MTR-3 protein or an MTR-3 target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated MTR-3 protein or target molecules can be prepared from
biotin-NHS (N-hydroxy-succinimide) using techniques known in the
art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),
and immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with MTR-3
protein or target molecules but which do not interfere with binding
of the MTR-3 protein to its target molecule can be derivatized to
the wells of the plate, and unbound target or MTR-3 protein trapped
in the wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the MTR-3 protein or target
molecule, as well as enzyme-linked assays which rely on detecting
an enzymatic activity associated with the MTR-3 protein or target
molecule.
[0701] In another embodiment, modulators of MTR-3 expression are
identified in a method wherein a cell is contacted with a candidate
compound and the expression of MTR-3 mRNA or protein in the cell is
determined. The level of expression of MTR-3 mRNA or protein in the
presence of the candidate compound is compared to the level of
expression of MTR-3 mRNA or protein in the absence of the candidate
compound. The candidate compound can then be identified as a
modulator of MTR-3 expression based on this comparison. For
example, when expression of MTR-3 mRNA or protein is greater
(statistically significantly greater) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of MTR-3 mRNA or protein expression.
Alternatively, when expression of MTR-3 mRNA or protein is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of MTR-3 mRNA or protein expression. The level of
MTR-3 mRNA or protein expression in the cells can be determined by
methods described herein for detecting MTR-3 mRNA or protein.
[0702] In yet another aspect of the invention, the MTR-3 proteins
can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with MTR-3
("MTR-3-binding proteins" or "MTR-3-6-bp") and are involved in
MTR-3 activity. Such MTR-3-binding proteins are also likely to be
involved in the propagation of signals by the MTR-3 proteins or
MTR-3 targets as, for example, downstream elements of an
MTR-3-mediated signaling pathway. Alternatively, such MTR-3-binding
proteins are likely to be MTR-3 inhibitors.
[0703] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for an MTR-3
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming an MTR-3-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the MTR-3 protein.
[0704] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating compound can be identified using a cell-based or a cell
free assay, and the ability of the compound to modulate the
activity of an MTR-3 protein can be confirmed in vivo, e.g., in an
animal such as an animal model for cellular tumorigenesis or a
cellular growth or proliferation disorder.
[0705] For example, a modulating compound identified as described
herein (e.g., an antisense MTR-3 nucleic acid molecule, a ribozyme,
an MTR-3-specific antibody, or an MTR-3-binding compound) can be
used in an animal model to determine the efficacy, toxicity, or
side effects of treatment with such a compound. Alternatively, a
modulating compound identified as described herein can be used in
an animal model to determine the mechanism of action of such an
agent. Examples of animal models of cancer include transplantable
models (e.g., xenografts of colon tumors such as Co-3, AC3603 or
WiDr or into immunocompromised mice such as SCID or nude mice);
transgenic models (e.g., B66-Min/+ mouse); chemical induction
models, e.g., carcinogen (e.g., azoxymethane, 2-dimethylhydrazine,
or N-nitrosodimethylamine) treated rats or mice; models of liver
metastasis from colon cancer such as that described by Rashidi et
al. (2000) Anticancer Res 20(2A):715; and cancer cell implantation
or inoculation models as described in, for example, Fingert, et al.
(1987) Cancer Res 46(14):3824-9 and Teraoka, et al. (1995) Jpn J
Cancer Res 86(5):419-23.
[0706] Furthermore, this invention pertains to uses of novel
compounds identified by the above-described screening assays for
treatments as described herein. In one embodiment, the invention
features a method of treating a subject having a cellular growth or
proliferation disorder that involves administering to the subject
an MTR-3 modulator such that treatment occurs. In another
embodiment, the invention features a method of treating a subject
having cancer, e.g., colon cancer, that involves treating a subject
with an MTR-3 modulator, such that treatment occurs. Preferred
MTR-3 modulators include, but are not limited to, MTR-3 proteins or
biologically active fragments, MTR-3 nucleic acid molecules, MTR-3
antibodies, ribozymes, and MTR-3 antisense oligonucleotides
designed based on the MTR-3 nucleotide sequences disclosed herein,
as well as peptides, organic and non-organic small molecules
identified as being capable of modulating MTR-3 expression and/or
activity, for example, according to at least one of the screening
assays described herein.
[0707] Any of the compounds, including but not limited to compounds
such as those identified in the foregoing assay systems, may be
tested for the ability to ameliorate cellular growth or
proliferation disorder symptoms. Cell-based and animal model-based
assays for the identification of compounds exhibiting such an
ability to ameliorate cellular growth or proliferation disorder
systems are described herein.
[0708] In one aspect, cell-based systems, as described herein, may
be used to identify compounds which may act to ameliorate cellular
growth or proliferation disorder symptoms, for example, reduction
in tumor burden, tumor size, tumor cell growth, differentiation,
and/or proliferation, and invasive and/or metastatic potential
before and after treatment. For example, such cell systems may be
exposed to a compound, suspected of exhibiting an ability to
ameliorate cellular growth or proliferation disorder symptoms, at a
sufficient concentration and for a time sufficient to elicit such
an amelioration of cellular growth or proliferation disorder
symptoms in the exposed cells. After exposure, the cells are
examined to determine whether one or more of the cellular growth or
proliferation disorder cellular phenotypes has been altered to
resemble a more normal or more wild type, non-cellular growth or
proliferation disorder phenotype. Cellular phenotypes that are
associated with cellular growth and/or proliferation disorders
include aberrant proliferation, growth, and migration, anchorage
independent growth, and loss of contact inhibition.
[0709] In addition, animal-based cellular growth or proliferation
disorder systems, such as those described herein, may be used to
identify compounds capable of ameliorating cellular growth or
proliferation disorder 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
cellular growth or proliferation disorders. For example, animal
models may be exposed to a compound, suspected of exhibiting an
ability to ameliorate cellular growth or proliferation disorder
symptoms, at a sufficient concentration and for a time sufficient
to elicit such an amelioration of cellular growth or proliferation
disorder symptoms in the exposed animals. The response of the
animals to the exposure may be monitored by assessing the reversal
of cellular growth or proliferation disorders, or symptoms
associated therewith, for example, reduction in tumor burden, tumor
size, and invasive and/or metastatic potential before and after
treatment.
[0710] With regard to intervention, any treatments which reverse
any aspect of cellular growth or proliferation disorder symptoms
should be considered as candidates for human cellular growth or
proliferation disorder therapeutic intervention. Dosages of test
compounds may be determined by deriving dose-response curves.
[0711] Additionally, gene expression patterns may be utilized to
assess the ability of a compound to ameliorate cellular growth
and/or proliferation disorder symptoms. For example, the expression
pattern of one or more genes may form part of a "gene expression
profile" or "transcriptional profile" which may be then be used in
such an assessment. "Gene expression profile" or "transcriptional
profile", as used herein, includes 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,
cell growth, proliferation, differentiation, transformation,
tumorigenesis, metastasis, and carcinogen exposure. Gene expression
profiles may be generated, for example, by utilizing a differential
display procedure, Northern analysis and/or RT-PCR. In one
embodiment, MTR-3 gene sequences may be used as probes and/or PCR
primers for the generation and corroboration of such gene
expression profiles. Gene expression profiles may be characterized
for known states within the cell- and/or animal-based model
systems. Subsequently, these known gene expression profiles may be
compared to ascertain the effect a test compound has to modify such
gene expression profiles, and to cause the profile to more closely
resemble that of a more desirable profile. For example,
administration of a compound may cause the gene expression profile
of a cellular growth or proliferation disorder model system to more
closely resemble the control system. Administration of a compound
may, alternatively, cause the gene expression profile of a control
system to begin to mimic a cellular growth and/or proliferation
disorder 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.
Predictive Medicine
[0712] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to
diagnostic assays for determining MTR-3 protein and/or nucleic acid
expression as well as MTR-3 activity, in the context of a
biological sample (e.g., blood, serum, cells, tissue, e.g., tumor
cells or colon tissue) to thereby determine whether an individual
is afflicted with a disorder, or is at risk of developing a
cellular growth or proliferation disorder, associated with aberrant
or unwanted MTR-3 expression or activity. The invention also
provides for prognostic (or predictive) assays for determining
whether an individual is at risk of developing a disorder
associated with MTR-3 protein, nucleic acid expression or activity.
For example, mutations in an MTR-3 gene can be assayed in a
biological sample. Such assays can be used for prognostic or
predictive purpose to thereby prophylactically treat an individual
prior to the onset of a disorder characterized by or associated
with MTR-3 protein, nucleic acid expression or activity.
[0713] Another aspect of the invention pertains to monitoring the
influence of agents (e.g., drugs, compounds) on the expression or
activity of MTR-3 in clinical trials.
[0714] These and other agents are described in further detail in
the following sections.
Diagnostic Assays
[0715] The present invention encompasses methods for diagnostic and
prognostic evaluation of cellular growth or proliferation disorder
conditions, and for the identification of subjects exhibiting a
predisposition to such conditions.
[0716] An exemplary method for detecting the presence or absence of
MTR-3 protein or nucleic acid in a biological sample involves
obtaining a biological sample from a test subject and contacting
the biological sample with a compound or an agent capable of
detecting MTR-3 protein or nucleic acid (e.g., mRNA, or genomic
DNA) that encodes MTR-3 protein such that the presence of MTR-3
protein or nucleic acid is detected in the biological sample. A
preferred agent for detecting MTR-3 mRNA or genomic DNA is a
labeled nucleic acid probe capable of hybridizing to MTR-3 mRNA or
genomic DNA. The nucleic acid probe can be, for example, the MTR-3
nucleic acid set forth in SEQ ID NO:7 or 9, or a portion thereof,
such as an oligonucleotide of at least 15, 20, 25, 30, 35, 40, 45,
50, 100, 250 or 500 nucleotides in length and sufficient to
specifically hybridize under stringent conditions to MTR-3 mRNA or
genomic DNA. Other suitable probes for use in the diagnostic assays
of the invention are described herein.
[0717] A preferred agent for detecting MTR-3 protein is an antibody
capable of binding to MTR-3 protein, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab')2) can be used. The term "labeled", with regard to the probe
or antibody, is intended to encompass direct labeling of the probe
or antibody by coupling (i.e., physically linking) a detectable
substance to the probe or antibody, as well as indirect labeling of
the probe or antibody by reactivity with another reagent that is
directly labeled. Examples of indirect labeling include detection
of a primary antibody using a fluorescently labeled secondary
antibody and end-labeling of a DNA probe with biotin such that it
can be detected with fluorescently labeled streptavidin. The term
"biological sample" is intended to include tissues, cells and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject. That is, the detection
method of the invention can be used to detect MTR-3 mRNA, protein,
or genomic DNA in a biological sample in vitro as well as in vivo.
For example, in vitro techniques for detection of MTR-3 mRNA
include Northern hybridizations and in situ hybridizations. In
vitro techniques for detection of MTR-3 protein include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. In vitro techniques
for detection of MTR-3 genomic DNA include Southern hybridizations.
Furthermore, in vivo techniques for detection of MTR-3 protein
include introducing into a subject a labeled anti-MTR-3 antibody.
For example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[0718] In one embodiment, the biological sample contains protein
molecules from the test subject. Alternatively, the biological
sample can contain mRNA molecules from the test subject or genomic
DNA molecules from the test subject. A preferred biological sample
is a serum sample isolated by conventional means from a subject.
Also preferred are biological samples from tumors (e.g., tumor
biopsies). Additional preferred biological samples include lung
sample, prostate tissue, liver tissue, breast tissue, skeletal
muscle tissue, brain tissue, breast tissue, heart tissue, ovarian
tissue, kidney tissue, lung tissue, vascular tissue, aortic tissue,
thyroid tissue, placental tissue, intestinal tissue, cervical
tissue, splenic tissue, esophageal tissue, thymic tissue, tonsillar
tissue, lymph nodes and osteogenic cells. Particularly preferred
samples are from colon tissue.
[0719] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting MTR-3
protein, mRNA, or genomic DNA, such that the presence of MTR-3
protein, mRNA or genomic DNA is detected in the biological sample,
and comparing the presence of MTR-3 protein, mRNA or genomic DNA in
the control sample with the presence of MTR-3 protein, mRNA or
genomic DNA in the test sample.
[0720] The invention also encompasses kits for detecting the
presence of MTR-3 in a biological sample. For example, the kit can
comprise a labeled compound or agent capable of detecting MTR-3
protein or mRNA in a biological sample; means for determining the
amount of MTR-3 in the sample; and means for comparing the amount
of MTR-3 in the sample with a standard. The compound or agent can
be packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect MTR-3 protein or nucleic
acid.
Prognostic Assays
[0721] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
cellular growth or proliferation disorder associated with aberrant
or unwanted MTR-3 expression or activity. As used herein, the term
"aberrant" includes an MTR-3 expression or activity which deviates
from the wild type MTR-3 expression or activity. Aberrant
expression or activity includes increased or decreased expression
or activity, as well as expression or activity which does not
follow the wild type developmental pattern of expression or the
subcellular pattern of expression. For example, aberrant MTR-3
expression or activity is intended to include the cases in which a
mutation in the MTR-3 gene causes the MTR-3 gene to be
under-expressed or over-expressed and situations in which such
mutations result in a non-functional MTR-3 protein or a protein
which does not function in a wild-type fashion, e.g., a protein
which does not interact with an MTR-3 ligand or substrate, or one
which interacts with a non-MTR-3 ligand or substrate. As used
herein, the term "unwanted" includes an unwanted phenomenon
involved in a biological response such as cellular proliferation.
For example, the term unwanted includes an MTR-3 expression pattern
or an MTR-3 protein activity which is undesirable in a subject,
e.g., differential (e.g., increased) expression of MTR-3 in tumors,
e.g., colon tumors or liver tumors.
[0722] The assays described herein, such as the preceding
diagnostic assays or the following assays, can be utilized to
identify a subject having or at risk of developing a disorder
associated with a misregulation in MTR-3 protein activity or
nucleic acid expression, such as a cellular growth or proliferation
disorder. Alternatively, the prognostic assays can be utilized to
identify a subject having or at risk for developing a cellular
growth or proliferation disorder, associated with a misregulation
in MTR-3 protein activity or nucleic acid expression. Thus, the
present invention provides a method for identifying a disorder
associated with aberrant or unwanted MTR-3 expression or activity
in which a test sample is obtained from a subject and MTR-3 protein
or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein
the presence of MTR-3 protein or nucleic acid is diagnostic for a
subject having or at risk of developing a disorder associated with
aberrant or unwanted MTR-3 expression or activity. As used herein,
a "test sample" refers to a biological sample obtained from a
subject of interest. For example, a test sample can be a biological
fluid (e.g., serum), cell sample, or tissue, e.g., tumor sample or
colon cell or tissue sample.
[0723] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
disorder associated with aberrant or unwanted MTR-3 expression or
activity. For example, such methods can be used to determine
whether a subject can be effectively treated with an agent for a
cellular growth or proliferation disorder. Thus, the present
invention provides methods for determining whether a subject can be
effectively treated with an agent for a cellular growth or
proliferation disorder, associated with aberrant or unwanted MTR-3
expression or activity in which a test sample is obtained and MTR-3
protein or nucleic acid expression or activity is detected (e.g.,
wherein the abundance of MTR-3 protein or nucleic acid expression
or activity is diagnostic for a subject that can be administered
the agent to treat a disorder associated with aberrant or unwanted
MTR-3 expression or activity).
[0724] The methods of the invention can also be used to detect
genetic alterations in an MTR-3 gene, thereby determining if a
subject with the altered gene is at risk for a disorder
characterized by misregulation in MTR-3 protein activity or nucleic
acid expression, such as a cellular growth or proliferation
disorder. In preferred embodiments, the methods include detecting,
in a sample of cells from the subject, the presence or absence of a
genetic alteration characterized by at least one of an alteration
affecting the integrity of a gene encoding an MTR-3-protein, or the
mis-expression of the MTR-3 gene. For example, such genetic
alterations can be detected by ascertaining the existence of at
least one of 1) a deletion of one or more nucleotides from an MTR-3
gene; 2) an addition of one or more nucleotides to an MTR-3 gene;
3) a substitution of one or more nucleotides of an MTR-3 gene, 4) a
chromosomal rearrangement of an MTR-3 gene; 5) an alteration in the
level of a messenger RNA transcript of an MTR-3 gene, 6) aberrant
modification of an MTR-3 gene, such as of the methylation pattern
of the genomic DNA, 7) the presence of a non-wild type splicing
pattern of a messenger RNA transcript of an MTR-3 gene, 8) a
non-wild type level of an MTR-3-protein, 9) allelic loss of an
MTR-3 gene, and 10) inappropriate post-translational modification
of an MTR-3-protein. As described herein, there are a large number
of assays known in the art which can be used for detecting
alterations in an MTR-3 gene. A preferred biological sample is a
tissue or serum sample isolated by conventional means from a
subject.
[0725] In certain embodiments, detection of the alteration involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364),
the latter of which can be particularly useful for detecting point
mutations in the MTR-3-gene (see Abravaya et al. (1995) Nucleic
Acids Res. 23:675-682). This method can include the steps of
collecting a sample of cells from a subject, isolating nucleic acid
(e.g., genomic, mRNA or both) from the cells of the sample,
contacting the nucleic acid sample with one or more primers which
specifically hybridize to an MTR-3 gene under conditions such that
hybridization and amplification of the MTR-3-gene (if present)
occurs, and detecting the presence or absence of an amplification
product, or detecting the size of the amplification product and
comparing the length to a control sample. It is anticipated that
PCR and/or LCR may be desirable to use as a preliminary
amplification step in conjunction with any of the techniques used
for detecting mutations described herein.
[0726] Other amplification methods include: 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.
[0727] In an alternative embodiment, mutations in an MTR-3 gene
from a sample cell can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
for example, U.S. Pat. No. 5,498,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[0728] In other embodiments, genetic mutations in MTR-3 can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, to high density arrays containing hundreds or thousands
of oligonucleotides probes (Cronin, M. T. et al. (1996) Human
Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2:
753-759). For example, genetic mutations in MTR-3 can be identified
in two dimensional arrays containing light-generated DNA probes as
described in Cronin, M. T. et al. supra. Briefly, a first
hybridization array of probes can be used to scan through long
stretches of DNA in a sample and control to identify base changes
between the sequences by making linear arrays of sequential
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene.
[0729] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
MTR-3 gene and detect mutations by comparing the sequence of the
sample MTR-3 with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques
developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA
74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It
is also contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
((1995) Biotechniques 19:448), including sequencing by mass
spectrometry (see, e.g., PCT International Publication No. WO
94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and
Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
[0730] Other methods for detecting mutations in the MTR-3 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230:1242). In general, the art technique of
"mismatch cleavage" starts by providing heteroduplexes of formed by
hybridizing (labeled) RNA or DNA containing the wild-type MTR-3
sequence with potentially mutant RNA or DNA obtained from a tissue
sample. The double-stranded duplexes are treated with an agent
which cleaves single-stranded regions of the duplex such as which
will exist due to basepair mismatches between the control and
sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically
digesting the mismatched regions. In other embodiments, either
DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or
osmium tetroxide and with piperidine in order to digest mismatched
regions. After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing polyacrylamide
gels to determine the site of mutation. See, for example, Cotton et
al. (1988) Proc. Natl. Acad Sci USA 85:4397; Saleeba et al. (1992)
Methods Enzymol. 217:286-295. In a preferred embodiment, the
control DNA or RNA can be labeled for detection.
[0731] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in MTR-3
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15:1657-1662). According to an exemplary
embodiment, a probe based on an MTR-3 sequence, e.g., a wild-type
MTR-3 sequence, is hybridized to a cDNA or other DNA product from a
test cell(s). The duplex is treated with a DNA mismatch repair
enzyme, and the cleavage products, if any, can be detected from
electrophoresis protocols or the like (described in, for example,
U.S. Pat. No. 5,459,039).
[0732] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in MTR-3 genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144;
and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79).
Single-stranded DNA fragments of sample and control MTR-3 nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al. (1991) Trends Genet 7:5).
[0733] In yet another embodiment the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265:12753).
[0734] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki
et al. (1989) Proc. Natl. Acad. Sci USA 86:6230). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA or a number of different mutations when the oligonucleotides
are attached to the hybridizing membrane and hybridized with
labeled target DNA.
[0735] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner (1993) Tibtech 11:238). In
addition it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88:189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0736] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving an MTR-3 gene.
[0737] Furthermore, any cell type or tissue in which MTR-3 is
expressed may be utilized in the prognostic assays described
herein.
Monitoring of Effects During Clinical Trials
[0738] The present invention provides methods for evaluating the
efficacy of drugs and monitoring the progress of patients involved
in clinical trials for the treatment of cellular growth or
proliferation disorders.
[0739] Monitoring the influence of compounds (e.g., drugs) on the
expression or activity of an MTR-3 protein (e.g., the modulation of
cell growth, proliferation and/or migration) can be applied not
only in basic drug screening, but also in clinical trials. For
example, the effectiveness of a compound determined by a screening
assay as described herein to increase MTR-3 gene expression,
protein levels, or upregulate MTR-3 activity, can be monitored in
clinical trials of subjects exhibiting decreased MTR-3 gene
expression, protein levels, or downregulated MTR-3 activity.
Alternatively, the effectiveness of an agent determined by a
screening assay to decrease MTR-3 gene expression, protein levels,
or downregulate MTR-3 activity, can be monitored in clinical trials
of subjects exhibiting increased MTR-3 gene expression, protein
levels, or upregulated MTR-3 activity. In such clinical trials, the
expression or activity of an MTR-3 gene, and preferably, other
genes that have been implicated in, for example, an
MTR-3-associated disorder can be used as a "read out" or markers of
the phenotype a particular cell, e.g., an endothelial cell or a
tumor cell. In addition, the expression of an MTR-3 gene, or the
level of MTR-3 protein activity may be used as a read out of a
particular drug or agent's effect on a cellular growth or
proliferation disorder.
[0740] For example, and not by way of limitation, genes, including
MTR-3, that are modulated in cells by treatment with an agent
(e.g., compound, drug or small molecule) which modulates MTR-3
activity (e.g., identified in a screening assay as described
herein) can be identified. Thus, to study the effect of agents on
MTR-3-associated disorders (e.g., cellular growth or proliferation
disorders), for example, in a clinical trial, cells can be isolated
and RNA prepared and analyzed for the levels of expression of MTR-3
and other genes implicated in the MTR-3-associated disorder,
respectively. The levels of gene expression (e.g., a gene
expression pattern) can be quantified by northern blot analysis or
RT-PCR, as described herein, or alternatively by measuring the
amount of protein produced, by one of the methods as described
herein, or by measuring the levels of activity of MTR-3 or other
genes. In this way, the gene expression pattern can serve as a
marker, indicative of the physiological response of the cells to
the agent. Accordingly, this response state may be determined
before, and at various points during treatment of the individual
with the agent.
[0741] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
including the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of an MTR-3 protein, mRNA, or genomic DNA
in the preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the MTR-3 protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the MTR-3 protein, mRNA, or
genomic DNA in the pre-administration sample with the MTR-3
protein, mRNA, or genomic DNA in the post administration sample or
samples; and (vi) altering the administration of the agent to the
subject accordingly. For example, increased administration of the
agent may be desirable to increase the expression or activity of
MTR-3 to higher levels than detected, i.e., to increase the
effectiveness of the agent. Alternatively, decreased administration
of the agent may be desirable to decrease expression or activity of
MTR-3 to lower levels than detected, i.e. to decrease the
effectiveness of the agent. According to such an embodiment, MTR-3
expression or activity may be used as an indicator of the
effectiveness of an agent, even in the absence of an observable
phenotypic response.
Methods of Treatment
[0742] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted MTR-3 expression or activity, e.g. a cellular
growth or proliferation disorder. With regards to both prophylactic
and therapeutic methods of treatment, such treatments may be
specifically tailored or modified, based on knowledge obtained from
the field of pharmacogenomics. "Pharmacogenomics", as used herein,
refers to the application of genomics technologies such as gene
sequencing, statistical genetics, and gene expression analysis to
drugs in clinical development and on the market. More specifically,
the term refers the study of how a patient's genes determine his or
her response to a drug (e.g., a patient's "drug response
phenotype", or "drug response genotype".) Thus, another aspect of
the invention provides methods for tailoring an individual's
prophylactic or therapeutic treatment with either the MTR-3
molecules of the present invention or MTR-3 modulators according to
that individual's drug response genotype. Pharmacogenomics allows a
clinician or physician to target prophylactic or therapeutic
treatments to patients who will most benefit from the treatment and
to avoid treatment of patients who will experience toxic
drug-related side effects.
Prophylactic Methods
[0743] In one aspect, the invention provides a method for
preventing in a subject, a cellular growth or proliferation
disorder associated with an aberrant or unwanted MTR-3 expression
or activity, by administering to the subject an MTR-3 or an agent
which modulates MTR-3 expression or at least one MTR-3 activity.
Subjects at risk for a cellular growth or proliferation disorder
which is caused or contributed to by aberrant or unwanted MTR-3
expression or activity can be identified by, for example, any or a
combination of diagnostic or prognostic assays as described herein.
Administration of a prophylactic agent can occur prior to the
manifestation of symptoms characteristic of the MTR-3 aberrancy,
such that a disorder is prevented or, alternatively, delayed in its
progression. Depending on the type of MTR-3 aberrancy, for example,
an MTR-3, MTR-3 agonist or MTR-3 antagonist agent can be used for
treating the subject. The appropriate agent can be determined based
on screening assays described herein.
Therapeutic Methods
[0744] Described herein are methods and compositions whereby
cellular growth or proliferation disorder symptoms may be
ameliorated. Certain cellular growth or proliferation disorders are
brought about, at least in part, by an excessive level of a 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 cellular growth or proliferation disorder symptoms.
Techniques for the reduction of gene expression levels or the
activity of a protein are discussed below.
[0745] Alternatively, certain other cellular growth or
proliferation disorders 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 protein's activity. As such, an
increase in the level of gene expression and/or the activity of
such proteins would bring about the amelioration of cellular growth
or proliferation disorder symptoms.
[0746] 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 gene's
expression, or the activity of the gene product, will reinforce the
protective effect it exerts. Some cellular growth or proliferation
disorder 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 cellular growth or
proliferation disorder symptoms. Techniques for increasing target
gene expression levels or target gene product activity levels are
discussed herein.
[0747] Accordingly, another aspect of the invention pertains to
methods of modulating MTR-3 expression or activity for therapeutic
purposes. Accordingly, in an exemplary embodiment, the modulatory
method of the invention involves contacting a cell with an MTR-3 or
agent that modulates one or more of the activities of MTR-3 protein
activity associated with the cell (e.g., an endothelial cell, such
as a colon cell, or a tumor cell). An agent that modulates MTR-3
protein activity can be an agent as described herein, such as a
nucleic acid or a protein, a naturally-occurring target molecule of
an MTR-3 protein (e.g., an MTR-3 ligand or substrate), an MTR-3
antibody, a MTR-3 agonist or antagonist, a peptidomimetic of a
MTR-3 agonist or antagonist, or other small molecule. In one
embodiment, the agent stimulates one or more MTR-3 activities.
Examples of such stimulatory agents include active MTR-3 protein
and a nucleic acid molecule encoding MTR-3 that has been introduced
into the cell. In another embodiment, the agent inhibits one or
more MTR-3 activities. Examples of such inhibitory agents include
antisense MTR-3 nucleic acid molecules, ribozymes, anti-MTR-3
antibodies, and MTR-3 inhibitors. These modulatory methods can be
performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g., by administering the agent to a
subject). As such, the present invention provides methods of
treating an individual afflicted with a disease or disorder
characterized by aberrant or unwanted expression or activity of a
MTR-3 protein or nucleic acid molecule. In one embodiment, the
method involves administering an agent (e.g., an agent identified
by a screening assay described herein), or combination of agents
that modulates (e.g., upregulates or downregulates) MTR-3
expression or activity. In another embodiment, the method involves
administering a MTR-3 protein or nucleic acid molecule as therapy
to compensate for reduced, aberrant, or unwanted MTR-3 expression
or activity, e.g., increased expression in tumors, e.g., colon
tumors.
[0748] Stimulation of MTR-3 activity is desirable in situations in
which MTR-3 is abnormally downregulated and/or in which increased
MTR-3 activity is likely to have a beneficial effect. Likewise,
inhibition of MTR-3 activity is desirable in situations in which
MTR-3 is abnormally upregulated and/or in which decreased MTR-3
activity is likely to have a beneficial effect.
Methods for Inhibiting Target Gene Expression, Synthesis, or
Activity
[0749] As discussed above, genes involved cellular growth or
proliferation disorders, including tumorigenic disorders, may cause
such disorders via an increased level of gene activity. In some
cases, such up-regulation may have a causative or exacerbating
effect on the disease state. A variety of techniques may be used to
inhibit the expression, synthesis, or activity of such genes and/or
proteins.
[0750] For example, compounds such as those identified through
assays described above, which exhibit inhibitory activity, may be
used in accordance with the invention to ameliorate cellular growth
or proliferation disease symptoms. Such molecules may include, but
are not limited to, small organic molecules, peptides, antibodies,
and the like.
[0751] For example, compounds can be administered that compete with
endogenous ligand for the MTR-3 protein. The resulting reduction in
the amount of ligand-bound MTR-3 protein will modulate endothelial
cell physiology. Compounds that can be particularly useful for this
purpose include, for example, soluble proteins or peptides or
portions and/or analogs thereof, of the MTR-3 protein, including,
for example, soluble fusion proteins such as Ig-tailed fusion
proteins. (For a discussion of the production of Ig-tailed fusion
proteins, see, for example, U.S. Pat. No. 5,116,964).
Alternatively, compounds, such as ligand analogs or antibodies,
that bind to MTR-3, but do not activate the protein can be
effective in inhibiting MTR-3 protein activity.
[0752] Further, antisense and ribozyme molecules which inhibit
expression of the MTR-3 genes of the present invention may also be
used in accordance with the invention to inhibit aberrant MTR-3
gene activity. Still further, triple helix molecules may be
utilized in inhibiting aberrant MTR-3 gene activity.
[0753] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a MTR-3 protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of
a route of administration of antisense nucleic acid molecules of
the invention include direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to
target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be
modified such that they specifically bind to receptors or antigens
expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies which
bind to cell surface receptors or antigens. The antisense nucleic
acid molecules can also be delivered to cells using the vectors
described herein. To achieve sufficient intracellular
concentrations of the antisense molecules, vector constructs in
which the antisense nucleic acid molecule is placed under the
control of a strong pol II or pol III promoter are preferred.
[0754] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0755] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591) or
hairpin ribozymes (described in Fedor (2000) J Mol Biol
297(2):269)) can be used to catalytically cleave MTR-3 mRNA
transcripts to thereby inhibit translation of MTR-3 mRNA. A
ribozyme having specificity for a MTR-3-encoding nucleic acid can
be designed based upon the nucleotide sequence of a MTR-3 cDNA
disclosed herein (i.e., SEQ ID NO:7 or 9). For example, a
derivative of a Tetrahymena L-19 IVS RNA can be constructed in
which the nucleotide sequence of the active site is complementary
to the nucleotide sequence to be cleaved in a MTR-3-encoding mRNA
(see, for example, Cech et al. U.S. Pat. No. 4,987,071; and Cech et
al. U.S. Pat. No. 5,116,742). Alternatively, MTR-3 mRNA can be used
to select a catalytic RNA having a specific ribonuclease activity
from a pool of RNA molecules (see, for example, Bartel, D. and
Szostak, J. W. (1993) Science 261:1411-1418).
[0756] MTR-3 gene expression can also be inhibited by targeting
nucleotide sequences complementary to the regulatory region of the
MTR-3 (e.g., the MTR-3 promoter and/or enhancers) to form triple
helical structures that prevent transcription of the MTR-3 gene in
target cells (see, for example, 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).
[0757] Antibodies that are both specific for the MTR-3 protein and
interfere with its activity may also be used to modulate or inhibit
MTR-3 protein function. Such antibodies may be generated using
standard techniques described herein, against the MTR-3 protein
itself or against peptides corresponding to portions of the
protein. Such antibodies include but are not limited to polyclonal,
monoclonal, Fab fragments, single chain antibodies, or chimeric
antibodies.
[0758] In instances where the target gene protein is intracellular
and whole antibodies are used, internalizing antibodies may be
preferred. Lipofectin liposomes may be used to deliver the antibody
or a fragment of the Fab region which binds to the target 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 (described in,
for example, Creighton (1983), supra; and Sambrook et al. (1989)
supra). 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. (1993) Proc.
Natl. Acad. Sci. USA 90:7889-7893).
[0759] In some instances, the target gene protein is extracellular,
or is a transmembrane protein. Antibodies that are specific for one
or more extracellular domains of the MTR-3 protein, for example,
and that interfere with its activity, are particularly useful in
treating cellular growth or proliferation disorders. Such
antibodies are especially efficient because they can access the
target domains directly from the bloodstream. Any of the
administration techniques described below which are appropriate for
peptide administration may be utilized to effectively administer
inhibitory target gene antibodies to their site of action.
Methods for Restoring or Enhancing Target Gene Activity
[0760] Genes that cause cellular growth or proliferation disorders
may be underexpressed within cellular growth or proliferation
disorder situations. Alternatively, the activity of the protein
products of such genes may be decreased, leading to the development
of cellular growth or proliferation disorder symptoms. Such
down-regulation of gene expression or decrease of protein activity
might have a causative or exacerbating effect on the disease
state.
[0761] In some cases, genes that are up-regulated in the disease
state might be exerting a protective effect. A variety of
techniques may be used to increase the expression, synthesis, or
activity of genes and/or proteins that exert a protective effect in
response to cellular growth or proliferation disorder
conditions.
[0762] Described in this section are methods whereby the level
MTR-3 activity may be increased to levels wherein cellular growth
or proliferation disorder symptoms are ameliorated. The level of
MTR-3 activity may be increased, for example, by either increasing
the level of MTR-3 gene expression or by increasing the level of
active MTR-3 protein which is present.
[0763] For example, a MTR-3 protein, at a level sufficient to
ameliorate cellular growth or proliferation disorder symptoms may
be administered to a patient exhibiting such symptoms. Any of the
techniques discussed below may be used for such administration. One
of skill in the art will readily know how to determine the
concentration of effective, non-toxic doses of the MTR-3 protein,
utilizing techniques such as those described below.
[0764] Additionally, RNA sequences encoding a MTR-3 protein may be
directly administered to a patient exhibiting cellular growth or
proliferation disorder symptoms, at a concentration sufficient to
produce a level of MTR-3 protein such that cellular growth or
proliferation disorder symptoms are ameliorated. Any of the
techniques discussed below, which achieve intracellular
administration of compounds, such as, for example, liposome
administration, may be used for the administration of such RNA
molecules. The RNA molecules may be produced, for example, by
recombinant techniques such as those described herein.
[0765] Further, subjects may be treated by gene replacement
therapy. One or more copies of a MTR-3 gene, or a portion thereof,
that directs the production of a normal MTR-3 protein with MTR-3
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 used for the introduction of MTR-3
gene sequences into human cells.
[0766] Cells, preferably, autologous cells, containing MTR-3
expressing gene sequences may then be introduced or reintroduced
into the subject at positions which allow for the amelioration of
cellular growth or proliferation disorder symptoms. Such cell
replacement techniques may be preferred, for example, when the gene
product is a secreted, extracellular gene product.
Pharmacogenomics
[0767] The MTR-3 molecules of the present invention, as well as
agents, or modulators which have a stimulatory or inhibitory effect
on MTR-3 activity (e.g., MTR-3 gene expression) as identified by a
screening assay described herein can be administered to individuals
to treat (prophylactically or therapeutically) MTR-3-associated
disorders (e.g., cellular growth and proliferation disorders)
associated with aberrant or unwanted MTR-3 activity. In conjunction
with such treatment, pharmacogenomics (i.e., the study of the
relationship between an individual's genotype and that individual's
response to a foreign compound or drug) may be considered.
Differences in metabolism of therapeutics can lead to severe
toxicity or therapeutic failure by altering the relation between
dose and blood concentration of the pharmacologically active drug.
Thus, a physician or clinician may consider applying knowledge
obtained in relevant pharmacogenomics studies in determining
whether to administer a MTR-3 molecule or a MTR-3 modulator as well
as tailoring the dosage and/or therapeutic regimen of treatment
with a MTR-3 molecule or MTR-3 modulator.
[0768] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin.
Chem. 43(2):254-266. In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body (altered
drug action) or genetic conditions transmitted as single factors
altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare genetic
defects or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0769] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[0770] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drugs
target is known (e.g., a MTR-3 protein of the present invention),
all common variants of that gene can be fairly easily identified in
the population and it can be determined if having one version of
the gene versus another is associated with a particular drug
response.
[0771] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[0772] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a MTR-3 molecule or MTR-3 modulator of the present
invention) can give an indication whether gene pathways related to
toxicity have been turned on.
[0773] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment an individual. This knowledge, when applied to dosing or
drug selection, can avoid adverse reactions or therapeutic failure
and thus enhance therapeutic or prophylactic efficiency when
treating a subject with a MTR-3 molecule or MTR-3 modulator, such
as a modulator identified by one of the exemplary screening assays
described herein.
Detection Assays
[0774] Portions or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. For example, these
sequences can be used to: (i) map their respective genes on a
chromosome; and, thus, locate gene regions associated with genetic
disease; (ii) identify an individual from a minute biological
sample (tissue typing); and (iii) aid in forensic identification of
a biological sample. These applications are described in the
subsections below.
Chromosome Mapping
[0775] Once the sequence (or a portion of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. This process is called chromosome
mapping. Accordingly, portions or fragments of the MTR-3 nucleotide
sequences, described herein, can be used to map the location of the
MTR-3 genes on a chromosome. The mapping of the MTR-3 sequences to
chromosomes is an important first step in correlating these
sequences with genes associated with disease.
[0776] Briefly, MTR-3 genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the
MTR-3 nucleotide sequences. Computer analysis of the MTR-3
sequences can be used to predict primers that do not span more than
one exon in the genomic DNA, thus complicating the amplification
process. These primers can then be used for PCR screening of
somatic cell hybrids containing individual human chromosomes. Only
those hybrids containing the human gene corresponding to the MTR-3
sequences will yield an amplified fragment.
[0777] Somatic cell hybrids are prepared by fusing somatic cells
from different mammals (e.g., human and mouse cells). As hybrids of
human and mouse cells grow and divide, they gradually lose human
chromosomes in random order, but retain the mouse chromosomes. By
using media in which mouse cells cannot grow, because they lack a
particular enzyme, but human cells can, the one human chromosome
that contains the gene encoding the needed enzyme, will be
retained. By using various media, panels of hybrid cell lines can
be established. Each cell line in a panel contains either a single
human chromosome or a small number of human chromosomes, and a full
set of mouse chromosomes, allowing easy mapping of individual genes
to specific human chromosomes. (D'Eustachio P. et al. (1983)
Science 220:919-924). Somatic cell hybrids containing only
fragments of human chromosomes can also be produced by using human
chromosomes with translocations and deletions.
[0778] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the MTR-3 nucleotide sequences to design
oligonucleotide primers, sublocalization can be achieved with
panels of fragments from specific chromosomes. Other mapping
strategies which can similarly be used to map a MTR-3 sequence to
its chromosome include in situ hybridization (described in Fan, Y.
et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),
pre-screening with labeled flow-sorted chromosomes, and
pre-selection by hybridization to chromosome specific cDNA
libraries.
[0779] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. Chromosome spreads can be
made using cells whose division has been blocked in metaphase by a
chemical such as colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then stained
with Giemsa. A pattern of light and dark bands develops on each
chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500
or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location with
sufficient signal intensity for simple detection. Preferably 1,000
bases, and more preferably 2,000 bases will suffice to get good
results at a reasonable amount of time. For a review of this
technique, see Verma et al., Human Chromosomes: A Manual of Basic
Techniques (Pergamon Press, New York 1988).
[0780] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0781] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland, J. et al. (1987) Nature, 325:783-787.
[0782] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the MTR-3 gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
Tissue Typing
[0783] The MTR-3 sequences of the present invention can also be
used to identify individuals from minute biological samples. The
United States military, for example, is considering the use of
restriction fragment length polymorphism (RFLP) for identification
of its personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identification. This method
does not suffer from the current limitations of "Dog Tags" which
can be lost, switched, or stolen, making positive identification
difficult. The sequences of the present invention are useful as
additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0784] Furthermore, the sequences of the present invention can be
used to provide an alternative technique which determines the
actual base-by-base DNA sequence of selected portions of an
individual's genome. Thus, the MTR-3 nucleotide sequences described
herein can be used to prepare two PCR primers from the 5' and 3'
ends of the sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0785] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
present invention can be used to obtain such identification
sequences from individuals and from tissue. The MTR-3 nucleotide
sequences of the invention uniquely represent portions of the human
genome. Allelic variation occurs to some degree in the coding
regions of these sequences, and to a greater degree in the
noncoding regions. It is estimated that allelic variation between
individual humans occurs with a frequency of about once per each
500 bases. Each of the sequences described herein can, to some
degree, be used as a standard against which DNA from an individual
can be compared for identification purposes. Because greater
numbers of polymorphisms occur in the noncoding regions, fewer
sequences are necessary to differentiate individuals. The noncoding
sequences of MTR-3 gene sequences can comfortably provide positive
individual identification with a panel of perhaps 10 to 1,000
primers which each yield a noncoding amplified sequence of 100
bases. If predicted coding sequences, such as those in SEQ ID NO:7
are used, a more appropriate number of primers for positive
individual identification would be 500-2,000.
[0786] If a panel of reagents from MTR-3 nucleotide sequences
described herein is used to generate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
Use of Partial MTR-3 Sequences in Forensic Biology
[0787] DNA-based identification techniques can also be used in
forensic biology. Forensic biology is a scientific field employing
genetic typing of biological evidence found at a crime scene as a
means for positively identifying, for example, a perpetrator of a
crime. To make such an identification, PCR technology can be used
to amplify DNA sequences taken from very small biological samples
such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, or semen found at a crime scene. The amplified sequence can
then be compared to a standard, thereby allowing identification of
the origin of the biological sample.
[0788] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
noncoding regions of MTR-3 gene sequences are particularly
appropriate for this use as greater numbers of polymorphisms occur
in the noncoding regions, making it easier to differentiate
individuals using this technique. Examples of polynucleotide
reagents include the MTR-3 nucleotide sequences or portions
thereof, e.g., fragments derived from the noncoding regions having
a length of at least 20 bases, preferably at least 30 bases.
[0789] The MTR-3 nucleotide sequences described herein can further
be used to provide polynucleotide reagents, e.g., labeled or
labelable probes which can be used in, for example, an in situ
hybridization technique, to identify a specific tissue, e.g., brain
tissue. This can be very useful in cases where a forensic
pathologist is presented with a tissue of unknown origin. Panels of
such MTR-3 probes can be used to identify tissue by species and/or
by organ type.
[0790] In a similar fashion, these reagents, e.g., MTR-3 primers or
probes can be used to screen tissue culture for contamination (i.e.
screen for the presence of a mixture of different types of cells in
a culture).
Recombinant Expression Vectors and Host Cells
[0791] Another aspect of the invention pertains to vectors, for
example recombinant expression vectors, containing a nucleic acid
containing an MTR-3 nucleic acid molecule or vectors containing a
nucleic acid molecule which encodes an MTR-3 polypeptide (or a
portion thereof). As used herein, the term "vector" refers to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments can be ligated. Another type of vector is a
viral vector, wherein additional DNA segments can be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) are integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "expression
vectors". In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" can be used interchangeably
as the plasmid is the most commonly used form of vector. However,
the methods of the invention may include other forms of expression
vectors, such as viral vectors (e.g., replication defective
retroviruses, adenoviruses and adeno-associated viruses), which
serve equivalent functions.
[0792] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operatively linked to the nucleic acid
sequence to be expressed. Within a recombinant expression vector,
"operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
which allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cells and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, and the
like. The expression vectors of the invention can be introduced
into host cells to thereby produce proteins or peptides, including
fusion proteins or peptides, encoded by nucleic acids as described
herein (e.g., MTR-3 proteins, mutant forms of MTR-3 proteins,
fusion proteins, and the like).
[0793] Accordingly, an exemplary embodiment provides a method for
producing a polypeptide, preferably an MTR-3 polypeptide, by
culturing in a suitable medium a host cell of the invention (e.g.,
a mammalian host cell such as a non-human mammalian cell)
containing a recombinant expression vector, such that the
polypeptide is produced.
[0794] The recombinant expression vectors of the invention can be
designed for expression of MTR-3 proteins in prokaryotic or
eukaryotic cells, e.g., for use in the cell-based assays of the
invention. For example, MTR-3 proteins can be expressed in
bacterial cells such as E. coli, insect cells (using baculovirus
expression vectors) yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0795] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein.
[0796] Purified fusion proteins can be utilized in MTR-3 activity
assays, (e.g., direct assays or competitive assays described in
detail below), or to generate antibodies specific for MTR-3
proteins, for example. In a preferred embodiment, a MTR-3 fusion
protein expressed in a retroviral expression vector of the present
invention can be utilized to infect bone marrow cells which are
subsequently transplanted into irradiated recipients. The pathology
of the subject recipient is then examined after sufficient time has
passed (e.g., six (6) weeks).
[0797] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
prophage harboring a T7 gn1 gene under the transcriptional control
of the lacUV 5 promoter.
[0798] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid
to be inserted into an expression vector so that the individual
codons for each amino acid are those preferentially utilized in E.
coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
[0799] In another embodiment, the MTR-3 expression vector is a
yeast expression vector. Examples of vectors for expression in
yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO
J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell
30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2
(Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen
Corp, San Diego, Calif.).
[0800] Alternatively, MTR-3 proteins can be expressed in insect
cells using baculovirus expression vectors. Baculovirus vectors
available for expression of proteins in cultured insect cells
(e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol.
Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers
(1989) Virology 170:31-39).
[0801] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)
EMBO J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd,
ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989.
[0802] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), endothelial cell-specific promoters
(e.g., KDR/flk promoter; U.S. Pat. No. 5,888,765),
pancreas-specific promoters (Edlund et al. (1985) Science
230:912-916), and mammary gland-specific promoters (e.g., milk whey
promoter; U.S. Pat. No. 4,873,316 and European Application
Publication No. 264,166). Developmentally-regulated promoters are
also encompassed, for example the murine hox promoters (Kessel and
Gruss (1990) Science 249:374-379) and the .alpha.-fetoprotein
promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
[0803] The expression characteristics of an endogenous MTR-3 gene
within a cell line or microorganism may be modified by inserting a
heterologous DNA regulatory element into the genome of a stable
cell line or cloned microorganism such that the inserted regulatory
element is operatively linked with the endogenous MTR-3 gene. For
example, an endogenous MTR-3 gene which is normally
"transcriptionally silent", i.e., a MTR-3 gene which is normally
not expressed, or is expressed only at very low levels in a cell
line or microorganism, may be activated by inserting a regulatory
element which is capable of promoting the expression of a normally
expressed gene product in that cell line or microorganism.
Alternatively, a transcriptionally silent, endogenous MTR-3 gene
may be activated by insertion of a promiscuous regulatory element
that works across cell types.
[0804] A heterologous regulatory element may be inserted into a
stable cell line or cloned microorganism, such that it is
operatively linked with an endogenous MTR-3 gene, using techniques,
such as targeted homologous recombination, which are well known to
those of skill in the art, and described, e.g., in Chappel, U.S.
Pat. No. 5,272,071; PCT publication No. WO 91/06667, published May
16, 1991.
[0805] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to MTR-3 mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0806] Another aspect of the invention pertains to the use of host
cells into which a MTR-3 nucleic acid molecule of the invention is
introduced, e.g., a MTR-3 nucleic acid molecule within a
recombinant expression vector or a MTR-3 nucleic acid molecule
containing sequences which allow it to homologously recombine into
a specific site of the host cell's genome. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is
understood that such terms refer not only to the particular subject
cell but to the progeny or potential progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term as used herein.
[0807] A host cell can be any prokaryotic or eukaryotic cell. For
example, a MTR-3 protein can be expressed in bacterial cells such
as E. coli, insect cells, yeast or mammalian cells (such as human
umbilical vein endothelial cells (HUVEC), human microvascular
endothelial cells (HMVEC), Chinese hamster ovary cells (CHO), human
ovarian surface epithelial (HOSE) cells, or COS cells). Other
suitable host cells are known to those skilled in the art.
[0808] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0809] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin, puromycin, zeomycin and methotrexate. Nucleic acid
encoding a selectable marker can be introduced into a host cell on
the same vector as that encoding a MTR-3 protein or can be
introduced on a separate vector. Cells stably transfected with the
introduced nucleic acid can be identified by drug selection (e.g.,
cells that have incorporated the selectable marker gene will
survive, while the other cells die).
[0810] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) a MTR-3 protein. Accordingly, the invention further
provides methods for producing a MTR-3 protein using the host cells
of the invention. In one embodiment, the method comprises culturing
the host cell of the invention (into which a recombinant expression
vector encoding a MTR-3 protein has been introduced) in a suitable
medium such that a MTR-3 protein is produced. In another
embodiment, the method further comprises isolating a MTR-3 protein
from the medium or the host cell.
Cell- and Animal-Based Model Systems
[0811] Described herein are cell- and animal-based systems which
act as models for cellular growth or proliferation disorders. 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 genes associated with
cellular growth or proliferation disorder, e.g., MTR-3. In
addition, animal- and cell-based assays may be used as part of
screening strategies designed to identify compounds which are
capable of ameliorating cellular growth or proliferation disorder
symptoms, as described, below. Thus, the animal- and cell-based
models may be used to identify drugs, pharmaceuticals, therapies
and interventions which may be effective in treating cellular
growth or proliferation disorders.
Animal-Based Systems
[0812] Animal-based model systems of cellular growth or
proliferation disorders may include, but are not limited to,
non-recombinant and engineered transgenic animals.
[0813] Animal based models for studying tumorigenesis in vivo are
well known in the art (reviewed in Animal Models of Cancer
Predisposition Syndromes, Hiai, H and Hino, O (eds.) 1999, Progress
in Experimental Tumor Research, Vol. 35; Clarke A R Carcinogenesis
(2000) 21:435-41) and include, for example, carcinogen-induced
tumors (Rithidech, K et al. Mutat Res (1999) 428:33-39; Miller, M L
et al. Environ Mol Mutagen (2000) 35:319-327), injection and/or
transplantation of tumor cells into an animal, as well as animals
bearing mutations in growth regulatory genes, for example,
oncogenes (e.g., ras) (Arbeit, J M et al. Am J Pathol (1993)
142:1187-1197; Sinn, E et al. Cell (1987) 49:465-475; Thorgeirsson,
S S et al. Toxicol Lett (2000) 112-113:553-555) and tumor
suppressor genes (e.g., p53) (Vooijs, M et al. Oncogene (1999)
18:5293-5303; Clark A R Cancer Metast Rev (1995) 14:125-148; Kumar,
T R et al. J Intern Med (1995) 238:233-238; Donehower, L A et al.
(1992) Nature 356215-221). Furthermore, experimental model systems
are available for the study of, for example, colon cancer (Heyer J,
et al. (1999) Oncogene 18(38):5325-33), ovarian cancer (Hamilton, T
C et al. Semin Oncol (1984) 11:285-298; Rahman, N A et al. Mol Cell
Endocrinol (1998) 145:167-174; Beamer, W G et al. Toxicol Pathol
(1998) 26:704-710), gastric cancer (Thompson, J et al. Int J Cancer
(2000) 86:863-869; Fodde, R et al. Cytogenet Cell Genet (1999)
86:105-111), breast cancer (Li, M et al. Oncogene (2000)
19:1010-1019; Green, J E et al. Oncogene (2000) 19:1020-1027),
melanoma (Satyamoorthy, K et al. Cancer Metast Rev (1999)
18:401-405), and prostate cancer (Shirai, T et al. Mutat Res (2000)
462:219-226; Bostwick, D G et al. Prostate (2000) 43:286-294).
[0814] Additionally, animal models exhibiting cellular growth or
proliferation disorder symptoms may be engineered by using, for
example, MTR-3 gene sequences described above, in conjunction with
techniques for producing transgenic animals that are well known to
those of skill in the art. For example, MTR-3 gene sequences may be
introduced into, and overexpressed in, the genome of the animal of
interest, or, if endogenous MTR-3 gene sequences are present, they
may either be overexpressed or, alternatively, be disrupted in
order to underexpress or inactivate MTR-3 gene expression, such as
described for the disruption of apoE in mice (Plump et al., 1992,
Cell 71: 343-353).
[0815] The host cells of the invention can also be used to produce
non-human transgenic animals. For example, in one embodiment, a
host cell of the invention is a fertilized oocyte or an embryonic
stem cell into which MTR-3-coding sequences have been introduced.
Such host cells can then be used to create non-human transgenic
animals in which exogenous MTR-3 sequences have been introduced
into their genome or homologous recombinant animals in which
endogenous MTR-3 sequences have been altered. Such animals are
useful for studying the function and/or activity of a MTR-3 and for
identifying and/or evaluating modulators of MTR-3 activity. As used
herein, a "transgenic animal" is a non-human animal, preferably a
mammal, more preferably a rodent such as a rat or mouse, in which
one or more of the cells of the animal includes a transgene. Other
examples of transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, amphibians, and the like. A transgene
is exogenous DNA which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal, thereby directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal. As used herein, a "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably
a mouse, in which an endogenous MTR-3 gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0816] A transgenic animal of the invention can be created by
introducing a MTR-3-encoding nucleic acid into the male pronuclei
of a fertilized oocyte, e.g., by microinjection, retroviral
infection, and allowing the oocyte to develop in a pseudopregnant
female foster animal. The MTR-3 cDNA sequence of SEQ ID NO:7 or 9
can be introduced as a transgene into the genome of a non-human
animal. Alternatively, a nonhuman homologue of a human MTR-3 gene,
such as a mouse or rat MTR-3 gene, can be used as a transgene.
Alternatively, a MTR-3 gene homologue, such as another MTR-3 family
member, can be isolated based on hybridization to the MTR-3 cDNA
sequences of SEQ ID NO:7 or 9 and used as a transgene. Intronic
sequences and polyadenylation signals can also be included in the
transgene to increase the efficiency of expression of the
transgene. A tissue-specific regulatory sequence(s) can be operably
linked to a MTR-3 transgene to direct expression of a MTR-3 protein
to particular cells. Methods for generating transgenic animals via
embryo manipulation and microinjection, particularly animals such
as mice, have become conventional in the art and are described, for
example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder
et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of a MTR-3
transgene in its genome and/or expression of MTR-3 mRNA in tissues
or cells of the animals. A transgenic founder animal can then be
used to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene encoding a MTR-3 protein
can further be bred to other transgenic animals carrying other
transgenes.
[0817] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of a MTR-3 gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the MTR-3 gene. The
MTR-3 gene can be a human gene (e.g., the cDNA of SEQ ID NO:7 or
9), but more preferably, is a non-human homologue of a human MTR-3
gene (e.g., a cDNA isolated by stringent hybridization with the
nucleotide sequence of SEQ ID NO:7 or 9). For example, a mouse
MTR-3 gene can be used to construct a homologous recombination
nucleic acid molecule, e.g., a vector, suitable for altering an
endogenous MTR-3 gene in the mouse genome. In a preferred
embodiment, the homologous recombination nucleic acid molecule is
designed such that, upon homologous recombination, the endogenous
MTR-3 gene is functionally disrupted (i.e., no longer encodes a
functional protein; also referred to as a "knock out" vector).
Alternatively, the homologous recombination nucleic acid molecule
can be designed such that, upon homologous recombination, the
endogenous MTR-3 gene is mutated or otherwise altered but still
encodes functional protein (e.g., the upstream regulatory region
can be altered to thereby alter the expression of the endogenous
MTR-3 protein). In the homologous recombination nucleic acid
molecule, the altered portion of the MTR-3 gene is flanked at its
5' and 3' ends by additional nucleic acid sequence of the MTR-3
gene to allow for homologous recombination to occur between the
exogenous MTR-3 gene carried by the homologous recombination
nucleic acid molecule and an endogenous MTR-3 gene in a cell, e.g.,
an embryonic stem cell. The additional flanking MTR-3 nucleic acid
sequence is of sufficient length for successful homologous
recombination with the endogenous gene. Typically, several
kilobases of flanking DNA (both at the 5' and 3' ends) are included
in the homologous recombination nucleic acid molecule (see, e.g.,
Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a
description of homologous recombination vectors). The homologous
recombination nucleic acid molecule is introduced into a cell,
e.g., an embryonic stem cell line (e.g., by electroporation) and
cells in which the introduced MTR-3 gene has homologously
recombined with the endogenous MTR-3 gene are selected (see e.g.,
Li, E. et al. (1992) Cell 69:915). The selected cells can then
injected into a blastocyst of an animal (e.g., a mouse) to form
aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and
Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.
(IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be
implanted into a suitable pseudopregnant female foster animal and
the embryo brought to term. Progeny harboring the homologously
recombined DNA in their germ cells can be used to breed animals in
which all cells of the animal contain the homologously recombined
DNA by germline transmission of the transgene. Methods for
constructing homologous recombination nucleic acid molecules, e.g.,
vectors, or homologous recombinant animals are described further in
Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and
in PCT International Publication Nos.: WO 90/11354 by Le Mouellec
et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et
al.; and WO 93/04169 by Berns et al.
[0818] In another embodiment, transgenic non-human animals of the
invention can be produced which contain selected systems which
allow for regulated expression of the transgene. One example of
such a system is the cre/loxP recombinase system of bacteriophage
P1. For a description of the cre/loxP recombinase system, see,
e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236.
Another example of a recombinase system is the FLP recombinase
system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science
251:1351-1355. If a cre/loxP recombinase system is used to regulate
expression of the transgene, animals containing transgenes encoding
both the Cre recombinase and a selected protein are required. Such
animals can be provided through the construction of "double"
transgenic animals, e.g., by mating two transgenic animals, one
containing a transgene encoding a selected protein and the other
containing a transgene encoding a recombinase.
[0819] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. (1997) Nature 385:810-813 and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.o phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyte and then transferred to pseudopregnant female
foster animal. The offspring borne of this female foster animal
will be a clone of the animal from which the cell, e.g., the
somatic cell, is isolated.
[0820] The MTR-3 transgenic animals that express MTR-3 mRNA or a
MTR-3 peptide (detected immunocytochemically, using antibodies
directed against MTR-3 epitopes) at easily detectable levels should
then be further evaluated to identify those animals which display
characteristic cellular growth or proliferation disorder symptoms.
Tumorigenic disease symptoms include, for example, tumor burden,
invasion and/or metastasis.
[0821] Additionally, specific cell types (e.g., tumor cells, colon
cells) within the transgenic animals may be analyzed and assayed
for cellular phenotypes characteristic of cellular growth or
proliferation disorders. In the case of endothelial cells, such
phenotypes include, but are not limited to cell proliferation,
growth and migration. Cellular phenotypes associated with a
tumorigenic disorder include, for example, dysregulated
proliferation and migration, anchorage independent growth, and loss
of contact inhibition. Cellular phenotypes may include a particular
cell type's pattern of expression of genes associated with cellular
growth or proliferation disorders as compared to known expression
profiles of the particular cell type in animals exhibiting cellular
growth or proliferation disorder symptoms.
Cell-Based Systems
[0822] Cells that contain and express MTR-3 gene sequences which
encode a MTR-3 protein, and, further, exhibit cellular phenotypes
associated with cellular growth or proliferation disorders, may be
used to identify compounds that exhibit anti-tumorigenic disease
activity. Such cells may include endothelial cells such as human
umbilical vein endothelial cells (HUVECs), human microvascular
endothelial cells (HMVEC); tumor cell lines such as HT-1080 (ATCC#
CCL-121), HCT-15 (ATCC# CCL-225), HCC70 (ATCC# CRL-2315), M059J
(ATCC# CRL-2366), and NCI-N417 (ATCC# CRL-5809); 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 cellular growth or
proliferation disorder animal models of the invention, discussed
above, may be used to generate cell lines, containing one or more
cell types involved in cellular growth or proliferation disorders,
that can be used as cell culture models for this disorder. While
primary cultures derived from the cellular growth or proliferation
disorder 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.
[0823] Alternatively, cells of a cell type known to be involved in
cellular growth or proliferation disorders may be transfected with
sequences capable of increasing or decreasing the amount of MTR-3
gene expression within the cell. For example, MTR-3 gene sequences
may be introduced into, and overexpressed in, the genome of the
cell of interest, or, if endogenous MTR-3 gene sequences are
present, they may be either overexpressed or, alternatively
disrupted in order to underexpress or inactivate MTR-3 gene
expression.
[0824] In order to overexpress an MTR-3 gene, the coding portion of
the MTR-3 gene may be ligated to a regulatory sequence which is
capable of driving gene expression in the cell type of interest,
e.g., a tumor cell or a colon cell. 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 above.
[0825] For underexpression of an endogenous MTR-3 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 MTR-3 alleles will be inactivated. Preferably, the
engineered MTR-3 sequence is introduced via gene targeting such
that the endogenous MTR-3 sequence is disrupted upon integration of
the engineered MTR-3 sequence into the cell's genome. Transfection
of host cells with MTR-3 genes is discussed, above.
[0826] Cells treated with compounds or transfected with MTR-3 genes
can be examined for phenotypes associated with cellular growth or
proliferation disorders. Cells (e.g., tumor cells) can be treated
with test compounds or transfected with genetically engineered
MTR-3 genes and examined for phenotypes associated with tumorigenic
disease, including, but not limited to changes in cellular
morphology, cell proliferation, cell migration, cell
transformation, anchorage independent growth, and loss of contact
inhibition.
[0827] Transfection of MTR-3 nucleic acid may be accomplished by
using standard techniques (described in, for example, Ausubel
(1989) supra). Transfected cells should be evaluated for the
presence of the recombinant MTR-3 gene sequences, for expression
and accumulation of MTR-3 mRNA, and for the presence of recombinant
MTR-3 protein production. In instances wherein a decrease in MTR-3
gene expression is desired, standard techniques may be used to
demonstrate whether a decrease in endogenous MTR-3 gene expression
and/or in MTR-3 protein production is achieved.
[0828] Cellular models for the study of tumorigenesis are known in
the art, and include cell lines derived from clinical tumors, cells
exposed to chemotherapeutic agents, cells exposed to carcinogenic
agents, and cell lines with genetic alterations in growth
regulatory genes, for example, oncogenes (e.g., ras) and tumor
suppressor genes (e.g., p53).
Pharmaceutical Compositions
[0829] Active compounds of the invention can be incorporated into
pharmaceutical compositions suitable for administration. As used
herein, the language "active compounds" includes MTR-3 nucleic acid
molecules, fragments of MTR-3 proteins, and anti-MTR-3 antibodies,
as well as identified compounds that modulate MTR-3 gene
expression, synthesis, and/or activity. Such compositions typically
comprise the compound, nucleic acid molecule, protein, or antibody
and a pharmaceutically acceptable carrier. As used herein the
language "pharmaceutically acceptable carrier" is intended to
include any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical
administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0830] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0831] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0832] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a fragment of a MTR-3
protein or a MTR-3 substrate) in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0833] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0834] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0835] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0836] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0837] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0838] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals. In one embodiment, a therapeutically effective dose
refers to that amount of an active compound sufficient to result in
amelioration of symptoms of cellular growth or proliferation
disorders.
[0839] 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 LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. 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.
[0840] 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 ED50 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
IC50 (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.
[0841] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
skilled artisan will appreciate that certain factors may influence
the dosage required to effectively treat a subject, including but
not limited to the severity of the disease or disorder, previous
treatments, the general health and/or age of the subject, and other
diseases present. Moreover, treatment of a subject with a
therapeutically effective amount of a protein, polypeptide, or
antibody can include a single treatment or, preferably, can include
a series of treatments.
[0842] In a preferred example, a subject is treated with antibody,
protein, or polypeptide in the range of between about 0.1 to 20
mg/kg body weight, one time per week for between about 1 to 10
weeks, preferably between 2 to 8 weeks, more preferably between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. It will also be appreciated that the effective dosage of
antibody, protein, or polypeptide used for treatment may increase
or decrease over the course of a particular treatment. Changes in
dosage may result and become apparent from the results of
diagnostic assays as described herein.
[0843] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds. It is understood that appropriate doses of small
molecule agents depends upon a number of factors within the ken of
the ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention.
[0844] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. Such appropriate doses may be determined using the
assays described herein. When one or more of these small molecules
is to be administered to an animal (e.g., a human) in order to
modulate expression or activity of a polypeptide or nucleic acid of
the invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0845] In certain embodiments of the invention, a modulator of
MTR-3 activity is administered in combination with other agents
(e.g., a small molecule), or in conjunction with another,
complementary treatment regime. For example, in one embodiment, a
modulator of MTR-3 activity is used to treat a tumorigenic
disorder, e.g., colon cancer. Accordingly, modulation of MTR-3
activity may be used in conjunction with, for example,
chemotherapeutic agents and/or radiation treatment.
[0846] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (CDDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0847] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
.alpha.-interferon, .beta.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[0848] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Helistrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2.sup.nd Ed.),
Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);
Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
[0849] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[0850] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
Isolated Nucleic Acid Molecules
[0851] One aspect of the invention pertains to isolated nucleic
acid molecules that encode MTR-3 proteins or biologically active
portions thereof, as well as nucleic acid fragments sufficient for
use as hybridization probes to identify MTR-3-encoding nucleic acid
molecules (e.g., MTR-3 mRNA) and fragments for use as PCR primers
for the amplification or mutation of MTR-3 nucleic acid molecules.
As used herein, the term "nucleic acid molecule" is intended to
include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules
(e.g., mRNA) and analogs of the DNA or RNA generated using
nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0852] The term "isolated nucleic acid molecule" includes nucleic
acid molecules which are separated from other nucleic acid
molecules which are present in the natural source of the nucleic
acid. For example, with regards to genomic DNA, the term "isolated"
includes nucleic acid molecules which are separated from the
chromosome with which the genomic DNA is naturally associated.
Preferably, an "isolated" nucleic acid is free of sequences which
naturally flank the nucleic acid (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. For example, in various
embodiments, the isolated MTR-3 nucleic acid molecule can contain
less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of
nucleotide sequences which naturally flank the nucleic acid
molecule in genomic DNA of the cell from which the nucleic acid is
derived. Moreover, an "isolated" nucleic acid molecule, such as a
cDNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized.
[0853] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:7
or 9, or a portion thereof, can be isolated using standard
molecular biology techniques and the sequence information provided
herein. Using all or a portion of the nucleic acid sequence of SEQ
ID NO:7 or 9 as hybridization probes, MTR-3 nucleic acid molecules
can be isolated using standard hybridization and cloning techniques
(e.g., as described in Sambrook, J. et al. Molecular Cloning: A
Laboratory Manual. 2.sup.nd, ed., Cold Spring Harbor Laboratory,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989).
[0854] Moreover, a nucleic acid molecule encompassing all or a
portion of SEQ ID NO:7 or 9, can be isolated by the polymerase
chain reaction (PCR) using synthetic oligonucleotide primers
designed based upon the sequence of SEQ ID NO:7 or 9.
[0855] A nucleic acid of the invention can be amplified using cDNA,
mRNA or alternatively, genomic DNA, as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to MTR-3 nucleotide
sequences can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0856] In one embodiment, an isolated nucleic acid molecule of the
invention comprises the nucleotide sequence shown in SEQ ID NO:7 or
9. This cDNA may comprise sequences encoding the human MTR-3
protein (e.g., the "coding region", from nucleotides), as well as
5' untranslated sequence (nucleotides 1-39) and 3' untranslated
sequences (nucleotides 1864-2898) of SEQ ID NO:7. Alternatively,
the nucleic acid molecule can comprise only the coding region of
SEQ ID NO:7 (e.g., nucleotides 40-1863, corresponding to SEQ ID
NO:9). Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention comprises SEQ ID NO:9 and nucleotides
1-39 of SEQ ID NO:9. In yet another embodiment, the isolated
nucleic acid molecule comprises SEQ ID NO:9 and nucleotides
1864-2898 of SEQ ID NO:7. In yet another embodiment, the nucleic
acid molecule consists of the nucleotide sequence set forth as SEQ
ID NO:7 or SEQ ID NO:9.
[0857] In still another embodiment, an isolated nucleic acid
molecule of the invention comprises a nucleic acid molecule which
is a complement of the nucleotide sequence shown in SEQ ID NO:7 or
9, or a portion of any of these nucleotide sequences. A nucleic
acid molecule which is complementary to the nucleotide sequence
shown in SEQ ID NO:7 or 9, is one which is sufficiently
complementary to the nucleotide sequence shown in SEQ ID NO:7 or 9,
such that it can hybridize to the nucleotide sequence shown in SEQ
ID NO:7 or 9, thereby forming a stable duplex.
[0858] In still another embodiment, an isolated nucleic acid
molecule of the present invention comprises a nucleotide sequence
which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to the nucleotide sequence shown in SEQ ID NO:7 or 9 (e.g., to the
entire length of the nucleotide sequence), or a portion or
complement of any of these nucleotide sequences. In one embodiment,
a nucleic acid molecule of the present invention comprises a
nucleotide sequence which is at least (or no greater than) 50-100,
100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500,
1500-1750, 1750-2000, 2000-2250, 2250-2500, 2500-2750, 2750-3000,
3000-3210 or more nucleotides in length and hybridizes under
stringent hybridization conditions to a complement of a nucleic
acid molecule of SEQ ID NO:7 or 9.
[0859] Moreover, the nucleic acid molecule of the invention can
comprise only a portion of the nucleic acid sequence of SEQ ID NO:7
or 9, for example, a fragment which can be used as a probe or
primer or a fragment encoding a portion of an MTR-3 protein, e.g.,
a biologically active portion of an MTR-3 protein. The nucleotide
sequence determined from the cloning of the MTR-3 gene allows for
the generation of probes and primers designed for use in
identifying and/or cloning other MTR-3 family members, as well as
MTR-3 homologues from other species. The probe/primer (e.g.,
oligonucleotide) typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12 or 15, preferably about 20 or 25, more
preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive
nucleotides of a sense sequence of SEQ ID NO:7 or 9, of an
anti-sense sequence of SEQ ID NO:7 or 9, or of a naturally
occurring allelic variant or mutant of SEQ ID NO:7 or 9.
[0860] Exemplary probes or primers are at least (or no greater
than) 12 or 15, 20 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or
more nucleotides in length and/or comprise consecutive nucleotides
of an isolated nucleic acid molecule described herein. Also
included within the scope of the present invention are probes or
primers comprising contiguous or consecutive nucleotides of an
isolated nucleic acid molecule described herein, but for the
difference of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases within the
probe or primer sequence. Probes based on the MTR-3 nucleotide
sequences can be used to detect (e.g., specifically detect)
transcripts or genomic sequences encoding the same or homologous
proteins. In preferred embodiments, the probe further comprises a
label group attached thereto, e.g., the label group can be a
radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. In another embodiment a set of primers is provided,
e.g., primers suitable for use in a PCR, which can be used to
amplify a selected region of an MTR-3 sequence, e.g., a domain,
region, site or other sequence described herein. The primers should
be at least 5, 10, or 50 base pairs in length and less than 100, or
less than 200, base pairs in length. The primers should be
identical, or differ by no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10 bases when compared to a sequence disclosed herein or to the
sequence of a naturally occurring variant. Such probes can be used
as a part of a diagnostic test kit for identifying cells or tissue
which misexpress an MTR-3 protein, such as by measuring a level of
an MTR-3-encoding nucleic acid in a sample of cells from a subject,
e.g., detecting MTR-3 mRNA levels or determining whether a genomic
MTR-3 gene has been mutated or deleted.
[0861] A nucleic acid fragment encoding a "biologically active
portion of an MTR-3 protein" can be prepared by isolating a portion
of the nucleotide sequence of SEQ ID NO:7 or 9, which encodes a
polypeptide having an MTR-3 biological activity (the biological
activities of the MTR-3 proteins are described herein), expressing
the encoded portion of the MTR-3 protein (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of the MTR-3 protein. In an exemplary embodiment, the
nucleic acid molecule is at least 50-100, 100-250, 250-500,
500-700, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000,
2000-2250, 2250-2500, 2500-2750, 2750-3000, 3000-3210 or more
nucleotides in length and encodes a protein having an MTR-3
activity (as described herein).
[0862] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO:7 or 9,
due to degeneracy of the genetic code and thus encode the same
MTR-3 proteins as those encoded by the nucleotide sequence shown in
SEQ ID NO:7 or 9. In another embodiment, an isolated nucleic acid
molecule of the invention has a nucleotide sequence encoding a
protein having an amino acid sequence which differs by at least 1,
but no greater than 5, 10, 20, 50 or 100 amino acid residues from
the amino acid sequence shown in SEQ ID NO:8. In yet another
embodiment, the nucleic acid molecule encodes the amino acid
sequence of human MTR-3. If an alignment is needed for this
comparison, the sequences should be aligned for maximum
homology.
[0863] Nucleic acid variants can be naturally occurring, such as
allelic variants (same locus), homologues (different locus), and
orthologues (different organism) or can be non-naturally occurring.
Non-naturally occurring variants can be made by mutagenesis
techniques, including those applied to polynucleotides, cells, or
organisms. The variants can contain nucleotide substitutions,
deletions, inversions and insertions. Variation can occur in either
or both the coding and non-coding regions. The variations can
produce both conservative and non-conservative amino acid
substitutions (as compared in the encoded product).
[0864] Allelic variants result, for example, from DNA sequence
polymorphisms within a population (e.g., the human population) that
lead to changes in the amino acid sequences of the MTR-3 proteins.
Such genetic polymorphism in the MTR-3 genes may exist among
individuals within a population due to natural allelic variation.
As used herein, the terms "gene" and "recombinant gene" refer to
nucleic acid molecules which include an open reading frame encoding
an MTR-3 protein, preferably a mammalian MTR-3 protein, and can
further include non-coding regulatory sequences, and introns.
[0865] Accordingly, in one embodiment, the invention features
isolated nucleic acid molecules which encode a naturally occurring
allelic variant of a polypeptide comprising the amino acid sequence
of SEQ ID NO:8, wherein the nucleic acid molecule hybridizes to a
complement of a nucleic acid molecule comprising SEQ ID NO:7 or 9,
for example, under stringent hybridization conditions.
[0866] Allelic variants of MTR-3, e.g., human MTR-3, include both
functional and non-functional MTR-3 proteins. Functional allelic
variants are naturally occurring amino acid sequence variants of
the MTR-3 protein that maintain the ability to, e.g., bind or
interact with an MTR-3 substrate or target molecule, transfer a
methyl group to or from an MTR-3 substrate or target molecule,
and/or modulate transcriptional activation. Functional allelic
variants will typically contain only conservative substitution of
one or more amino acids of SEQ ID NO:8, or substitution, deletion
or insertion of non-critical residues in non-critical regions of
the protein.
[0867] Non-functional allelic variants are naturally occurring
amino acid sequence variants of the MTR-3 protein, e.g., human
MTR-3, that do not have the ability to, e.g., bind or interact with
an MTR-3 substrate or target molecule, transfer a methyl group to
or from an MTR-3 substrate or target molecule, and/or modulate
transcriptional activation. Non-functional allelic variants will
typically contain a non-conservative substitution, a deletion, or
insertion, or premature truncation of the amino acid sequence of
SEQ ID NO:8, or a substitution, insertion, or deletion in critical
residues or critical regions of the protein.
[0868] The present invention further provides non-human orthologues
(e.g., non-human orthologues of the human MTR-3 protein).
Orthologues of the human MTR-3 protein are proteins that are
isolated from non-human organisms and possess the same MTR-3
substrate or target molecule binding mechanisms, methyltransferase
activity, and/or modulation of transcriptional activation
mechanisms of the human MTR-3 protein. Orthologues of the human
MTR-3 protein can readily be identified as comprising an amino acid
sequence that is substantially homologous to SEQ ID NO:8. The mouse
orthologue of human MTR-3 has been identified by Chen, et al.
(1999) Science 284:2174-2177.
[0869] Moreover, nucleic acid molecules encoding other MTR-3 family
members and, thus, which have a nucleotide sequence which differs
from the MTR-3 sequences of SEQ ID NO:7 or 9, are intended to be
within the scope of the invention. For example, another MTR-3 cDNA
can be identified based on the nucleotide sequence of human MTR-3.
Moreover, nucleic acid molecules encoding MTR-3 proteins from
different species, and which, thus, have a nucleotide sequence
which differs from the MTR-3 sequences of SEQ ID NO:7 or 9, are
intended to be within the scope of the invention. For example, a
monkey MTR-3 cDNA can be identified based on the nucleotide
sequence of a human MTR-3.
[0870] Nucleic acid molecules corresponding to natural allelic
variants and homologues of the MTR-3 cDNAs of the invention can be
isolated based on their homology to the MTR-3 nucleic acids
disclosed herein using the cDNAs disclosed herein, or a portion
thereof, as a hybridization probe according to standard
hybridization techniques under stringent hybridization conditions.
Nucleic acid molecules corresponding to natural allelic variants
and homologues of the MTR-3 cDNAs of the invention can further be
isolated by mapping to the same chromosome or locus as the MTR-3
gene.
[0871] Orthologues, homologues and allelic variants can be
identified using methods known in the art (e.g., by hybridization
to an isolated nucleic acid molecule of the present invention, for
example, under stringent hybridization conditions). In one
embodiment, an isolated nucleic acid molecule of the invention is
at least 15, 20, 25, 30 or more nucleotides in length and
hybridizes under stringent conditions to the nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:7 or 9. In other
embodiment, the nucleic acid is at least 50-100, 100-250, 250-500,
500-700, 750-1000, 1000-1250, 1250-1500, 1500-1750, 1750-2000,
2000-2250, 2250-2500, 2500-2750, 2750-3000, 3000-3210 or more
nucleotides in length.
[0872] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences that are significantly
identical or homologous to each other remain hybridized to each
other. Preferably, the conditions are such that sequences at least
about 70%, more preferably at least about 80%, even more preferably
at least about 85% or 90% identical to each other remain hybridized
to each other. Such stringent conditions are known to those skilled
in the art and can be found in Current Protocols in Molecular
Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995),
sections 2, 4, and 6. Additional stringent conditions can be found
in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7,
9, and 11. A preferred, non-limiting example of stringent
hybridization conditions includes hybridization in 4.times. sodium
chloride/sodium citrate (SSC), at about 65-70.degree. C. (or
alternatively hybridization in 4.times.SSC plus 50% formamide at
about 42-50.degree. C.) followed by one or more washes in
1.times.SSC, at about 65-70.degree. C. A preferred, non-limiting
example of highly stringent hybridization conditions includes
hybridization in 1.times.SSC, at about 65-70.degree. C. (or
alternatively hybridization in 1.times.SSC plus 50% formamide at
about 42-50.degree. C.) followed by one or more washes in
0.3.times.SSC, at about 65-70.degree. C. A preferred, non-limiting
example of reduced stringency hybridization conditions includes
hybridization in 4.times.SSC, at about 50-60.degree. C. (or
alternatively hybridization in 6.times.SSC plus 50% formamide at
about 40-45.degree. C.) followed by one or more washes in
2.times.SSC, at about 50-60.degree. C. Ranges intermediate to the
above-recited values, e.g., at 65-70.degree. C. or at 42-50.degree.
C. are also intended to be encompassed by the present invention.
SSPE (1.times.SSPE is 0.15M NaCl, 10 mM NaH.sub.2PO.sub.4, and 1.25
mM EDTA, pH 7.4) can be substituted for SSC (1.times.SSC is 0.15M
NaCl and 15 mM sodium citrate) in the hybridization and wash
buffers; washes are performed for 15 minutes each after
hybridization is complete. The hybridization temperature for
hybrids anticipated to be less than 50 base pairs in length should
be 5-10.degree. C. less than the melting temperature (T.sub.m) of
the hybrid, where T.sub.m is determined according to the following
equations. For hybrids less than 18 base pairs in length,
T.sub.m(.degree. C.)=2(# of A+T bases)+4(# of G+C bases). For
hybrids between 18 and 49 base pairs in length, T.sub.m(.degree.
C.)=81.5+16.6(log.sub.10[Na.sup.+])+0.41(% G+C)-(600/N), where N is
the number of bases in the hybrid, and [Na.sup.+] is the
concentration of sodium ions in the hybridization buffer
([Na.sup.+] for 1.times.SSC=0.165 M). It will also be recognized by
the skilled practitioner that additional reagents may be added to
hybridization and/or wash buffers to decrease non-specific
hybridization of nucleic acid molecules to membranes, for example,
nitrocellulose or nylon membranes, including but not limited to
blocking agents (e.g., BSA or salmon or herring sperm carrier DNA),
detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP
and the like. When using nylon membranes, in particular, an
additional preferred, non-limiting example of stringent
hybridization conditions is hybridization in 0.25-0.5M
NaH.sub.2PO.sub.4, 7% SDS at about 65.degree. C., followed by one
or more washes at 0.02M NaH.sub.2PO.sub.4, 1% SDS at 65.degree. C.
(see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA
81:1991-1995), or alternatively 0.2.times.SSC, 1% SDS.
[0873] Preferably, an isolated nucleic acid molecule of the
invention that hybridizes under stringent conditions to the
sequence of SEQ ID NO:7 or 9 corresponds to a naturally-occurring
nucleic acid molecule. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein).
[0874] In addition to naturally-occurring allelic variants of the
MTR-3 sequences that may exist in the population, the skilled
artisan will further appreciate that changes can be introduced by
mutation into the nucleotide sequences of SEQ ID NO:7 or 9, thereby
leading to changes in the amino acid sequence of the encoded MTR-3
proteins, without altering the functional ability of the MTR-3
proteins. For example, nucleotide substitutions leading to amino
acid substitutions at "non-essential" amino acid residues can be
made in the sequence of SEQ ID NO:7 or 9, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number ______. A "non-essential" amino acid residue is a
residue that can be altered from the wild-type sequence of MTR-3
(e.g., the sequence of SEQ ID NO:8) without altering the biological
activity, whereas an "essential" amino acid residue is required for
biological activity. For example, amino acid residues that are
conserved among the MTR-3 proteins of the present invention, e.g.,
those present in a VLD binding domain or a methyltransferase
domain, are predicted to be particularly unamenable to alteration.
Furthermore, additional amino acid residues that are conserved
between the MTR-3 proteins of the present invention and other
members of the methyltransferase family are not likely to be
amenable to alteration.
[0875] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding MTR-3 proteins that contain changes
in amino acid residues that are not essential for activity. Such
MTR-3 proteins differ in amino acid sequence from SEQ ID NO:8, yet
retain biological activity. In one embodiment, the isolated nucleic
acid molecule comprises a nucleotide sequence encoding a protein,
wherein the protein comprises an amino acid sequence at least about
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% 99% or more identical to SEQ ID NO:8, e.g., to
the entire length of SEQ ID NO:8.
[0876] An isolated nucleic acid molecule encoding an MTR-3 protein
homologous to the protein of SEQ ID NO:8 can be created by
introducing one or more nucleotide substitutions, additions or
deletions into the nucleotide sequence of SEQ ID NO:7 or 9, such
that one or more amino acid substitutions, additions or deletions
are introduced into the encoded protein. Mutations can be
introduced into SEQ ID NO:7 or 9, such as site-directed mutagenesis
and PCR-mediated mutagenesis. Preferably, conservative amino acid
substitutions are made at one or more predicted non-essential amino
acid residues. A "conservative amino acid substitution" is one in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in an MTR-3 protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of an MTR-3 coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for MTR-3 biological activity to identify
mutants that retain activity. Following mutagenesis of SEQ ID NO:7
or 9, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, the encoded protein
can be expressed recombinantly and the activity of the protein can
be determined.
[0877] In a preferred embodiment, a mutant MTR-3 protein can be
assayed for the ability to (i) modulate transcriptional activation
(e.g., either directly or indirectly); (ii) modulate (directly or
indirectly) chromatin structure to, for example, regulate the
recruitment of an RNA polymerase II transcription initiation
complex to a gene promoter; (iii) modulate the methylation state of
proteins in the transcription machinery; (iv) interact with an
MTR-3 substrate or target molecule (e.g., a non-MTR-3 protein); (v)
convert an MTR-3 substrate or target molecule to a product (e.g.,
transfer of a methyl group to or from the substrate or target
molecule); (vi) interact with and/or methyl transfer to a second
non-MTR-3 protein; (vii) transfer a methyl group to an arginine
residue; (viii) modulate protein-protein interaction (e.g.,
MTR-3-MTR-3 and/or MTR-3-non-MTR-3 interaction); (ix) modulate
and/or coordination of protein complex formation (e.g.,
MTR-3-containing complex formation); (x) regulate substrate or
target molecule activity; (xi) modulate intra- or inter-cellular
signaling, (xii) modulate cellular targeting and/or transport of
proteins; and/or (xiii) modulate cellular proliferation, growth, or
differentiation.
[0878] In addition to the nucleic acid molecules encoding MTR-3
proteins described above, another aspect of the invention pertains
to isolated nucleic acid molecules which are antisense thereto. In
an exemplary embodiment, the invention provides an isolated nucleic
acid molecule which is antisense to an MTR-3 nucleic acid molecule
(e.g., is antisense to the coding strand of an MTR-3 nucleic acid
molecule). An "antisense" nucleic acid comprises a nucleotide
sequence which is complementary to a "sense" nucleic acid encoding
a protein, e.g., complementary to the coding strand of a
double-stranded cDNA molecule or complementary to an mRNA sequence.
Accordingly, an antisense nucleic acid can hydrogen bond to a sense
nucleic acid. The antisense nucleic acid can be complementary to an
entire MTR-3 coding strand, or to only a portion thereof. In one
embodiment, an antisense nucleic acid molecule is antisense to
"coding region sequences" of the coding strand of a nucleotide
sequence encoding MTR-3. The term "coding region sequences" refers
to the region of the nucleotide sequence comprising codons which
are translated into amino acid residues (e.g., the coding region
sequences of human MTR-3 corresponding to SEQ ID NO:9). In another
embodiment, the antisense nucleic acid molecule is antisense to a
"noncoding region" of the coding strand of a nucleotide sequence
encoding MTR-3. The term "noncoding region" refers to 5' and/or 3'
sequences which flank the coding region sequences that are not
translated into amino acids (also referred to as 5' and 3'
untranslated regions).
[0879] Given the coding strand sequences encoding MTR-3 disclosed
herein (e.g., SEQ ID NO:9), antisense nucleic acids of the
invention can be designed according to the rules of Watson and
Crick base pairing. The antisense nucleic acid molecule can be
complementary to coding region sequences of MTR-3 mRNA, but more
preferably is an oligonucleotide which is antisense to only a
portion of the MTR-3 mRNA. An antisense oligonucleotide can be, for
example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, or more nucleotides in length. An antisense nucleic
acid of the invention can be constructed using chemical synthesis
and enzymatic ligation reactions using procedures known in the art.
For example, an antisense nucleic acid (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense
and sense nucleic acids, e.g., phosphorothioate derivatives and
acridine substituted nucleotides can be used. Examples of modified
nucleotides which can be used to generate the antisense nucleic
acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, 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-isopentenyladenine,
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-N2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0880] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule or a ribozyme as described above in section 3.
[0881] Alternatively, MTR-3 gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the MTR-3 (e.g., the MTR-3 promoter and/or enhancers;
e.g., nucleotides 1-347 of SEQ ID NO:7) to form triple helical
structures that prevent transcription of the MTR-3 gene in target
cells. 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.
[0882] In yet another embodiment, the MTR-3 nucleic acid molecules
of the present invention can be modified at the base moiety, sugar
moiety or phosphate backbone to improve, e.g., the stability,
hybridization, or solubility of the molecule. For example, the
deoxyribose phosphate backbone of the nucleic acid molecules can be
modified to generate peptide nucleic acids (see Hyrup, B. and
Nielsen, P. E. (1996) Bioorg. Med. Chem. 4(1):5-23). As used
herein, the terms "peptide nucleic acids" or "PNAs" refer to
nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only
the four natural nucleobases are retained. The neutral backbone of
PNAs has been shown to allow for specific hybridization to DNA and
RNA under conditions of low ionic strength. The synthesis of PNA
oligomers can be performed using standard solid phase peptide
synthesis protocols as described in Hyrup and Nielsen (1996) supra
and Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA
93:14670-675.
[0883] PNAs of MTR-3 nucleic acid molecules can be used in
therapeutic and diagnostic applications. For example, PNAs can be
used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of MTR-3 nucleic acid molecules can also be used in the analysis of
single base pair mutations in a gene, (e.g., by PNA-directed PCR
clamping); as `artificial restriction enzymes` when used in
combination with other enzymes, (e.g., S1 nucleases (Hyrup and
Nielsen (1996) supra)); or as probes or primers for DNA sequencing
or hybridization (Hyrup and Nielsen (1996) supra; Perry-O'Keefe et
al. (1996) supra).
[0884] In another embodiment, PNAs of MTR-3 can be modified, (e.g.,
to enhance their stability or cellular uptake), by attaching
lipophilic or other helper groups to PNA, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of
drug delivery known in the art. For example, PNA-DNA chimeras of
MTR-3 nucleic acid molecules can be generated which may combine the
advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes (e.g., RNase H and DNA polymerases) to interact
with the DNA portion while the PNA portion would provide high
binding affinity and specificity. PNA-DNA chimeras can be linked
using linkers of appropriate lengths selected in terms of base
stacking, number of bonds between the nucleobases, and orientation
(Hyrup and Nielsen (1996) supra). The synthesis of PNA-DNA chimeras
can be performed as described in Hyrup and Nielsen (1996) supra and
Finn, P. J. et al. (1996) Nucleic Acids Res. 24(17):3357-63. For
example, a DNA chain can be synthesized on a solid support using
standard phosphoramidite coupling chemistry and modified nucleoside
analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite, can be used as a between the PNA and the 5' end of
DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17:5973-88). PNA
monomers are then coupled in a stepwise manner to produce a
chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn,
P. J. et al. (1996) supra). Alternatively, chimeric molecules can
be synthesized with a 5' DNA segment and a 3' PNA segment
(Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett.
5:1119-11124).
[0885] In other embodiments, 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. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (See, e.g., Krol et al.
(1988) Bio-Techniques 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, or hybridization-triggered cleavage agent).
Isolated MTR-3 Proteins and Anti-MTR-3 Antibodies
[0886] One aspect of the invention pertains to isolated or
recombinant MTR-3 proteins and polypeptides, and biologically
active portions thereof, as well as polypeptide fragments suitable
for use as immunogens to raise anti-MTR-3 antibodies. In one
embodiment, native MTR-3 proteins can be isolated from cells or
tissue sources by an appropriate purification scheme using standard
protein purification techniques. In another embodiment, MTR-3
proteins are produced by recombinant DNA techniques. Alternative to
recombinant expression, an MTR-3 protein or polypeptide can be
synthesized chemically using standard peptide synthesis
techniques.
[0887] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the MTR-3 protein is derived, or substantially free from chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of MTR-3 protein in which the protein is separated
from cellular components of the cells from which it is isolated or
recombinantly produced. In one embodiment, the language
"substantially free of cellular material" includes preparations of
MTR-3 protein having less than about 30% (by dry weight) of
non-MTR-3 protein (also referred to herein as a "contaminating
protein"), more preferably less than about 20% of non-MTR-3
protein, still more preferably less than about 10% of non-MTR-3
protein, and most preferably less than about 5% non-MTR-3 protein.
When the MTR-3 protein or biologically active portion thereof is
recombinantly produced, it is also preferably substantially free of
culture medium, i.e., culture medium represents less than about
20%, more preferably less than about 10%, and most preferably less
than about 5% of the volume of the protein preparation.
[0888] The language "substantially free of chemical precursors or
other chemicals" includes preparations of MTR-3 protein in which
the protein is separated from chemical precursors or other
chemicals which are involved in the synthesis of the protein. In
one embodiment, the language "substantially free of chemical
precursors or other chemicals" includes preparations of MTR-3
protein having less than about 30% (by dry weight) of chemical
precursors or non-MTR-3 chemicals, more preferably less than about
20% chemical precursors or non-MTR-3 chemicals, still more
preferably less than about 10% chemical precursors or non-MTR-3
chemicals, and most preferably less than about 5% chemical
precursors or non-MTR-3 chemicals.
[0889] As used herein, a "biologically active portion" of an MTR-3
protein includes a fragment of an MTR-3 protein which participates
in an interaction between an MTR-3 molecule and a non-MTR-3
molecule (e.g., an MTR-3 substrate). Biologically active portions
of an MTR-3 protein include peptides comprising amino acid
sequences sufficiently identical to or derived from the MTR-3 amino
acid sequences, e.g., the amino acid sequences shown in SEQ ID
NO:8, which include sufficient amino acid residues to exhibit at
least one activity of an MTR-3 protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the MTR-3 protein, e.g., MTR-3 activity,
methyltransferase activity, modulation of protein transport,
modulation of intra- or inter-cellular signaling, modulation of
transcriptional activation, and/or modulation of cell growth,
proliferation, and/or differentiation mechanisms. A biologically
active portion of an MTR-3 protein can be a polypeptide which is,
for example, 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300,
350, 400, 450, 500, 550, 600 or more amino acids in length.
Biologically active portions of an MTR-3 protein can be used as
targets for developing agents which modulate an MTR-3 mediated
activity, e.g., methyltransferase activity, modulation of protein
transport, modulation of intra- or inter-cellular signaling,
modulation of transcriptional activation, and/or modulation of cell
growth, proliferation, and/or differentiation mechanisms.
[0890] In one embodiment, a biologically active portion of an MTR-3
protein comprises at least one MTR-3 domain, one VLD binding
domain, and/or one transmembrane domain. Moreover, other
biologically active portions, in which other regions of the protein
are deleted, can be prepared by recombinant techniques and
evaluated for one or more of the functional activities of a native
MTR-3 protein.
[0891] Another aspect of the invention features fragments of the
protein having the amino acid sequence of SEQ ID NO:8, for example,
for use as immunogens. In one embodiment, a fragment comprises at
least 5 amino acids (e.g., contiguous or consecutive amino acids)
of the amino acid sequence of SEQ ID NO:8. In another embodiment, a
fragment comprises at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50
or more amino acids (e.g., contiguous or consecutive amino acids)
of the amino acid sequence of SEQ ID NO:8.
[0892] In a preferred embodiment, an MTR-3 protein has an amino
acid sequence shown in SEQ ID NO:8. In other embodiments, the MTR-3
protein is substantially identical to SEQ ID NO:8, and retains the
functional activity of the protein of SEQ ID NO:8, yet differs in
amino acid sequence due to natural allelic variation or
mutagenesis, as described in detail in subsection I above. In
another embodiment, the MTR-3 protein is a protein which comprises
an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to SEQ ID NO:8.
[0893] In another embodiment, the invention features an MTR-3
protein which is encoded by a nucleic acid molecule consisting of a
nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to a nucleotide sequence of SEQ ID NO:7 or 9, or a
complement thereof. This invention further features an MTR-3
protein which is encoded by a nucleic acid molecule consisting of a
nucleotide sequence which hybridizes under stringent hybridization
conditions to a complement of a nucleic acid molecule comprising
the nucleotide sequence of SEQ ID NO:7 or 9, or a complement
thereof.
[0894] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence (e.g., when aligning a second sequence to
the MTR-3 amino acid sequence of SEQ ID NO:8 having 608 amino acid
residues, at least 182, preferably at least 243, more preferably at
least 304, even more preferably at least 364, and even more
preferably at least 425, 486, or 547 amino acid residues are
aligned). The amino acid residues or nucleotides at corresponding
amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino
acid residue or nucleotide as the corresponding position in the
second sequence, then the molecules are identical at that position
(as used herein amino acid or nucleic acid "identity" is equivalent
to amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[0895] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm
which has been incorporated into the GAP program in the GCG
software package, using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package, using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A preferred, non-limiting
example of parameters to be used in conjunction with the GAP
program include a Blosum 62 scoring matrix with a gap penalty of
12, a gap extend penalty of 4, and a frameshift gap penalty of
5.
[0896] In another embodiment, the percent identity between two
amino acid or nucleotide sequences is determined using the
algorithm of Meyers and Miller (Comput. Appl. Biosci., 4:11-17
(1988)) which has been incorporated into the ALIGN program (version
2.0 or version 2.0U), using a PAM120 weight residue table, a gap
length penalty of 12 and a gap penalty of 4.
[0897] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against public databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul et
al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can
be performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to MTR-3 nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to MTR-3 protein molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al. (1997)
Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and
Gapped BLAST programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used.
[0898] The invention also provides MTR-3 chimeric or fusion
proteins. As used herein, an MTR-3 "chimeric protein" or "fusion
protein" comprises an MTR-3 polypeptide operatively linked to a
non-MTR-3 polypeptide. A "MTR-3 polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to MTR-3,
whereas a "non-MTR-3 polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to a protein which is not
substantially homologous to the MTR-3 protein, e.g., a protein
which is different from the MTR-3 protein and which is derived from
the same or a different organism. Within an MTR-3 fusion protein
the MTR-3 polypeptide can correspond to all or a portion of an
MTR-3 protein. In a preferred embodiment, an MTR-3 fusion protein
comprises at least one biologically active portion of an MTR-3
protein. In another preferred embodiment, an MTR-3 fusion protein
comprises at least two biologically active portions of an MTR-3
protein. Within the fusion protein, the term "operatively linked"
is intended to indicate that the MTR-3 polypeptide and the
non-MTR-3 polypeptide are fused in-frame to each other. The
non-MTR-3 polypeptide can be fused to the N-terminus or C-terminus
of the MTR-3 polypeptide.
[0899] For example, in one embodiment, the fusion protein is a
GST-MTR-3 fusion protein in which the MTR-3 sequences are fused to
the C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant MTR-3. In another
embodiment, the fusion protein is an MTR-3 protein containing a
heterologous signal sequence at its N-terminus. In certain host
cells (e.g., mammalian host cells), expression and/or secretion of
MTR-3 can be increased through use of a heterologous signal
sequence.
[0900] The MTR-3 fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject in vivo. The MTR-3 fusion proteins can be used to affect
the bioavailability of an MTR-3 substrate. Use of MTR-3 fusion
proteins may be useful therapeutically for the treatment of
disorders caused by, for example, (i) aberrant modification or
mutation of a gene encoding an MTR-3 protein; (ii) mis-regulation
of the MTR-3 gene; and (iii) aberrant post-translational
modification of an MTR-3 protein.
[0901] Moreover, the MTR-3-fusion proteins of the invention can be
used as immunogens to produce anti-MTR-3 antibodies in a subject,
to purify MTR-3 substrates, and in screening assays to identify
molecules which inhibit or enhance the interaction of MTR-3 with an
MTR-3 substrate.
[0902] Preferably, an MTR-3 chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). An MTR-3-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the MTR-3 protein.
[0903] The present invention also pertains to variants of the MTR-3
proteins which function as either MTR-3 agonists (mimetics) or as
MTR-3 antagonists. Variants of the MTR-3 proteins can be generated
by mutagenesis, e.g., discrete point mutation or truncation of an
MTR-3 protein. An agonist of the MTR-3 proteins can retain
substantially the same, or a subset, of the biological activities
of the naturally occurring form of an MTR-3 protein. An antagonist
of an MTR-3 protein can inhibit one or more of the activities of
the naturally occurring form of the MTR-3 protein by, for example,
competitively modulating an MTR-3-mediated activity of an MTR-3
protein. Thus, specific biological effects can be elicited by
treatment with a variant of limited function. In one embodiment,
treatment of a subject with a variant having a subset of the
biological activities of the naturally occurring form of the
protein has fewer side effects in a subject relative to treatment
with the naturally occurring form of the MTR-3 protein.
[0904] In one embodiment, variants of an MTR-3 protein which
function as either MTR-3 agonists (mimetics) or as MTR-3
antagonists can be identified by screening combinatorial libraries
of mutants, e.g., truncation mutants, of an MTR-3 protein for MTR-3
protein agonist or antagonist activity. In one embodiment, a
variegated library of MTR-3 variants is generated by combinatorial
mutagenesis at the nucleic acid level and is encoded by a
variegated gene library. A variegated library of MTR-3 variants can
be produced by, for example, enzymatically ligating a mixture of
synthetic oligonucleotides into gene sequences such that a
degenerate set of potential MTR-3 sequences is expressible as
individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display) containing the set of
MTR-3 sequences therein. There are a variety of methods which can
be used to produce libraries of potential MTR-3 variants from a
degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an
appropriate expression vector. Use of a degenerate set of genes
allows for the provision, in one mixture, of all of the sequences
encoding the desired set of potential MTR-3 sequences. Methods for
synthesizing degenerate oligonucleotides are known in the art (see,
e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984)
Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056;
Ike et al. (1983) Nucleic Acid Res. 11:477.
[0905] In addition, libraries of fragments of an MTR-3 protein
coding sequence can be used to generate a variegated population of
MTR-3 fragments for screening and subsequent selection of variants
of an MTR-3 protein. In one embodiment, a library of coding
sequence fragments can be generated by treating a double stranded
PCR fragment of an MTR-3 coding sequence with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form
double stranded DNA which can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. By this
method, an expression library can be derived which encodes
N-terminal, C-terminal and internal fragments of various sizes of
the MTR-3 protein.
[0906] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of MTR-3 proteins. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recursive ensemble mutagenesis
(REM), a new technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify MTR-3 variants (Arkin and Youvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al.
(1993) Protein Engineering 6(3):327-331).
[0907] In one embodiment, cell based assays can be exploited to
analyze a variegated MTR-3 library. For example, a library of
expression vectors can be transfected into a cell line which
ordinarily responds to MTR-3 in a particular MTR-3
substrate-dependent manner. The transfected cells are then
contacted with MTR-3 and the effect of the expression of the mutant
on signaling by the MTR-3 substrate can be detected, e.g., by
measuring levels methylated amino acid residues in the substrate,
gene transcription, and/or cell proliferation, growth or
differentiation. Plasmid DNA can then be recovered from the cells
which score for inhibition, or alternatively, potentiation of
signaling by the MTR-3 substrate, or which score for increased or
decreased levels of methylation of the substrate, and the
individual clones further characterized.
[0908] An isolated MTR-3 protein, or a portion or fragment thereof,
can be used as an immunogen to generate antibodies that bind MTR-3
using standard techniques for polyclonal and monoclonal antibody
preparation. A full-length MTR-3 protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of MTR-3 for use as immunogens. The antigenic peptide of MTR-3
comprises at least 8 amino acid residues of the amino acid sequence
shown in SEQ ID NO:8 and encompasses an epitope of MTR-3 such that
an antibody raised against the peptide forms a specific immune
complex with MTR-3. Preferably, the antigenic peptide comprises at
least 10 amino acid residues, more preferably at least 15 amino
acid residues, even more preferably at least 20 amino acid
residues, and most preferably at least 30 amino acid residues.
[0909] Preferred epitopes encompassed by the antigenic peptide are
regions of MTR-3 that are located on the surface of the protein,
e.g., hydrophilic regions, as well as regions with high
antigenicity.
[0910] An MTR-3 immunogen typically is used to prepare antibodies
by immunizing a suitable subject (e.g., rabbit, goat, mouse, or
other mammal) with the immunogen. An appropriate immunogenic
preparation can contain, for example, recombinantly expressed MTR-3
protein or a chemically-synthesized MTR-3 polypeptide. The
preparation can further include an adjuvant, such as Freund's
complete or incomplete adjuvant, or similar immunostimulatory
agent. Immunization of a suitable subject with an immunogenic MTR-3
preparation induces a polyclonal anti-MTR-3 antibody response.
[0911] Accordingly, another aspect of the invention pertains to
anti-MTR-3 antibodies. The term "antibody" as used herein refers to
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site which specifically binds (immunoreacts with) an
antigen, such as MTR-3. Examples of immunologically active portions
of immunoglobulin molecules include F(ab) and F(ab').sub.2
fragments which can be generated by treating the antibody with an
enzyme such as pepsin. The invention provides polyclonal and
monoclonal antibodies that bind MTR-3. The term "monoclonal
antibody" or "monoclonal antibody composition", as used herein,
refers to a population of antibody molecules that contain only one
species of an antigen binding site capable of immunoreacting with a
particular epitope of MTR-3. A monoclonal antibody composition thus
typically displays a single binding affinity for a particular MTR-3
protein with which it immunoreacts.
[0912] Polyclonal anti-MTR-3 antibodies can be prepared as
described above by immunizing a suitable subject with an MTR-3
immunogen. The anti-MTR-3 antibody titer in the immunized subject
can be monitored over time by standard techniques, such as with an
enzyme linked immunosorbent assay (ELISA) using immobilized MTR-3.
If desired, the antibody molecules directed against MTR-3 can be
isolated from the mammal (e.g., from the blood) and further
purified by well known techniques, such as protein A chromatography
to obtain the IgG fraction. At an appropriate time after
immunization, e.g., when the anti-MTR-3 antibody titers are
highest, antibody-producing cells can be obtained from the subject
and used to prepare monoclonal antibodies by standard techniques,
such as the hybridoma technique originally described by Kohler and
Milstein (1975) Nature 256:495-497 (see also Brown et al. (1981) J.
Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.
255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA
76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the
more recent human B cell hybridoma technique (Kozbor et al. (1983)
Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al.
(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
monoclonal antibody hybridomas is well known (see generally
Kenneth, R. H. in Monoclonal Antibodies: A New Dimension In
Biological Analyses, Plenum Publishing Corp., New York, N.Y.
(1980); Lerner, E. A. (1981) Yale J. Biol. Med., 54:387-402;
Gefter, M. L. et al. (1977) Somatic Cell Genet. 3:231-36). Briefly,
an immortal cell line (typically a myeloma) is fused to lymphocytes
(typically splenocytes) from a mammal immunized with an MTR-3
immunogen as described above, and the culture supernatants of the
resulting hybridoma cells are screened to identify a hybridoma
producing a monoclonal antibody that binds MTR-3.
[0913] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-MTR-3 monoclonal antibody (see, e.g.,
Galfre, G. et al. (1977) Nature 266:55052; Gefter et al. Somatic
Cell Genet., supra; Lerner (1981) supra; Kenneth, Monoclonal
Antibodies, supra). Moreover, the ordinarily skilled worker will
appreciate that there are many variations of such methods which
also would be useful. Typically, the immortal cell line (e.g., a
myeloma cell line) is derived from the same mammalian species as
the lymphocytes. For example, murine hybridomas can be made by
fusing lymphocytes from a mouse immunized with an immunogenic
preparation of the present invention with an immortalized mouse
cell line. Preferred immortal cell lines are mouse myeloma cell
lines that are sensitive to culture medium containing hypoxanthine,
aminopterin and thymidine ("HAT medium"). Any of a number of
myeloma cell lines can be used as a fusion partner according to
standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or
Sp2/O-Ag14 myeloma lines. These myeloma lines are available from
ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to
mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma
cells resulting from the fusion are then selected using HAT medium,
which kills unfused and unproductively fused myeloma cells (unfused
splenocytes die after several days because they are not
transformed). Hybridoma cells producing a monoclonal antibody of
the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind MTR-3, e.g., using a standard
ELISA assay.
[0914] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-MTR-3 antibody can be identified and
isolated by screening a recombinant combinatorial immunoglobulin
library (e.g., an antibody phage display library) with MTR-3 to
thereby isolate immunoglobulin library members that bind MTR-3.
Kits for generating and screening phage display libraries are
commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the Stratagene
SurfZAP.TM. Phage Display Kit, Catalog No. 240612). Additionally,
examples of methods and reagents particularly amenable for use in
generating and screening antibody display library can be found in,
for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT
International Publication No. WO 92/18619; Dower et al. PCT
International Publication No. WO 91/17271; Winter et al. PCT
International Publication WO 92/20791; Markland et al. PCT
International Publication No. WO 92/15679; Breitling et al. PCT
International Publication WO 93/01288; McCafferty et al. PCT
International Publication No. WO 92/01047; Garrad et al. PCT
International Publication No. WO 92/09690; Ladner et al. PCT
International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992)
J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature
352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA
89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377;
Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al.
(1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et
al. (1990) Nature 348:552-554.
[0915] Additionally, recombinant anti-MTR-3 antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in Robinson et al. International Application No.
PCT/US86/02269; Akira, et al. European Patent Application 184,187;
Taniguchi, M., European Patent Application 171,496; Morrison et al.
European Patent Application 173,494; Neuberger et al. PCT
International Publication No. WO 86/01533; Cabilly et al. U.S. Pat.
No. 4,816,567; Cabilly et al. European Patent Application 125,023;
Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc.
Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.
139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA
84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et
al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl.
Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science
229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S.
Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525;
Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988)
J. Immunol. 141:4053-4060.
[0916] An anti-MTR-3 antibody (e.g., monoclonal antibody) can be
used to isolate MTR-3 by standard techniques, such as affinity
chromatography or immunoprecipitation. An anti-MTR-3 antibody can
facilitate the purification of natural MTR-3 from cells and of
recombinantly produced MTR-3 expressed in host cells. Moreover, an
anti-MTR-3 antibody can be used to detect MTR-3 protein (e.g., in a
cellular lysate or cell supernatant) in order to evaluate the
abundance and pattern of expression of the MTR-3 protein.
Anti-MTR-3 antibodies can be used diagnostically to monitor protein
levels in tissue as part of a clinical testing procedure, e.g., to,
for example, determine the efficacy of a given treatment regimen.
Detection can be facilitated by coupling (i.e., physically linking)
the antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0917] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the Sequence Listing, are
incorporated herein by reference.
EXAMPLES
Example 1
Identification and Characterization of Human MTR-3 cDNA
[0918] In this example, the identification and characterization of
the gene encoding human MTR-3 (clone Fbh27420) also referred to
herein as "27420" is described.
Isolation of the MTR-3 cDNA
[0919] The invention is based, at least in part, on the discovery
of a human gene encoding a novel protein, referred to herein as
MTR-3. The entire sequence of human clone Fbh27420 was determined
and found to contain an open reading frame termed human "MTR-3".
The amino acid sequence of the human MTR-3 expression product is
described herein. The MTR-3 protein sequence set forth in SEQ ID
NO:8 comprises about 608 amino acids. The coding region (open
reading frame) of SEQ ID NO:7 is set forth as SEQ ID NO:9.
Analysis of the Human MTR-3 Molecule
[0920] A BLASTN 2.0 search against the PATENT.sub.--2 nucleotide
database, using a score of 100 and a word length of 12 (Altschul et
al. (1990) J. Mol. Biol. 215:403) and of the nucleotide sequence of
human MTR-3 as a query sequence revealed a number of nucleotides
with some similarity to that of the invention, including a human
transferase (PCT Publication No. 00/00594) with 99% identity over
nucleotides 1399-2520 of SEQ ID NO:7, 99% identity over nucleotides
396-1401 of SEQ ID NO:7, and 100% identity over nucleotides 302-388
of SEQ ID NO:7.
[0921] A BLASTX 2.0 search against the PATENT.sub.--2/gsprot
protein database, using a wordlength of 3, a score of 100, and a
BLOSUM62 matrix, and using the amino acid sequence of human MTR-3
as a query sequence, identified a number of proteins with some
similarity to human MTR-3 protein. For example, a human transferase
(PCT Publication No. 00/00594) is 99% identical to human MTR-3 over
amino acid residues 162-608 of SEQ ID NO:8.
[0922] The amino acid sequence of human MTR-3 was analyzed using
the program PSORT to predict the localization of the protein within
the cell. This program assesses the presence of different targeting
and localization amino acid sequences within the query sequence.
The results of the analysis predict that human MTR-3 (SEQ ID NO:8)
is localized to the cytoplasm, mitochondria, and nucleus).
[0923] A search of the amino acid sequence of MTR-3 was performed
against the HMM database. This search resulted in the
identification of a potential "cellulose binding domain" in the
amino acid sequence of MTR-3 (SEQ ID NO:8) at about residues
563-585 (score=5.0).
[0924] A MEMSAT analysis of the polypeptide sequence of SEQ ID NO:8
was also performed predicting four potential transmembrane domains
in the amino acid sequence of human MTR-3 (SEQ ID NO:8) at about
residues 18-41 (score=0.4), 97-113 (score=1.1), 187-203
(score=2.0), and 382-404 (score=2.7).
[0925] A search of the amino acid sequence of MTR-3 was also
performed against the ProSite database. This search resulted in the
identification in the amino acid sequence of human MTR-3 of a
number of potential N-glycosylation sites at about residues
179-182, 230-233, 504-507, and 545-548, a potential
glycosaminoglycan attachment site at about residues 569-572, a
potential cAMP- and cGMP-dependent protein kinase phosphorylation
site at about residues 444-447, a number of potential protein
kinase C phosphorylation sites at about residues 126-128, 138-140,
232-234, and 352-254, a number of potential casein kinase II
phosphorylation sites at about residues 39-42, 73-76, 113-116,
246-249, 288-291, 491-494, and 516-519 and a number of potential
N-myristoylation sites at about residues 11-16, 22-27, 192-197,
320-325, 382-387, 397-402, 460-465, 486-491, 500-505, 508-513,
527-532, 552-557, 558-563, 565-570, and 571-576.
[0926] A search of the amino acid sequence of human MTR-3 was also
performed against the ProDom database. The search resulted in the
identification of a potential "arginine N-methyltransferase domain"
in the amino acid sequence of MTR-3 at about residues 292-460.
Tissue Distribution of Human MTR-3 mRNA by RT-PCR
[0927] RT-PCR was used to detect the presence of MTR-3 mRNA in
various tumor and metastatic tissue samples as compared to normal
tissue samples. RT-PCR was also used to detect the presence of
MTR-3 mRNA in various xenograft cell lines. In breast tissue, MTR-3
mRNA was detected in 1/1 normal tissue samples as compared to 4/4
tumor clinical samples after 30 cycles of PCR. In xenograft cell
lines isolated from breast tissue, MTR-3 mRNA was detected in 1/1
normal and 5/5 xenograft cell lines. Positive breast cell lines
were: ZR-75, T47D, MCF-7, MDA-MB-435, and MDA-MB-231.
[0928] In colon tissue, MTR-3 mRNA was detected in 2/2 normal
tissue samples as compared to 5/5 tumor tissue samples after 30
cycles of PCR. Positive colon cell lines were: HCT116, HCT15, HT29,
SW620, DLD1, KM12, and SW 480. In liver tissue, MTR-3 mRNA was
detected in 2/2 normal samples and in 5/5 colon metastases to the
liver after 30 cycles of PCR.
Tissue Distribution of Human MTR-3 mRNA by Northern Analysis
[0929] This example describes the tissue distribution of MTR-3
mRNA, as determined by Northern analysis.
[0930] Northern blot hybridizations with the various RNA samples
are performed under standard conditions and washed under stringent
conditions, i.e., 0.2.times.SSC at 65.degree. C. The DNA probe is
radioactively labeled with .sup.32P-dCTP using the Prime-It kit
(Stratagene, La Jolla, Calif.) according to the instructions of the
supplier. Filters containing human mRNA (MultiTissue Northern I and
MultiTissue Northern II from Clontech, Palo Alto, Calif.) are
probed in ExpressHyb hybridization solution (Clontech) and washed
at high stringency according to manufacturer's recommendations.
[0931] Electronic Northern analysis was carried out by
identification of the MTR-3 sequence in various libraries using
BLAST. Electronic Northern analysis indicated expression in many
tissues, including lung, heart, kidney, t-cells, and placenta.
Tissue Distribution of MTR-3 by In Situ Analysis
[0932] For in situ analysis, various tissues, e.g., tissues
obtained from normal colon, colon tumors, and colon metastases to
the liver were first frozen on dry ice. Ten-micrometer-thick
sections of the tissues were post-fixed with 4% formaldehyde in
DEPC treated 1.times. phosphate-buffered saline at room temperature
for 10 minutes before being rinsed twice in DEPC 1.times.
phosphate-buffered saline and once in 0.1 M triethanolamine-HCl (pH
8.0). Following incubation in 0.25% acetic anhydride-0.1 M
triethanolamine-HCl for 10 minutes, sections were rinsed in DEPC
2.times.SSC (1.times.SSC is 0.15M NaCl plus 0.015M sodium citrate).
Tissue was then dehydrated through a series of ethanol washes,
incubated in 100% chloroform for 5 minutes, and then rinsed in 100%
ethanol for 1 minute and 95% ethanol for 1 minute and allowed to
air dry.
[0933] Hybridizations were performed with .sup.35S-radiolabeled
(5.times.10.sup.7 cpm/ml) cRNA probes. Probes were incubated in the
presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5),
1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05%
yeast total RNA type X1, 1.times.Denhardt's solution, 50%
formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium
dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at
55.degree. C.
[0934] After hybridization, slides were washed with 2.times.SSC.
Sections were then sequentially incubated at 37.degree. C. in TNE
(a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1
mM EDTA), for 10 minutes, in TNE with 10 .mu.g of RNase A per ml
for 30 minutes, and finally in TNE for 10 minutes. Slides were then
rinsed with 2.times.SSC at room temperature, washed with
2.times.SSC at 50.degree. C. for 1 hour, washed with 0.2.times.SSC
at 55.degree. C. for 1 hour, and 0.2.times.SSC at 60.degree. C. for
1 hour. Sections were then dehydrated rapidly through serial
ethanol-0.3 M sodium acetate concentrations before being air dried
and exposed to Kodak Biomax MR scientific imaging film for 24 hours
and subsequently dipped in NB-2 photoemulsion and exposed at
4.degree. C. for 7 days before being developed and counter
stained.
[0935] In situ hybridization results indicated expression in 0/2
normal colon cells, in 3/3 colon tumor cells, and in 2/2 colon
metastases to the liver. Results further indicated negative
expression in normal or tumor cells from breast tissue and normal
or tumor cells from liver tissue.
Tissue Expression Analysis of MTR-3 mRNA Using Taqman Analysis
[0936] This example describes the tissue distribution of human
MTR-3 mRNA in a variety of cells and tissues, as determined using
the TaqMan.TM. procedure. The Taqman.TM. procedure is a
quantitative, reverse transcription PCR-based approach for
detecting mRNA. The RT-PCR reaction exploits the 5' nuclease
activity of AmpliTaq Gold.TM. DNA Polymerase to cleave a TaqMan.TM.
probe during PCR. Briefly, cDNA was generated from the samples of
interest, e.g., lung tumor samples, normal lung samples, colon
tumor samples, and normal colon samples, and used as the starting
material for PCR amplification. In addition to the 5' and 3'
gene-specific primers, a gene-specific oligonucleotide probe
(complementary to the region being amplified) was included in the
reaction (i.e., the Taqman.TM. probe). The TaqMan.TM. probe
includes the oligonucleotide with a fluorescent reporter dye
covalently linked to the 5' end of the probe (such as FAM
(6-carboxyfluorescein), TET
(6-carboxy-4,7,2',7'-tetrachlorofluorescein), JOE
(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and a
quencher dye (TAMRA (6-carboxy-N,N,N',N'-tetramethylrhodamine) at
the 3' end of the probe.
[0937] During the PCR reaction, cleavage of the probe separates the
reporter dye and the quencher dye, resulting in increased
fluorescence of the reporter. Accumulation of PCR products is
detected directly by monitoring the increase in fluorescence of the
reporter dye. When the probe is intact, the proximity of the
reporter dye to the quencher dye results in suppression of the
reporter fluorescence. During PCR, if the target of interest is
present, the probe specifically anneals between the forward and
reverse primer sites. The 5'-3' nucleolytic activity of the
AmpliTaq.TM. Gold DNA Polymerase cleaves the probe between the
reporter and the quencher only if the probe hybridizes to the
target. The probe fragments are then displaced from the target, and
polymerization of the strand continues. The 3' end of the probe is
blocked to prevent extension of the probe during PCR. This process
occurs in every cycle and does not interfere with the exponential
accumulation of product. RNA was prepared using the trizol method
and treated with DNase to remove contaminating genomic DNA. cDNA
was synthesized using standard techniques. Mock cDNA synthesis in
the absence of reverse transcriptase resulted in samples with no
detectable PCR amplification of the control gene confirms efficient
removal of genomic DNA contamination.
[0938] A human normal tissue panel indicated broad distribution of
human MTR-3 expression, with highest expression in testis.
Increased expression of human MTR-3 was detected in colon tumor
samples (T) versus normal colon tissue samples (N). Increased
expression of human MTR-3 was detected in colon metastases to the
liver (Liver Met) versus normal liver tissue samples (N).
Overexpression in breast and lung tumors versus respective normal
tissue samples was also detected.
[0939] These data reveal a significant up-regulation of MTR-3 mRNA
in carcinomas, colon carcinomas in particular. Given that the mRNA
for MTR-3 is expressed in a variety of tumors, with significant
up-regulation in carcinoma samples in comparison to normal samples,
it is believed that inhibition of MTR-3 activity may inhibit tumor
progression by, for example, inhibiting transcriptional activation
and cellular growth and proliferation.
Example 2
Expression of Recombinant MTR-3 Protein in Bacterial Cells
[0940] In this example, human MTR-3 is expressed as a recombinant
glutathione-S-transferase (GST) fusion polypeptide in E. coli and
the fusion polypeptide is isolated and characterized. Specifically,
MTR-3 is fused to GST and this fusion polypeptide is expressed in
E. coli, e.g., strain PEB199. Expression of the GST-MTR-3 fusion
protein in PEB199 is induced with IPTG. The recombinant fusion
polypeptide is purified from crude bacterial lysates of the induced
PEB199 strain by affinity chromatography on glutathione beads.
Using polyacrylamide gel electrophoretic analysis of the
polypeptide purified from the bacterial lysates, the molecular
weight of the resultant fusion polypeptide is determined.
Example 3
Expression of Recombinant MTR-3 Protein in COS Cells
[0941] To express the human MTR-3 gene in COS cells, the pcDNA/Amp
vector by Invitrogen Corporation (San Diego, Calif.) is used. This
vector contains an SV40 origin of replication, an ampicillin
resistance gene, an E. coli replication origin, a CMV promoter
followed by a polylinker region, and an SV40 intron and
polyadenylation site. A DNA fragment encoding the entire MTR-3
protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG
tag fused in-frame to its 3' end of the fragment is cloned into the
polylinker region of the vector, thereby placing the expression of
the recombinant protein under the control of the CMV promoter.
[0942] To construct the plasmid, the MTR-3 DNA sequence is
amplified by PCR using two primers. The 5' primer contains the
restriction site of interest followed by approximately twenty
nucleotides of the MTR-3 coding sequence starting from the
initiation codon; the 3' end sequence contains complementary
sequences to the other restriction site of interest, a translation
stop codon, the HA tag or FLAG tag and the last 20 nucleotides of
the MTR-3 coding sequence. The PCR amplified fragment and the
pCDNA/Amp vector are digested with the appropriate restriction
enzymes and the vector is dephosphorylated using the CIAP enzyme
(New England Biolabs, Beverly, Mass.). Preferably the two
restriction sites chosen are different so that the MTR-3 gene is
inserted in the correct orientation. The ligation mixture is
transformed into E. coli cells (strains HB101, DH5.alpha., SURE,
available from Stratagene Cloning Systems, La Jolla, Calif., can be
used), the transformed culture is plated on ampicillin media
plates, and resistant colonies are selected. Plasmid DNA is
isolated from transformants and examined by restriction analysis
for the presence of the correct fragment.
[0943] COS cells are subsequently transfected with the
MTR-3-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium
chloride co-precipitation methods, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Other suitable
methods for transfecting host cells can be found in Sambrook, J.,
Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of
the MTR-3 polypeptide is detected by radiolabelling
(.sup.35S-methionine or .sup.35S-cysteine available from NEN,
Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and
Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA
specific monoclonal antibody. Briefly, the cells are labeled for 8
hours with .sup.35S-methionine (or .sup.35S-cysteine). The culture
media are then collected and the cells are lysed using detergents
(RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM
Tris, pH 7.5). Both the cell lysate and the culture media are
precipitated with an HA-specific monoclonal antibody. Precipitated
polypeptides are then analyzed by SDS-PAGE.
[0944] Alternatively, DNA containing the MTR-3 coding sequence is
cloned directly into the polylinker of the pCDNA/Amp vector using
the appropriate restriction sites. The resulting plasmid is
transfected into COS cells in the manner described above, and the
expression of the MTR-3 polypeptide is detected by radiolabelling
and immunoprecipitation using an MTR-3 specific monoclonal
antibody.
IV. METHOD OF TREATING BONE DISEASE USING 17906
Background of the Invention
Carboxypeptidases
[0945] Proteolytic enzymes are involved in many cellular processes.
The carboxypeptidase family of enzymes catalyzes the cleavage of
C-terminal amino acids of peptides and proteins, altering their
biological activity. Lysosomal carboxypeptidase enzymes are highly
concentrated in lysosomes, but may also be active extracellularly
after their release from lysosomes in soluble form or bound to
transmembrane or other membrane-associated proteins.
Carboxypeptidases may cleave peptides in a sequence-specific
manner. For example, prolylcarboxypeptidases cleave only peptides
linked to proline residues (for example, des-Arg9-bradykinin,
angiotensin II). There is also evidence that these enzymes are
involved in terminating signal transduction by inactivating peptide
ligands after receptor endocytosis.
[0946] In contrast to endoproteases which cleave internal peptide
bonds of proteins and polypeptides, carboxypeptidases (CPs)
catalyze the cleavage of only the C-terminal peptide bond,
releasing one amino acid at a time. The two main groups of CPs
include serine CPs and metallo-CPs, the serine CPs containing a
signature trio of Ser, Asp, His in the active site. This trio is
also contained in prolylendopeptidase serine proteases. Serine CPs
include polycarboxypeptidase (PRCP) also referred to as
angiotensinase C; and deamidase, also referred to as cathepsin A
and lysosomal protective protein. See Skidgel et al. (1998)
Immunological Reviews 161:129-141.
[0947] Metallo-CPs contain a signature glutamic acid as the primary
catalytic residue and require zinc-binding for activity.
Metallo-CPs can be grouped by substrate specificity into CPA and
CPB types; the CPA type preferentially cleaving C-terminal
hydrophobic residues, and the CPB type cleaving only peptides with
C-terminal basic Arg or Lys residues. See R. A. Skidgel (1993) In:
Hooper N M, ed. Zinc Metalloproteases in Health and Disease,
London: Taylor & Francis, Ltd., p. 241-283.
[0948] CPM is a B type carboxypeptidase which is anchored on cell
membranes via gylcosylphosphatidylinositol (GPI) association with
its mildly hydrophobic stretch of 15 C-terminal amino acids. As in
many other proteins sharing this anchoring mechanism, CPM is
released from the membrane by bacterial
phosphatidylinositol-specific phospholipase C. Human CPM is a
glycoprotein of 426 amino acid residues with 43% identity to human
intracellular secretory granular CP (CPE), 41% with the active 50
kDa subunit of human plasma CPN, and 15% with bovine pancreatic CPA
or CPB. The active sites of these CPs contain conserved amino acid
residues corresponding to the zinc binding residues
His.sup.66Glu.sup.69 and His.sup.173, substrate binding residues
Arg.sup.137 and Tyr.sup.242, and the catalytic Glu.sup.264, as
designated for CPM. Sequence homologies around these conserved
residues is high, with an identity between CPs M, E and N of
approximately 70-90%. See Tan et al. (1989) J. Biol. Chem.
264:13165-13170; Deddish et al. (1990) J. Biol. Chem.
265:15083-15089; R. A. Skidgel (1993) In: Hooper N M, ed. Zinc
Metalloproteases in Health and Disease, London: Taylor &
Francis, Ltd., p. 241-283. CPM has been mapped to the chromosomal
location of chromosome 12q13-q15 which is associated with a variety
of solid tumors.
[0949] The optimal pH range of CPM is in the neutral range of
6.5-7.5. As no endogenous inhibitors are known for CPM, the enzyme
is considered to be constitutively active. Synthetic inhibitors
including Arg analogs DL-2
mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA) and
guanidinoethylmercaptosuccinic acid (GEMSA) inhibit CPM. See R. A.
Skidgel (1991) In: Conn P M, ed. Methods in Neurosciences: Peptide
Technology Vol. 6, Orlando: Academic Press, p. 373-385; Plummer et
al. (1981) Biochem. Biophys. Res. Comm. 98: 448-254.
[0950] As with other B type regulatory CPs, CPM cleaves only
C-terminal Arg or Lys residues; however, CPM has a preference for
the C-terminal Arg. The penultimate amino acid also affects the
rate of hydrolysis. Naturally occurring peptide substrates of CPM
include bradykinin, Arg.sup.6- and Lys.sup.6 enkephalins, dynorphin
A.sup.1-13 and epidermal growth factor (EGF). See Sidgel et al.
(1989) J. Biol. Chem. 264:2236-2241; McGwire et al. (1995) J. Biol.
Chem. 270:17154-17158.
[0951] CPM is primarily found on the plasma membrane, with highest
levels found in lung and placenta. It is also present in kidney,
blood vessels, intestine, brain and peripheral nerves. See R. A.
Skidgel (1988) Trends Pharm. Sci. 9:299-304; Skidgel et al. (1984)
Biochem. Pharmacol. 33: 3471-3478; Skidgel et al. (1991) FASEB J.
5: 1578; Nagae et al. (1992) J. Neurochem. 59:2201-2212; Nagae et
al. (1993) Am. J. Respir. Cell Mol. Biol. 9:221-229. Expression of
CPM is responsive to differentiation of monocytes and lymphocytes.
See de Saint-V is et al. (1995) Blood 86:1098-1105; Rehli et al.
(1995) J. Biol. Chem. 270:15644-15649.
[0952] CPM participates in the control of peptide hormone activity
at the cell surface and degradation of extracellular proteins and
peptides. It catalyzes the second step in prohormone processing and
removes C-terminal Arg or Lys residues from peptides released from
prohormones. CPM functions as a soluble enzyme after its release
from the plasma membrane and may function in the plasma membrane
form to control peptide receptor activities. CPM can regulate
receptor specificity of kinins by cleaving the C-terminal
ARG.sup.9, for example, from bradykinin. The intact bradykinin
binds the B2 receptor. The cleaved bradykinin
(des-ARG.sup.9-bradykinin). Des-ARG.sup.9-bradykinin also binds the
B1 receptors: stimulates IL-1 and tumor necrosis factor release
from macrophages. Regulation of the B1 receptor is associated with
injury or inflammation. CPM may also be involved with other
inflammatory mediators, such as anaphylatoxin C5a which mediates
histamine release. In addition, CPM may metabolize growth factors
containing terminal Arg or Lys, such as EGF, EGF-like peptides,
nerve growth factor (NGF) amphiregulin, hepatocyte growth factor,
erythropoietin, and macrophage-stimulating protein. In the lung,
varying levels of CPM are associated with pneumocystic or bacterial
pneumonia or lung cancer, and in the placenta, CPM may protect the
fetus from maternally derived peptides. See R. A. Skidgel (1992) J.
Cardiovasc. Pharmacol. 20 (Suppl. 9):S4-S9; Bhoola et al. (1992)
Pharmacol. Rev. 44:1-80; R. A. Skidgel (1993) In: Hooper N M, ed.
Zinc Metalloproteases in Health and Disease, London: Taylor &
Francis, Ltd., p. 241-283; Dragovic et al. (1995) Am. J. Respir.
Crit. Care Med. 152:760-764; Nagae et al. (1992) J. Neurochem.
59:2201-2212; MacFadden et al. (1988) FASEB J. 2:1179
(Abstract).
[0953] Another B-type regulatory CP metalloprotein is CPD, a
membrane-bound glycoprotein. Human CPD is a protein of 1,377 amino
acids with 75% identity with duck GP180 and 90% identity with rat
CPD. Human CPD contains two hydrophobic regions located at the C-
and N-termini. A 55-60 residue cytoplasmic domain is highly
conserved among duck, human and rat sequences and may be
significant in intracellular sorting, protein-protein interactions
or endocytosis. CPD contains three tandem CP homology domains
numbered sequentially from the N- to the C-terminus, and thereby
may contain more than one active site. See Tan et al. (1997)
Biochem. J. 327:81-87; Skidgel et al. (1993) In: Robertson J L S,
Nicholls M G, eds. The Renin Angiotensin System, Vol. 1, London:
Gower Medical Publishing, p. 10.1-10.10. CPD is located on human
chromosome 17, 17P, 11.1-17q, 11.2.
[0954] CPD is primarily found on intracellular membranes, mainly in
the Golgi, with some CPD found on the plasma membrane. The tissue
distribution of CPD is wide and includes most duck tissues and
mammalian tissues as well, including brain, pituitary, placenta,
pancreas, adrenal, kidney, lung, heart, spleen, intestine, ovary,
and testes. See McGwire et al. (1997) Life Sci. 60:715-724; Song et
al. (1995) J. Biol. Chem. 270:25007-25013; Xin et al. (1997) DNA
Cell Biol. 16:897-909; Tan et al. (1997) Biochem. J. 327:81-87;
Song et al. (1996) J. Biol. Chem. 271:28884-28889.
[0955] The function of CPD is speculated to include peptide and
protein processing in the constitutive secretory pathway after
endoprotease cleavage of precursor proteins. The enzyme has an
acidic pH optimum. Mammalian CPD may act as a hepatitis B virus
binding protein, similar to the duck CPD. See R. A. Skidgel (1998)
Immunological Reviews 161:129-141.
[0956] Serine CPs include PRCP and deamidase. PRCP cloned from a
human kidney library indicates a glycoprotein of 51 kDa.sup.3; and
containing 496 amino acids, including a 30 residue signal peptide
and a 15 residue propeptide. See Tan et al. (1993) J. Biol. Chem.
268:16631-16638. A serine repeat is found in the C-terminal half,
similar to the serine repeat of a yeast CP encoded by the KEX1
gene.
[0957] PRCP has an acidic pH optimum for synthetic peptide
substrates, but retains activity at neutral ranges with longer
naturally occurring peptides. PRCP cleaves peptides only if the
penultimate residue is proline. The enzyme does not cleave
Pro-Pro-COOH or (OH)-Pro-Pro-COOH bond. See Odya et al. (1978) J.
Biol. Chem. 253:5927-5931. Substrates of PRCP include
des-Arg.sup.9-bradykinin and angiotensin II.
[0958] PRCP may be involved in terminating signal transduction by
inactivating peptide ligands after receptor endocytosis. PRCP is
contained in lysosomes and released in response to stimulation. The
enzyme is widely distributed and found in human placenta, lung,
liver, and kidney.
[0959] Another serine CP, deamidase, is likely a 94 kDa homodimer
of 52 kDa subunits. Human platelet deamidase is activated by
cleavage of a 14 amino acid fragment from the C-terminus. The
enzyme binds and maintains activity and stability of
.beta.-galactocidase and neuraminidase in lysosomes, a defect of
which is associated with severe galactosialidosis. See Bonten et
al. (1995) J. Biol. Chem. 270:26441-26445; Galjart et al. (1988)
Cell 54:755-764; D'Azzo et al. (1982) Proc. Natl. Acad. Sci.
79:4535-4539. The gene for the human deamidase is mapped to
chromosome 20 at q13.1.
[0960] Deamidase cleaves various peptides containing C-terminal or
penultimate hydrophobic residues including substance P, angiotensin
I, bradykinin, endothelin, and fMet-Leu-Phe. Like PRCP, deamidase
is also found in lysosomes, and distributed in human placenta,
lung, liver, and kidney. Like PRCP, deamidase is implicated in
blocking part of the signal transduction pathway stimulated by
peptides. Bradykinin, containing a C-terminal Arg.sup.9 and a
penultimate hydrophobic amino acid Phe.sup.8, is cleaved by
deamidase. Similarly, angiotensin, containing a C-terminal His and
a penultimate Phe, is cleaved by deamidase. Accordingly, deamidase
is implicated in termination of bradykinin activity on the B2
receptor to generate a B1 receptor agonist. Deamidase may also have
a role in chemotaxis and in metabolism of the anti-cancer growth
factor antagonist. See Skidgel et al. (1998) Immunological Reviews
161:129-141; Jackman et al. (1990) J. Biol. Chem. 265:11265-11272;
Jackman et al. (1995) Am. J. Respir. Cell Mol. Biol. 13:196-204;
Hinek et al. (1996) Biol. Chem. 377:471-480; Jones et al. (1995)
Peptides 16:777-783; Cummings et al. (1995) Biochem Pharmacol.
49:1709-1712.
[0961] Given the wide distribution and various physiological and
pathological roles of carboxypeptidases, methods and compositions
directed at regulating levels of these enzymes are useful for
regulating peptide hormone activity, modulating metabolism of
substance P, angiotensin I, angiotensin II, bradykinin, and
endothelin, and regulation of signal transduction by inactivation
of peptide ligands subsequent to receptor endocytosis.
[0962] Accordingly, carboxypeptidases are a major target for drug
action and development.
[0963] The carboxypeptidase gene used in the methods of the
invention (GenBank Accession AF095719) was purported to be involved
in the histone hyperacetylation signaling pathway relating to
prostate cancer differentiation. (Huang H. et al. Cancer Res.
(1999) "Carboxypeptidase A3 (CPA3): a novel gene highly induced by
histone deacetylase inhibitors during differentiation of prostate
epithelial cancer cells" 15; 59(12):2981-8). It was suggested that
the CPA3 gene is involved in the histone hyperacetylation signaling
pathway activated during NaBu-mediated differentiation of the
androgen-independent prostate cancer cell line, PC-3 cells.
Bone Disorders
[0964] Human bone is subject to constant breakdown and re-synthesis
in a complex process mediated by two cell types: osteoblasts, which
produce new bone, and osteoclasts, which destroy bone. The
activities of these two cell types are kept under control and in
proper balance by a complex network of cytokines, growth factors
and other cellular signals. It is understood that a number of known
bone disorders may have their genesis in aberrant control of these
cells. Likewise, a considerable amount of medical research has
focussed on identifying the aspects of this control network which
can be exploited to re-generate bone in patients with bone
diseases.
[0965] Osteoporosis is one of several known degenerative bone
disorders which can cause significant risk and hardship to those
affected. It is generally defined as the gradual decrease in bone
strength and density that occurs with advancing age, particularly
among post-menopausal women. The clinical manifestations of
osteoporosis include fractures of the vertebral bodies, the neck,
and intertrochanteric regions of the femur, and the distal radius.
Osteoporotic individuals may fracture any bone more easily than
their non-osteoporotic counterparts. As many as many as 15-20
million individuals in the United States are afflicted with
osteoporosis. About 1.3 million fractures attributable to
osteoporosis occur annually in people age 45 and older. Among those
who live to be age 90, 32 percent of women and 17 percent of men
will suffer a hip fracture, mostly due to osteoporosis.
[0966] In addition to osteoporosis, there is a plethora of other
conditions which are characterized by the need to enhance bone
formation. Perhaps the most obvious is in the case of bone
fractures, where it would be desirable to stimulate bone growth and
to hasten and complete bone repair. Agents that enhance bone
formation would also be useful in certain surgical procedures
(e.g., facial reconstruction). Other conditions which result in a
deficit or abnormal formation of bone include osteogenesis
imperfecta (brittle bone disease), hypophosphatasia, Paget's
disease, fibrous dysplasia, osteopetrosis, myeloma bone disease,
and the depletion of calcium in bone which is related to primary
hyperparathyroidism.
[0967] There are currently no pharmaceutical approaches to managing
any of these conditions that is completely satisfactory. Bone
deterioration associated with osteoporosis and other bone
conditions may be treated with estrogens or bisphosphonates, which
have known side effects, or with further invasive surgical
procedures. Bone fractures are still treated exclusively using
casts, braces, anchoring devices and other strictly mechanical
means. More recently, surgical approaches to these types of injury
utilize bovine or human cadaver bone which is chemically treated
(to remove proteins) in order to prevent rejection. However, such
bone implants, while mechanically important, are biologically dead
(they do not contain bone-forming cells, growth factors, or other
regulatory proteins). Thus, they do not greatly modulate the repair
process. All of these concerns demonstrate a great need for new or
novel forms of bone therapy.
Summary of the Invention
[0968] The present invention provides methods for the diagnosis and
treatment of bone associated disease, including but not limited to,
osteogenesis imperfecta (brittle bone disease), osteoporosis,
Paget's disease (enlarged bones), fibrous dysplasia (uneven bone
growth), hypophosphatasia, osteopetrosis, primary hyperthyroidism,
or myeloma bone disease. The present invention is based, at least
in part, on the discovery that the 17906 gene is down-regulated
during osteoblast differentiation, and, thus, may be associated
with a bone disorder.
[0969] In one aspect, the invention provides a method for
identifying the presence of a nucleic acid molecule associated with
a bone associated disorder in a sample by contacting a sample
comprising nucleic acid molecules with a hybridization probe
comprising at least 25 contiguous nucleotides of SEQ ID NO:10, and
detecting the presence of a nucleic acid molecule associated with a
bone associated disorder when the sample contains a nucleic acid
molecule that hybridizes to the nucleic acid probe. In one
embodiment, the hybridization probe is detectably labeled. In
another embodiment the sample comprising nucleic acid molecules is
subjected to agarose gel electrophoresis and southern blotting
prior to contacting with the hybridization probe. In a further
embodiment, the sample comprising nucleic acid molecules is
subjected to agarose gel electrophoresis and northern blotting
prior to contacting with the hybridization probe. In yet another
embodiment, the detecting is by in situ hybridization. In other
embodiments, the method is used to detect mRNA or genomic DNA in
the sample.
[0970] The invention also provides a method for identifying a
nucleic acid associated with a bone associated disorder in a
sample, by contacting a sample comprising nucleic acid molecules
with a first and a second amplification primer, the first primer
comprising at least 25 contiguous nucleotides of SEQ ID NO:10 and
the second primer comprising at least 25 contiguous nucleotides
from the complement of SEQ ID NO:10, incubating the sample under
conditions that allow for nucleic acid amplification, and detecting
the presence of a nucleic acid molecule associated with a bone
associated disorder when the sample contains a nucleic acid
molecule that is amplified. In one embodiment, the sample
comprising nucleic acid molecules is subjected to agarose gel
electrophoresis after the incubation step.
[0971] In addition, the invention provides a method for identifying
a polypeptide associated with a bone associated disorder in a
sample by contacting a sample comprising polypeptide molecules with
a binding substance specific for a 17906 polypeptide, and detecting
the presence of a polypeptide associated with a bone associated
disorder when the sample contains a polypeptide molecule that binds
to the binding substance. In one embodiment the binding substance
is an antibody. In another embodiment, the binding substance is a
17906 ligand. In a further embodiment, the binding substance is
detectably labeled.
[0972] In another aspect, the invention provides a method of
identifying a subject at risk for a bone associated disorder by
contacting a sample obtained from the subject comprising nucleic
acid molecules with a hybridization probe comprising at least 25
contiguous nucleotides of SEQ ID NO:10, and detecting the presence
of a nucleic acid molecule which identifies a subject a risk for a
bone associated disorder when the sample contains a nucleic acid
molecule that hybridizes to the nucleic acid probe.
[0973] In a further aspect, the invention provides a method for
identifying a subject at risk for a bone associated disorder by
contacting a sample obtained from a subject comprising nucleic acid
molecules with a first and a second amplification primer, the first
primer comprising at least 25 contiguous nucleotides of SEQ ID
NO:10 and the second primer comprising at least 25 contiguous
nucleotides from the complement of SEQ ID NO:10, incubating the
sample under conditions that allow for nucleic acid amplification,
and detecting a nucleic acid molecule which identifies a subject at
risk for a bone associated disorder when the sample contains a
nucleic acid molecule that is amplified.
[0974] In yet another aspect, the invention provides a method of
identifying a subject at risk for a bone associated disorder by
contacting a sample obtained from the subject comprising
polypeptide molecules with a binding substance specific for a 17906
polypeptide, and identifying a subject at risk for a bone
associated disorder by detecting the presence of a polypeptide
molecule in the sample that binds to the binding substance.
[0975] In another aspect, the invention provides a method for
identifying a compound capable of treating a bone associated
disorder characterized by aberrant 17906 nucleic acid expression or
17906 protein activity by assaying the ability of the compound to
modulate the expression of a 17906 nucleic acid or the activity of
a 17906 protein. In one embodiment, the disorder is osteoporosis.
In a further embodiment, the ability of the compound to modulate
the activity of the 17906 protein is determined by detecting the
induction of an intracellular second messenger.
[0976] In addition, the invention provides a method for treating a
subject having a bone associated disorder characterized by aberrant
17906 protein activity or aberrant 17906 nucleic acid expression by
administering to the subject a 17906 modulator. In one embodiment,
the 17906 modulator is administered in a pharmaceutically
acceptable formulation. In another embodiment the 17906 modulator
is administered using a gene therapy vector. In a further
embodiment, the 17906 modulator is a small molecule.
[0977] In one embodiment, a modulator is capable of modulating
17906 polypeptide activity. In another embodiment, the 17906
modulator is an anti-17906 antibody. In a further embodiment, the
17906 modulator is a 17906 polypeptide comprising the amino acid
sequence of SEQ ID NO:11, or a fragment thereof. In yet another
embodiment, the 17906 modulator is a 17906 polypeptide comprising
an amino acid sequence which is at least 90 percent identical to
the amino acid sequence of SEQ ID NO:11, wherein the percent
identity is calculated using the ALIGN program for comparing amino
acid sequences, a PAM120 weight residue table, a gap length penalty
of 12, and a gap penalty of 4. In a further embodiment, the 17906
modulator is an isolated naturally occurring allelic variant of a
polypeptide consisting of the amino acid sequence of SEQ ID NO:11,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a complement of a nucleic acid molecule consisting of
SEQ ID NO:10 at 6.times.SSC at 45.degree. C., followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 50-65.degree. C.
[0978] In one embodiment, the 17906 modulator is capable of
modulating 17906 nucleic acid expression. In another embodiment,
the 17906 modulator is an antisense 17906 nucleic acid molecule. In
yet another embodiment, the 17906 modulator is a ribozyme. In a
further embodiment, the 17906 modulator comprises the nucleotide
sequence of SEQ ID NO:10, or a fragment thereof. In another
embodiment, the 17906 modulator comprises a nucleic acid molecule
encoding a polypeptide comprising an amino acid sequence which is
at least 90 percent identical to the amino acid sequence of SEQ ID
NO:11, wherein the percent identity is calculated using the ALIGN
program for comparing amino acid sequences, a PAM120 weight residue
table, a gap length penalty of 12, and a gap penalty of 4. In yet
another embodiment, the 17906 modulator comprises a nucleic acid
molecule encoding a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:11,
wherein the nucleic acid molecule which hybridizes to a complement
of a nucleic acid molecule consisting of SEQ ID NO:10 at
6.times.SSC at 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 50-65.degree. C.
[0979] In another aspect, the invention provides a method for
identifying a compound capable of modulating a osteocyte activity
by contacting a osteocyte with a test compound and assaying the
ability of the test compound to modulate the expression of a 17906
nucleic acid or the activity of a 17906 protein. In certain
embodiments, a compound that modulates the expression of a 17906
nucleic acid or the activity of a 17906 protein modulates osteocyte
proliferation, migration, or the expression of cell surface
adhesion molecules.
[0980] Furthermore, the invention provides a method for modulating
a osteocyte activity comprising contacting a osteocyte with a 17906
modulator.
[0981] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
Detailed Description of the Invention
[0982] The present invention provides methods and compositions for
the diagnosis and treatment of bone associated disease, including
but not limited to, osteogenesis imperfecta (brittle bone disease),
osteoporosis, Paget's disease (enlarged bones), fibrous dysplasia
(uneven bone growth), hypophosphatasia, osteopetrosis, primary
hyperthyroidism, or myeloma bone disease. The present invention is
based, at least in part, on the discovery that carboxypepsidase
genes, referred to herein as "carboxypepsidase 17906" or "17906"
nucleic acid and protein molecules, are down-regulated during
osteoblast differentiation, and, thus, may be associated with a
bone disorder.
[0983] As used herein, "differential expression" includes both
quantitative as well as qualitative differences in the temporal
and/or tissue expression pattern of a gene. Thus, a differentially
expressed gene may have its expression activated or inactivated in
normal versus bone associated disease conditions. The degree to
which expression differs in normal versus bone associated disease
or control versus experimental states need only be large enough to
be visualized via standard characterization techniques, e.g.,
quantitative PCR, Northern analysis, or subtractive hybridization.
The expression pattern of a differentially expressed gene may be
used as part of a prognostic or diagnostic bone associated disease
evaluation, or may be used in methods for identifying compounds
useful for the treatment of bone associated disease. In addition, a
differentially expressed gene involved in bone associated disease
may represent a target gene such that modulation of the level of
target gene expression or of target gene product activity may act
to ameliorate a bone associated disease condition. Compounds that
modulate target gene expression or activity of the target gene
product can be used in the treatment of bone associated disease.
Although the 17906 genes described herein may be differentially
expressed with respect to bone associated disease, and/or their
products may interact with gene products important to bone
associated disease, the genes may also be involved in mechanisms
important to additional bone associated processes.
[0984] The 17906 molecules of the present invention may be involved
in signal transduction and, thus, may that function to modulate
cell proliferation, differentiation, and motility. Thus, the 17906
molecules of the present invention may play a role in cellular
growth signaling mechanisms. As used herein, the term "cellular
growth signaling mechanisms" includes signal transmission from cell
receptors, e.g., G protein coupled receptors, which regulates 1)
cell transversal through the cell cycle, 2) cell differentiation,
3) cell survival, and/or 4) cell migration and patterning.
[0985] Accordingly, the 17906 molecules of the present invention
may be involved in cellular signal transduction pathways that
modulate bone cell activity. As used herein, a "bone cell
activity", "osteocyte activity", or "bone cell function" includes
cell proliferation differentiation, migration, and expression of
cell surface adhesion molecules, as well as cellular process that
contribute to the physiological role of bone cells (e.g., the
regulation of calcium secretion).
[0986] Thus, the 17906 molecules, by participating in cellular
growth signaling mechanisms, may modulate cell behavior and act as
targets and therapeutic agents for controlling cellular
proliferation and differentiation of bone cells.
[0987] The 17906 molecules of the present invention may also act as
novel diagnostic targets and therapeutic agents for bone associated
diseases or disorders. As used herein, a "bone associated disease
or disorder" includes a disease or disorder which affects bones.
The term bone associated disorder includes a disorder affecting the
normal function of the bones. For example, a bone associated
disorder includes osteogenesis imperfecta (brittle bone disease),
osteoporosis, Paget's disease (enlarged bones), fibrous dysplasia
(uneven bone growth), hypophosphatasia, osteopetrosis, primary
hyperthyroidism, or myeloma bone disease. bone associated disorders
are described in, for example, Lamber et al. (2000) Pharmacotherapy
20:34-51; Eisman et al. (1999) Endocrine Reviews 20:788-804; Byers
et al. (1992) Annual Rev. Med., 43:269-282.
[0988] A bone associated disorder also includes a bone cell
disorder. As used herein a "bone cell disorder" includes a disorder
characterized by aberrant or unwanted bone cell activity, e.g.,
proliferation, migration, angiogenesis, or aberrant expression of
cell surface adhesion molecules.
[0989] The present invention provides methods for identifying the
presence of a 17906 nucleic acid or polypeptide molecule associated
with a bone associated disorder. In addition, the invention
provides methods for identifying a subject at risk for a bone
associated disorder by detecting the presence of a 17906 nucleic
acid or polypeptide molecule.
[0990] The invention also provides a method for identifying a
compound capable of treating a bone associated disorder
characterized by aberrant 17906 nucleic acid expression or 17906
protein activity by assaying the ability of the compound to
modulate the expression of a 17906 nucleic acid or the activity of
a 17906 protein. Furthermore, the invention provides a method for
treating a subject having a bone associated disorder characterized
by aberrant 17906 protein activity or aberrant 17906 nucleic acid
expression by administering to the subject a 17906 modulator which
is capable of modulating 17906 protein activity or 17906 nucleic
acid expression.
[0991] Moreover, the invention provides a method for identifying a
compound capable of modulating an bone cell activity by modulating
the expression of a 17906 nucleic acid or the activity of a 17906
protein. The invention provides a method for modulating an bone
cell activity comprising contacting an bone cell with a 17906
modulator.
[0992] Various aspects of the invention are described in further
detail in the following subsections.
Screening Assays
[0993] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules (organic or inorganic) or other drugs) which bind to
17906 proteins, have a stimulatory or inhibitory effect on, for
example, 17906 expression or 17906 activity, or have a stimulatory
or inhibitory effect on, for example, the expression or activity of
a 17906 substrate.
[0994] These assays are designed to identify compounds that bind to
a 17906 protein, bind to other cellular or extracellular proteins
that interact with a 17906 protein, and interfere with the
interaction of the 17906 protein with other cellular or
extracellular proteins. For example, in the case of the 17906
protein, which is a transmembrane receptor-type protein, such
techniques can identify ligands for such a receptor. A 17906
protein ligand can, for example, act as the basis for amelioration
of bone associated diseases, such as, for example, osteoporosis.
Such compounds may include, but are not limited to peptides,
antibodies, or small organic or inorganic compounds. Such compounds
may also include other cellular proteins.
[0995] Compounds identified via assays such as those described
herein may be useful, for example, for ameliorating bone associated
disease. In instances whereby a bone associated disease condition
results from an overall lower level of 17906 gene expression and/or
17906 protein in a cell or tissue, compounds that interact with the
17906 protein may include compounds which accentuate or amplify the
activity of the bound 17906 protein. Such compounds would bring
about an effective increase in the level of 17906 protein activity,
thus ameliorating symptoms.
[0996] In other instances mutations within the 17906 gene may cause
aberrant types or excessive amounts of 17906 proteins to be made
which have a deleterious effect that leads to bone associated
disease. Similarly, physiological conditions may cause an excessive
increase in 17906 gene expression leading to bone associated
disease. In such cases, compounds that bind to a 17906 protein may
be identified that inhibit the activity of the 17906 protein.
Assays for testing the effectiveness of compounds identified by
techniques such as those described in this section are discussed
herein.
[0997] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
17906 protein or polypeptide or biologically active portion
thereof. In another embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of a 17906 protein or polypeptide or biologically active
portion thereof. The test compounds of the present invention can be
obtained using any of the numerous approaches in combinatorial
library methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K. S. (1997) Anticancer
Drug Des. 12:145).
[0998] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0999] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra.).
[1000] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a 17906 protein or biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to modulate 17906 activity is determined. Determining
the ability of the test compound to modulate 17906 activity can be
accomplished by monitoring, for example, intracellular calcium,
IP.sub.3, cAMP, or diacylglycerol concentration, the
phosphorylation profile of intracellular proteins, cell
proliferation and/or migration, the expression of cell surface
adhesion molecules, or the activity of a 17906-regulated
transcription factor. The cell can be of mammalian origin, e.g., a
bone cell. In one embodiment, compounds that interact with a 17906
receptor domain can be screened for their ability to function as
ligands, i.e., to bind to the 17906 receptor and modulate a signal
transduction pathway. Identification of 17906 ligands, and
measuring the activity of the ligand-receptor complex, leads to the
identification of modulators (e.g., antagonists) of this
interaction. Such modulators may be useful in the treatment of bone
associated disease.
[1001] The ability of the test compound to modulate 17906 binding
to a substrate or to bind to 17906 can also be determined.
Determining the ability of the test compound to modulate 17906
binding to a substrate can be accomplished, for example, by
coupling the 17906 substrate with a radioisotope or enzymatic label
such that binding of the 17906 substrate to 17906 can be determined
by detecting the labeled 17906 substrate in a complex. 17906 could
also be coupled with a radioisotope or enzymatic label to monitor
the ability of a test compound to modulate 17906 binding to a 17906
substrate in a complex. Determining the ability of the test
compound to bind 17906 can be accomplished, for example, by
coupling the compound with a radioisotope or enzymatic label such
that binding of the compound to 17906 can be determined by
detecting the labeled 17906 compound in a complex. For example,
compounds (e.g., 17906 ligands or substrates) can be labeled with
.sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Compounds can further
be enzymatically labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product.
[1002] The presence of 17906 in the serum of the transgenic and
wild type animals can be determined by, for example, a
carboxipeptidase assay. Briefly, 5 .mu.l of serum of mice, for
example, can be combined with 45 .mu.l of 55 .mu.M of an
appropriate 17906 substrate including but not limited to e.g.,
angiotensin I, a kinin, or kinetensin, in 17906 buffer. Then, the
rate of proteolytic degradation of the substrate can be measured by
measuring the production of fluorescence (in fluororescence units)
per second for 30 minutes at room temperature at a gain setting of
10. The average rate of fluoresence units per second (FU/sec)
correlates directly with the amount of 17906 in the serum. As a
control for the specificity of 17906, a standard carboxypeptidase
assay can be performed (Holmquist and Riordan, Carboxypeptidase A,
pp 44-60, Peptidase and their Inhibitors in Method of Enzymatic
Analysis (1984)). Further, an additional carboxypeptidase assay can
be performed in accordance with that described in Ostrowska, H. et
al. (1998) Rocz Akad. Med. Bialymst., 43:39-55, which is
incorporated herein by reference.
[1003] It is also within the scope of this invention to determine
the ability of a compound (e.g., a 17906 ligand or substrate) to
interact with 17906 without the labeling of any of the
interactants. For example, a microphysiometer can be used to detect
the interaction of a compound with 17906 without the labeling of
either the compound or the 17906 (McConnell, H. M. et al. (1992)
Science 257:1906-1912). As used herein, a "microphysiometer" (e.g.,
Cytosensor) is an analytical instrument that measures the rate at
which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate
can be used as an indicator of the interaction between a compound
and 17906.
[1004] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a 17906 target molecule
(e.g., a 17906 substrate) with a test compound and determining the
ability of the test compound to modulate (e.g., stimulate or
inhibit) the activity of the 17906 target molecule. Determining the
ability of the test compound to modulate the activity of a 17906
target molecule can be accomplished, for example, by determining
the ability of the 17906 protein to bind to or interact with the
17906 target molecule.
[1005] Determining the ability of the 17906 protein or a
biologically active fragment thereof, to bind to or interact with a
17906 target molecule can be accomplished by one of the methods
described above for determining direct binding. In a preferred
embodiment, determining the ability of the 17906 protein to bind to
or interact with a 17906 target molecule can be accomplished by
determining the activity of the target molecule. For example, the
activity of the target molecule can be determined by detecting
induction of a cellular second messenger of the target (i.e.,
intracellular Ca.sup.2+, diacylglycerol, IP.sub.3, cAMP), detecting
catalytic/enzymatic activity of the target an appropriate
substrate, detecting the induction of a reporter gene (comprising a
target-responsive regulatory element operatively linked to a
nucleic acid encoding a detectable marker, e.g., luciferase), or
detecting a target-regulated cellular response (e.g., cell
proliferation or migration).
[1006] In yet another embodiment, an assay of the present invention
is a cell-free assay in which a 17906 protein or biologically
active portion thereof, is contacted with a test compound and the
ability of the test compound to bind to the 17906 protein or
biologically active portion thereof is determined. Preferred
biologically active portions of the 17906 proteins to be used in
assays of the present invention include fragments which participate
in interactions with non-17906 molecules, e.g., fragments with high
surface probability scores. Binding of the test compound to the
17906 protein can be determined either directly or indirectly as
described above. In a preferred embodiment, the assay includes
contacting the 17906 protein or biologically active portion thereof
with a known compound which binds 17906 to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with a 17906 protein,
wherein determining the ability of the test compound to interact
with a 17906 protein comprises determining the ability of the test
compound to preferentially bind to 17906 or biologically active
portion thereof as compared to the known compound. Compounds that
modulate the interaction of 17906 with a known target protein may
be useful in regulating the activity of a 17906 protein, especially
a mutant 17906 protein.
[1007] In another embodiment, the assay is a cell-free assay in
which a 17906 protein or biologically active portion thereof is
contacted with a test compound and the ability of the test compound
to modulate (e.g., stimulate or inhibit) the activity of the 17906
protein or biologically active portion thereof is determined.
Determining the ability of the test compound to modulate the
activity of a 17906 protein can be accomplished, for example, by
determining the ability of the 17906 protein to bind to a 17906
target molecule by one of the methods described above for
determining direct binding. Determining the ability of the 17906
protein to bind to a 17906 target molecule can also be accomplished
using a technology such as real-time Biomolecular Interaction
Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem.
63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.
5:699-705). As used herein, "BIA" is a technology for studying
biospecific interactions in real time, without labeling any of the
interactants (e.g., BIAcore). Changes in the optical phenomenon of
surface plasmon resonance (SPR) can be used as an indication of
real-time reactions between biological molecules.
[1008] In another embodiment, determining the ability of the test
compound to modulate the activity of a 17906 protein can be
accomplished by determining the ability of the 17906 protein to
further modulate the activity of a downstream effector of a 17906
target molecule. For example, the activity of the effector molecule
on an appropriate target can be determined or the binding of the
effector to an appropriate target can be determined as previously
described.
[1009] In yet another embodiment, the cell-free assay involves
contacting a 17906 protein or biologically active portion thereof
with a known compound which binds the 17906 protein to form an
assay mixture, contacting the assay mixture with a test compound,
and determining the ability of the test compound to interact with
the 17906 protein, wherein determining the ability of the test
compound to interact with the 17906 protein comprises determining
the ability of the 17906 protein to preferentially bind to or
modulate the activity of a 17906 target molecule.
[1010] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either
17906 or its target molecule to facilitate separation of complexed
from uncomplexed forms of one or both of the proteins, as well as
to accommodate automation of the assay. Binding of a test compound
to a 17906 protein, or interaction of a 17906 protein with a target
molecule in the presence and absence of a candidate compound, can
be accomplished in any vessel suitable for containing the
reactants. Examples of such vessels include microtitre plates, test
tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/17906 fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or 17906 protein, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtitre plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of 17906 binding or activity
determined using standard techniques.
[1011] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either a 17906 protein or a 17906 target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated 17906 protein or target molecules can be prepared from
biotin-NHS (N-hydroxy-succinimide) using techniques known in the
art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),
and immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with 17906
protein or target molecules but which do not interfere with binding
of the 17906 protein to its target molecule can be derivatized to
the wells of the plate, and unbound target or 17906 protein trapped
in the wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the 17906 protein or target
molecule, as well as enzyme-linked assays which rely on detecting
an enzymatic activity associated with the 17906 protein or target
molecule.
[1012] In another embodiment, modulators of 17906 expression are
identified in a method wherein a cell is contacted with a candidate
compound and the expression of 17906 mRNA or protein in the cell is
determined. The level of expression of 17906 mRNA or protein in the
presence of the candidate compound is compared to the level of
expression of 17906 mRNA or protein in the absence of the candidate
compound. The candidate compound can then be identified as a
modulator of 17906 expression based on this comparison. For
example, when expression of 17906 mRNA or protein is greater
(statistically significantly greater) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of 17906 mRNA or protein expression.
Alternatively, when expression of 17906 mRNA or protein is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of 17906 mRNA or protein expression. The level of
17906 mRNA or protein expression in the cells can be determined by
methods described herein for detecting 17906 mRNA or protein.
[1013] In yet another aspect of the invention, the 17906 proteins
can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with 17906
("17906-binding proteins" or "17906-bp") and are involved in 17906
activity. Such 17906-binding proteins are also likely to be
involved in the propagation of signals by the 17906 proteins or
17906 targets as, for example, downstream elements of a
17906-mediated signaling pathway. Alternatively, such 17906-binding
proteins are likely to be 17906 inhibitors.
[1014] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a 17906
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact; in
vivo, forming a 17906-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the 17906 protein.
[1015] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a 17906 protein can be confirmed in vivo, e.g., in an animal
such as an animal model for bone associated disease, as described
herein.
[1016] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein (e.g., a 17906 modulating
agent, an antisense 17906 nucleic acid molecule, a 17906-specific
antibody, or a 17906-binding partner) can be used in an animal
model to determine the efficacy, toxicity, or side effects of
treatment with such an agent. Alternatively, an agent identified as
described herein can be used in an animal model to determine the
mechanism of action of such an agent. Furthermore, this invention
pertains to uses of novel agents identified by the above-described
screening assays for treatments as described herein.
[1017] Any of the compounds, including but not limited to compounds
such as those identified in the foregoing assay systems, may be
tested for the ability to ameliorate bone associated disease
symptoms. Cell-based and animal model-based assays for the
identification of compounds exhibiting such an ability to
ameliorate bone associated disease systems are described
herein.
[1018] In one aspect, cell-based systems, as described herein, may
be used to identify compounds which may act to ameliorate bone
associated disease symptoms. For example, such cell systems may be
exposed to a compound, suspected of exhibiting an ability to
ameliorate bone associated disease symptoms, at a sufficient
concentration and for a time sufficient to elicit such an
amelioration of bone associated disease symptoms in the exposed
cells. After exposure, the cells are examined to determine whether
one or more of the bone associated disease cellular phenotypes has
been altered to resemble a more normal or more wild type phenotype.
Cellular phenotypes that are associated with bone associated
disease states include aberrant proliferation and migration,
deposition of extracellular matrix components, and expression of
growth factors, cytokines, and other inflammatory mediators.
[1019] In addition, animal-based bone associated disease systems,
such as those described herein, may be used to identify compounds
capable of ameliorating bone associated 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 bone associated disease. For example,
animal models may be exposed to a compound, suspected of exhibiting
an ability to ameliorate bone associated disease symptoms, at a
sufficient concentration and for a time sufficient to elicit such
an amelioration of bone associated 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
bone associated disease, for example, by measuring bone loss and/or
measuring bone loss before and after treatment.
[1020] With regard to intervention, any treatments which reverse
any aspect of bone associated disease symptoms should be considered
as candidates for human bone associated disease therapeutic
intervention. Dosages of test agents may be determined by deriving
dose-response curves.
[1021] Additionally, gene expression patterns may be utilized to
assess the ability of a compound to ameliorate bone associated
disease symptoms. For example, the expression pattern of one or
more genes may form part of a "gene expression profile" or
"transcriptional profile" which may be then be used in such an
assessment. "Gene expression profile" or "transcriptional profile",
as used herein, includes 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 herein. Gene expression profiles
may be generated, for example, by utilizing a differential display
procedure, Northern analysis and/or real-time quantitative RT-PCR.
In one embodiment, 17906 gene sequences may be used as probes
and/or PCR primers for the generation and corroboration of such
gene expression profiles.
[1022] Gene expression profiles may be characterized for known
states, either bone associated disease or normal, within the cell-
and/or animal-based model systems. Subsequently, these known gene
expression profiles may be compared to ascertain the effect a test
compound has to modify such gene expression profiles, and to cause
the profile to more closely resemble that of a more desirable
profile.
[1023] For example, administration of a compound may cause the gene
expression profile of a bone associated disease model system to
more closely resemble the control system. Administration of a
compound may, alternatively, cause the gene expression profile of a
control system to begin to mimic a bone associated 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.
Predictive Medicine
[1024] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to
diagnostic assays for determining 17906 protein and/or nucleic acid
expression as well as 17906 activity, in the context of a
biological sample (e.g., blood, serum, cells, tissue) to thereby
determine whether an individual is afflicted with a disease or
disorder, or is at risk of developing a bone associated disorder,
associated with aberrant or unwanted 17906 expression or activity.
The invention also provides for prognostic (or predictive) assays
for determining whether an individual is at risk of developing a
disorder associated with 17906 protein, nucleic acid expression or
activity. For example, mutations in a 17906 gene can be assayed in
a biological sample. Such assays can be used for prognostic or
predictive purpose to thereby prophylactically treat an individual
prior to the onset of a disorder characterized by or associated
with 17906 protein, nucleic acid expression or activity.
[1025] Another aspect of the invention pertains to monitoring the
influence of agents (e.g., drugs, compounds) on the expression or
activity of 17906 in clinical trials.
[1026] These and other agents are described in further detail in
the following sections.
Diagnostic Assays
[1027] The present invention encompasses methods for diagnostic and
prognostic evaluation of bone associated disease conditions, and
for the identification of subjects exhibiting a predisposition to
such conditions.
[1028] An exemplary method for detecting the presence or absence of
17906 protein or nucleic acid in a biological sample involves
obtaining a biological sample from a test subject and contacting
the biological sample with a compound or an agent capable of
detecting 17906 protein or nucleic acid (e.g., mRNA, or genomic
DNA) that encodes 17906 protein such that the presence of 17906
protein or nucleic acid is detected in the biological sample. A
preferred agent for detecting 17906 mRNA or genomic DNA is a
labeled nucleic acid probe capable of hybridizing to 17906 mRNA or
genomic DNA. The nucleic acid probe can be, for example, the 17906
nucleic acid set forth in SEQ ID NO:10, or a portion thereof, such
as an oligonucleotide of at least 15, 20, 25, 30, 35, 40, 45, 50,
100, 250 or 500 nucleotides in length and sufficient to
specifically hybridize under stringent conditions to 17906 mRNA or
genomic DNA. Other suitable probes for use in the diagnostic assays
of the invention are described herein.
[1029] A preferred agent for detecting 17906 protein is an antibody
capable of binding to 17906 protein, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab')2) can be used. The term "labeled", with regard to the probe
or antibody, is intended to encompass direct labeling of the probe
or antibody by coupling (i.e., physically linking) a detectable
substance to the probe or antibody, as well as indirect labeling of
the probe or antibody by reactivity with another reagent that is
directly labeled. Examples of indirect labeling include detection
of a primary antibody using a fluorescently labeled secondary
antibody and end-labeling of a DNA probe with biotin such that it
can be detected with fluorescently labeled streptavidin. The term
"biological sample" is intended to include tissues, cells and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject. That is, the detection
method of the invention can be used to detect 17906 mRNA, protein,
or genomic DNA in a biological sample in vitro as well as in vivo.
For example, in vitro techniques for detection of 17906 mRNA
include Northern hybridizations and in situ hybridizations. In
vitro techniques for detection of 17906 protein include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. In vitro techniques
for detection of 17906 genomic DNA include Southern hybridizations.
Furthermore, in vivo techniques for detection of 17906 protein
include introducing into a subject a labeled anti-17906 antibody.
For example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[1030] In one embodiment, the biological sample contains protein
molecules from the test subject. Alternatively, the biological
sample can contain mRNA molecules from the test subject or genomic
DNA molecules from the test subject. A preferred biological sample
is a serum sample isolated by conventional means from a
subject.
[1031] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting 17906
protein, mRNA, or genomic DNA, such that the presence of 17906
protein, mRNA or genomic DNA is detected in the biological sample,
and comparing the presence of 17906 protein, mRNA or genomic DNA in
the control sample with the presence of 17906 protein, mRNA or
genomic DNA in the test sample.
[1032] The invention also encompasses kits for detecting the
presence of 17906 in a biological sample. For example, the kit can
comprise a labeled compound or agent capable of detecting 17906
protein or mRNA in a biological sample; means for determining the
amount of 17906 in the sample; and means for comparing the amount
of 17906 in the sample with a standard. The compound or agent can
be packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect 17906 protein or nucleic
acid.
Prognostic Assays
[1033] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
bone associated disease or disorder associated with aberrant or
unwanted 17906 expression or activity. As used herein, the term
"aberrant" includes a 17906 expression or activity which deviates
from the wild type 17906 expression or activity. Aberrant
expression or activity includes increased or decreased expression
or activity, as well as expression or activity which does not
follow the wild type developmental pattern of expression or the
subcellular pattern of expression. For example, aberrant 17906
expression or activity is intended to include the cases in which a
mutation in the 17906 gene causes the 17906 gene to be
under-expressed or over-expressed and situations in which such
mutations result in a non-functional 17906 protein or a protein
which does not function in a wild-type fashion, e.g., a protein
which does not interact with a 17906 ligand or substrate, or one
which interacts with a non-17906 ligand or substrate. As used
herein, the term "unwanted" includes an unwanted phenomenon
involved in a biological response such as cellular proliferation.
For example, the term unwanted includes a 17906 expression pattern
or a 17906 protein activity which is undesirable in a subject.
[1034] The assays described herein, such as the preceding
diagnostic assays or the following assays, can be utilized to
identify a subject having or at risk of developing a disorder
associated with a misregulation in 17906 protein activity or
nucleic acid expression, such as a bone associated disorder.
Alternatively, the prognostic assays can be utilized to identify a
subject having or at risk for developing a bone associated disorder
associated with a misregulation in 17906 protein activity or
nucleic acid expression. Thus, the present invention provides a
method for identifying a disease or disorder associated with
aberrant or unwanted 17906 expression or activity in which a test
sample is obtained from a subject and 17906 protein or nucleic acid
(e.g., mRNA or genomic DNA) is detected, wherein the presence of
17906 protein or nucleic acid is diagnostic for a subject having or
at risk of developing a disease or disorder associated with
aberrant or unwanted 17906 expression or activity. As used herein,
a "test sample" refers to a biological sample obtained from a
subject of interest. For example, a test sample can be a biological
fluid (e.g., serum), cell sample, or tissue.
[1035] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder associated with aberrant or unwanted 17906
expression or activity. For example, such methods can be used to
determine whether a subject can be effectively treated with an
agent for a bone associated disorder. Thus, the present invention
provides methods for determining whether a subject can be
effectively treated with an agent for a bone associated disorder
associated with aberrant or unwanted 17906 expression or activity
in which a test sample is obtained and 17906 protein or nucleic
acid expression or activity is detected (e.g., wherein the
abundance of 17906 protein or nucleic acid expression or activity
is diagnostic for a subject that can be administered the agent to
treat a disorder associated with aberrant or unwanted 17906
expression or activity).
[1036] The methods of the invention can also be used to detect
genetic alterations in a 17906 gene, thereby determining if a
subject with the altered gene is at risk for a disorder
characterized by misregulation in 17906 protein activity or nucleic
acid expression, such as a proliferative disorder. In preferred
embodiments, the methods include detecting, in a sample of cells
from the subject, the presence or absence of a genetic alteration
characterized by at least one of an alteration affecting the
integrity of a gene encoding a 17906-protein, or the mis-expression
of the 17906 gene. For example, such genetic alterations can be
detected by ascertaining the existence of at least one of 1) a
deletion of one or more nucleotides from a 17906 gene; 2) an
addition of one or more nucleotides to a 17906 gene; 3) a
substitution of one or more nucleotides of a 17906 gene, 4) a
chromosomal rearrangement of a 17906 gene; 5) an alteration in the
level of a messenger RNA transcript of a 17906 gene, 6) aberrant
modification of a 17906 gene, such as of the methylation pattern of
the genomic DNA, 7) the presence of a non-wild type splicing
pattern of a messenger RNA transcript of a 17906 gene, 8) a
non-wild type level of a 17906-protein, 9) allelic loss of a 17906
gene, and 10) inappropriate post-translational modification of a
17906-protein. As described herein, there are a large number of
assays known in the art which can be used for detecting alterations
in a 17906 gene. A preferred biological sample is a tissue or serum
sample isolated by conventional means from a subject.
[1037] In certain embodiments, detection of the alteration involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364),
the latter of which can be particularly useful for detecting point
mutations in the 17906-gene (see Abravaya et al. (1995) Nucleic
Acids Res. 23:675-682). This method can include the steps of
collecting a sample of cells from a subject, isolating nucleic acid
(e.g., genomic, mRNA or both) from the cells of the sample,
contacting the nucleic acid sample with one or more primers which
specifically hybridize to a 17906 gene under conditions such that
hybridization and amplification of the 17906-gene (if present)
occurs, and detecting the presence or absence of an amplification
product, or detecting the size of the amplification product and
comparing the length to a control sample. It is anticipated that
PCR and/or LCR may be desirable to use as a preliminary
amplification step in conjunction with any of the techniques used
for detecting mutations described herein.
[1038] Other amplification methods include: 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.
[1039] In an alternative embodiment, mutations in a 17906 gene from
a sample cell can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
for example, U.S. Pat. No. 5,498,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[1040] In other embodiments, genetic mutations in 17906 can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, to high density arrays containing hundreds or thousands
of oligonucleotides probes (Cronin, M. T. et al. (1996) Human
Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2:
753-759). For example, genetic mutations in 17906 can be identified
in two dimensional arrays containing light-generated DNA probes as
described in Cronin, M. T. et al. supra. Briefly, a first
hybridization array of probes can be used to scan through long
stretches of DNA in a sample and control to identify base changes
between the sequences by making linear arrays of sequential
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene.
[1041] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
17906 gene and detect mutations by comparing the sequence of the
sample 17906 with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques
developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA
74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It
is also contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
((1995) Biotechniques 19:448), including sequencing by mass
spectrometry (see, e.g., PCT International Publication No. WO
94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and
Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
[1042] Other methods for detecting mutations in the 17906 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230:1242). In general, the art technique of
"mismatch cleavage" starts by providing heteroduplexes of formed by
hybridizing (labeled) RNA or DNA containing the wild-type 17906
sequence with potentially mutant RNA or DNA obtained from a tissue
sample. The double-stranded duplexes are treated with an agent
which cleaves single-stranded regions of the duplex such as which
will exist due to basepair mismatches between the control and
sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically
digesting the mismatched regions. In other embodiments, either
DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or
osmium tetroxide and with piperidine in order to digest mismatched
regions. After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing polyacrylamide
gels to determine the site of mutation. See, for example, Cotton et
al. (1988) Proc. Natl. Acad Sci USA 85:4397; Saleeba et al. (1992)
Methods Enzymol. 217:286-295. In a preferred embodiment, the
control DNA or RNA can be labeled for detection.
[1043] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in 17906
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15:1657-1662). According to an exemplary
embodiment, a probe based on a 17906 sequence, e.g., a wild-type
17906 sequence, is hybridized to a cDNA or other DNA product from a
test cell(s). The duplex is treated with a DNA mismatch repair
enzyme, and the cleavage products, if any, can be detected from
electrophoresis protocols or the like (described in, for example,
U.S. Pat. No. 5,459,039).
[1044] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in 17906 genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144;
and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79).
Single-stranded DNA fragments of sample and control 17906 nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al. (1991) Trends Genet 7:5).
[1045] In yet another embodiment the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265:12753).
[1046] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki
et al. (1989) Proc. Natl. Acad. Sci USA 86:6230). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA or a number of different mutations when the oligonucleotides
are attached to the hybridizing membrane and hybridized with
labeled target DNA.
[1047] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner (1993) Tibtech 11:238). In
addition it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88:189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[1048] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a 17906 gene.
[1049] Furthermore, any cell type or tissue in which 17906 is
expressed may be utilized in the prognostic assays described
herein.
Monitoring of Effects During Clinical Trials
[1050] The present invention provides methods for evaluating the
efficacy of drugs and monitoring the progress of patients involved
in clinical trials for the treatment of bone associated
disease.
[1051] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a 17906 protein (e.g., the modulation of
cell proliferation and/or migration) can be applied not only in
basic drug screening, but also in clinical trials. For example, the
effectiveness of an agent determined by a screening assay as
described herein to increase 17906 gene expression, protein levels,
or upregulate 17906 activity, can be monitored in clinical trials
of subjects exhibiting decreased 17906 gene expression, protein
levels, or downregulated 17906 activity. Alternatively, the
effectiveness of an agent determined by a screening assay to
decrease 17906 gene expression, protein levels, or downregulate
17906 activity, can be monitored in clinical trials of subjects
exhibiting increased 17906 gene expression, protein levels, or
upregulated 17906 activity. In such clinical trials, the expression
or activity of a 17906 gene, and preferably, other genes that have
been implicated in, for example, a 17906-associated disorder can be
used as a "read out" or markers of the phenotype a particular cell,
e.g., a bone cell. In addition, the expression of a 17906 gene, or
the level of 17906 protein activity may be used as a read out of a
particular drug or agent's effect on a bone associated disease
state.
[1052] For example, and not by way of limitation, genes, including
17906, that are modulated in cells by treatment with an agent
(e.g., compound, drug or small molecule) which modulates 17906
activity (e.g., identified in a screening assay as described
herein) can be identified. Thus, to study the effect of agents on
17906-associated disorders (e.g., bone associated disorders
characterized by deregulated bone cell activity), for example, in a
clinical trial, cells can be isolated and RNA prepared and analyzed
for the levels of expression of 17906 and other genes implicated in
the 17906-associated disorder, respectively. The levels of gene
expression (e.g., a gene expression pattern) can be quantified by
northern blot analysis or real-time quantitative RT-PCR, as
described herein, or alternatively by measuring the amount of
protein produced, by one of the methods as described herein, or by
measuring the levels of activity of 17906 or other genes. In this
way, the gene expression pattern can serve as a marker, indicative
of the physiological response of the cells to the agent.
Accordingly, this response state may be determined before, and at
various points during treatment of the individual with the
agent.
[1053] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
including the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of a 17906 protein, mRNA, or genomic DNA in
the preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the 17906 protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the 17906 protein, mRNA, or
genomic DNA in the pre-administration sample with the 17906
protein, mRNA, or genomic DNA in the post administration sample or
samples; and (vi) altering the administration of the agent to the
subject accordingly. For example, increased administration of the
agent may be desirable to increase the expression or activity of
17906 to higher levels than detected, i.e., to increase the
effectiveness of the agent. Alternatively, decreased administration
of the agent may be desirable to decrease expression or activity of
17906 to lower levels than detected, i.e. to decrease the
effectiveness of the agent. According to such an embodiment, 17906
expression or activity may be used as an indicator of the
effectiveness of an agent, even in the absence of an observable
phenotypic response.
Methods of Treatment
[1054] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted 17906 expression or activity, e.g. a bone
associated disorder. With regards to both prophylactic and
therapeutic methods of treatment, such treatments may be
specifically tailored or modified, based on knowledge obtained from
the field of pharmacogenomics. "Pharmacogenomics", as used herein,
refers to the application of genomics technologies such as gene
sequencing, statistical genetics, and gene expression analysis to
drugs in clinical development and on the market. More specifically,
the term refers the study of how a patient's genes determine his or
her response to a drug (e.g., a patient's "drug response
phenotype", or "drug response genotype".) Thus, another aspect of
the invention provides methods for tailoring an individual's
prophylactic or therapeutic treatment with either the 17906
molecules of the present invention or 17906 modulators according to
that individual's drug response genotype. Pharmacogenomics allows a
clinician or physician to target prophylactic or therapeutic
treatments to patients who will most benefit from the treatment and
to avoid treatment of patients who will experience toxic
drug-related side effects.
Prophylactic Methods
[1055] In one aspect, the invention provides a method for
preventing in a subject, a bone associated disease or condition
associated with an aberrant or unwanted 17906 expression or
activity, by administering to the subject a 17906 or an agent which
modulates 17906 expression or at least one 17906 activity. Subjects
at risk for a bone associated disease which is caused or
contributed to by aberrant or unwanted 17906 expression or activity
can be identified by, for example, any or a combination of
diagnostic or prognostic assays as described herein. Administration
of a prophylactic agent can occur prior to the manifestation of
symptoms characteristic of the 17906 aberrancy, such that a disease
or disorder is prevented or, alternatively, delayed in its
progression. Depending on the type of 17906 aberrancy, for example,
a 17906, 17906 agonist or 17906 antagonist agent can be used for
treating the subject. The appropriate agent can be determined based
on screening assays described herein.
Therapeutic Methods
[1056] Described herein are methods and compositions whereby bone
associated disease symptoms may be ameliorated. Certain bone
associated diseases are brought about, at least in part, by an
excessive level of a 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 bone associated disease symptoms.
Techniques for the reduction of gene expression levels or the
activity of a protein are discussed below.
[1057] Alternatively, certain other bone associated 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
protein's activity. As such, an increase in the level of gene
expression and/or the activity of such proteins would bring about
the amelioration of bone associated disease symptoms.
[1058] 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 gene's
expression, or the activity of the gene product, will reinforce the
protective effect it exerts. Some bone associated 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 bone associated disease symptoms.
Techniques for increasing target gene expression levels or target
gene product activity levels are discussed herein.
[1059] Accordingly, another aspect of the invention pertains to
methods of modulating 17906 expression or activity for therapeutic
purposes. Accordingly, in an exemplary embodiment, the modulatory
method of the invention involves contacting a cell with a 17906 or
agent that modulates one or more of the activities of 17906 protein
activity associated with the cell (e.g., a bone cell). An agent
that modulates 17906 protein activity can be an agent as described
herein, such as a nucleic acid or a protein, a naturally-occurring
target molecule of a 17906 protein (e.g., a 17906 ligand or
substrate), a 17906 antibody, a 17906 agonist or antagonist, a
peptidomimetic of a 17906 agonist or antagonist, or other small
molecule. In one embodiment, the agent stimulates one or more 17906
activities. Examples of such stimulatory agents include active
17906 protein and a nucleic acid molecule encoding 17906 that has
been introduced into the cell. In another embodiment, the agent
inhibits one or more 17906 activities. Examples of such inhibitory
agents include antisense 17906 nucleic acid molecules, anti-17906
antibodies, and 17906 inhibitors. These modulatory methods can be
performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g., by administering the agent to a
subject). As such, the present invention provides methods of
treating an individual afflicted with a disease or disorder
characterized by aberrant or unwanted expression or activity of a
17906 protein or nucleic acid molecule. In one embodiment, the
method involves administering an agent (e.g., an agent identified
by a screening assay described herein), or combination of agents
that modulates (e.g., upregulates or downregulates) 17906
expression or activity. In another embodiment, the method involves
administering a 17906 protein or nucleic acid molecule as therapy
to compensate for reduced, aberrant, or unwanted 17906 expression
or activity.
[1060] Stimulation of 17906 activity is desirable in situations in
which 17906 is abnormally downregulated and/or in which increased
17906 activity is likely to have a beneficial effect. Likewise,
inhibition of 17906 activity is desirable in situations in which
17906 is abnormally upregulated and/or in which decreased 17906
activity is likely to have a beneficial effect.
Methods for Inhibiting Target Gene Expression, Synthesis, or
Activity
[1061] As discussed above, genes involved in bone associated
disorders may cause such disorders via an increased level of gene
activity. In some cases, such up-regulation may have a causative or
exacerbating effect on the disease state. A variety of techniques
may be used to inhibit the expression, synthesis, or activity of
such genes and/or proteins.
[1062] For example, compounds such as those identified through
assays described above, which exhibit inhibitory activity, may be
used in accordance with the invention to ameliorate bone associated
disease symptoms. Such molecules may include, but are not limited
to, small organic molecules, peptides, antibodies, and the
like.
[1063] For example, compounds can be administered that compete with
endogenous ligand for the 17906 protein. The resulting reduction in
the amount of ligand-bound 17906 protein will modulate bone 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 17906 protein, including,
for example, soluble fusion proteins such as Ig-tailed fusion
proteins. (For a discussion of the production of Ig-tailed fusion
proteins, see, for example, U.S. Pat. No. 5,116,964).
Alternatively, compounds, such as ligand analogs or antibodies,
that bind to the 17906 receptor site, but do not activate the
protein, (e.g., receptor-ligand antagonists) can be effective in
inhibiting 17906 protein activity.
[1064] Further, antisense and ribozyme molecules which inhibit
expression of the 17906 gene may also be used in accordance with
the invention to inhibit aberrant 17906 gene activity. Still
further, triple helix molecules may be utilized in inhibiting
aberrant 17906 gene activity.
[1065] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a 17906 protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of
a route of administration of antisense nucleic acid molecules of
the invention include direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to
target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be
modified such that they specifically bind to receptors or antigens
expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies which
bind to cell surface receptors or antigens. The antisense nucleic
acid molecules can also be delivered to cells using the vectors
described herein. To achieve sufficient intracellular
concentrations of the antisense molecules, vector constructs in
which the antisense nucleic acid molecule is placed under the
control of a strong pol II or pol III promoter are preferred.
[1066] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[1067] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can
be used to catalytically cleave 17906 mRNA transcripts to thereby
inhibit translation of 17906 mRNA. A ribozyme having specificity
for a 17906-encoding nucleic acid can be designed based upon the
nucleotide sequence of a 17906 cDNA disclosed herein (i.e., SEQ ID
NO:10). For example, a derivative of a Tetrahymena L-19 IVS RNA can
be constructed in which the nucleotide sequence of the active site
is complementary to the nucleotide sequence to be cleaved in a
17906-encoding mRNA (see, for example, Cech et al. U.S. Pat. No.
4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Alternatively,
17906 mRNA can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules (see, for
example, Bartel, D. and Szostak, J. W. (1993) Science
261:1411-1418).
[1068] 17906 gene expression can also be inhibited by targeting
nucleotide sequences complementary to the regulatory region of the
17906 (e.g., the 17906 promoter and/or enhancers) to form triple
helical structures that prevent transcription of the 17906 gene in
target cells (see, for example, 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).
[1069] Antibodies that are both specific for the 17906 protein and
interfere with its activity may also be used to modulate or inhibit
17906 protein function. Such antibodies may be generated using
standard techniques described herein, against the 17906 protein
itself or against peptides corresponding to portions of the
protein. Such antibodies include but are not limited to polyclonal,
monoclonal, Fab fragments, single chain antibodies, or chimeric
antibodies.
[1070] In instances where the target gene protein is intracellular
and whole antibodies are used, internalizing antibodies may be
preferred. Lipofectin liposomes may be used to deliver the antibody
or a fragment of the Fab region which binds to the target 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 (described in,
for example, Creighton (1983), supra; and Sambrook et al. (1989)
supra). 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. (1993) Proc.
Natl. Acad. Sci. USA 90:7889-7893).
[1071] In some instances, the target gene protein is extracellular,
or is a transmembrane protein, such as the 17906 protein.
Antibodies that are specific for one or more extracellular domains
of the 17906 protein, for example, and that interfere with its
activity, are particularly useful in treating bone associated
disease. Such antibodies are especially efficient because they can
access the target domains directly from the bloodstream. Any of the
administration techniques described below which are appropriate for
peptide administration may be utilized to effectively administer
inhibitory target gene antibodies to their site of action.
Methods for Restoring, Enhancing or Inhibiting Target Gene
Activity
[1072] Described in this section are methods whereby the level
17906 activity may be modulated to levels wherein bone associated
disease symptoms are ameliorated. The level of 17906 activity may
be modulated, for example, by either modulating the level of 17906
gene expression or by modulating the level of active 17906 protein
which is present.
[1073] Specifically, 17906 is down-regulated in osteoblast
differentiation, thus 17906 may be used to modulate osteoblast
activity, either by increasing 17906 activity and promoting bone
cell proliferation or inhibiting 17906 activity and promoting bone
cell differentiation, for example. Modulation to further decrease
differentiation and to allow bone cells to proliferate is useful
for bone regeneration and thus useful for treating diseases such as
osteoporosis. Modulation to increase differentiation and reduce
proliferation is useful for reducing bone cell growth and thus is
useful for treating diseases such as myeloma bone disease.
[1074] Genes that cause bone associated disease may be
underexpressed within bone associated disease situations. Bone
associated disease symptoms may also develop due to the decrease of
activity of the protein products of such genes. Such
down-regulation of gene expression or decrease of protein activity
might have a causative or exacerbating effect on the disease
state.
[1075] In some cases, genes that are down-regulated in the disease
state might be exerting a protective effect. A variety of
techniques may be used to decrease the expression, synthesis, or
activity of 17906 genes and/or proteins that exert a causatory
effect on bone associated disease conditions.
[1076] In contrast, an inhibitor of a 17906 protein, at a level
sufficient to ameliorate bone associated disease symptoms may be
administered to a patient exhibiting such symptoms. Any of the
techniques discussed below may be used for such administration. One
of skill in the art will readily know how to determine the
concentration of effective, non-toxic doses of an inhibitor of the
17906 protein, utilizing techniques such as those described
below.
[1077] Additionally, antisense 17906 DNA sequences may be directly
administered to a patient exhibiting bone associated disease
symptoms, at a concentration sufficient to reduce the level of
17906 protein such that bone associated disease symptoms are
ameliorated. Any of the techniques discussed below, which achieve
intracellular administration of compounds, such as, for example,
liposome administration, may be used for the administration of such
antisense DNA molecules. The DNA molecules may be produced, for
example, by recombinant techniques such as those described
herein.
[1078] Further, subjects may be treated by gene replacement
therapy. One or more copies of an antagonist of the 17906 molecule,
e.g., a portion of the 17906 gene, 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 used
for the introduction of 17906 gene sequences into human cells.
[1079] Cells, preferably, autologous cells, containing 17906
antagonist expressing gene sequences may then be introduced or
reintroduced into the subject at positions which allow for the
amelioration of bone associated disease symptoms. Such cell
replacement techniques may be preferred, for example, when the gene
product is a secreted, extracellular gene product.
Pharmacogenomics
[1080] The 17906 molecules of the present invention, as well as
agents, or modulators which have a stimulatory or inhibitory effect
on 17906 activity (e.g., 17906 gene expression) as identified by a
screening assay described herein can be administered to individuals
to treat (prophylactically or therapeutically) 17906-associated
disorders (e.g., bone associated disorders) associated with
aberrant or unwanted 17906 activity. In conjunction with such
treatment, pharmacogenomics (i.e., the study of the relationship
between an individual's genotype and that individual's response to
a foreign compound or drug) may be considered. Differences in
metabolism of therapeutics can lead to severe toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus, a
physician or clinician may consider applying knowledge obtained in
relevant pharmacogenomics studies in determining whether to
administer a 17906 molecule or a 17906 modulator as well as
tailoring the dosage and/or therapeutic regimen of treatment with a
17906 molecule or 17906 modulator.
[1081] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin.
Chem. 43(2):254-266. In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body (altered
drug action) or genetic conditions transmitted as single factors
altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare genetic
defects or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[1082] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[1083] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drugs
target is known (e.g., a 17906 protein of the present invention),
all common variants of that gene can be fairly easily identified in
the population and it can be determined if having one version of
the gene versus another is associated with a particular drug
response.
[1084] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[1085] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a 17906 molecule or 17906 modulator of the present
invention) can give an indication whether gene pathways related to
toxicity have been turned on.
[1086] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment an individual. This knowledge, when applied to dosing or
drug selection, can avoid adverse reactions or therapeutic failure
and thus enhance therapeutic or prophylactic efficiency when
treating a subject with a 17906 molecule or 17906 modulator, such
as a modulator identified by one of the exemplary screening assays
described herein.
Detection Assays
[1087] Portions or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. For example, these
sequences can be used to: (i) map their respective genes on a
chromosome; and, thus, locate gene regions associated with genetic
disease; (ii) identify an individual from a minute biological
sample (tissue typing); and (iii) aid in forensic identification of
a biological sample. These applications are described in the
subsections below.
Chromosome Mapping
[1088] Once the sequence (or a portion of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. This process is called chromosome
mapping. Accordingly, portions or fragments of the 17906 nucleotide
sequences, described herein, can be used to map the location of the
17906 genes on a chromosome. The mapping of the 17906 sequences to
chromosomes is an important first step in correlating these
sequences with genes associated with disease. The 17906 gene has
been mapped to human chromosome position 15q14-15.
[1089] Briefly, 17906 genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the
17906 nucleotide sequences. Computer analysis of the 17906
sequences can be used to predict primers that do not span more than
one exon in the genomic DNA, thus complicating the amplification
process. These primers can then be used for PCR screening of
somatic cell hybrids containing individual human chromosomes. Only
those hybrids containing the human gene corresponding to the 17906
sequences will yield an amplified fragment.
[1090] Somatic cell hybrids are prepared by fusing somatic cells
from different mammals (e.g., human and mouse cells). As hybrids of
human and mouse cells grow and divide, they gradually lose human
chromosomes in random order, but retain the mouse chromosomes. By
using media in which mouse cells cannot grow, because they lack a
particular enzyme, but human cells can, the one human chromosome
that contains the gene encoding the needed enzyme, will be
retained. By using various media, panels of hybrid cell lines can
be established. Each cell line in a panel contains either a single
human chromosome or a small number of human chromosomes, and a full
set of mouse chromosomes, allowing easy mapping of individual genes
to specific human chromosomes. (D'Eustachio P. et al. (1983)
Science 220:919-924). Somatic cell hybrids containing only
fragments of human chromosomes can also be produced by using human
chromosomes with translocations and deletions.
[1091] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the 17906 nucleotide sequences to design
oligonucleotide primers, sublocalization can be achieved with
panels of fragments from specific chromosomes. Other mapping
strategies which can similarly be used to map a 17906 sequence to
its chromosome include in situ hybridization (described in Fan, Y.
et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),
pre-screening with labeled flow-sorted chromosomes, and
pre-selection by hybridization to chromosome specific cDNA
libraries.
[1092] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. Chromosome spreads can be
made using cells whose division has been blocked in metaphase by a
chemical such as colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then stained
with Giemsa. A pattern of light and dark bands develops on each
chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500
or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location with
sufficient signal intensity for simple detection. Preferably 1,000
bases, and more preferably 2,000 bases will suffice to get good
results at a reasonable amount of time. For a review of this
technique, see Verma et al., Human Chromosomes: A Manual of Basic
Techniques (Pergamon Press, New York 1988).
[1093] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[1094] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland, J. et al. (1987) Nature, 325:783-787.
[1095] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the 17906 gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
Tissue Typing
[1096] The 17906 sequences of the present invention can also be
used to identify individuals from minute biological samples. The
United States military, for example, is considering the use of
restriction fragment length polymorphism (RFLP) for identification
of its personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identification. This method
does not suffer from the current limitations of "Dog Tags" which
can be lost, switched, or stolen, making positive identification
difficult. The sequences of the present invention are useful as
additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[1097] Furthermore, the sequences of the present invention can be
used to provide an alternative technique which determines the
actual base-by-base DNA sequence of selected portions of an
individual's genome. Thus, the 17906 nucleotide sequences described
herein can be used to prepare two PCR primers from the 5' and 3'
ends of the sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[1098] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
present invention can be used to obtain such identification
sequences from individuals and from tissue. The 17906 nucleotide
sequences of the invention uniquely represent portions of the human
genome. Allelic variation occurs to some degree in the coding
regions of these sequences, and to a greater degree in the
noncoding regions. It is estimated that allelic variation between
individual humans occurs with a frequency of about once per each
500 bases. Each of the sequences described herein can, to some
degree, be used as a standard against which DNA from an individual
can be compared for identification purposes. Because greater
numbers of polymorphisms occur in the noncoding regions, fewer
sequences are necessary to differentiate individuals. The noncoding
sequences of 17906 gene sequences can comfortably provide positive
individual identification with a panel of perhaps 10 to 1,000
primers which each yield a noncoding amplified sequence of 100
bases. If predicted coding sequences, such as those in SEQ ID NO:10
are used, a more appropriate number of primers for positive
individual identification would be 500-2,000.
[1099] If a panel of reagents from 17906 nucleotide sequences
described herein is used to generate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
Use of Partial 17906 Sequences in Forensic Biology
[1100] DNA-based identification techniques can also be used in
forensic biology. Forensic biology is a scientific field employing
genetic typing of biological evidence found at a crime scene as a
means for positively identifying, for example, a perpetrator of a
crime. To make such an identification, PCR technology can be used
to amplify DNA sequences taken from very small biological samples
such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, or semen found at a crime scene. The amplified sequence can
then be compared to a standard, thereby allowing identification of
the origin of the biological sample.
[1101] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
noncoding regions of 17906 gene sequences are particularly
appropriate for this use as greater numbers of polymorphisms occur
in the noncoding regions, making it easier to differentiate
individuals using this technique. Examples of polynucleotide
reagents include the 17906 nucleotide sequences or portions
thereof, e.g., fragments derived from the noncoding regions having
a length of at least 20 bases, preferably at least 30 bases.
[1102] The 17906 nucleotide sequences described herein can further
be used to provide polynucleotide reagents, e.g., labeled or
labelable probes which can be used in, for example, an in situ
hybridization technique, to identify a specific tissue, e.g., brain
tissue. This can be very useful in cases where a forensic
pathologist is presented with a tissue of unknown origin. Panels of
such 17906 probes can be used to identify tissue by species and/or
by organ type.
[1103] In a similar fashion, these reagents, e.g., 17906 primers or
probes can be used to screen tissue culture for contamination (i.e.
screen for the presence of a mixture of different types of cells in
a culture).
Recombinant Expression Vectors and Host Cells
[1104] The methods of the invention include the use of vectors,
preferably expression vectors, containing a nucleic acid encoding a
17906 protein (or a portion thereof). As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. One type of
vector is a "plasmid", which refers to a circular double stranded
DNA loop into which additional DNA segments can be ligated. Another
type of vector is a viral vector, wherein additional DNA segments
can be ligated into the viral genome. Certain vectors are capable
of autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the methods of the invention may include other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[1105] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operatively linked to the nucleic acid
sequence to be expressed. Within a recombinant expression vector,
"operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
which allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cells and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, and the
like. The expression vectors of the invention can be introduced
into host cells to thereby produce proteins or peptides, including
fusion proteins or peptides, encoded by nucleic acids as described
herein (e.g., 17906 proteins, mutant forms of 17906 proteins,
fusion proteins, and the like).
[1106] The recombinant expression vectors of the invention can be
designed for expression of 17906 proteins in prokaryotic or
eukaryotic cells, e.g., for use in the cell-based assays of the
invention. For example, 17906 proteins can be expressed in
bacterial cells such as E. coli, insect cells (using baculovirus
expression vectors) yeast cells or mammalian cells. Suitable host
cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[1107] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein.
[1108] Purified fusion proteins can be utilized in 17906 activity
assays, (e.g., direct assays or competitive assays described in
detail below), or to generate antibodies specific for 17906
proteins, for example. In a preferred embodiment, a 17906 fusion
protein expressed in a retroviral expression vector of the present
invention can be utilized to infect bone marrow cells which are
subsequently transplanted into irradiated recipients. The pathology
of the subject recipient is then examined after sufficient time has
passed (e.g., six (6) weeks).
[1109] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
prophage harboring a T7 gn1 gene under the transcriptional control
of the lacUV 5 promoter.
[1110] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid
to be inserted into an expression vector so that the individual
codons for each amino acid are those preferentially utilized in E.
coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
[1111] In another embodiment, the 17906 expression vector is a
yeast expression vector. Examples of vectors for expression in
yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO
J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell
30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2
(Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen
Corp, San Diego, Calif.).
[1112] Alternatively, 17906 proteins can be expressed in insect
cells using baculovirus expression vectors. Baculovirus vectors
available for expression of proteins in cultured insect cells
(e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol.
Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers
(1989) Virology 170:31-39).
[1113] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)
EMBO J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd,
ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989.
[1114] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al. (1985) Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[1115] The expression characteristics of an endogenous 17906 gene
within a cell line or microorganism may be modified by inserting a
heterologous DNA regulatory element into the genome of a stable
cell line or cloned microorganism such that the inserted regulatory
element is operatively linked with the endogenous 17906 gene. For
example, an endogenous 17906 gene which is normally
"transcriptionally silent", i.e., a 17906 gene which is normally
not expressed, or is expressed only at very low levels in a cell
line or microorganism, may be activated by inserting a regulatory
element which is capable of promoting the expression of a normally
expressed gene product in that cell line or microorganism.
Alternatively, a transcriptionally silent, endogenous 17906 gene
may be activated by insertion of a promiscuous regulatory element
that works across cell types.
[1116] A heterologous regulatory element may be inserted into a
stable cell line or cloned microorganism, such that it is
operatively linked with an endogenous 17906 gene, using techniques,
such as targeted homologous recombination, which are well known to
those of skill in the art, and described, e.g., in Chappel, U.S.
Pat. No. 5,272,071; PCT publication No. WO 91/06667, published May
16, 1991.
[1117] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to 17906 mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[1118] Another aspect of the invention pertains to host cells into
which a 17906 nucleic acid molecule of the invention is introduced,
e.g., a 17906 nucleic acid molecule within a recombinant expression
vector or a 17906 nucleic acid molecule containing sequences which
allow it to homologously recombine into a specific site of the host
cell's genome. The terms "host cell" and "recombinant host cell"
are used interchangeably herein. It is understood that such terms
refer not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[1119] A host cell can be any prokaryotic or eukaryotic cell. For
example, a 17906 protein can be expressed in bacterial cells such
as E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[1120] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[1121] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding a 17906 protein or can be introduced on a separate
vector. Cells stably transfected with the introduced nucleic acid
can be identified by drug selection (e.g., cells that have
incorporated the selectable marker gene will survive, while the
other cells die).
[1122] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) a 17906 protein. Accordingly, the invention further
provides methods for producing a 17906 protein using the host cells
of the invention. In one embodiment, the method comprises culturing
the host cell of the invention (into which a recombinant expression
vector encoding a 17906 protein has been introduced) in a suitable
medium such that a 17906 protein is produced. In another
embodiment, the method further comprises isolating a 17906 protein
from the medium or the host cell.
Cell- and Animal-Based Model Systems
[1123] Described herein are cell- and animal-based systems which
act as models for bone associated 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 genes associated with bone associated
disease, e.g., 17906. In addition, animal- and cell-based assays
may be used as part of screening strategies designed to identify
compounds which are capable of ameliorating bone associated disease
symptoms, as described, below. Thus, the animal- and cell-based
models may be used to identify drugs, pharmaceuticals, therapies
and interventions which may be effective in treating bone
associated disease. Furthermore, such animal models may be used to
determine the LD50 and the ED50 in animal subjects, and such data
can be used to determine the in vivo efficacy of potential bone
associated disease treatments.
Animal-Based Systems
[1124] Animal-based model systems of bone associated disease may
include, but are not limited to, non-recombinant and engineered
transgenic animals.
[1125] Non-recombinant animal models for bone associated disease
may include, for example, genetic models.
[1126] Additionally, animal models exhibiting bone associated
disease symptoms may be engineered by using, for example, 17906
gene sequences described above, in conjunction with techniques for
producing transgenic animals that are well known to those of skill
in the art. For example, 17906 gene sequences may be introduced
into, and overexpressed in, the genome of the animal of interest,
or, if endogenous 17906 gene sequences are present, they may either
be overexpressed or, alternatively, be disrupted in order to
underexpress or inactivate 17906 gene expression.
[1127] The host cells of the invention can also be used to produce
non-human transgenic animals. For example, in one embodiment, a
host cell of the invention is a fertilized oocyte or an embryonic
stem cell into which 17906-coding sequences have been introduced.
Such host cells can then be used to create non-human transgenic
animals in which exogenous 17906 sequences have been introduced
into their genome or homologous recombinant animals in which
endogenous 17906 sequences have been altered. Such animals are
useful for studying the function and/or activity of a 17906 and for
identifying and/or evaluating modulators of 17906 activity. As used
herein, a "transgenic animal" is a non-human animal, preferably a
mammal, more preferably a rodent such as a rat or mouse, in which
one or more of the cells of the animal includes a transgene. Other
examples of transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, amphibians, and the like. A transgene
is exogenous DNA which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal, thereby directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal. As used herein, a "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably
a mouse, in which an endogenous 17906 gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[1128] A transgenic animal of the invention can be created by
introducing a 17906-encoding nucleic acid into the male pronuclei
of a fertilized oocyte, e.g., by microinjection, retroviral
infection, and allowing the oocyte to develop in a pseudopregnant
female foster animal. The 17906 cDNA sequence of SEQ ID NO:10 can
be introduced as a transgene into the genome of a non-human animal.
Alternatively, a nonhuman homologue of a human 17906 gene, such as
a mouse or rat 17906 gene, can be used as a transgene.
Alternatively, a 17906 gene homologue, such as another 17906 family
member, can be isolated based on hybridization to the 17906 cDNA
sequences of SEQ ID NO:10 and used as a transgene. Intronic
sequences and polyadenylation signals can also be included in the
transgene to increase the efficiency of expression of the
transgene. A tissue-specific regulatory sequence(s) can be operably
linked to a 17906 transgene to direct expression of a 17906 protein
to particular cells. Methods for generating transgenic animals via
embryo manipulation and microinjection, particularly animals such
as mice, have become conventional in the art and are described, for
example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder
et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of a 17906
transgene in its genome and/or expression of 17906 mRNA in tissues
or cells of the animals. A transgenic founder animal can then be
used to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene encoding a 17906 protein
can further be bred to other transgenic animals carrying other
transgenes.
[1129] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of a 17906 gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the 17906 gene. The
17906 gene can be a human gene (e.g., the cDNA of SEQ ID NO:10),
but more preferably, is a non-human homologue of a human 17906 gene
(e.g., a cDNA isolated by stringent hybridization with the
nucleotide sequence of SEQ ID NO:10). For example, a mouse 17906
gene can be used to construct a homologous recombination nucleic
acid molecule, e.g., a vector, suitable for altering an endogenous
17906 gene in the mouse genome. In a preferred embodiment, the
homologous recombination nucleic acid molecule is designed such
that, upon homologous recombination, the endogenous 17906 gene is
functionally disrupted (i.e., no longer encodes a functional
protein; also referred to as a "knock out" vector). Alternatively,
the homologous recombination nucleic acid molecule can be designed
such that, upon homologous recombination, the endogenous 17906 gene
is mutated or otherwise altered but still encodes functional
protein (e.g., the upstream regulatory region can be altered to
thereby alter the expression of the endogenous 17906 protein). In
the homologous recombination nucleic acid molecule, the altered
portion of the 17906 gene is flanked at its 5' and 3' ends by
additional nucleic acid sequence of the 17906 gene to allow for
homologous recombination to occur between the exogenous 17906 gene
carried by the homologous recombination nucleic acid molecule and
an endogenous 17906 gene in a cell, e.g., an embryonic stem cell.
The additional flanking 17906 nucleic acid sequence is of
sufficient length for successful homologous recombination with the
endogenous gene. Typically, several kilobases of flanking DNA (both
at the 5' and 3' ends) are included in the homologous recombination
nucleic acid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R.
(1987) Cell 51:503 for a description of homologous recombination
vectors). The homologous recombination nucleic acid molecule is
introduced into a cell, e.g., an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced 17906 gene has
homologously recombined with the endogenous 17906 gene are selected
(see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells
can then injected into a blastocyst of an animal (e.g., a mouse) to
form aggregation chimeras (see e.g., Bradley, A. in
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric
embryo can then be implanted into a suitable pseudopregnant female
foster animal and the embryo brought to term. Progeny harboring the
homologously recombined DNA in their germ cells can be used to
breed animals in which all cells of the animal contain the
homologously recombined DNA by germline transmission of the
transgene. Methods for constructing homologous recombination
nucleic acid molecules, e.g., vectors, or homologous recombinant
animals are described further in Bradley, A. (1991) Current Opinion
in Biotechnology 2:823-829 and in PCT International Publication
Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et
al.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et
al.
[1130] In another embodiment, transgenic non-human animals can be
produced which contain selected systems which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a
recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a
cre/loxP recombinase system is used to regulate expression of the
transgene, animals containing transgenes encoding both the Cre
recombinase and a selected protein are required. Such animals can
be provided through the construction of "double" transgenic
animals, e.g., by mating two transgenic animals, one containing a
transgene encoding a selected protein and the other containing a
transgene encoding a recombinase.
[1131] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. (1997) Nature 385:810-813 and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.O phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyte and then transferred to pseudopregnant female
foster animal. The offspring borne of this female foster animal
will be a clone of the animal from which the cell, e.g., the
somatic cell, is isolated.
[1132] The 17906 transgenic animals that express 17906 mRNA or a
17906 peptide (detected immunocytochemically, using antibodies
directed against 17906 epitopes) at easily detectable levels should
then be further evaluated to identify those animals which display
characteristic bone associated disease symptoms. Such symptoms may
include, for example, increased prevalence and size of fatty
streaks and/or bone associated disease plaques.
[1133] Additionally, specific cell types within the transgenic
animals may be analyzed and assayed for cellular phenotypes
characteristic of bone associated disease. In the case of
monocytes, such phenotypes may include but are not limited to
increases in rates of LDL uptake, adhesion to bone cells,
transmigration, foam cell formation, fatty streak formation, and
production of foam cell specific products. Cellular phenotypes may
include a particular cell type's pattern of expression of genes
associated with bone associated disease as compared to known
expression profiles of the particular cell type in animals
exhibiting bone associated disease symptoms.
[1134] An alternative animal-based model system of bone associated
disease useful in the present invention is found in ovariectomized
rats as described by Dunstan et al. (Dunstan, C. R. et al. J. Bone
Miner Res. Vol. 14(6):953-9, 1999). After ovariectomy (OVX), adult
female rats begin losing bone density, which can lead to conditions
similar to severe osteoporosis. As such the ovariectomized rats may
be examined for the prevention of bone density decreases or for new
bone formation after various treatments, including those of the
present invention.
[1135] Ovariectomized rats may also be used as a model for orally
administered agents to assay for effects on bone loss, as shown by
Mundy et al. (Mundy, G. et al. Science, Vol. 386:1946-1949, 1999).
Mundy et al. also describe another animal-based model system of
detecting bone growth by injection into the subcutaneous tissue
overlying the murine calvaria in mice (Mundy, G. et al. Science,
Vol. 386:1946, 1999). Lastly, Mundy et al. describe a model system
based on neonatal murine calvarial (skullcap) bones in organ
culture as well as an in vitro model for bone formation based on a
murine osteoblast cell line. Both of these may be used as described
below for cell-based model systems.
Cell-Based Systems
[1136] Cells that contain and express 17906 gene sequences which
encode a 17906 protein, and/or exhibit cellular phenotypes
associated with bone associated disease, may be used to identify
compounds that exhibit anti-bone associated disease activity. Such
cells may include non-recombinant monocyte cell lines, such as U937
(ATCC# CRL-1593), THP-1 (ATCC#TIB-202), and P388D1 (ATCC# TIB-63);
hepatic cells such as human Hepa; 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 bone associated disease animal models
of the invention, discussed above, may be used to generate cell
lines, containing one or more cell types involved in bone
associated disease, that can be used as cell culture models for
this disorder. While primary cultures derived from the bone
associated 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.
[1137] Alternatively, cells of a cell type known to be involved in
bone associated disease may be transfected with sequences capable
of increasing or decreasing the amount of 17906 gene expression
within the cell. For example, 17906 gene sequences may be
introduced into, and overexpressed in, the genome of the cell of
interest, or, if endogenous 17906 gene sequences are present, they
may be either overexpressed or, alternatively disrupted in order to
underexpress or inactivate 17906 gene expression.
[1138] In order to overexpress a 17906 gene, the coding portion of
the 17906 gene may be ligated to a regulatory sequence which is
capable of driving gene expression in the cell type of interest,
e.g., a bone cell. 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 above.
[1139] For underexpression of an endogenous 17906 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 17906 alleles will be inactivated. Preferably, the
engineered 17906 sequence is introduced via gene targeting such
that the endogenous 17906 sequence is disrupted upon integration of
the engineered 17906 sequence into the cell's genome. Transfection
of host cells with 17906 genes is discussed, above.
[1140] Cells treated with compounds or transfected with 17906 genes
can be examined for phenotypes associated with bone associated
disease. In the case of osteocytes, such phenotypes include but are
not limited to expression of cytokines or growth factors.
Expression of cytokines or growth factors may be measured using any
of the assays described herein.
[1141] Similarly, bone cells can be treated with test compounds or
transfected with genetically engineered 17906 genes. The bone cells
can then be examined for phenotypes associated with bone associated
disease, including, but not limited to changes in cellular
morphology, cell proliferation, and cell migration; or for the
effects on production of other proteins involved in bone associated
disease such as adhesion molecules (e.g., ICAM, VCAM), PDGF, and
E-selectin.
[1142] Transfection of 17906 nucleic acid may be accomplished by
using standard techniques (described in, for example, Ausubel
(1989) supra). Transfected cells should be evaluated for the
presence of the recombinant 17906 gene sequences, for expression
and accumulation of 17906 mRNA, and for the presence of recombinant
17906 protein production. In instances wherein a decrease in 17906
gene expression is desired, standard techniques may be used to
demonstrate whether a decrease in endogenous 17906 gene expression
and/or in 17906 protein production is achieved.
Pharmaceutical Compositions
[1143] Active compounds for use in the methods of the invention can
be incorporated into pharmaceutical compositions suitable for
administration. As used herein, the language "active compounds"
includes 17906 nucleic acid molecules, fragments of 17906 proteins,
and anti-17906 antibodies, as well as identified compounds that
modulate 17906 gene expression, synthesis, and/or activity. Such
compositions typically comprise the compound, nucleic acid
molecule, protein, or antibody and a pharmaceutically acceptable
carrier. As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[1144] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[1145] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[1146] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a fragment of a 17906
protein or a 17906 ligand) in the required amount in an appropriate
solvent with one or a combination of ingredients enumerated above,
as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[1147] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[1148] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[1149] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[1150] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[1151] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[1152] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals. In one embodiment, a therapeutically effective dose
refers to that amount of an active compound sufficient to result in
amelioration of symptoms of bone associated disease.
[1153] 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 LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. 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.
[1154] 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 ED50 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
IC50 (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.
[1155] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
skilled artisan will appreciate that certain factors may influence
the dosage required to effectively treat a subject, including but
not limited to the severity of the disease or disorder, previous
treatments, the general health and/or age of the subject, and other
diseases present. Moreover, treatment of a subject with a
therapeutically effective amount of a protein, polypeptide, or
antibody can include a single treatment or, preferably, can include
a series of treatments.
[1156] In a preferred example, a subject is treated with antibody,
protein, or polypeptide in the range of between about 0.1 to 20
mg/kg body weight, one time per week for between about 1 to 10
weeks, preferably between 2 to 8 weeks, more preferably between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. It will also be appreciated that the effective dosage of
antibody, protein, or polypeptide used for treatment may increase
or decrease over the course of a particular treatment. Changes in
dosage may result and become apparent from the results of
diagnostic assays as described herein.
[1157] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds. It is understood that appropriate doses of small
molecule agents depends upon a number of factors within the ken of
the ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention.
[1158] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. Such appropriate doses may be determined using the
assays described herein. When one or more of these small molecules
is to be administered to an animal (e.g., a human) in order to
modulate expression or activity of a polypeptide or nucleic acid of
the invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[1159] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[1160] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
.alpha.-interferon, .beta.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[1161] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Helistrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2.sup.nd Ed.),
Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);
Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
[1162] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[1163] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
Isolated Nucleic Acid Molecules
[1164] The nucleotide sequence of the isolated human 17906 cDNA and
the predicted amino acid sequence of the human 17906 polypeptide
are shown in SEQ ID NOs:10 and 11, respectively. The nucleotide
sequence encoding human 17906 is identical to the nucleic acid
molecule with GenBank Accession Number AF095719 (Huang, H. et al.
Cancer Res. (1999) 59(12):2981-2988).
[1165] The human 17906 gene, which is approximately 2795
nucleotides in length, encodes a protein having a molecular weight
of approximately 46.4 kD and which is approximately 422 amino acid
residues in length.
[1166] The methods of the invention include the use of isolated
nucleic acid molecules that encode 17906 proteins or biologically
active portions thereof, as well as nucleic acid fragments
sufficient for use as hybridization probes to identify
17906-encoding nucleic acid molecules (e.g., 17906 mRNA) and
fragments for use as PCR primers for the amplification or mutation
of 17906 nucleic acid molecules. As used herein, the term "nucleic
acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA
or RNA generated using nucleotide analogs. The nucleic acid
molecule can be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[1167] The term "isolated nucleic acid molecule" includes nucleic
acid molecules which are separated from other nucleic acid
molecules which are present in the natural source of the nucleic
acid. For example, with regards to genomic DNA, the term "isolated"
includes nucleic acid molecules which are separated from the
chromosome with which the genomic DNA is naturally associated.
Preferably, an "isolated" nucleic acid is free of sequences which
naturally flank the nucleic acid (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. For example, in various
embodiments, the isolated 17906 nucleic acid molecule can contain
less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of
nucleotide sequences which naturally flank the nucleic acid
molecule in genomic DNA of the cell from which the nucleic acid is
derived. Moreover, an "isolated" nucleic acid molecule, such as a
cDNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized.
[1168] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having the nucleotide sequence of SEQ ID
NO:10, or a portion thereof, can be isolated using standard
molecular biology techniques and the sequence information provided
herein. Using all or portion of the nucleic acid sequence of SEQ ID
NO:10, as a hybridization probe, 17906 nucleic acid molecules can
be isolated using standard hybridization and cloning techniques
(e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis,
T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989).
[1169] Moreover, a nucleic acid molecule encompassing all or a
portion of SEQ ID NO:10 can be isolated by the polymerase chain
reaction (PCR) using synthetic oligonucleotide primers designed
based upon the sequence of SEQ ID NO:10.
[1170] A nucleic acid of the invention can be amplified using cDNA,
mRNA or alternatively, genomic DNA, as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to 17906 nucleotide
sequences can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[1171] In a preferred embodiment, an isolated nucleic acid molecule
of the invention comprises the nucleotide sequence shown in SEQ ID
NO:10. The sequence of SEQ ID NO:10 corresponds to the human 17906
cDNA. This cDNA comprises sequences encoding the human 17906
protein (i.e., "the coding region of SEQ ID NO:10").
[1172] In another preferred embodiment, an isolated nucleic acid
molecule of the invention comprises a nucleic acid molecule which
is a complement of the nucleotide sequence shown in SEQ ID NO:10,
or a portion of any of this nucleotide sequence. A nucleic acid
molecule which is complementary to the nucleotide sequence shown in
SEQ ID NO:10 is one which is sufficiently complementary to the
nucleotide sequence shown in SEQ ID NO:10 such that it can
hybridize to the nucleotide sequence shown in SEQ ID NO:10, thereby
forming a stable duplex.
[1173] In still another preferred embodiment, an isolated nucleic
acid molecule of the present invention comprises a nucleotide
sequence which is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to the
entire length of the nucleotide sequence shown in SEQ ID NO:10, or
a portion of any of this nucleotide sequence.
[1174] Moreover, the nucleic acid molecule of the invention can
comprise only a portion of the nucleic acid sequence of SEQ ID
NO:10, for example, a fragment which can be used as a probe or
primer or a fragment encoding a portion of a 17906 protein, e.g., a
biologically active portion of a 17906 protein. The nucleotide
sequence determined from the cloning of the 17906 gene allows for
the generation of probes and primers designed for use in
identifying and/or cloning other 17906 family members, as well as
17906 homologues from other species. The probe/primer typically
comprises substantially purified oligonucleotide. The
oligonucleotide typically comprises a region of nucleotide sequence
that hybridizes under stringent conditions to at least about 12 or
15, preferably about 20 or 25, more preferably about 30, 35, 40,
45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense
sequence of SEQ ID NO:10, of an anti-sense sequence of SEQ ID
NO:10, or of a naturally occurring allelic variant or mutant of SEQ
ID NO:10. In one embodiment, a nucleic acid molecule of the present
invention comprises a nucleotide sequence which is greater than
100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800,
or more nucleotides in length and hybridizes under stringent
hybridization conditions to a nucleic acid molecule of SEQ ID
NO:10.
[1175] Probes based on the 17906 nucleotide sequence can be used to
detect transcripts or genomic sequences encoding the same or
homologous proteins. In preferred embodiments, the probe further
comprises a label group attached thereto, e.g., the label group can
be a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells or tissue which misexpress a 17906
protein, such as by measuring a level of a 17906-encoding nucleic
acid in a sample of cells from a subject e.g., detecting 17906 mRNA
levels or determining whether a genomic 17906 gene has been mutated
or deleted.
[1176] A nucleic acid fragment encoding a "biologically active
portion of a 17906 protein" can be prepared by isolating a portion
of the nucleotide sequence of SEQ ID NO:10 which encodes a
polypeptide having a 17906 biological activity (the biological
activities of the 17906 protein is described herein), expressing
the encoded portion of the 17906 protein (e.g., by recombinant
expression in vitro) and assessing the activity of the encoded
portion of the 17906 protein.
[1177] The methods of the invention further encompass nucleic acid
molecules that differ from the nucleotide sequence shown in SEQ ID
NO:10, due to degeneracy of the genetic code and thus encode the
same 17906 protein as those encoded by the nucleotide sequence
shown in SEQ ID NO:10. In another embodiment, an isolated nucleic
acid molecule of the invention has a nucleotide sequence encoding a
protein having an amino acid sequence shown in SEQ ID NO:11.
[1178] In addition to the 17906 nucleotide sequence shown in SEQ ID
NO:10, it will be appreciated by those skilled in the art that DNA
sequence polymorphisms that lead to changes in the amino acid
sequences of the 17906 protein may exist within a population (e.g.,
the human population). Such genetic polymorphism in the 17906 gene
may exist among individuals within a population due to natural
allelic variation. As used herein, the terms "gene" and
"recombinant gene" refer to nucleic acid molecules which include an
open reading frame encoding a 17906 protein, preferably a mammalian
17906 protein, and can further include non-coding regulatory
sequences, and introns.
[1179] Allelic variants of human 17906 include both functional and
non-functional 17906 proteins. Functional allelic variants are
naturally occurring amino acid sequence variants of the human 17906
protein that maintain the ability to bind a 17906 ligand or
substrate and/or modulate cell proliferation and/or migration
mechanisms. Functional allelic variants will typically contain only
conservative substitution of one or more amino acids of SEQ ID
NO:11, or substitution, deletion or insertion of non-critical
residues in non-critical regions of the protein.
[1180] Non-functional allelic variants are naturally occurring
amino acid sequence variants of the human 17906 protein that do not
have the ability to either bind a 17906 ligand or substrate and/or
modulate cell proliferation and/or migration mechanisms.
Non-functional allelic variants will typically contain a
non-conservative substitution, a deletion, or insertion or
premature truncation of the amino acid sequence of SEQ ID NO:10, or
a substitution, insertion or deletion in critical residues or
critical regions.
[1181] The methods of the present invention may further use
non-human orthologues of the human 17906 protein. Orthologues of
the human 17906 protein are proteins that are isolated from
non-human organisms and possess the same 17906 ligand binding
and/or modulation of cell proliferation and/or migration mechanisms
of the human 17906 protein. Orthologues of the human 17906 protein
can readily be identified as comprising an amino acid sequence that
is substantially identical to SEQ ID NO:11.
[1182] Moreover, nucleic acid molecules encoding other 17906 family
members and, thus, which have a nucleotide sequence which differs
from the 17906 sequence of SEQ ID NO:10 are intended to be within
the scope of the invention. For example, another 17906 cDNA can be
identified based on the nucleotide sequence of human 17906.
Moreover, nucleic acid molecules encoding 17906 proteins from
different species, and which, thus, have a nucleotide sequence
which differs from the 17906 sequence of SEQ ID NO:10 are intended
to be within the scope of the invention. For example, a mouse 17906
cDNA can be identified based on the nucleotide sequence of human
17906.
[1183] Nucleic acid molecules corresponding to natural allelic
variants and homologues of the 17906 cDNA of the invention can be
isolated based on their homology to the 17906 nucleic acid
disclosed herein using the cDNAs disclosed herein, or a portion
thereof, as a hybridization probe according to standard
hybridization techniques under stringent hybridization conditions.
Nucleic acid molecules corresponding to natural allelic variants
and homologues of the 17906 cDNA of the invention can further be
isolated by mapping to the same chromosome or locus as the 17906
gene.
[1184] Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention is at least 15, 20, 25, 30 or more
nucleotides in length and hybridizes under stringent conditions to
the nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO:10. In other embodiment, the nucleic acid is at least 30, 50,
100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,
750, 800, 1000, 1200, or more nucleotides in length. As used
herein, the term "hybridizes under stringent conditions" is
intended to describe conditions for hybridization and washing under
which nucleotide sequences at least 60% identical to each other
typically remain hybridized to each other. Preferably, the
conditions are such that sequences at least about 70%, more
preferably at least about 80%, even more preferably at least about
85% or 90% identical to each other typically remain hybridized to
each other. Such stringent conditions are known to those skilled in
the art and can be found in Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred,
non-limiting example of stringent hybridization conditions are
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 50.degree. C., preferably at 55.degree.
C., more preferably at 60.degree. C., and even more preferably at
65.degree. C. Ranges intermediate to the above-recited values,
e.g., at 60-65.degree. C. or at 55-60.degree. C. are also intended
to be encompassed by the present invention. Preferably, an isolated
nucleic acid molecule of the invention that hybridizes under
stringent conditions to the sequence of SEQ ID NO:10 corresponds to
a naturally-occurring nucleic acid molecule. As used herein, a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA
molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein).
[1185] In addition to naturally-occurring allelic variants of the
17906 sequences that may exist in the population, the skilled
artisan will further appreciate that changes can be introduced by
mutation into the nucleotide sequence of SEQ ID NO:10, thereby
leading to changes in the amino acid sequence of the encoded 17906
protein, without altering the functional ability of the 17906
protein. For example, nucleotide substitutions leading to amino
acid substitutions at "non-essential" amino acid residues can be
made in the sequence of SEQ ID NO:10. A "non-essential" amino acid
residue is a residue that can be altered from the wild-type
sequence of 17906 (e.g., the sequence of SEQ ID NO:11) without
altering the biological activity, whereas an "essential" amino acid
residue is required for biological activity. For example, amino
acid residues that are conserved among the 17906 proteins of the
present invention are predicted to be particularly unamenable to
alteration. Furthermore, additional amino acid residues that are
conserved between the 17906 proteins of the present invention and
other members of the G protein-coupled receptor family are not
likely to be amenable to alteration.
[1186] Accordingly, the methods of the invention may include the
use of nucleic acid molecules encoding 17906 proteins that contain
changes in amino acid residues that are not essential for activity.
Such 17906 proteins differ in amino acid sequence from SEQ ID
NO:11, yet retain biological activity. In one embodiment, the
isolated nucleic acid molecule comprises a nucleotide sequence
encoding a protein, wherein the protein comprises an amino acid
sequence at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to SEQ ID
NO:11.
[1187] An isolated nucleic acid molecule encoding a 17906 protein
identical to the protein of SEQ ID NO:11 can be created by
introducing one or more nucleotide substitutions, additions or
deletions into the nucleotide sequence of SEQ ID NO:10 such that
one or more amino acid substitutions, additions or deletions are
introduced into the encoded protein. Mutations can be introduced
into SEQ ID NO:10 by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Preferably, conservative
amino acid substitutions are made at one or more predicted
non-essential amino acid residues. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in
the art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a predicted nonessential amino acid residue in a
17906 protein is preferably replaced with another amino acid
residue from the same side chain family. Alternatively, in another
embodiment, mutations can be introduced randomly along all or part
of a 17906 coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for 17906 biological activity
to identify mutants that retain activity. Following mutagenesis of
SEQ ID NO:10, the encoded protein can be expressed recombinantly
and the activity of the protein can be determined.
[1188] In a preferred embodiment, a mutant 17906 protein can be
assayed for the ability to (1) interact with a non-17906 protein
molecule, e.g., a 17906 ligand or substrate; (2) activate a
17906-dependent signal transduction pathway; or (3) modulate cell
proliferation and/or migration mechanisms, or modulate the
expression of cell surface adhesion molecules.
[1189] In addition to the nucleic acid molecules encoding 17906
proteins described herein, another aspect of the invention pertains
to isolated nucleic acid molecules which are antisense thereto. An
"antisense" nucleic acid comprises a nucleotide sequence which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. Accordingly, an
antisense nucleic acid can hydrogen bond to a sense nucleic acid.
The antisense nucleic acid can be complementary to an entire 17906
coding strand, or to only a portion thereof. In one embodiment, an
antisense nucleic acid molecule is antisense to a "coding region"
of the coding strand of a nucleotide sequence encoding 17906. The
term "coding region" refers to the region of the nucleotide
sequence comprising codons which are translated into amino acid
residues (e.g., the coding region of human 17906 corresponds to
nucleotides 8-1273 of SEQ ID NO:10). In another embodiment, the
antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding
17906. The term "noncoding region" refers to 5' and 3' sequences
which flank the coding region that are not translated into amino
acids (i.e., also referred to as 5' and 3' untranslated
regions).
[1190] Given the coding strand sequences encoding 17906 disclosed
herein (e.g., nucleotides 8-1273 of SEQ ID NO:10), antisense
nucleic acids of the invention can be designed according to the
rules of Watson and Crick base pairing. The antisense nucleic acid
molecule can be complementary to the entire coding region of 17906
mRNA, but more preferably is an oligonucleotide which is antisense
to only a portion of the coding or noncoding region of 17906 mRNA.
For example, the antisense oligonucleotide can be complementary to
the region surrounding the translation start site of 17906 mRNA. An
antisense oligonucleotide can be, for example, about 5, 10, 15, 20,
25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense
nucleic acid of the invention can be constructed using chemical
synthesis and enzymatic ligation reactions using procedures known
in the art. For example, an antisense nucleic acid (e.g., an
antisense oligonucleotide) can be chemically synthesized using
naturally occurring nucleotides or variously modified nucleotides
designed to increase the biological stability of the molecules or
to increase the physical stability of the duplex formed between the
antisense and sense nucleic acids, e.g., phosphorothioate
derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides which can be used to generate the
antisense nucleic acid include 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, 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-isopentenyladenine,
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. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest).
[1191] In yet another embodiment, the 17906 nucleic acid molecules
of the present invention can be modified at the base moiety, sugar
moiety or phosphate backbone to improve, e.g., the stability,
hybridization, or solubility of the molecule. For example, the
deoxyribose phosphate backbone of the nucleic acid molecules can be
modified to generate peptide nucleic acids (see Hyrup B. et al.
(1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used
herein, the terms "peptide nucleic acids" or "PNAs" refer to
nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only
the four natural nucleobases are retained. The neutral backbone of
PNAs has been shown to allow for specific hybridization to DNA and
RNA under conditions of low ionic strength. The synthesis of PNA
oligomers can be performed using standard solid phase peptide
synthesis protocols as described in Hyrup B. et al. (1996) supra;
Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
[1192] PNAs of 17906 nucleic acid molecules can be used in
therapeutic and diagnostic applications. For example, PNAs can be
used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of 17906 nucleic acid molecules can also be used in the analysis of
single base pair mutations in a gene, (e.g., by PNA-directed PCR
clamping); as `artificial restriction enzymes` when used in
combination with other enzymes, (e.g., S1 nucleases (Hyrup B.
(1996) supra)); or as probes or primers for DNA sequencing or
hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe
supra).
[1193] In another embodiment, PNAs of 17906 can be modified, (e.g.,
to enhance their stability or cellular uptake), by attaching
lipophilic or other helper groups to PNA, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of
drug delivery known in the art. For example, PNA-DNA chimeras of
17906 nucleic acid molecules can be generated which may combine the
advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, (e.g., RNAse H and DNA polymerases), to
interact with the DNA portion while the PNA portion would provide
high binding affinity and specificity. PNA-DNA chimeras can be
linked using linkers of appropriate lengths selected in terms of
base stacking, number of bonds between the nucleobases, and
orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA
chimeras can be performed as described in Hyrup B. (1996) supra and
Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For
example, a DNA chain can be synthesized on a solid support using
standard phosphoramidite coupling chemistry and modified nucleoside
analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite, can be used as a between the PNA and the 5' end of
DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA
monomers are then coupled in a stepwise manner to produce a
chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn
P. J. et al. (1996) supra). Alternatively, chimeric molecules can
be synthesized with a 5' DNA segment and a 3' PNA segment
(Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5:
1119-11124).
[1194] In other embodiments, 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. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (See, e.g., Krol et al.
(1988) Bio-Techniques 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, or hybridization-triggered cleavage agent).
Isolated 17906 Proteins and Anti-17906 Antibodies
[1195] The methods of the invention include the use of isolated
17906 proteins, and biologically active portions thereof, as well
as polypeptide fragments suitable for use as immunogens to raise
anti-17906 antibodies.
[1196] Isolated proteins of the present invention, preferably 17906
proteins, have an amino acid sequence sufficiently identical to the
amino acid sequence of SEQ ID NO:11, or are encoded by a nucleotide
sequence sufficiently identical to SEQ ID NO:10. As used herein,
the term "sufficiently identical" refers to a first amino acid or
nucleotide sequence which contains a sufficient or minimum number
of identical or equivalent (e.g., an amino acid residue which has a
similar side chain) amino acid residues or nucleotides to a second
amino acid or nucleotide sequence such that the first and second
amino acid or nucleotide sequences share common structural domains
or motifs and/or a common functional activity. For example, amino
acid or nucleotide sequences which share common structural domains
have at least 30%, 40%, or 50% homology, preferably 60% homology,
more preferably 70%-80%, and even more preferably 90-95% homology
across the amino acid sequences of the domains and contain at least
one and preferably two structural domains or motifs, are defined
herein as sufficiently identical. Furthermore, amino acid or
nucleotide sequences which share at least 30%, 40%, or 50%,
preferably 60%, more preferably 70-80%, or 90-95% homology and
share a common functional activity are defined herein as
sufficiently identical.
[1197] As used interchangeably herein, a "17906 activity",
"biological activity of 17906" or "functional activity of 17906",
refers to an activity exerted by a 17906 protein, polypeptide or
nucleic acid molecule on a 17906 responsive cell (e.g., a bone
cell) or tissue, or on a 17906 protein substrate, as determined in
vivo, or in vitro, according to standard techniques. In one
embodiment, a 17906 activity is a direct activity, such as an
association with a 17906 target molecule. As used herein, a "target
molecule" or "binding partner" is a molecule with which a 17906
protein binds or interacts in nature, such that 17906-mediated
function is achieved. A 17906 target molecule can be a non-17906
molecule or a 17906 protein or polypeptide of the present
invention. In an exemplary embodiment, a 17906 target molecule is a
17906 ligand. Alternatively, a 17906 activity is an indirect
activity, such as a cellular signaling activity mediated by
interaction of the 17906 protein with a 17906 ligand. Preferably, a
17906 activity is the ability to act as a signal transduction
molecule and to modulate bone cell proliferation, differentiation,
and/or migration. Accordingly, another embodiment of the invention
features isolated 17906 proteins and polypeptides having a 17906
activity.
[1198] In one embodiment, native 17906 proteins can be isolated
from cells or tissue sources by an appropriate purification scheme
using standard protein purification techniques. In another
embodiment, 17906 proteins are produced by recombinant DNA
techniques. Alternative to recombinant expression, a 17906 protein
or polypeptide can be synthesized chemically using standard peptide
synthesis techniques.
[1199] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the 17906 protein is derived, or substantially free from chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of 17906 protein in which the protein is separated
from cellular components of the cells from which it is isolated or
recombinantly produced. In one embodiment, the language
"substantially free of cellular material" includes preparations of
17906 protein having less than about 30% (by dry weight) of
non-17906 protein (also referred to herein as a "contaminating
protein"), more preferably less than about 20% of non-17906
protein, still more preferably less than about 10% of non-17906
protein, and most preferably less than about 5% non-17906 protein.
When the 17906 protein or biologically active portion thereof is
recombinantly produced, it is also preferably substantially free of
culture medium, i.e., culture medium represents less than about
20%, more preferably less than about 10%, and most preferably less
than about 5% of the volume of the protein preparation.
[1200] The language "substantially free of chemical precursors or
other chemicals" includes preparations of 17906 protein in which
the protein is separated from chemical precursors or other
chemicals which are involved in the synthesis of the protein. In
one embodiment, the language "substantially free of chemical
precursors or other chemicals" includes preparations of 17906
protein having less than about 30% (by dry weight) of chemical
precursors or non-17906 chemicals, more preferably less than about
20% chemical precursors or non-17906 chemicals, still more
preferably less than about 10% chemical precursors or non-17906
chemicals, and most preferably less than about 5% chemical
precursors or non-17906 chemicals.
[1201] As used herein, a "biologically active portion" of a 17906
protein includes a fragment of a 17906 protein which participates
in an interaction between a 17906 molecule and a non-17906
molecule. Biologically active portions of a 17906 protein include
peptides comprising amino acid sequences sufficiently identical to
or derived from the amino acid sequence of the 17906 protein, e.g.,
the amino acid sequence shown in SEQ ID NO:11, which include less
amino acids than the full length 17906 protein, and exhibit at
least one activity of a 17906 protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the 17906 protein, e.g., modulating cell proliferation
mechanisms. A biologically active portion of a 17906 protein can be
a polypeptide which is, for example, 10, 25, 50, 100, 200, or more
amino acids in length. Biologically active portions of a 17906
protein can be used as targets for developing agents which modulate
a 17906 mediated activity, e.g., a cell proliferation mechanism. A
biologically active portion of a 17906 protein comprises a protein
in which regions of the protein are deleted, can be prepared by
recombinant techniques and evaluated for one or more of the
functional activities of a native 17906 protein.
[1202] In a preferred embodiment, the 17906 protein has an amino
acid sequence shown in SEQ ID NO:11. In other embodiments, the
17906 protein is substantially identical to SEQ ID NO:11, and
retains the functional activity of the protein of SEQ ID NO:11, yet
differs in amino acid sequence due to natural allelic variation or
mutagenesis, as described in detail in subsection I above.
Accordingly, in another embodiment, the 17906 protein is a protein
which comprises an amino acid sequence at least about 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or
more identical to SEQ ID NO:11.
[1203] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-identical
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence (e.g., when aligning a second sequence to
the 17906 amino acid sequence of SEQ ID NO:11 having 516 amino acid
residues, at least 136, preferably at least 181, more preferably at
least 227, even more preferably at least 272, and even more
preferably at least 317, 362 or 408 amino acid residues are
aligned). The amino acid residues or nucleotides at corresponding
amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same amino
acid residue or nucleotide as the corresponding position in the
second sequence, then the molecules are identical at that position
(as used herein amino acid or nucleic acid "identity" is equivalent
to amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[1204] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm
which has been incorporated into the GAP program in the GCG
software package, using either a Blosum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package, using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the
percent identity between two amino acid or nucleotide sequences is
determined using the algorithm of E. Meyers and W. Miller (Myers
and Miller, Comput. Appl. Biosci. 4:11-17 (1988)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
[1205] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against public databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can
be performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to 17906 nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=100, wordlength=3 to obtain amino
acid sequences homologous to 17906 protein molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used.
[1206] The methods of the invention may also use 17906 chimeric or
fusion proteins. As used herein, a 17906 "chimeric protein" or
"fusion protein" comprises a 17906 polypeptide operatively linked
to a non-17906 polypeptide. A "17906 polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to 17906,
whereas a "non-17906 polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to a protein which is not
substantially homologous to the 17906 protein, e.g., a protein
which is different from the 17906 protein and which is derived from
the same or a different organism. Within a 17906 fusion protein the
17906 polypeptide can correspond to all or a portion of a 17906
protein. In a preferred embodiment, a 17906 fusion protein
comprises at least one biologically active portion of a 17906
protein. In another preferred embodiment, a 17906 fusion protein
comprises at least two biologically active portions of a 17906
protein. Within the fusion protein, the term "operatively linked"
is intended to indicate that the 17906 polypeptide and the
non-17906 polypeptide are fused in-frame to each other. The
non-17906 polypeptide can be fused to the N-terminus or C-terminus
of the 17906 polypeptide.
[1207] For example, in one embodiment, the fusion protein is a
GST-17906 fusion protein in which the 17906 sequences are fused to
the C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant 17906. In another
embodiment, the fusion protein is a 17906 protein containing a
heterologous signal sequence at its N-terminus. In certain host
cells (e.g., mammalian host cells), expression and/or secretion of
17906 can be increased through use of a heterologous signal
sequence.
[1208] The 17906 fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject in vivo. The 17906 fusion proteins can be used to affect
the bioavailability of a 17906 ligand. Use of 17906 fusion proteins
may be useful therapeutically for the treatment of disorders caused
by, for example, (i) aberrant modification or mutation of a gene
encoding a 17906 protein; (ii) mis-regulation of the 17906 gene;
and (iii) aberrant post-translational modification of a 17906
protein. In one embodiment, a 17906 fusion protein may be used to
treat a bone associated disorder. In another embodiment, a 17906
fusion protein may be used to treat a bone cell disorder.
[1209] Moreover, the 17906-fusion proteins of the invention can be
used as immunogens to produce anti-17906 antibodies in a subject,
to purify 17906 ligands and in screening assays to identify
molecules which inhibit the interaction of 17906 with a 17906
substrate.
[1210] Preferably, a 17906 chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A 17906-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the 17906 protein.
[1211] The methods of the present invention may also include the
use of variants of the 17906 protein which function as either 17906
agonists (mimetics) or as 17906 antagonists. Variants of the 17906
protein can be generated by mutagenesis, e.g., discrete point
mutation or truncation of a 17906 protein. An agonist of the 17906
protein can retain substantially the same, or a subset, of the
biological activities of the naturally occurring form of a 17906
protein. An antagonist of a 17906 protein can inhibit one or more
of the activities of the naturally occurring form of the 17906
protein by, for example, competitively modulating a 17906-mediated
activity of a 17906 protein. Thus, specific biological effects can
be elicited by treatment with a variant of limited function. In one
embodiment, treatment of a subject with a variant having a subset
of the biological activities of the naturally occurring form of the
protein has fewer side effects in a subject relative to treatment
with the naturally occurring form of the 17906 protein.
[1212] In one embodiment, variants of a 17906 protein which
function as either 17906 agonists (mimetics) or as 17906
antagonists can be identified by screening combinatorial libraries
of mutants, e.g., truncation mutants, of a 17906 protein for 17906
protein agonist or antagonist activity. In one embodiment, a
variegated library of 17906 variants is generated by combinatorial
mutagenesis at the nucleic acid level and is encoded by a
variegated gene library. A variegated library of 17906 variants can
be produced by, for example, enzymatically ligating a mixture of
synthetic oligonucleotides into gene sequences such that a
degenerate set of potential 17906 sequences is expressible as
individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display) containing the set of
17906 sequences therein. There are a variety of methods which can
be used to produce libraries of potential 17906 variants from a
degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an
appropriate expression vector. Use of a degenerate set of genes
allows for the provision, in one mixture, of all of the sequences
encoding the desired set of potential 17906 sequences. Methods for
synthesizing degenerate oligonucleotides are known in the art (see,
e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984)
Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056;
Ike et al. (1983) Nucleic Acid Res. 11:477.
[1213] In addition, libraries of fragments of a 17906 protein
coding sequence can be used to generate a variegated population of
17906 fragments for screening and subsequent selection of variants
of a 17906 protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of a 17906 coding sequence with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form
double stranded DNA which can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. By this
method, an expression library can be derived which encodes
N-terminal, C-terminal and internal fragments of various sizes of
the 17906 protein.
[1214] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of 17906 proteins. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recrusive ensemble mutagenesis
(REM), a new technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify 17906 variants (Arkin and Yourvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.
(1993) Protein Engineering 6(3):327-331).
[1215] In one embodiment, cell based assays can be exploited to
analyze a variegated 17906 library. For example, a library of
expression vectors can be transfected into a cell line, e.g., a
bone cell line, which ordinarily responds to a 17906 ligand in a
particular 17906-dependent manner. The transfected cells are then
contacted with a 17906 ligand and the effect of expression of the
mutant on signaling by the 17906 receptor can be detected, e.g., by
monitoring the generation of an intracellular second messenger
(e.g., calcium, cAMP, IP.sub.3, or diacylglycerol), the
phosphorylation profile of intracellular proteins, cell
proliferation and/or migration, the expression profile of cell
surface adhesion molecules, or the activity of a 17906-regulated
transcription factor. Plasmid DNA can then be recovered from the
cells which score for inhibition, or alternatively, potentiation of
signaling by the 17906 receptor, and the individual clones further
characterized.
[1216] An isolated 17906 protein, or a portion or fragment thereof,
can be used as an immunogen to generate antibodies that bind 17906
using standard techniques for polyclonal and monoclonal antibody
preparation. A full-length 17906 protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of 17906 for use as immunogens. The antigenic peptide of 17906
comprises at least 8 amino acid residues of the amino acid sequence
shown in SEQ ID NO:11 and encompasses an epitope of 17906 such that
an antibody raised against the peptide forms a specific immune
complex with 17906. Preferably, the antigenic peptide comprises at
least 10 amino acid residues, more preferably at least 15 amino
acid residues, even more preferably at least 20 amino acid
residues, and most preferably at least 30 amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions
of 17906 that are located on the surface of the protein, e.g.,
hydrophilic regions, as well as regions with high antigenicity.
[1217] A 17906 immunogen typically is used to prepare antibodies by
immunizing a suitable subject, (e.g., rabbit, goat, mouse or other
mammal) with the immunogen. An appropriate immunogenic preparation
can contain, for example, recombinantly expressed 17906 protein or
a chemically synthesized 17906 polypeptide. The preparation can
further include an adjuvant, such as Freund's complete or
incomplete adjuvant, or similar immunostimulatory agent.
Immunization of a suitable subject with an immunogenic 17906
preparation induces a polyclonal anti-17906 antibody response.
[1218] Accordingly, another aspect of the invention pertains to
anti-17906 antibodies. The term "antibody" as used herein refers to
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site which specifically binds (immunoreacts with) an
antigen, such as 17906. Examples of immunologically active portions
of immunoglobulin molecules include F(ab) and F(ab').sub.2
fragments which can be generated by treating the antibody with an
enzyme such as pepsin. The invention provides polyclonal and
monoclonal antibodies that bind 17906. The term "monoclonal
antibody" or "monoclonal antibody composition", as used herein,
refers to a population of antibody molecules that contain only one
species of an antigen binding site capable of immunoreacting with a
particular epitope of 17906. A monoclonal antibody composition thus
typically displays a single binding affinity for a particular 17906
protein with which it immunoreacts.
[1219] Polyclonal anti-17906 antibodies can be prepared as
described above by immunizing a suitable subject with a 17906
immunogen. The anti-17906 antibody titer in the immunized subject
can be monitored over time by standard techniques, such as with an
enzyme linked immunosorbent assay (ELISA) using immobilized 17906.
If desired, the antibody molecules directed against 17906 can be
isolated from the mammal (e.g., from the blood) and further
purified by well known techniques, such as protein A chromatography
to obtain the IgG fraction. At an appropriate time after
immunization, e.g., when the anti-17906 antibody titers are
highest, antibody-producing cells can be obtained from the subject
and used to prepare monoclonal antibodies by standard techniques,
such as the hybridoma technique originally described by Kohler and
Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981)
J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.
255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA
76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the
more recent human B cell hybridoma technique (Kozbor et al. (1983)
Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.
(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
monoclonal antibody hybridomas is well known (see generally R. H.
Kenneth, in Monoclonal Antibodies: A New Dimension In Biological
Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A.
Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al.
(1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell
line (typically a myeloma) is fused to lymphocytes (typically
splenocytes) from a mammal immunized with a 17906 immunogen as
described above, and the culture supernatants of the resulting
hybridoma cells are screened to identify a hybridoma producing a
monoclonal antibody that binds 17906.
[1220] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-17906 monoclonal antibody (see, e.g.,
G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic
Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra;
Kenneth, Monoclonal Antibodies, cited supra). Moreover, the
ordinarily skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines can be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from ATCC. Typically, HAT-sensitive mouse myeloma cells
are fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using
HAT medium, which kills unfused and unproductively fused myeloma
cells (unfused splenocytes die after several days because they are
not transformed). Hybridoma cells producing a monoclonal antibody
of the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind 17906, e.g., using a standard
ELISA assay.
[1221] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-17906 antibody can be identified and
isolated by screening a recombinant combinatorial immunoglobulin
library (e.g., an antibody phage display library) with 17906 to
thereby isolate immunoglobulin library members that bind 17906.
Kits for generating and screening phage display libraries are
commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the Stratagene
SurfZAP.TM. Phage Display Kit, Catalog No. 240612). Additionally,
examples of methods and reagents particularly amenable for use in
generating and screening antibody display library can be found in,
for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT
International Publication No. WO 92/18619; Dower et al. PCT
International Publication No. WO 91/17271; Winter et al. PCT
International Publication WO 92/20791; Markland et al. PCT
International Publication No. WO 92/15679; Breitling et al. PCT
International Publication WO 93/01288; McCafferty et al. PCT
International Publication No. WO 92/01047; Garrard et al. PCT
International Publication No. WO 92/09690; Ladner et al. PCT
International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J.
Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628;
Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad
et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991)
Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad.
Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990)
348:552-554.
[1222] Additionally, recombinant anti-17906 antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, can also be used in the methods of the
present invention. Such chimeric and humanized monoclonal
antibodies can be produced by recombinant DNA techniques known in
the art, for example using methods described in Robinson et al.
International Application No. PCT/US86/02269; Akira, et al.
European Patent Application 184,187; Taniguchi, M., European Patent
Application 171,496; Morrison et al. European Patent Application
173,494; Neuberger et al. PCT International Publication No. WO
86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.
European Patent Application 125,023; Better et al. (1988) Science
240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA
84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et
al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.
(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature
314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.
80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et
al. (1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539;
Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[1223] An anti-17906 antibody (e.g., monoclonal antibody) can be
used to isolate 17906 by standard techniques, such as affinity
chromatography or immunoprecipitation. An anti-17906 antibody can
facilitate the purification of natural 17906 from cells and of
recombinantly produced 17906 expressed in host cells. Moreover, an
anti-17906 antibody can be used to detect 17906 protein (e.g., in a
cellular lysate or cell supernatant) in order to evaluate the
abundance and pattern of expression of the 17906 protein.
Anti-17906 antibodies can be used diagnostically to monitor protein
levels in tissue as part of a clinical testing procedure, e.g., to,
for example, determine the efficacy of a given treatment regimen.
Detection can be facilitated by coupling (i.e., physically linking)
the antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include [1224] .sup.125I, .sup.131I, .sup.35S or
.sup.3H.
[1225] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the Sequence Listing, are
incorporated herein by reference.
EXAMPLES
Example 1
Regulation of 17906 Expression in Cells Involved in
Osteogenesis
[1226] TaqMan real-time quantitative RT-PCR was used to detect the
presence of RNA transcript corresponding to human 17906 in several
tissues. It was found that the corresponding orthologs of 17906 are
expressed in a variety of tissues such as epithelial, fibroblast,
osteoblast and glial cells, as well as, breast tumor, brain cortex
and brain hypothalamus tissues.
[1227] Reverse Transcriptase PCR (RT-PCR) was used to detect the
presence of RNA transcript corresponding to human 17906 in RNA
prepared from cells and tissues related to osteoblasts. Expression
of 17906 was assessed in several tissues. A relatively low
expression of the transcript was found in differentiated
osteoblasts, and relatively high expression of the transcript was
found in primary cultured osteoblasts.
[1228] Relative expression levels of the 17906 was assessed in
osteogenic cells and adipogenic cells using TaqMan PCR.
[1229] TaqMan PCR was also used to assess the expression of 17906
in several cellular models of osteoporosis.
[1230] Relative mRNA expression levels of the 17906 gene was also
assessed in osteoblasts stimulated with parathyroid hormone (PTH),
interleukin-1.alpha. (IL-1.alpha.), and dexamethasone (DEX).
Example 2
Expression of Recombinant 17906 Protein in Bacterial Cells
[1231] In this example, 17906 is expressed as a recombinant
glutathione-S-transferase (GST) fusion polypeptide in E. coli and
the fusion polypeptide is isolated and characterized. Specifically,
17906 is fused to GST and this fusion polypeptide is expressed in
E. coli, e.g., strain PEB199. Expression of the GST-17906 fusion
protein in PEB199 is induced with IPTG. The recombinant fusion
polypeptide is purified from crude bacterial lysates of the induced
PEB199 strain by affinity chromatography on glutathione beads.
Using polyacrylamide gel electrophoretic analysis of the
polypeptide purified from the bacterial lysates, the molecular
weight of the resultant fusion polypeptide is determined.
Example 3
Expression of Recombinant 17906 Protein in COS Cells
[1232] To express the 17906 gene in COS cells, the pcDNA/Amp vector
by Invitrogen Corporation (San Diego, Calif.) is used. This vector
contains an SV40 origin of replication, an ampicillin resistance
gene, an E. coli replication origin, a CMV promoter followed by a
polylinker region, and an SV40 intron and polyadenylation site. A
DNA fragment encoding the entire 17906 protein and an HA tag
(Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to
its 3' end of the fragment is cloned into the polylinker region of
the vector, thereby placing the expression of the recombinant
protein under the control of the CMV promoter.
[1233] To construct the plasmid, the 17906 DNA sequence is
amplified by PCR using two primers. The 5' primer contains the
restriction site of interest followed by approximately twenty
nucleotides of the 17906 coding sequence starting from the
initiation codon; the 3' end sequence contains complementary
sequences to the other restriction site of interest, a translation
stop codon, the HA tag or FLAG tag and the last 20 nucleotides of
the 17906 coding sequence. The PCR amplified fragment and the
pCDNA/Amp vector are digested with the appropriate restriction
enzymes and the vector is dephosphorylated using the CIAP enzyme
(New England Biolabs, Beverly, Mass.). Preferably the two
restriction sites chosen are different so that the 17906 gene is
inserted in the correct orientation. The ligation mixture is
transformed into E. coli cells (strains HB101, DH5.quadrature.,
SURE, available from Stratagene Cloning Systems, La Jolla, Calif.,
can be used), the transformed culture is plated on ampicillin media
plates, and resistant colonies are selected. Plasmid DNA is
isolated from transformants and examined by restriction analysis
for the presence of the correct fragment.
[1234] COS cells are subsequently transfected with the
17906-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium
chloride co-precipitation methods, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Other suitable
methods for transfecting host cells can be found in Sambrook, J.,
Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of
the VR-3 or VR-5 polypeptide is detected by radiolabelling
(.sup.35S-methionine or .sup.35S-cysteine available from NEN,
Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and
Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA
specific monoclonal antibody. Briefly, the cells are labelled for 8
hours with .sup.35S-methionine (or .sup.35S-cysteine). The culture
media are then collected and the cells are lysed using detergents
(RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM
Tris, pH 7.5). Both the cell lysate and the culture media are
precipitated with an HA specific monoclonal antibody. Precipitated
polypeptides are then analyzed by SDS-PAGE.
[1235] Alternatively, DNA containing the 17906 coding sequence is
cloned directly into the polylinker of the pCDNA/Amp vector using
the appropriate restriction sites. The resulting plasmid is
transfected into COS cells in the manner described above, and the
expression of the 17906 polypeptide is detected by radiolabelling
and immunoprecipitation using a 17906 specific monoclonal
antibody.
V. METHODS AND COMPOSITIONS FOR THE DIAGNOSIS AND TREATMENT OF
HEMATOLOGICAL DISORDERS USING 16319
Background of the Invention
[1236] Hematological disorders are blood associated disorders.
Blood is a highly specialized tissue which carries oxygen and
nutrients to all parts of the body and waste products back to the
lungs, kidneys and liver for disposal. Thus, blood maintains
communication between different parts of the body. Blood is also an
essential part of the immune system, crucial to fluid and
temperature balance, a hydraulic fluid for certain functions and a
highway for hormonal messages.
[1237] All blood cells in adults are produced in the bone marrow.
Red cells, white cells and platelets are produced in the marrow of
bones, especially the vertebrae, ribs, hips, skull and sternum.
These essential blood cells fight infection, carry oxygen and help
control bleeding. Specifically, red blood cells are disc-shaped
cells containing hemoglobin, which enables these cells to pick up
and deliver oxygen to all parts of the body. White blood cells are
the body's primary defense against infection. They can move out of
the blood stream and reach tissues being invaded. Platelets are
small blood cells that control bleeding by forming clusters to plug
small holes in blood vessels and assist in the clotting
process.
[1238] Each day the bone marrow generates and releases into the
circulation several billion fully-differentiated, functional blood
cells. Hematopoiesis is the process by which blood cells develop
and differentiate from pluripotent stem cells in the bone marrow.
Production of these cells derives from a small stock of quiescent
progenitor cells (including the most primitive stem cells and other
less primitive but still immature progenitors). The most primitive
stem cells have the capacity to generate several billion cells
containing all blood lineages. The production of such a large
number of cells is achieved by extensive proliferation coupled with
successive differentiation steps leading to a balanced production
of mature cells.
[1239] The production of mature blood cells by the hematopoietic
system involves complex interactions between soluble factors, the
marrow microenvironment, and hematopoietic progenitors. In
particular, hematopoiesis involves a complex interplay of
polypeptide growth factors acting via membrane-bound receptors on
their target cells. Signaling by growth factors results in cellular
proliferation and differentiation, with a response to a particular
growth factor often being lineage-specific and/or stage-specific.
Development of a single cell type, such as a red blood cell, from a
stem cell may require the coordinated action of a plurality of
growth factors acting in the proper sequence.
[1240] Impaired blood cell production occurs when the proliferation
and differentiation of the stem cells or committed cells is
disturbed. Impaired blood cell production is the root of
hematological disorders. Some of the more common diseases caused by
impaired blood cell production, i.e., hematological disorders,
include aplastic anemia, hypoplastic anemia, pure red cell aplasia
and anemia associated with renal failure or endocrine disorders.
Disturbances in the proliferation and differentiation of
erythroblasts include defects in DNA synthesis such as impaired
utilization of vitamin B12 or folic acid and the megaloblastic
anemias, defects in heme or globin synthesis, and anemias of
unknown origins such as sideroblastic anemia, anemia associated
with chronic infections such as malaria, trypanosomiasis, HIV,
hepatitis virus or other viruses, and myelophthisic anemias caused
by marrow deficiencies. Impaired blood cell production also affects
cancer patients and other autoimmune disease patients who receive
bone marrow irradiation or chemotherapy treatment.
[1241] Hematological disorders are, thus, a diverse family of
disorders embracing clinical and laboratory aspects of a large
number of diseases, both malignant and non-malignant. Although some
progress has been made in diagnostic and therapeutic strategies to
combat hematological disorders, molecular advances are continuing
at a rate exceeding the rate of progress in therapeutics. Thus,
novel methods for diagnosis and treatment of hematological
disorders based on known molecular advances are urgently needed in
the field.
Summary of the Invention
[1242] The present invention provides methods and compositions for
the diagnosis and treatment of hematological disorders. The present
invention is based, at least in part, on the discovery that the
16319 gene, is expressed at high levels in hematopoietic cells of
various lineages and stages of differentiation. Specifically, 16319
is expressed in high levels in CD34+ progenitor cells (including
stem cells from bone marrow, cord blood and peripheral blood) and
this high level of expression is maintained in erythroid cells in
vitro and Glycophorin A positive cells in vivo. Thus, the 16319
molecules, by participating in the TGF-.beta. downstream signaling
pathway, modulate hematopoietic cell behavior and are useful as
targets and therapeutic agents for the modulation of hematopoietic
cell activity, e.g., cell proliferation or apoptosis, and the
treatment of hematological disorders.
[1243] Accordingly, the present invention provides methods for the
diagnosis and treatment of hematological diseases including but not
limited to apalstic anemia, hemophilia, sickle cell anemia,
thalassemia, blood loss and other blood disorders, e.g., blood
disorders related to bone marrow irradiation treatments,
chemotherapy treatments or compromised kidney function.
[1244] In one aspect, the invention provides methods for
identifying a compound capable of treating a hematological
disorder, e.g., anemia or thalassemia. The method includes assaying
the ability of the compound to modulate 16319 nucleic acid
expression or 16319 polypeptide activity. In one embodiment, the
ability of the compound to modulate nucleic acid expression or
16319 polypeptide activity is determined by detecting modulation of
proliferation of a hematopoietic cell. In another embodiment, the
ability of the compound to modulate nucleic acid expression or
16319 polypeptide activity is determined by detecting modulation of
apoptosis of a hematopoietic cell.
[1245] In another aspect, the invention provides methods for
identifying a compound capable of modulating a hematological
activity, e.g., hematopoietic cell proliferation, differentiation,
or cell death. The method includes contacting a cell expressing a
16319 nucleic acid or polypeptide (e.g., a hematopoietic cell) with
a test compound and assaying the ability of the test compound to
modulate the expression of a 16319 nucleic acid or the activity of
a 16319 polypeptide.
[1246] Another aspect of the invention provides a method for
modulating a hematological activity, e.g., hematopoietic cell
proliferation, cell differentiation, or cell death. The method
includes contacting a hematopoietic cell with an effective amount
of a 16319 modulator, for example, an anti-16319 antibody, a 16319
polypeptide comprising the amino acid sequence of SEQ ID NO:13 or a
fragment thereof, a 16319 polypeptide comprising an amino acid
sequence which is at least 90 percent identical to the amino acid
sequence of SEQ ID NO:13, an isolated naturally occurring allelic
variant of a polypeptide consisting of the amino acid sequence of
SEQ ID NO:13, a small molecule, an antisense 16319 nucleic acid
molecule, a nucleic acid molecule of SEQ ID NO:12 or a fragment
thereof, or a ribozyme.
[1247] In yet another aspect, the invention features a method for
treating a subject having a hematological disorder, e.g., a
hematological disorder characterized by aberrant 16319 polypeptide
activity or aberrant 16319 nucleic acid expression, e.g., anemia or
thalessemia. The method includes administering to the subject a
therapeutically effective amount of a 16319 modulator, e.g., in a
pharmaceutically acceptable formulation or by using a gene therapy
vector. Embodiments of this aspect of the invention include the
16319 modulator being a small molecule, an anti-16319 antibody, a
16319 polypeptide comprising the amino acid sequence of SEQ ID
NO:13 or a fragment thereof, a 16319 polypeptide comprising an
amino acid sequence which is at least 90 percent identical to the
amino acid sequence of SEQ ID NO:13, an isolated naturally
occurring allelic variant of a polypeptide consisting of the amino
acid sequence of SEQ ID NO:13, an antisense 16319 nucleic acid
molecule, a nucleic acid molecule of SEQ ID NO:12 or a fragment
thereof, or a ribozyme.
[1248] In another aspect, the invention provides a method for
modulating, e.g., increasing or decreasing, hematopoietic cell
apoptosis in a subject by administering to the subject a 16319
modulator in an amount effective for modulating hematopoietic cell
apoptosis.
[1249] In another aspect, the invention provides a method for
modulating, e.g., increasing or decreasing, hematopoietic cell
proliferation in a subject by administering to the subject a 16319
modulator in an amount effective for modulating hematopoietic cell
proliferation.
[1250] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
Detailed Description of the Invention
[1251] In recent years, studies of growth factors have considerably
changed our understanding of hematopoietic regulation and clinical
therapeutic strategies. These growth factors often act in cascade,
directing cells toward either the cell cycle, cell differentiation
or cell death. Transforming growth factor-.beta. (TGF-.beta.) is
one of the key regulatory elements of the hematopoietic system.
(Jacobsen et al., (1991) Blood 78:2239; Snoeck H W et al., (1996)
J. Exp. Med. 183:705; Van Ranst P C et al., (1996) Exp. Hematol.
24:1509) TGF-.beta.s are generally potent growth inhibitors,
although they can act in a stimulatory manner on some cell types.
Hematopoietic cells, in particular, are potently growth inhibited
by TGF-.beta.. (Martin et al., Ann. N.Y. Acad. Sci. (1995)
752:300-8.)
[1252] TGF-.beta.'s anti-proliferative effect on cells is mediated
in part by inhibition of phosphorylation of the retinoblastoma
protein (RB) and blockage of the cell cycle at the G1/S phase.
(Terada et al. (1999) Kidney International 56:1378-1390.) The G1
phase of the cell cycle represents the interval in which cells
respond maximally to extracellular signals, including mitogens,
anti-proliferative factors, matrix adhesive substances, and
intercellular contacts. The R point is when cells become committed
to duplicating their DNA and undergoing mitosis. Phosphorylation of
RB temporally coincides with passage through the R point of the
cell cycle. TGF-.beta.'s inhibition of RB phosphorylation prevents
the cell from exiting the G1 phase and proceeding to the R point
for initiation of the determinative stages of replication.
(Sundershan, C., et al. (1998) J. Cell. Physiol. 176:67-75.)
Studies have shown that phosphorylation of RB is initially
triggered by holoenzymes composed of cyclin-D subunits. The
cyclin-D subunits are induced and assembled into holoenzymes as
cells enter the replicative cycle in response to mitogenic
stimulation. (Terada et al. (1999) Kidney International
56:1378-1390.)
[1253] The TGF-.beta.-associated-kinase-1 ("16319") has been shown
to be intimately involved in the TGF-.beta. signaling pathway.
(Yamaguchi et al., Science (1995) 270: 2008-11.) 16319 is a member
of the mitogen-activated protein kinase kinase kinase (MAPKKK)
family. MAPKs play an important role in transducing extracellular
signals into a cellular response and are classically activated by
growth factors. (Terada et al., (1999) Neph. Dial. Transplant. 14
(supl 1):45-47. As described in Yamaguchi et al. and Terada et al.,
16319 is known to participate in the signal transduction pathway of
TGF-.beta.. In particular, it has been shown that the
TGF-.beta.-16319 pathway significantly reduces the levels of
cyclin-D1 in cells by inhibiting cyclin-D1 promoter activity.
(Terada et al. (1999) Nephrol Dial Transplantation 14 [Suppl
1]:45-47). By reducing the levels of cyclin-D1 in cells, 16319
facilitates the anti-proliferative effects of TGF-.beta. by
preventing the phosphorylation of RB which prevents the cell from
exiting the G1 phase and proceeding with replication.
[1254] The present invention demonstrates that 16319 is expressed
at high levels in CD34+ progenitor cells from bone marrow, cord
blood and peripheral blood, and that these high levels of
expression are maintained in erythroid cells in vitro and in
Glycophorin A positive (GPA+) cells in vivo. CD34 is known to be
expressed on early lymphohematopoietic stem and progenitor cells
and on hematopoietic progenitors derived from fetal yolk sac,
embryonic liver, and extra-hepatic embryonic tissues including
aorta-associated hematopoietic progenitors in the 5 week human
embryo (Nishio et al., (2001) Exp. Hematol. 29(1):19-29).
Glycophorin A protein is known as a late erythroid lineage specific
protein. Thus, the present invention demonstrates a novel
association of the 16319 protein with hematopoietic cells of
various lineages and at various stages of differentiation. Since
16319 is a known modulator of cell cycle progression, modulation of
16319 allows for the modulation of hematopoietic cell cycle
progression. Accordingly, the present invention provides methods
and compositions for the diagnosis and treatment of hematological
disorders.
[1255] As used herein, a "hematological disorder" includes a
disease, disorder, or condition which affects a hematopoietic cell
or tissue. Hematological disorders include diseases, disorders, or
conditions associated with aberrant hematological content or
function. Hematological disorders can be characterized by a
misregulation (e.g., downregulation or upregulation) of 16319
activity. Examples of hematological disorders include disorders
resulting from bone marrow irradiation or chemotherapy treatments
for cancer, disorders such as Pernicious Anemia, Hemorrhagic
Anemia, Hemolytic Anemia, Aplastic Anemia, Sickle Cell Anemia,
Sideroblastic Anemia, Anemia associated with chronic infections
such as Malaria, Trypanosomiasis, HIV, Hepatitis virus or other
viruses, Myelophthisic Anemias caused by marrow deficiencies, renal
failure resulting from Anemia, Anemia, Polycethemia, Infectious
Mononucleosis (IM), Acute Non-Lymphocytic Leukemia (ANLL), Acute
Myeloid Leukemia (AML), Acute Promyelocytic Leukemia (APL), Acute
Myelomonocytic Leukemia (AMMoL), Polycethemia Vera, Lymphoma, Acute
Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia, Wilm's
Tumor, Ewing's Sarcoma, Retinoblastoma, Hemophilia, disorders
associated with an increased risk of Thrombosis, Herpes,
Thalessemia, antibody-mediated disorders such as transfusion
reactions and Erythroblastosis, mechanical trauma to red blood
cells such as micro-angiopathic hemolytic anemias, Thrombotic
Thrombocytopenic Purpura and disseminated intravascular
coagulation, infections by parasites such as Plasmodium, chemical
injuries from, e.g., lead poisoning, and Hypersplenism.
[1256] As used herein, "16319" encompasses proteins characterized
by their ability to modulate signal transduction to thereby
modulate hematopoietic cell proliferation or apoptosis in vitro or
in vivo. A representative human 16319 cDNA sequence is shown herein
in SEQ ID NO:12, and the corresponding amino acid sequence is shown
in SEQ ID NO:13. Those skilled in the art will recognize that the
illustrated sequences correspond to a single allele of the human
16319 gene, and that allelic variation is expected to exist.
Allelic variants include those containing silent mutations and
those in which mutations result in amino acid sequence changes. It
will also be evident that one skilled in the art could create
additional variants, such as by engineering sites that would
facilitate manipulation of the nucleotide sequence using
alternative codons, by substitution of codons to produce
conservative changes in amino acid sequence, etc. The use of
allelic and engineered variant 16319s is contemplated by the
present invention. The use of 16319 molecules from non-human
species are also contemplated by the present invention.
[1257] The present invention provides methods and compositions for
the diagnosis and treatment of hematological disorders. The 16319
modulators identified according to the methods of the invention can
be used to modulate hematopoietic cell proliferation and are,
therefore, useful in treating or diagnosing hematological
disorders. For example, inhibition of the activity of a 16319
molecule can cause increased hematopoietic cell proliferation and,
therefore, increased blood cell production in a subject, thereby
preventing hematological disorders, e.g., aplastic anemia or sickle
cell anemia in the subject. Thus, the 16319 modulators used in the
methods of the of the invention can be used to treat hematological
disorders. The 16319 modulators identified according to the methods
of the invention can also be used to inhibit apoptosis of
hematopoietic cells, e.g., by inhibiting 16319, thus increasing
blood cell production in a subject, thereby preventing
hematological disorders, e.g., aplastic anemia or sickle cell
anemia in the subject.
[1258] Alternatively, stimulation of the activity of a 16319
molecule can cause decreased hematopoietic cell proliferation and,
therefore, decreased blood cell production in a subject, thereby
preventing hematological disorders, e.g., hemorrhagic anemia,
polycethemia, infectious mononucleosis or leukemia in the subject.
Thus, the 16319 modulators used in the methods of the of the
invention can be used to treat hematological disorders. 16319
modulators can also increase apoptosis of hematopoietic cells, thus
decreasing blood cell production in a subject, thereby inhibiting
hematological disorders, e.g., hemorrhagic anemia, polycethimia,
infectious mononucleosis or leukemia in the subject.
[1259] As used interchangeably herein, "16319 activity,"
"biological activity of 16319" or "functional activity of 16319,"
includes an activity exerted by a 16319 protein, polypeptide or
nucleic acid molecule on a 16319 responsive cell or tissue, e.g., a
hematopoietic cell, or on a 16319 protein substrate, as determined
in vivo, or in vitro, according to standard techniques. 16319
activity can be a direct activity, such as an association with a
16319-target molecule e.g., RB. As used herein, a "substrate" or
"target molecule" or "binding partner" is a molecule with which a
16319 protein binds or interacts in nature, such that
16319-mediated function, e.g., modulation of apoptosis or
modulation of cell proliferation, is achieved. A 16319 target
molecule can be a non-16319 molecule or a 16319 protein or
polypeptide. Examples of such target molecules include proteins in
the same signaling path as the 16319 protein, e.g., proteins which
may function upstream (including both stimulators and inhibitors of
activity) or downstream of the 16319 protein in a pathway involving
regulation of hematopoietic cell proliferation or apoptosis.
Alternatively, a 16319 activity is an indirect activity, such as a
cellular signaling activity mediated by interaction of the 16319
protein with a 16319 target molecule. The biological activities of
16319 are described herein. For example, the 16319 proteins can
have one or more of the following activities: (1) they modulate
hematopoietic cell proliferation; (2) they modulate apoptosis of
hematopoietic cells; (3) they modulate cyclin D levels in a cell;
(4) they modulate the phosphorylation state of RB; and (5) they
modulate the anti-proliferative effects of TGF-.beta..
[1260] As used herein, the term "hematopoietic cell" includes yolk
sac stem cells, primitive erythroid cells, fetal liver cells, fetal
spleen cells, fetal bone marrow cells, non-fetal bone marrow cells,
megakaryocytes, stem cells, lymphoid stem cells, myeloid stem
cells, progenitor cells, progenitor lymphocytes, progenitor T
lymphocytes, progenitor B lymphocytes, progenitor erythrocytes,
progenitor neutrophils, progenitor eosinophils, progenitor
basophils, progenitor monocytes, progenitor mast cells, progenitor
platelets, committed lymphocytes, committed T lymphocytes,
committed B lymphocytes, committed erythrocytes, committed
neutrophils, committed eosinophils, committed basophils, committed
monocytes, committed mast cells, committed platelets,
differentiated lymphocytes, differentiated T lymphocytes,
differentiated B lymphocytes, differentiated erythrocytes,
differentiated neutrophils, differentiated eosinophils,
differentiated basophils, differentiated monocytes, differentiated
mast cells, differentiated platelets, mature lymphocytes, mature T
lymphocytes, mature B lymphocytes, mature erythrocytes, mature
neutrophils, mature eosinophils, mature basophils, mature
monocytes, mature mast cells, and mature platelets.
[1261] As used herein, the term "progenitor cell" includes any
somatic cell which has the capacity to generate fully
differentiated, functional progeny by differentiation and
proliferation. Progenitor cells include progenitors from any tissue
or organ system, including, but not limited to, blood, nerve,
muscle, skin, gut, bone, kidney, liver, pancreas, thymus, and the
like. Progenitor cells are distinguished from "committed cells" and
"differentiated cells," which are defined as those cells which may
or may not have the capacity to proliferate, i.e., self-replicate,
but which are unable to undergo further differentiation to a
different cell type under normal physiological conditions.
Moreover, progenitor cells are further distinguished from abnormal
cells such as cancer cells, especially leukemia cells, which
proliferate (self-replicate) but which generally do not further
differentiate, despite appearing to be immature or
undifferentiated.
[1262] Progenitor cells include all the cells in a lineage of
differentiation and proliferation prior to the most differentiated
or the fully mature cell. Thus, for example, progenitors include
the skin progenitor in the mature individual, which is capable of
differentiation to only one type of cell, but which is itself not
fully mature or fully differentiated. Production of mature,
functional blood cells results from proliferation and
differentiation of "unipotential progenitors," i.e., those
progenitors which have the capacity to make only one type of one
type of blood cell. For red blood cell production, a progenitor
called a "CFU-E" (colony forming unit-erythroid) has the capacity
to generate two to 32 progeny cells.
[1263] Various other hematopoietic progenitors have been
characterized. For example, hematopoietic progenitor cells include
those cells which are capable of successive cycles of
differentiating and proliferating to yield up to eight different
mature hematopoietic cell lineages. At the most primitive or
undifferentiated end of the hematopoietic spectrum, hematopoietic
progenitor cells include the hematopoietic "stem cells." These rare
cells, which represent 1 in 10,000 to 1 in 100,000 of cells in the
bone marrow, each have the capacity to generate a billion mature
blood cells of all lineages and are responsible for sustaining
blood cell production over the life of an animal. They reside in
the marrow primarily in a quiescent state and may form identical
daughter cells through a process called self-renewal. Accordingly,
such an uncommitted progenitor can be described as being
"totipotent," i.e., both necessary and sufficient for generating
all types of mature blood cells. Progenitor cells which retain a
capacity to generate all blood cell lineages but which can not
self-renew are termed "pluripotent." Cells which can produce some
but not all blood lineages and can not self-renew are termed
"multipotent."
[1264] As used herein, "hematopoietic cell activity" includes an
activity exerted by a hematopoietic cell, or an activity that takes
place in a hematopoietic cell. For example, such activities include
cellular processes that contribute to the physiological role of
hematopoietic cells, such as hematopoiesis, but are not limited to,
cell proliferation, differentiation, growth, migration and
programmed cell death.
[1265] As used herein, the term "modulate" includes alteration of,
e.g., by increasing or decreasing the particular parameter being
described, e.g., 16319 activity.
[1266] As used herein the term "apoptosis" includes programmed cell
death which can be characterized using techniques which are known
in the art. Apoptotic cell death can be characterized, e.g., by
cell shrinkage, membrane blebbing and chromatin condensation
culminating in cell fragmentation. Cells undergoing apoptosis also
display a characteristic pattern of internucleosomal DNA
cleavage.
I. Screening Assays
[1267] The invention provides methods (also referred to herein as
"screening assays") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules, ribozymes, or 16319 antisense molecules) which bind to
16319 proteins, have a stimulatory or inhibitory effect on 16319
expression or 16319 activity, or have a stimulatory or inhibitory
effect on the expression or activity of a 16319 target molecule.
Compounds identified using the assays described herein may be
useful for treating hematological disorders.
[1268] Candidate/test compounds include, for example, 1) peptides
such as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam, K. S. et al.
(1991) Nature 354:82-84; Houghten, R. et al. (1991) Nature
354:84-86) and combinatorial chemistry-derived molecular libraries
made of D- and/or L-configuration amino acids; 2) phosphopeptides
(e.g., members of random and partially degenerate, directed
phosphopeptide libraries, see, e.g., Songyang, Z. et al. (1993)
Cell 72:767-778); 3) antibodies (e.g., polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric, and single chain antibodies as
well as Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[1269] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K. S. (1997) Anticancer
Drug Des. 12:145).
[1270] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med.
Chem. 37:1233.
[1271] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or phage (Scott and Smith (1990) Science 249:386-390;
Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.
Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310;
Ladner supra.).
[1272] Assays that may be used to identify compounds that modulate
16319 activity include assays for cytochrome C release from
mitochondria during cell apoptosis, e.g., hematopoietic cell
apoptosis (as described in, for example, Bossy-Wetzel E. et al.
(2000) Methods in Enzymol. 322:235-42); cytofluorometric
quantitation of nuclear apoptosis induced in a cell-free system (as
described in, for example, Lorenzo H. K. et al. (2000) Methods in
Enzymol. 322:198-201); apoptotic nuclease assays (as described in,
for example, Hughes F. M. (2000) Methods in Enzymol. 322:47-62);
analysis of apoptotic cells, e.g., apoptotic hematopoietic cells,
by flow and laser scanning cytometry (as described in, for example,
Darzynkiewicz Z. et al. (2000) Methods in Enzymol. 322:18-39);
detection of apoptosis by annexin V labeling (as described in, for
example, Bossy-Wetzel E. et al. (2000) Methods in Enzymol.
322:15-18); transient transfection assays for cell death genes (as
described in, for example, Miura M. et al. (2000) Methods in
Enzymol. 322:480-92); and assays that detect DNA cleavage in
apoptotic cells, e.g., apoptotic hematopoietic cells (as described
in, for example, Kauffman S. H. et al. (2000) Methods in Enzymol.
322:3-15).
[1273] Proliferation assays that may be used to identify compounds
that modulate 16319 activity include assays using 32D cells (a
multi-lineage murine hematopoietic cell line) as described in U.S.
Pat. No. 6,231,880, the contents of which are incorporated herein
by reference. Cell proliferation assays which measure the growth
phenotype of cells with an ablated growth regulatory gene of
interest, e.g., 16319 are described in Sudershan, C., et al. (1998)
J. Cell. Physiol. 176:67-75. The ability of a test compound to
modulate 16319 activity may also be determined by monitoring
cellular processes such as cell division, protein synthesis,
nucleic acid (DNA or RNA) synthesis, nucleic acid (principally DNA)
fragmentation and apoptosis.
[1274] In one aspect, an assay is a cell-based assay in which a
cell which expresses a 16319 protein or biologically active portion
thereof (e.g., the 16319 gene lacking the twenty-two amino terminal
amino acid residues) of the 16319 protein that is believed to be
involved in the modulation of hematopoietic cell proliferation, or
modulation of apoptosis of hematopoietic cells, is contacted with a
test compound and the ability of the test compound to modulate
16319 activity is determined. In a preferred embodiment, the
biologically active portion of the 16319 protein includes a domain
or motif that can modulate apoptosis of hematopoietic cells and/or
which can modulate hematopoietic cell proliferation. Determining
the ability of the test compound to modulate 16319 activity can be
accomplished by monitoring, for example, the production of one or
more specific metabolites in a cell which expresses 16319 (see,
e.g., Saada et al. (2000) Biochem Biophys. Res. Commun. 269:
382-386) or by monitoring cell death, cell proliferation, or cell
differentiation in the cell. The cell, for example, can be of
mammalian origin, e.g., a hematopoietic cell such as a committed
erythrocyte or a progenitor cell.
[1275] The ability of the test compound to modulate 16319 binding
to a substrate or to bind to 16319 can also be determined.
Determining the ability of the test compound to modulate 16319
binding to a substrate can be accomplished, for example, by
coupling the 16319 substrate with a radioisotope or enzymatic label
such that binding of the 16319 substrate to 16319 can be determined
by detecting the labeled 16319 substrate in a complex.
Alternatively, 16319 could be coupled with a radioisotope or
enzymatic label to monitor the ability of a test compound to
modulate 16319 binding to a 16319 substrate in a complex.
Determining the ability of the test compound to bind 16319 can be
accomplished, for example, by coupling the compound with a
radioisotope or enzymatic label such that binding of the compound
to 16319 can be determined by detecting the labeled 16319 compound
in a complex. For example, 16319 substrates can be labeled with
.sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Alternatively,
compounds can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic label detected by determination of conversion of an
appropriate substrate to product.
[1276] It is also within the scope of this invention to determine
the ability of a compound to interact with 16319 without the
labeling of any of the interactants. For example, a
microphysiometer can be used to detect the interaction of a
compound with 16319 without the labeling of either the compound or
the 16319 (McConnell, H. M. et al. (1992) Science 257:1906-1912).
As used herein, a "microphysiometer" (e.g., Cytosensor) is an
analytical instrument that measures the rate at which a cell
acidifies its environment using a light-addressable potentiometric
sensor (LAPS). Changes in this acidification rate can be used as an
indicator of the interaction between a compound and 16319.
[1277] The ability of a 16319 modulator to modulate, e.g., inhibit
or increase, 16319 activity can also be determined through
screening assays which identify modulators which either increase or
decrease apoptosis and cell proliferation. In one embodiment, the
invention provides for a screening assay involving contacting cells
which express a 16319 protein or polypeptide with a test compound,
and examining the cells for the morphological features of
apoptosis. For example, cells expressing a 16319 protein or
polypeptide can be contacted with a test compound and nuclearly
stained with acridine orange. Subsequently, nuclear DNA can be
extracted and analyzed for DNA fragmentation as described in
Inohora et al., (1997) EMBO J. 16:1686-1694.
[1278] To determine whether a test compound modulates 16319
expression, in vitro transcriptional assays can be performed. To
perform such an assay, the full length promoter and enhancer of
16319 can be linked to a reporter gene such as chloramphenicol
acetyltransferase (CAT) and introduced into host cells. The same
host cells can then be transfected with the test compound. The
effect of the test compound can be measured by testing CAT activity
and comparing it to CAT activity in cells which do not contain the
test compound. An increase or decrease in CAT activity indicates a
modulation of 16319 expression and is, therefore, an indicator of
the ability of the test compound to modulate hematopoietic cell
proliferation or apoptosis.
[1279] In yet another embodiment, an assay of the present invention
is a cell-free assay in which a 16319 protein or biologically
active portion thereof (e.g., (e.g., the 16319 gene without the
twenty-two amino terminus amino acids) is contacted with a test
compound and the ability of the test compound to bind to or to
modulate (e.g., stimulate or inhibit) the activity of the 16319
protein or biologically active portion thereof is determined.
Preferred biologically active portions of the 16319 proteins to be
used in assays of the present invention include fragments which
participate in interactions with non-16319 molecules, e.g.,
fragments with high surface probability scores. Binding of the test
compound to the 16319 protein can be determined either directly or
indirectly as described above. Determining the ability of the 16319
protein to bind to a test compound can also be accomplished using a
technology such as real-time Biomolecular Interaction Analysis
(BIA) (Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem.
63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol.
5:699-705). As used herein, "BIA" is a technology for studying
biospecific interactions in real time, without labeling any of the
interactants (e.g., BIAcore). Changes in the optical phenomenon of
surface plasmon resonance (SPR) can be used as an indication of
real-time reactions between biological molecules.
[1280] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either
16319 or a 16319 target molecule to facilitate separation of
complexed from uncomplexed forms of one or both of the proteins, as
well as to accommodate automation of the assay. Binding of a test
compound to a 16319 protein, or interaction of a 16319 protein with
a 16319 target molecule in the presence and absence of a test
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtitre plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/16319 fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or 16319 protein, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtitre plate wells are washed to remove any
unbound components, the matrix is immobilized in the case of beads,
and complex formation is determined either directly or indirectly,
for example, as described above. Alternatively, the complexes can
be dissociated from the matrix, and the level of 16319 binding or
activity determined using standard techniques.
[1281] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either a 16319 protein or a 16319 target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated 16319 protein or target molecules can be prepared from
biotin-NHS (N-hydroxy-succinimide) using techniques known in the
art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),
and immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies which are reactive
with 16319 protein or target molecules but which do not interfere
with binding of the 16319 protein to its target molecule can be
derivatized to the wells of the plate, and unbound target or 16319
protein is trapped in the wells by antibody conjugation. Methods
for detecting such complexes, in addition to those described above
for the GST-immobilized complexes, include immunodetection of
complexes using antibodies reactive with the 16319 protein or
target molecule, as well as enzyme-linked assays which rely on
detecting an enzymatic activity associated with the 16319 protein
or target molecule.
[1282] In yet another aspect of the invention, the 16319 protein or
fragments thereof (e.g., the N-terminal region of the 16319 protein
that is believed to be involved in the regulation of apoptotic
activity) can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with 16319
("16319-binding proteins" or "16319-bp) and are involved in 16319
activity. Such 16319-binding proteins are also likely to be
involved in the propagation of signals by the 16319 proteins or
16319 targets as, for example, downstream elements of a
16319-mediated signaling pathway. Alternatively, such 16319-binding
proteins are likely to be 16319 inhibitors.
[1283] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a 16319
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a 16319-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the 16319 protein.
[1284] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a
cell-free assay, and the ability of the agent to modulate the
activity of a 16319 protein can be confirmed in vivo, e.g., in an
animal such as an animal model for aplastic anemia, sickle cell
anemia, thalessemia or sideroblastic anemia. Examples of animals
that can be used include, for example, the 16319-DN transgenic
mouse model described in EP 1127944; the C57 mouse model for
testing whether a compound has in vivo activity in stimulating
erythropoiesis as described in U.S. Pat. No. 6,231,880 (also
describes cell proliferation stimulation induced by hematopoietic
growth factors in baboons); the transgenic mouse model for bone
marrow transplantation for sickle cell anemia described in Iannone,
R. et al., (2001) Blood 97(12):3960-3965; a rat model for Aplastic
Anemia described in Santiago, S. et al. (2001) Transplant Proc.
33(4):2600-2602; transgenic animal models to screen for fetal
hemoglobin-stimulating compounds as described in Fibach, E. (2001)
Semin Hematol 38(4):374-381; mouse models for the treatment of
autoimmune diseases by hematopoietic stem cell transplantation is
described in Ikehara, S. (2001) Experimental Hematology 29:661-669
(specifically, mice with thrombocytic purpura, thrombocytopenia,
renal failure, hemolytic anemia, systemic lupus erythematosus,
hemolytic anemia, sjogren syndrome, rheumatoid arthritis,
pancreatitis, sialoadentis, autoimmune hepatitis, myocardial
infarcton, insulin-dependent diabetes mellitus,
non-insulin-dependent diabetes mellitus and fogal segmental
glomerular sclerosis are described); the three mouse models with
globin gene mutations resulting in human thalessemia as described
in Martinell, J., et al. (1981) Proc. Natl. Acad. Sci.
78(8):5056-5060; animal models for X-linked Sideroblastic Anemia
are described in Yamamoto, M. et al., (2000) Intl. J. Hematology
Review 72:157-164; the mouse model for anemic yolk sacs as
described in Martin, J. S., et al. (1995) Ann. N.Y. Acad. Sci.
752:300-8; various animal models for sickle cell anemia are
described in Nagel. R. L. (2001) Brit J. Hematol. 112:19-25
(specifically, models with a combination of murine globins and
human globin chains, the NYC1 model, the S+S Antilles model, and
transgenic models with exclusively human globin chains are
described); and animal models of cyclic hematopoiesis as described
in Jones, J. B. & Lange, R. D. (1983) Exp. Hematol.
11(7):571-580. Additionally, transgenic animals for the Human Beta
Globin Gene Locus as described in U.S. Pat. No. 6,231,880 may be
used.
[1285] Moreover, a 16319 modulator identified as described herein
(e.g., an antisense 16319 nucleic acid molecule, a 16319-specific
antibody, or a small molecule) can be used in an animal model to
determine the efficacy, toxicity, or side effects of treatment with
such a modulator. Alternatively, a 16319 modulator identified as
described herein can be used in an animal model to determine the
mechanism of action of such a modulator.
Predictive Medicine
[1286] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to
diagnostic assays for determining 16319 protein and/or nucleic acid
expression as well as 16319 activity, in the context of a
biological sample (e.g., blood) to thereby determine whether an
individual is afflicted with a hematological disorder. The
invention also provides for prognostic (or predictive) assays for
determining whether an individual is at risk of developing a
hematological disorder. For example, mutations in a 16319 gene can
be assayed for in a biological sample. Such assays can be used for
prognostic or predictive purpose to thereby prophylactically treat
an individual prior to the onset of a hematological disorder.
[1287] Another aspect of the invention pertains to monitoring the
influence of 16319 modulators (e.g., anti-16319 antibodies or 16319
ribozymes) on the expression or activity of 16319 in clinical
trials.
[1288] These and other agents are described in further detail in
the following sections.
Diagnostic Assays For Hematological Disorders
[1289] To determine whether a subject is afflicted with a
hematological disorder, a biological sample may be obtained from a
subject and the biological sample may be contacted with a compound
or an agent capable of detecting a 16319 protein or nucleic acid
(e.g., mRNA or genomic DNA) that encodes a 16319 protein, in the
biological sample. A preferred agent for detecting 16319 mRNA or
genomic DNA is a labeled nucleic acid probe capable of hybridizing
to 16319 mRNA or genomic DNA. The nucleic acid probe can be, for
example, the 16319 nucleic acid set forth in SEQ ID NO:12, or a
portion thereof, such as an oligonucleotide of at least 15, 20, 25,
30, 25, 40, 45, 50, 100, 250 or 500 nucleotides in length and
sufficient to specifically hybridize under stringent conditions to
16319 mRNA or genomic DNA. Other suitable probes for use in the
diagnostic assays of the invention are described herein.
[1290] A preferred agent for detecting 16319 protein in a sample is
an antibody capable of binding to 16319 protein, preferably an
antibody with a detectable label. Antibodies can be polyclonal, or
more preferably, monoclonal. An intact antibody, or a fragment
thereof (e.g., Fab or F(ab')2) can be used. The term "labeled",
with regard to the probe or antibody, is intended to encompass
direct labeling of the probe or antibody by coupling (i.e.,
physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently labeled streptavidin.
[1291] The term "biological sample" is intended to include tissues
(e.g., blood), cells, and biological fluids isolated from a
subject, as well as tissues, cells, and fluids present within a
subject. That is, the detection method of the invention can be used
to detect 16319 mRNA, protein, or genomic DNA in a biological
sample in vitro as well as in vivo. For example, in vitro
techniques for detection of 16319 mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detection of 16319 protein include enzyme linked immunosorbent
assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro techniques for detection of 16319
genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of 16319 protein include introducing into
a subject a labeled anti-16319 antibody. For example, the antibody
can be labeled with a radioactive marker whose presence and
location in a subject can be detected by standard imaging
techniques.
[1292] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting 16319
protein, mRNA, or genomic DNA, such that the presence of 16319
protein, mRNA or genomic DNA is detected in the biological sample,
and comparing the presence of 16319 protein, mRNA or genomic DNA in
the control sample with the presence of 16319 protein, mRNA or
genomic DNA in the test sample.
Prognostic Assays For Hematological Disorders
[1293] The present invention further pertains to methods for
identifying subjects having or at risk of developing a
hematological disorder associated with aberrant 16319 expression or
activity.
[1294] As used herein, the term "aberrant" includes a 16319
expression or activity which deviates from the wild type 16319
expression or activity. Aberrant expression or activity includes
increased or decreased expression or activity, as well as
expression or activity which does not follow the wild type
developmental pattern of expression or the subcellular pattern of
expression. For example, aberrant 16319 expression or activity is
intended to include the cases in which a mutation in the 16319 gene
causes the 16319 gene to be under-expressed or over-expressed and
situations in which such mutations result in a non-functional 16319
protein or a protein which does not function in a wild-type
fashion, e.g., a protein which does not interact with a 16319
substrate, or one which interacts with a non-16319 substrate.
[1295] The assays described herein, such as the preceding
diagnostic assays or the following assays, can be used to identify
a subject having or at risk of developing a hematological disorder,
e.g., aplastic anemia, Sickle Cell Anemia, polycythemia or
leukemia. A biological sample may be obtained from a subject and
tested for the presence or absence of a genetic alteration. For
example, such genetic alterations can be detected by ascertaining
the existence of at least one of 1) a deletion of one or more
nucleotides from a 16319 gene, 2) an addition of one or more
nucleotides to a 16319 gene, 3) a substitution of one or more
nucleotides of a 16319 gene, 4) a chromosomal rearrangement of a
16319 gene, 5) an alteration in the level of a messenger RNA
transcript of a 16319 gene, 6) aberrant modification of a 16319
gene, such as of the methylation pattern of the genomic DNA, 7) the
presence of a non-wild type splicing pattern of a messenger RNA
transcript of a 16319 gene, 8) a non-wild type level of a
16319-protein, 9) allelic loss of a 16319 gene, and 10)
inappropriate post-translational modification of a
16319-protein.
[1296] As described herein, there are a large number of assays
known in the art which can be used for detecting genetic
alterations in a 16319 gene. For example, a genetic alteration in a
16319 gene may be detected using a probe/primer in a polymerase
chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and
4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a
ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988)
Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad.
Sci. USA 91:360-364), the latter of which can be particularly
useful for detecting point mutations in a 16319 gene (see Abravaya
et al. (1995) Nucleic Acids Res. 23:675-682). This method includes
collecting a biological sample from a subject, isolating nucleic
acid (e.g., genomic DNA, mRNA or both) from the sample, contacting
the nucleic acid sample with one or more primers which specifically
hybridize to a 16319 gene under conditions such that hybridization
and amplification of the 16319 gene (if present) occurs, and
detecting the presence or absence of an amplification product, or
detecting the size of the amplification product and comparing the
length to a control sample. It is anticipated that PCR and/or LCR
may be desirable to use as a preliminary amplification step in
conjunction with any of the techniques used for detecting mutations
described herein.
[1297] Alternative amplification methods include: 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.
[1298] In an alternative embodiment, mutations in a 16319 gene from
a biological sample can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
for example, U.S. Pat. No. 5,498,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[1299] In other embodiments, genetic mutations in 16319 can be
identified by hybridizing biological sample derived and control
nucleic acids, e.g., DNA or RNA, to high density arrays containing
hundreds or thousands of oligonucleotide probes (Cronin, M. T. et
al. (1996) Human Mutation 7:244-255; Kozal, M. J. et al. (1996)
Nature Medicine 2:753-759). For example, genetic mutations in 16319
can be identified in two dimensional arrays containing
light-generated DNA probes as described in Cronin, M. T. et al.
(1996) supra. Briefly, a first hybridization array of probes can be
used to scan through long stretches of DNA in a sample and control
to identify base changes between the sequences by making linear
arrays of sequential, overlapping probes. This step allows for the
identification of point mutations. This step is followed by a
second hybridization array that allows for the characterization of
specific mutations by using smaller, specialized probe arrays
complementary to all variants or mutations detected. Each mutation
array is composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant gene.
[1300] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
16319 gene in a biological sample and detect mutations by comparing
the sequence of the 16319 in the biological sample with the
corresponding wild-type (control) sequence. Examples of sequencing
reactions include those based on techniques developed by Maxam and
Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger (1977)
Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that
any of a variety of automated sequencing procedures can be utilized
when performing the diagnostic assays (Naeve, C. W. (1995)
Biotechniques 19:448-53), including sequencing by mass spectrometry
(see, e.g., PCT International Publication No. WO 94/16101; Cohen et
al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993)
Appl. Biochem. Biotechnol. 38:147-159).
[1301] Other methods for detecting mutations in the 16319 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230:1242). In general, the art technique of
"mismatch cleavage" starts by providing heteroduplexes formed by
hybridizing (labeled) RNA or DNA containing the wild-type 16319
sequence with potentially mutant RNA or DNA obtained from a tissue
sample. The double-stranded duplexes are treated with an agent
which cleaves single-stranded regions of the duplex such as which
will exist due to base pair mismatches between the control and
sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically
digest the mismatched regions. In other embodiments, either DNA/DNA
or RNA/DNA duplexes can be treated with hydroxylamine or osmium
tetroxide and with piperidine in order to digest mismatched
regions. After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing polyacrylamide
gels to determine the site of mutation. See, for example, Cotton et
al. (1988) Proc. Natl. Acad Sci USA 85:4397 and Saleeba et al.
(1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the
control DNA or RNA can be labeled for detection.
[1302] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in 16319
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15:1657-1662). According to an exemplary
embodiment, a probe based on a 16319 sequence, e.g., a wild-type
16319 sequence, is hybridized to a cDNA or other DNA product from a
test cell(s). The duplex is treated with a DNA mismatch repair
enzyme, and the cleavage products, if any, can be detected from
electrophoresis protocols or the like. See, for example, U.S. Pat.
No. 5,459,039.
[1303] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in 16319 genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA: 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144
and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79).
Single-stranded DNA fragments of sample and control 16319 nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al. (1991) Trends Genet 7:5).
[1304] In yet another embodiment the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to ensure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265:12753).
[1305] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki
et al. (1989) Proc. Natl. Acad. Sci USA 86:6230). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA or a number of different mutations when the oligonucleotides
are attached to the hybridizing membrane and hybridized with
labeled target DNA.
[1306] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner (1993) Tibtech 11:238). In
addition it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88:189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[1307] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered a 16319
modulator (e.g., an agonist, antagonist, peptidomimetic, protein,
peptide, nucleic acid, or small molecule) to effectively treat a
hematological disorder.
Monitoring of Effects During Clinical Trials
[1308] The present invention further provides methods for
determining the effectiveness of a 16319 modulator (e.g., a 16319
modulator identified herein) in treating a hematological disorder
in a subject. For example, the effectiveness of a 16319 modulator
in increasing 16319 gene expression, protein levels, or in
upregulating 16319 activity, can be monitored in clinical trials of
subjects exhibiting decreased 16319 gene expression, protein
levels, or downregulated 16319 activity. Alternatively, the
effectiveness of a 16319 modulator in decreasing 16319 gene
expression, protein levels, or in downregulating 16319 activity,
can be monitored in clinical trials of subjects exhibiting
increased 16319 gene expression, protein levels, or 16319 activity.
In such clinical trials, the expression or activity of a 16319
gene, and preferably, other genes that have been implicated in, for
example, a hematological disorder can be used as a "read out" or
marker of the phenotype of a particular cell.
[1309] For example, and not by way of limitation, genes, including
16319, that are modulated in cells by treatment with an agent which
modulates 16319 activity (e.g., identified in a screening assay as
described herein) can be identified. Thus, to study the effect of
agents which modulate 16319 activity on subjects suffering from a
hematological disorder in, for example, a clinical trial, cells can
be isolated and RNA prepared and analyzed for the levels of
expression of 16319 and other genes implicated in the hematological
disorder. The levels of gene expression (e.g., a gene expression
pattern) can be quantified by Northern blot analysis or RT-PCR, as
described herein, or alternatively by measuring the amount of
protein produced, by one of the methods described herein, or by
measuring the levels of activity of 16319 or other genes. In this
way, the gene expression pattern can serve as a marker, indicative
of the physiological response of the cells to the agent which
modulates 16319 activity. This response state may be determined
before, and at various points during treatment of the individual
with the agent which modulates 16319 activity.
[1310] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent which modulates 16319 activity (e.g., an agonist,
antagonist, peptidomimetic, protein, peptide, nucleic acid, or
small molecule identified by the screening assays described herein)
including the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of a 16319 protein, mRNA, or genomic DNA in
the pre-administration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the 16319 protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the 16319 protein, mRNA, or
genomic DNA in the pre-administration sample with the 16319
protein, mRNA, or genomic DNA in the post administration sample or
samples; and (vi) altering the administration of the agent to the
subject accordingly. For example, increased administration of the
agent may be desirable to increase the expression or activity of
16319 to higher levels than detected, i.e., to increase the
effectiveness of the agent. Alternatively, decreased administration
of the agent may be desirable to decrease expression or activity of
16319 to lower levels than detected, i.e. to decrease the
effectiveness of the agent. According to such an embodiment, 16319
expression or activity may be used as an indicator of the
effectiveness of an agent, even in the absence of an observable
phenotypic response.
Methods of Treatment of Subjects Suffering From Hematological
Disorders
[1311] The present invention provides for both prophylactic and
therapeutic methods of treating a subject, e.g., a human, at risk
of (or susceptible to) a hematological disorder such as aplastic
anemia, Sickle Cell Anemia, polycythemia or leukemia. With regard
to both prophylactic and therapeutic methods of treatment, such
treatments may be specifically tailored or modified, based on
knowledge obtained from the field of pharmacogenomics.
"Pharmacogenomics," as used herein, refers to the application of
genomics technologies such as gene sequencing, statistical
genetics, and gene expression analysis to drugs in clinical
development and on the market. More specifically, the term refers
to the study of how a patient's genes determine his or her response
to a drug (e.g., a patient's "drug response phenotype", or "drug
response genotype").
[1312] Thus, another aspect of the invention provides methods for
tailoring an subject's prophylactic or therapeutic treatment with
either the 16319 molecules of the present invention or 16319
modulators according to that individual's drug response genotype.
Pharmacogenomics allows a clinician or physician to target
prophylactic or therapeutic treatments to patients who will most
benefit from the treatment and to avoid treatment of patients who
will experience toxic drug-related side effects.
Prophylactic Methods
[1313] In one aspect, the invention provides a method for
preventing in a subject, a hematological disorder by administering
to the subject an agent which modulates 16319 expression or 16319
activity, e.g., modulation of hematopoietic cell proliferation or
modulation of apoptosis of hematopoietic cells. Subjects at risk
for a hematological disorder can be identified by, for example, any
or a combination of the diagnostic or prognostic assays described
herein. Administration of a prophylactic agent can occur prior to
the manifestation of symptoms characteristic of aberrant 16319
expression or activity, such that a hematological disorder is
prevented or, alternatively, delayed in its progression. Depending
on the type of 16319 aberrancy, for example, a 16319, 16319 agonist
or 16319 antagonist agent can be used for treating the subject. The
appropriate agent can be determined based on screening assays
described herein.
Therapeutic Methods
[1314] Another aspect of the invention pertains to methods for
treating a subject suffering from a hematological disorder. These
methods involve administering to a subject an agent which modulates
16319 expression or activity (e.g., an agent identified by a
screening assay described herein), or a combination of such agents.
In another embodiment, the method involves administering to a
subject a 16319 protein or nucleic acid molecule as therapy to
compensate for reduced, aberrant, or unwanted 16319 expression or
activity.
[1315] Stimulation of 16319 activity is desirable in situations in
which 16319 is abnormally downregulated and/or in which increased
16319 activity is likely to have a beneficial effect, i.e., an
increase in induction of apoptosis or a decrease in the
proliferation of hematopoietic cells, thereby ameliorating
hematological disorders such as polycythemia or infectious
mononucleosis in a subject. Likewise, inhibition of 16319 activity
is desirable in situations in which 16319 is abnormally upregulated
and/or in which decreased 16319 activity is likely to have a
beneficial effect, e.g., inhibition of apoptosis in hematopoietic
cells and an increase in hematopoietic cell proliferation, thereby
ameliorating a hematological disorder such as aplastic anemia or
hemorrhagic anemia in a subject.
Pharmaceutical Compositions
[1316] The agents which modulate 16319 activity can be administered
to a subject using pharmaceutical compositions suitable for such
administration. Such compositions typically comprise the agent
(e.g., nucleic acid molecule, protein, or antibody) and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[1317] A pharmaceutical composition used in the therapeutic methods
of the invention is formulated to be compatible with its intended
route of administration. Examples of routes of administration
include parenteral, e.g., intravenous, intradermal, subcutaneous,
oral (e.g., inhalation), transdermal (topical), transmucosal, and
rectal administration. Solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include
the following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[1318] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, and sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[1319] Sterile injectable solutions can be prepared by
incorporating the agent that modulates 16319 activity (e.g., a
fragment of a 16319 protein or an anti-16319 antibody) in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[1320] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[1321] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[1322] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[1323] The agents that modulate 16319 activity can also be prepared
in the form of suppositories (e.g., with conventional suppository
bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
[1324] In one embodiment, the agents that modulate 16319 activity
are prepared with carriers that will protect the compound against
rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[1325] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the agent that modulates 16319 activity
and the particular therapeutic effect to be achieved, and the
limitations inherent in the art of compounding such an agent for
the treatment of subjects.
[1326] Toxicity and therapeutic efficacy of such agents can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
can be expressed as the ratio LD50/ED50. Agents which exhibit large
therapeutic indices are preferred. While agents that exhibit toxic
side effects may be used, care should be taken to design a delivery
system that targets such agents to the site of affected tissue in
order to minimize potential damage to uninfected cells and,
thereby, reduce side effects.
[1327] 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 16319 modulating agents lies preferably
within a range of circulating concentrations that include the ED50
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 agent used in the therapeutic
methods 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 IC50 (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.
[1328] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
skilled artisan will appreciate that certain factors may influence
the dosage required to effectively treat a subject, including but
not limited to the severity of the disease or disorder, previous
treatments, the general health and/or age of the subject, and other
diseases present. Moreover, treatment of a subject with a
therapeutically effective amount of a protein, polypeptide, or
antibody can include a single treatment or, preferably, can include
a series of treatments.
[1329] In a preferred example, a subject is treated with antibody,
protein, or polypeptide in the range of between about 0.1 to 20
mg/kg body weight, one time per week for between about 1 to 10
weeks, preferably between 2 to 8 weeks, more preferably between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. It will also be appreciated that the effective dosage of
antibody, protein, or polypeptide used for treatment may increase
or decrease over the course of a particular treatment. Changes in
dosage may result and become apparent from the results of
diagnostic assays as described herein.
[1330] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds. It is understood that appropriate doses of small
molecule agents depends upon a number of factors within the ken of
the ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention.
[1331] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram). It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. Such appropriate doses may be determined using the
assays described herein. When one or more of these small molecules
is to be administered to an animal (e.g., a human) in order to
modulate expression or activity of a polypeptide or nucleic acid of
the invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[1332] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[1333] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
alpha-interferon, beta-interferon, nerve growth factor, platelet
derived growth factor, tissue plasminogen activator; or biological
response modifiers such as, for example, lymphokines, interleukin-1
("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte macrophase colony stimulating factor ("GM-CSF"),
granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
[1334] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
[1335] The nucleic acid molecules used in the methods of the
invention can be inserted into vectors and used as gene therapy
vectors. Gene therapy vectors can be delivered to a subject by, for
example, intravenous injection, local administration (see U.S. Pat.
No. 5,328,470) or by stereotactic injection (see, e.g., Chen et al.
(1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system.
Transplantation and Transfusions
[1336] The present invention provides methods for increasing
hematopoietic cells in patients, particularly patients undergoing
radiation therapy and/or chemotherapy, e.g., in the treatment of
cancer. Such therapies kill dividing progenitor cells in the marrow
and peripheral blood, limiting therapy and often requiring
transfusions to restore circulating levels of platelets and other
blood cells. Of particular interest are those patients receiving
bone marrow and/or peripheral blood stem cell transplants following
radiation therapy and patients suffering from congenital metabolic
defects necessitating bone marrow transplant. Among these
indications are bone marrow transplants associated with the
treatment of breast cancer, leukemia, lymphoma, multiple myeloma,
and congenital defects such as severe combined immune deficiency,
thallasemia, and sickle cell anemia. Peripheral blood stem cell
transplantation may be preferred in conditions where a risk of
tumor cells in the blood is not present.
[1337] As used herein, the term "transplantation" includes the
process of removing cells from a donor subject and subsequently
administering the cells to a recipient subject. The term
encompasses both allogeneic transplantation, wherein the donor and
recipient are different subjects of the same species; and
autologous transplantation, wherein the donor and recipient are the
same subject.
[1338] Methods for carrying out bone marrow and peripheral blood
stem cell transplants are known in the art. (Snyder et al.,
"Transfusion Medicine" in Benz and McArthur, eds., Hematology 1994,
American Society of Hematology, 96-106, 1994.) For example,
peripheral blood stem cells are collected by leukapheresis
according to accepted clinical procedures. Hematopoietic progenitor
cells can be selected on the basis of cell surface markers (e.g.
CD34), allowing for enrichment of the desired cells and depletion
of contaminating tumor cells. The collected cells are stored frozen
in a suitable cryoprotectant (e.g. dimethyl sulfoxide, hydroxyethyl
starch) until needed. Marrow cells are collected from donors by
bone puncture under anesthesia. To reduce the volume, the collected
marrow is usually processed to separate plasma from the cellular
components. Removal of plasma can also eliminate red cell
incompatibilities in allogeneic transplantation. The cell fraction
can be enriched for mononuclear cells using density gradient
techniques or automated separation methods and depleted of T cells
using various cytotoxic agents. Collected marrow cells are
cryopreserved according to established procedures that include
controlled-rate freezing and the use of cryoprotectants. Stem cells
are thawed in a warm water bath immediately prior to use to
minimize loss associated with thawing. In the case of allogeneic
transplants, donors and recipients are tissue matched to minimize
the risk of graft-versus-host disease.
[1339] An increase in hematopoietic cells results from
transplantation into a recipient patient of stem cells,
particularly cells of the myeloid lineage, including CD34+ stem
cells and cells derived from CD34+ stem cells. Of particular
interest are cells in the megakaryocyte and erythrocyte lineages,
which reconstitute the recipient's platelet and erythrocyte
populations, respectively.
[1340] In one aspect of the invention, a donor is treated, prior to
donation of marrow or peripheral blood cells, with a compound that
inhibits 16319, in an amount sufficient to stimulate proliferation
of hematopoietic cells and/or in an amount sufficient to inhibit
apoptosis of hematopoietic cells. Treatment of the donor will be
carried out for a period of from one to several days, preferably
about 2-5 days, during a period of from 3 days to 2 weeks prior to
harvesting of bone marrow or peripheral blood stem cells. It is
preferred to treat the donor during a period of five to ten days
prior to harvesting of cells. The increase in CD34+ stem cells and
other cells of the myeloid lineage in the donor will be manifested
by improved recovery of hematopoietic cells in the transplant
recipient. In another aspect of the invention, the recipient is
treated with a compound that inhibits 16319 after transplantation
to further enhance hematopoietic cell recovery.
[1341] Another aspect of the invention features a method for
increasing the number of hematopoietic cells in a subject, for
example, a subject undergoing radiation therapy and/or
chemotherapy, e.g., for the treatment of cancer. The method
includes the process of removing cells from a donor and
subsequently administering the cells to a recipient. In one aspect
of the invention, a donor is treated, prior to donation of marrow
or peripheral blood cells, with a compound that inhibits 16319, in
an amount sufficient to stimulate proliferation of hematopoietic
cells and/or in an amount sufficient to inhibit apoptosis of
hematopoietic cells. In another aspect of the invention, the
recipient is treated with a compound that inhibits 16319 after
transplantation to further enhance hematopoietic cell recovery.
[1342] In another aspect, the invention provides methods for
increasing hematopoietic progenitor and committed erythroid cells
in a recipient subject in need of such an increase. The methods
include administering to a donor subject an amount of 16319
sufficient to inhibit induction of apoptosis and prevent inhibition
of cell proliferation of hematopoietic cells in the donor;
collecting cells from the donor, wherein the cells are bone marrow
cells or peripheral blood stem cells; and administering the bone
marrow cells or peripheral blood stem cells to a recipient subject.
The donor and recipient may be different or the same subject. In
one embodiment of the invention, the recipient subject has been
treated with chemotherapy or radiation therapy.
[1343] In another aspect, the invention provides methods of
preparing cells for transplantation comprising administering to a
donor subject an amount of 16319 or a 16319 modulator sufficient to
inhibit induction of apoptosis and prevent inhibition of cell
proliferation of hematopoietic cells in the donor subject, and
collecting cells from the donor subject, e.g., bone marrow cells or
peripheral blood stem cells.
[1344] In another aspect, the invention provides a method of
stimulating platelet recovery or erythrocyte recovery in a subject
receiving chemotherapy or radiation therapy. The method includes
administering to the subject an amount of 16319 or a 16319
modulator sufficient to stimulate proliferation of cells of the
myeloid lineage in the subject; collecting bone marrow cells or
peripheral blood stem cells from the subject prior to chemotherapy
or radiation therapy; and returning the collected cells to the
subject subsequent to chemotherapy or radiation therapy. Within one
embodiment this method further includes administering to the
subject, after or concurrently with returning the collected cells,
an amount of 16319 or a 16319 modulator sufficient to enhance
platelet recovery or erythrocyte recovery.
Pharmacogenomics
[1345] In conjunction with the therapeutic methods of the
invention, pharmacogenomics (i.e., the study of the relationship
between a subject's genotype and that subject's response to a
foreign compound or drug) may be considered. Differences in
metabolism of therapeutics can lead to severe toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus, a
physician or clinician may consider applying knowledge obtained in
relevant pharmacogenomics studies in determining whether to
administer an agent which modulates 16319 activity, as well as
tailoring the dosage and/or therapeutic regimen of treatment with
an agent which modulates 16319 activity.
[1346] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin.
Chem. 43(2):254-266. In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body (altered
drug action) or genetic conditions transmitted as single factors
altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare genetic
defects or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate aminopeptidase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[1347] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants). Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[1348] Alternatively, a method termed the "candidate gene approach"
can be utilized to identify genes that predict drug response.
According to this method, if a gene that encodes a drug target is
known (e.g., a 16319 protein of the present invention), all common
variants of that gene can be fairly easily identified in the
population and it can be determined if having one version of the
gene versus another is associated with a particular drug
response.
[1349] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and the cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[1350] Alternatively, a method termed the "gene expression
profiling" can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a 16319 molecule or 16319 modulator of the present
invention) can give an indication whether gene pathways related to
toxicity have been turned on.
[1351] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of a subject. This knowledge, when applied to dosing or
drug selection, can avoid adverse reactions or therapeutic failure
and, thus, enhance therapeutic or prophylactic efficiency when
treating a subject suffering from a hematological disorder with an
agent which modulates 16319 activity.
Recombinant Expression Vectors and Host Cells Used in the Methods
of the Invention
[1352] The methods of the invention (e.g., the screening assays
described herein) include the use of vectors, preferably expression
vectors, containing a nucleic acid encoding a 16319 protein (or a
portion thereof). As used herein, the term "vector" refers to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments can be ligated. Another type of vector is a
viral vector, wherein additional DNA segments can be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) are integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "expression
vectors". In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" can be used interchangeably
as the plasmid is the most commonly used form of vector. However,
the invention is intended to include such other forms of expression
vectors, such as viral vectors (e.g., replication defective
retroviruses, adenoviruses and adeno-associated viruses), which
serve equivalent functions.
[1353] The recombinant expression vectors to be used in the methods
of the invention comprise a nucleic acid of the invention in a form
suitable for expression of the nucleic acid in a host cell, which
means that the recombinant expression vectors include one or more
regulatory sequences, selected on the basis of the host cells to be
used for expression, which is operatively linked to the nucleic
acid sequence to be expressed. Within a recombinant expression
vector, "operably linked" is intended to mean that the nucleotide
sequence of interest is linked to the regulatory sequence(s) in a
manner which allows for expression of the nucleotide sequence
(e.g., in an in vitro transcription/translation system or in a host
cell when the vector is introduced into the host cell). The term
"regulatory sequence" is intended to include promoters, enhancers
and other expression control elements (e.g., polyadenylation
signals). Such regulatory sequences are described, for example, in
Goeddel (1990) Methods Enzymol. 185:3-7. Regulatory sequences
include those which direct constitutive expression of a nucleotide
sequence in many types of host cells and those which direct
expression of the nucleotide sequence only in certain host cells
(e.g., tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of protein
desired, and the like. The expression vectors of the invention can
be introduced into host cells to thereby produce proteins or
peptides, including fusion proteins or peptides, encoded by nucleic
acids as described herein (e.g., 16319 proteins, mutant forms of
16319 proteins, fusion proteins, and the like).
[1354] The recombinant expression vectors to be used in the methods
of the invention can be designed for expression of 16319 proteins
in prokaryotic or eukaryotic cells. For example, 16319 proteins can
be expressed in bacterial cells such as E. coli, insect cells
(using baculovirus expression vectors), yeast cells, or mammalian
cells. Suitable host cells are discussed further in Goeddel (1990)
supra. Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[1355] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein.
[1356] Purified fusion proteins can be utilized in 16319 activity
assays, (e.g., direct assays or competitive assays described in
detail below), or to generate antibodies specific for 16319
proteins. In a preferred embodiment, a 16319 fusion protein
expressed in a retroviral expression vector of the present
invention can be utilized to infect bone marrow cells which are
subsequently transplanted into irradiated recipients. The pathology
of the subject recipient is then examined after sufficient time has
passed (e.g., six weeks).
[1357] In another embodiment, a nucleic acid of the invention is
expressed in mammalian cells using a mammalian expression vector.
Examples of mammalian expression vectors include pCDM8 (Seed, B.
(1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.
6:187-195). When used in mammalian cells, the expression vector's
control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40. For other
suitable expression systems for both prokaryotic and eukaryotic
cells see chapters 16 and 17 of Sambrook, J. et al., Molecular
Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[1358] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
[1359] The methods of the invention may further use a recombinant
expression vector comprising a DNA molecule of the invention cloned
into the expression vector in an antisense orientation. That is,
the DNA molecule is operatively linked to a regulatory sequence in
a manner which allows for expression (by transcription of the DNA
molecule) of an RNA molecule which is antisense to 16319 mRNA.
Regulatory sequences operatively linked to a nucleic acid cloned in
the antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific,
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid, or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes, see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[1360] Another aspect of the invention pertains to the use of host
cells into which a 16319 nucleic acid molecule of the invention is
introduced, e.g., a 16319 nucleic acid molecule within a
recombinant expression vector or a 16319 nucleic acid molecule
containing sequences which allow it to homologously recombine into
a specific site of the host cell's genome. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is
understood that such terms refer not only to the particular subject
cell but to the progeny or potential progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term as used herein.
[1361] A host cell can be any prokaryotic or eukaryotic cell. For
example, a 16319 protein can be expressed in bacterial cells such
as E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[1362] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[1363] A host cell used in the methods of the invention, such as a
prokaryotic or eukaryotic host cell in culture, can be used to
produce (i.e., express) a 16319 protein. Accordingly, the invention
further provides methods for producing a 16319 protein using the
host cells of the invention. In one embodiment, the method
comprises culturing the host cell of the invention (into which a
recombinant expression vector encoding a 16319 protein has been
introduced) in a suitable medium such that a 16319 protein is
produced. In another embodiment, the method further comprises
isolating a 16319 protein from the medium or the host cell.
Isolated Nucleic Acid Molecules Used in the Methods of the
Invention
[1364] The coding sequence of the isolated human 16319 cDNA and the
predicted amino acid sequence of the human 16319 polypeptide are
shown SEQ ID NOs:12 and 13, respectively. The 16319 sequence is
also described in Yamaguchi, et al. (1995), supra), the contents of
which are incorporated herein by reference.
[1365] The methods of the invention include the use of isolated
nucleic acid molecules that encode 16319 proteins or biologically
active portions thereof, as well as nucleic acid fragments
sufficient for use as hybridization probes to identify
16319-encoding nucleic acid molecules (e.g., 16319 mRNA) and
fragments for use as PCR primers for the amplification or mutation
of 16319 nucleic acid molecules. As used herein, the term "nucleic
acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA
or RNA generated using nucleotide analogs. The nucleic acid
molecule can be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[1366] A nucleic acid molecule used in the methods of the present
invention, e.g., a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:12, or a portion thereof, can be isolated
using standard molecular biology techniques and the sequence
information provided herein. Using all or portion of the nucleic
acid sequence of SEQ ID NO:12 as a hybridization probe, 16319
nucleic acid molecules can be isolated using standard hybridization
and cloning techniques (e.g., as described in Sambrook, J., Fritsh,
E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.
2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989).
[1367] Moreover, a nucleic acid molecule encompassing all or a
portion of SEQ ID NO:12 can be isolated by the polymerase chain
reaction (PCR) using synthetic oligonucleotide primers designed
based upon the sequence of SEQ ID NO:12.
[1368] A nucleic acid used in the methods of the invention can be
amplified using cDNA, mRNA or, alternatively, genomic DNA as a
template and appropriate oligonucleotide primers according to
standard PCR amplification techniques. Furthermore,
oligonucleotides corresponding to 16319 nucleotide sequences can be
prepared by standard synthetic techniques, e.g., using an automated
DNA synthesizer.
[1369] In a preferred embodiment, the isolated nucleic acid
molecules used in the methods of the invention comprise the
nucleotide sequence shown in SEQ ID NO:12, a complement of the
nucleotide sequence shown in SEQ ID NO:12, or a portion of any of
these nucleotide sequences. A nucleic acid molecule which is
complementary to the nucleotide sequence shown in SEQ ID NO:12, is
one which is sufficiently complementary to the nucleotide sequence
shown in SEQ ID NO:12 such that it can hybridize to the nucleotide
sequence shown in SEQ ID NO:12 thereby forming a stable duplex.
[1370] In still another preferred embodiment, an isolated nucleic
acid molecule used in the methods of the present invention
comprises a nucleotide sequence which is at least about 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more
identical to the entire length of the nucleotide sequence shown in
SEQ ID NO:12 or a portion of any of this nucleotide sequence.
[1371] Moreover, the nucleic acid molecules used in the methods of
the invention can comprise only a portion of the nucleic acid
sequence of SEQ ID NO:12, for example, a fragment which can be used
as a probe or primer or a fragment encoding a portion of a 16319
protein, e.g., a biologically active portion of a 16319 protein.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12 or 15, preferably about 20 or 25, more
preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive
nucleotides of a sense sequence of SEQ ID NO:12 of an anti-sense
sequence of SEQ ID NO:12 or of a naturally occurring allelic
variant or mutant of SEQ ID NO:12. In one embodiment, a nucleic
acid molecule used in the methods of the present invention
comprises a nucleotide sequence which is greater than 100, 100-200,
200-300, 300-400, 400-500, 500-600, or more nucleotides in length
and hybridizes under stringent hybridization conditions to a
nucleic acid molecule of SEQ ID NO:12.
[1372] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences that are significantly
identical or homologous to each other remain hybridized to each
other. Preferably, the conditions are such that sequences at least
about 70%, more preferably at least about 80%, even more preferably
at least about 85% or 90% identical to each other remain hybridized
to each other. Such stringent conditions are known to those skilled
in the art and can be found in Current Protocols in Molecular
Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995),
sections 2, 4 and 6. Additional stringent conditions can be found
in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9
and 11. A preferred, non-limiting example of stringent
hybridization conditions includes hybridization in 4.times. sodium
chloride/sodium citrate (SSC), at about 65-70.degree. C. (or
hybridization in 4.times.SSC plus 50% formamide at about
42-50.degree. C.) followed by one or more washes in 1.times.SSC, at
about 65-70.degree. C. A preferred, non-limiting example of highly
stringent hybridization conditions includes hybridization in
1.times.SSC, at about 65-70.degree. C. (or hybridization in
1.times.SSC plus 50% formamide at about 42-50.degree. C.) followed
by one or more washes in 0.3.times.SSC, at about 65-70.degree. C. A
preferred, non-limiting example of reduced stringency hybridization
conditions includes hybridization in 4.times.SSC, at about
50-60.degree. C. (or alternatively hybridization in 6.times.SSC
plus 50% formamide at about 40-45.degree. C.) followed by one or
more washes in 2.times.SSC, at about 50-60.degree. C. Ranges
intermediate to the above-recited values, e.g., at 65-70.degree. C.
or at 42-50.degree. C. are also intended to be encompassed by the
present invention. SSPE (1.times.SSPE is 0.15M NaCl, 10 mM
NaH.sub.2PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be substituted for
SSC (1.times.SSC is 0.15M NaCl and 15 mM sodium citrate) in the
hybridization and wash buffers; washes are performed for 15 minutes
each after hybridization is complete. The hybridization temperature
for hybrids anticipated to be less than 50 base pairs in length
should be 5-10.degree. C. less than the melting temperature
(T.sub.m) of the hybrid, where T.sub.m is determined according to
the following equations. For hybrids less than 18 base pairs in
length, T.sub.m(.degree. C.)=2(# of A+T bases)+4(# of G+C bases).
For hybrids between 18 and 49 base pairs in length,
T.sub.m(.degree. C.)=81.5+16.6(log.sub.10[Na.sup.+])+0.41(%
G+C)-(600/N), where N is the number of bases in the hybrid, and
[Na.sup.+] is the concentration of sodium ions in the hybridization
buffer ([Na.sup.+] for 1.times.SSC=0.165 M). It will also be
recognized by the skilled practitioner that additional reagents may
be added to hybridization and/or wash buffers to decrease
non-specific hybridization of nucleic acid molecules to membranes,
for example, nitrocellulose or nylon membranes, including but not
limited to blocking agents (e.g., BSA or salmon or herring sperm
carrier DNA), detergents (e.g., SDS), chelating agents (e.g.,
EDTA), Ficoll, PVP and the like. When using nylon membranes, in
particular, an additional preferred, non-limiting example of
stringent hybridization conditions is hybridization in 0.25-0.5M
NaH.sub.2PO.sub.4, 7% SDS at about 65.degree. C., followed by one
or more washes at 0.02M NaH.sub.2PO.sub.4, 1% SDS at 65.degree. C.,
see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA
81:1991-1995, (or alternatively 0.2.times.SSC, 1% SDS).
[1373] In preferred embodiments, the probe further comprises a
label group attached thereto, e.g., the label group can be a
radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells or tissue which misexpress a 16319
protein, such as by measuring a level of a 16319-encoding nucleic
acid in a sample of cells from a subject e.g., detecting 16319 mRNA
levels or determining whether a genomic 16319 gene has been mutated
or deleted.
[1374] The methods of the invention further encompass the use of
nucleic acid molecules that differ from the nucleotide sequence
shown in SEQ ID NO:12 due to degeneracy of the genetic code and
thus encode the same 16319 proteins as those encoded by the
nucleotide sequence shown in SEQ ID NO:12. In another embodiment,
an isolated nucleic acid molecule included in the methods of the
invention has a nucleotide sequence encoding a protein having an
amino acid sequence shown in SEQ ID NO:13.
[1375] The methods of the invention further include the use of
allelic variants of human 16319, e.g., functional and
non-functional allelic variants. Functional allelic variants are
naturally occurring amino acid sequence variants of the human 16319
protein that maintain a 16319 activity. Functional allelic variants
will typically contain only conservative substitution of one or
more amino acids of SEQ ID NO:13, or substitution, deletion or
insertion of non-critical residues in non-critical regions of the
protein.
[1376] Non-functional allelic variants are naturally occurring
amino acid sequence variants of the human 16319 protein that do not
have a 16319 activity. Non-functional allelic variants will
typically contain a non-conservative substitution, deletion, or
insertion or premature truncation of the amino acid sequence of SEQ
ID NO:13, or a substitution, insertion or deletion in critical
residues or critical regions of the protein.
[1377] The methods of the present invention may further use
non-human orthologues of the human 16319 protein. Orthologues of
the human 16319 protein are proteins that are isolated from
non-human organisms and possess the same 16319 activity.
[1378] The methods of the present invention further include the use
of nucleic acid molecules comprising the nucleotide sequence of SEQ
ID NO:12 or a portion thereof, in which a mutation has been
introduced. The mutation may lead to amino acid substitutions at
"non-essential" amino acid residues or at "essential" amino acid
residues. A "non-essential" amino acid residue is a residue that
can be altered from the wild-type sequence of 16319 (e.g., the
sequence of SEQ ID NO:13) without altering the biological activity,
whereas an "essential" amino acid residue is required for
biological activity. For example, amino acid residues that are
conserved among the 16319 proteins of the present invention and
other members of the CIDE family (e.g., CIDE-B, FSP-27, and DFF45)
are not likely to be amenable to alteration.
[1379] Mutations can be introduced into SEQ ID NO:12 by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Preferably, conservative amino acid substitutions are
made at one or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., asparagine, glutamine,
serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
glycine, alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a 16319 protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of a 16319 coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for 16319 biological activity to identify
mutants that retain activity. Following mutagenesis of SEQ ID NO:12
the encoded protein can be expressed recombinantly and the activity
of the protein can be determined using the assay described
herein.
[1380] Another aspect of the invention pertains to the use of
isolated nucleic acid molecules which are antisense to the
nucleotide sequence of SEQ ID NO:12. An "antisense" nucleic acid
comprises a nucleotide sequence which is complementary to a "sense"
nucleic acid encoding a protein, e.g., complementary to the coding
strand of a double-stranded cDNA molecule or complementary to an
mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen
bond to a sense nucleic acid. The antisense nucleic acid can be
complementary to an entire 16319 coding strand, or to only a
portion thereof. In one embodiment, an antisense nucleic acid
molecule is antisense to a "coding region" of the coding strand of
a nucleotide sequence encoding a 16319. The term "coding region"
refers to the region of the nucleotide sequence comprising codons
which are translated into amino acid residues. In another
embodiment, the antisense nucleic acid molecule is antisense to a
"noncoding region" of the coding strand of a nucleotide sequence
encoding 16319. The term "noncoding region" refers to 5' and 3'
sequences which flank the coding region that are not translated
into amino acids (also referred to as 5' and 3' untranslated
regions).
[1381] Given the coding strand sequences encoding 16319 disclosed
herein, antisense nucleic acids of the invention can be designed
according to the rules of Watson and Crick base pairing. The
antisense nucleic acid molecule can be complementary to the entire
coding region of 16319 mRNA, but more preferably is an
oligonucleotide which is antisense to only a portion of the coding
or noncoding region of 16319 mRNA. For example, the antisense
oligonucleotide can be complementary to the region surrounding the
translation start site of 16319 mRNA. An antisense oligonucleotide
can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50
nucleotides in length. An antisense nucleic acid of the invention
can be constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. Examples of modified nucleotides which can
be used to generate the antisense nucleic acid include
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, 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-isopentenyladenine,
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. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[1382] The antisense nucleic acid molecules used in the methods of
the invention are typically administered to a subject or generated
in situ such that they hybridize with or bind to cellular mRNA
and/or genomic DNA encoding a 16319 protein to thereby inhibit
expression of the protein, e.g., by inhibiting transcription and/or
translation. The hybridization can be by conventional nucleotide
complementarity to form a stable duplex, or, for example, in the
case of an antisense nucleic acid molecule which binds to DNA
duplexes, through specific interactions in the major groove of the
double helix. An example of a route of administration of antisense
nucleic acid molecules of the invention include direct injection at
a tissue site. Alternatively, antisense nucleic acid molecules can
be modified to target selected cells and then administered
systemically. For example, for systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface, e.g.,
by linking the antisense nucleic acid molecules to peptides or
antibodies which bind to cell surface receptors or antigens. The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong pol II or pol III promoter are
preferred.
[1383] In yet another embodiment, the antisense nucleic acid
molecule used in the methods of the invention is an
.alpha.-anomeric nucleic acid molecule. An .alpha.-anomeric nucleic
acid molecule forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gaultier et al. (1987) Nucleic
Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can
also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987)
Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue
(Inoue et al. (1987) FEBS Lett. 215:327-330).
[1384] In still another embodiment, an antisense nucleic acid used
in the methods of the invention is a ribozyme. Ribozymes are
catalytic RNA molecules with ribonuclease activity which are
capable of cleaving a single-stranded nucleic acid, such as an
mRNA, to which they have a complementary region. Thus, ribozymes
(e.g., hammerhead ribozymes (described in Haselhoff and Gerlach
(1988) Nature 334:585-591)) can be used to catalytically cleave
16319 mRNA transcripts to thereby inhibit translation of 16319
mRNA. A ribozyme having specificity for a 16319-encoding nucleic
acid can be designed based upon the nucleotide sequence of a 16319
cDNA disclosed herein (i.e., SEQ ID NO:12). For example, a
derivative of a Tetrahymena L-19 IVS RNA can be constructed in
which the nucleotide sequence of the active site is complementary
to the nucleotide sequence to be cleaved in a 16319-encoding mRNA.
See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al.
U.S. Pat. No. 5,116,742. Alternatively, 16319 mRNA can be used to
select a catalytic RNA having a specific ribonuclease activity from
a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W.
(1993) Science 261:1411-1418.
[1385] Alternatively, 16319 gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the 16319 (e.g., the 16319 promoter and/or enhancers) to
form triple helical structures that prevent transcription of the
16319 gene in target cells. 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.
[1386] In yet another embodiment, the 16319 nucleic acid molecules
used in the methods of the present invention can be modified at the
base moiety, sugar moiety or phosphate backbone to improve, e.g.,
the stability, hybridization, or solubility of the molecule. For
example, the deoxyribose phosphate backbone of the nucleic acid
molecules can be modified to generate peptide nucleic acids (see
Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1):
5-23). As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics, e.g., DNA mimics, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. (1996) Proc.
Natl. Acad. Sci. 93:14670-675.
[1387] PNAs of 16319 nucleic acid molecules can be used in the
therapeutic and diagnostic applications described herein. For
example, PNAs can be used as antisense or antigene agents for
sequence-specific modulation of gene expression by, for example,
inducing transcription or translation arrest or inhibiting
replication. PNAs of 16319 nucleic acid molecules can also be used
in the analysis of single base pair mutations in a gene, (e.g., by
PNA-directed PCR clamping); as `artificial restriction enzymes`
when used in combination with other enzymes, (e.g., S1 nucleases
(Hyrup B. et al. (1996) supra)); or as probes or primers for DNA
sequencing or hybridization (Hyrup B. et al. (1996) supra;
Perry-O'Keefe et al. (1996) supra).
[1388] In another embodiment, PNAs of 16319 can be modified, (e.g.,
to enhance their stability or cellular uptake), by attaching
lipophilic or other helper groups to PNA, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of
drug delivery known in the art. For example, PNA-DNA chimeras of
16319 nucleic acid molecules can be generated which may combine the
advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, (e.g., RNAse H and DNA polymerases), to
interact with the DNA portion while the PNA portion would provide
high binding affinity and specificity. PNA-DNA chimeras can be
linked using linkers of appropriate lengths selected in terms of
base stacking, number of bonds between the nucleobases, and
orientation (Hyrup B. et al. (1996) supra). The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup B. et al.
(1996) supra and Finn P. J. et al. (1996) Nucleic Acids Res. 24
(17): 3357-63. For example, a DNA chain can be synthesized on a
solid support using standard phosphoramidite coupling chemistry and
modified nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can
be used as a between the PNA and the 5' end of DNA (Mag, M. et al.
(1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then
coupled in a stepwise manner to produce a chimeric molecule with a
5' PNA segment and a 3' DNA segment (Finn P. J. et al. (1996)
supra). Alternatively, chimeric molecules can be synthesized with a
5' DNA segment and a 3' PNA segment (Peterser, K. H. et al. (1975)
Bioorganic Med. Chem. Lett. 5: 1119-11124).
[1389] In other embodiments, the oligonucleotide used in the
methods of the invention 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. USA 86:6553-6556;
Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT
Publication No. WO88/09810) or the blood-brain barrier (see, e.g.,
PCT Publication No. WO89/10134). In addition, oligonucleotides can
be modified with hybridization-triggered cleavage agents (See,
e.g., Krol et al. (1988) Bio-Techniques 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, or hybridization-triggered cleavage agent).
Isolated 16319 Proteins and Anti-16319 Antibodies Used in the
Methods of the Invention
[1390] The methods of the invention include the use of isolated
16319 proteins, and biologically active portions thereof, as well
as polypeptide fragments suitable for use as immunogens to raise
anti-16319 antibodies. In one embodiment, native 16319 proteins can
be isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques.
In another embodiment, 16319 proteins are produced by recombinant
DNA techniques. Alternative to recombinant expression, a 16319
protein or polypeptide can be synthesized chemically using standard
peptide synthesis techniques.
[1391] As used herein, a "biologically active portion" of a 16319
protein includes a fragment of a 16319 protein having a 16319
activity. Biologically active portions of a 16319 protein include
peptides comprising amino acid sequences sufficiently identical to
or derived from the amino acid sequence of the 16319 protein, e.g.,
the amino acid sequence shown in SEQ ID NO:13, which include fewer
amino acids than the full length 16319 proteins, and exhibit at
least one activity of a 16319 protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the 16319 protein (e.g., the N-terminal region of the
16319 protein that is believed to be involved in the regulation of
apoptotic activity). A biologically active portion of a 16319
protein can be a polypeptide which is, for example, 25, 50, 75,
100, 125, 150, 175, 200, 250, 300 or more amino acids in length.
Biologically active portions of a 16319 protein can be used as
targets for developing agents which modulate a 16319 activity.
[1392] In a preferred embodiment, the 16319 protein used in the
methods of the invention has an amino acid sequence shown in SEQ ID
NO:13. In other embodiments, the 16319 protein is substantially
identical to SEQ ID NO:13, and retains the functional activity of
the protein of SEQ ID NO:13, yet differs in amino acid sequence due
to natural allelic variation or mutagenesis, as described in detail
in subsection V above. Accordingly, in another embodiment, the
16319 protein used in the methods of the invention is a protein
which comprises an amino acid sequence at least about 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more
identical to SEQ ID NO:13.
[1393] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-identical
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence (e.g., when aligning a second sequence to
the 16319 amino acid sequence of SEQ ID NO:13 having 500 amino acid
residues, at least 75, preferably at least 150, more preferably at
least 225, even more preferably at least 300, and even more
preferably at least 400 or more amino acid residues are aligned).
The amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position (as
used herein amino acid or nucleic acid "identity" is equivalent to
amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[1394] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm
which has been incorporated into the GAP program in the GCG
software package, using either a Blosum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the
percent identity between two amino acid or nucleotide sequences is
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci. 4:11-17 (1988)) which has been incorporated into the
ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[1395] The methods of the invention may also use 16319 chimeric or
fusion proteins. As used herein, a 16319 "chimeric protein" or
"fusion protein" comprises a 16319 polypeptide operatively linked
to a non-16319 polypeptide. An "16319 polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a 16319
molecule, whereas a "non-16319 polypeptide" refers to a polypeptide
having an amino acid sequence corresponding to a protein which is
not substantially homologous to the 16319 protein, e.g., a protein
which is different from the 16319 protein and which is derived from
the same or a different organism. Within a 16319 fusion protein the
16319 polypeptide can correspond to all or a portion of a 16319
protein. In a preferred embodiment, a 16319 fusion protein
comprises at least one biologically active portion of a 16319
protein. In another preferred embodiment, a 16319 fusion protein
comprises at least two biologically active portions of a 16319
protein. Within the fusion protein, the term "operatively linked"
is intended to indicate that the 16319 polypeptide and the
non-16319 polypeptide are fused in-frame to each other. The
non-16319 polypeptide can be fused to the N-terminus or C-terminus
of the 16319 polypeptide.
[1396] For example, in one embodiment, the fusion protein is a
GST-16319 fusion protein in which the 16319 sequences are fused to
the C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant 16319.
[1397] In another embodiment, this fusion protein is a 16319
protein containing a heterologous signal sequence at its
N-terminus. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of 16319 can be increased through use
of a heterologous signal sequence.
[1398] The 16319 fusion proteins used in the methods of the
invention can be incorporated into pharmaceutical compositions and
administered to a subject in vivo. The 16319 fusion proteins can be
used to affect the bioavailability of a 16319 substrate. Use of
16319 fusion proteins may be useful therapeutically for the
treatment of disorders caused by, for example, (i) aberrant
modification or mutation of a gene encoding a 16319 protein; (ii)
mis-regulation of the 16319 gene; and (iii) aberrant
post-translational modification of a 16319 protein.
[1399] Moreover, the 16319-fusion proteins used in the methods of
the invention can be used as immunogens to produce anti-16319
antibodies in a subject, to purify 16319 ligands and in screening
assays to identify molecules which inhibit the interaction of 16319
with a 16319 substrate.
[1400] Preferably, a 16319 chimeric or fusion protein used in the
methods of the invention is produced by standard recombinant DNA
techniques. For example, DNA fragments coding for the different
polypeptide sequences are ligated together in-frame in accordance
with conventional techniques, for example by employing blunt-ended
or stagger-ended termini for ligation, restriction enzyme digestion
to provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A 16319-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the 16319 protein.
[1401] The present invention also pertains to the use of variants
of the 16319 proteins which function as either 16319 agonists
(mimetics) or as 16319 antagonists. Variants of the 16319 proteins
can be generated by mutagenesis, e.g., discrete point mutation or
truncation of a 16319 protein. An agonist of the 16319 proteins can
retain substantially the same, or a subset, of the biological
activities of the naturally occurring form of a 16319 protein. An
antagonist of a 16319 protein can inhibit one or more of the
activities of the naturally occurring form of the 16319 protein by,
for example, competitively modulating a 16319-mediated activity of
a 16319 protein. Thus, specific biological effects can be elicited
by treatment with a variant of limited function. In one embodiment,
treatment of a subject with a variant having a subset of the
biological activities of the naturally occurring form of the
protein has fewer side effects in a subject relative to treatment
with the naturally occurring form of the 16319 protein.
[1402] In one embodiment, variants of a 16319 protein which
function as either 16319 agonists (mimetics) or as 16319
antagonists can be identified by screening combinatorial libraries
of mutants, e.g., truncation mutants, of a 16319 protein for 16319
protein agonist or antagonist activity. In one embodiment, a
variegated library of 16319 variants is generated by combinatorial
mutagenesis at the nucleic acid level and is encoded by a
variegated gene library. A variegated library of 16319 variants can
be produced by, for example, enzymatically ligating a mixture of
synthetic oligonucleotides into gene sequences such that a
degenerate set of potential 16319 sequences is expressible as
individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display) containing the set of
16319 sequences therein. There are a variety of methods which can
be used to produce libraries of potential 16319 variants from a
degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an
appropriate expression vector. Use of a degenerate set of genes
allows for the provision, in one mixture, of all of the sequences
encoding the desired set of potential 16319 sequences. Methods for
synthesizing degenerate oligonucleotides are known in the art (see,
e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984)
Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056;
Ike et al. (1983) Nucleic Acid Res. 11:477).
[1403] In addition, libraries of fragments of a 16319 protein
coding sequence can be used to generate a variegated population of
16319 fragments for screening and subsequent selection of variants
of a 16319 protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of a 16319 coding sequence with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form
double stranded DNA which can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. By this
method, an expression library can be derived which encodes
N-terminal, C-terminal and internal fragments of various sizes of
the 16319 protein.
[1404] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of 16319 proteins. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recursive ensemble mutagenesis
(REM), a new technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify 16319 variants (Arkin and Yourvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.
(1993) Protein Engineering 6(3):327-331).
[1405] The methods of the present invention further include the use
of anti-16319 antibodies. An isolated 16319 protein, or a portion
or fragment thereof, can be used as an immunogen to generate
antibodies that bind 16319 using standard techniques for polyclonal
and monoclonal antibody preparation. A full-length 16319 protein
can be used or, alternatively, antigenic peptide fragments of 16319
can be used as immunogens. The antigenic peptide of 16319 comprises
at least 8 amino acid residues of the amino acid sequence shown in
SEQ ID NO:13 and encompasses an epitope of 16319 such that an
antibody raised against the peptide forms a specific immune complex
with the 16319 protein. Preferably, the antigenic peptide comprises
at least 10 amino acid residues, more preferably at least 15 amino
acid residues, even more preferably at least 20 amino acid
residues, and most preferably at least 30 amino acid residues.
[1406] Preferred epitopes encompassed by the antigenic peptide are
regions of 16319 that are located on the surface of the protein,
e.g., hydrophilic regions, as well as regions with high
antigenicity.
[1407] A 16319 immunogen is typically used to prepare antibodies by
immunizing a suitable subject, (e.g., rabbit, goat, mouse, or other
mammal) with the immunogen. An appropriate immunogenic preparation
can contain, for example, recombinantly expressed 16319 protein or
a chemically synthesized 16319 polypeptide. The preparation can
further include an adjuvant, such as Freund's complete or
incomplete adjuvant, or similar immunostimulatory agent.
Immunization of a suitable subject with an immunogenic 16319
preparation induces a polyclonal anti-16319 antibody response.
[1408] The term "antibody" as used herein refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
which specifically binds (immunoreacts with) an antigen, such as a
16319. Examples of immunologically active portions of
immunoglobulin molecules include F(ab) and F(ab').sub.2 fragments
which can be generated by treating the antibody with an enzyme such
as pepsin. The invention provides polyclonal and monoclonal
antibodies that bind 16319 molecules. The term "monoclonal
antibody" or "monoclonal antibody composition", as used herein,
refers to a population of antibody molecules that contain only one
species of an antigen binding site capable of immunoreacting with a
particular epitope of 16319. A monoclonal antibody composition thus
typically displays a single binding affinity for a particular 16319
protein with which it immunoreacts.
[1409] Polyclonal anti-16319 antibodies can be prepared as
described above by immunizing a suitable subject with a 16319
immunogen. The anti-16319 antibody titer in the immunized subject
can be monitored over time by standard techniques, such as with an
enzyme linked immunosorbent assay (ELISA) using immobilized 16319.
If desired, the antibody molecules directed against 16319 can be
isolated from the mammal (e.g., from the blood) and further
purified by well known techniques, such as protein A chromatography
to obtain the IgG fraction. At an appropriate time after
immunization, e.g., when the anti-16319 antibody titers are
highest, antibody-producing cells can be obtained from the subject
and used to prepare monoclonal antibodies by standard techniques,
such as the hybridoma technique originally described by Kohler and
Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981)
J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.
255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA
76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the
more recent human B cell hybridoma technique (Kozbor et al. (1983)
Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.
(1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
monoclonal antibody hybridomas is well known (see generally
Kenneth, R. H. in Monoclonal Antibodies: A New Dimension In
Biological Analyses, Plenum Publishing Corp., New York, N.Y.
(1980); Lerner, E. A. (1981) Yale J. Biol. Med. 54:387-402; Gefter,
M. L. et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an
immortal cell line (typically a myeloma) is fused to lymphocytes
(typically splenocytes) from a mammal immunized with a 16319
immunogen as described above, and the culture supernatants of the
resulting hybridoma cells are screened to identify a hybridoma
producing a monoclonal antibody that binds 16319.
[1410] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-16319 monoclonal antibody (see, e.g.,
G. Galfre et al. (1977) Nature 266:55052; Gefter et al. (1977)
supra; Lerner (1981) supra; and Kenneth (1980) supra). Moreover,
the ordinarily skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines can be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from ATCC. Typically, HAT-sensitive mouse myeloma cells
are fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using
HAT medium, which kills unfused and unproductively fused myeloma
cells (unfused splenocytes die after several days because they are
not transformed). Hybridoma cells producing a monoclonal antibody
of the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind 16319, e.g., using a standard
ELISA assay.
[1411] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-16319 antibody can be identified and
isolated by screening a recombinant combinatorial immunoglobulin
library (e.g., an antibody phage display library) with 16319 to
thereby isolate immunoglobulin library members that bind 16319.
Kits for generating and screening phage display libraries are
commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the Stratagene
SurfZAP.TM. Phage Display Kit, Catalog No. 240612). Additionally,
examples of methods and reagents particularly amenable for use in
generating and screening antibody display library can be found in,
for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT
International Publication No. WO 92/18619; Dower et al. PCT
International Publication No. WO 91/17271; Winter et al. PCT
International Publication WO 92/20791; Markland et al. PCT
International Publication No. WO 92/15679; Breitling et al. PCT
International Publication WO 93/01288; McCafferty et al. PCT
International Publication No. WO 92/01047; Garrard et al. PCT
International Publication No. WO 92/09690; Ladner et al. PCT
International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J.
Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628;
Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad
et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991)
Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad.
Sci. USA 88:7978-7982; and McCafferty et al. (1990) Nature
348:552-554.
[1412] Additionally, recombinant anti-16319 antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the methods of
the invention. Such chimeric and humanized monoclonal antibodies
can be produced by recombinant DNA techniques known in the art, for
example using methods described in Robinson et al. International
Application No. PCT/US86/02269; Akira, et al. European Patent
Application 184,187; Taniguchi, M., European Patent Application
171,496; Morrison et al. European Patent Application 173,494;
Neuberger et al. PCT International Publication No. WO 86/01533;
Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European
Patent Application 125,023; Better et al. (1988) Science
240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA
84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et
al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.
(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature
314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559;
Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986)
BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.
(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science
239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.
[1413] An anti-16319 antibody can be used to detect 16319 protein
(e.g., in a cellular lysate or cell supernatant) in order to
evaluate the abundance and pattern of expression of the 16319
protein. Anti-16319 antibodies can be used diagnostically to
monitor protein levels in tissue as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling (i.e.,
physically linking) the antibody to a detectable substance.
Examples of detectable substances include various enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, and radioactive materials. Examples of
suitable enzymes include horseradish peroxidase, alkaline
phosphatase, .quadrature.-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin, and examples
of suitable radioactive material include .sup.125I, .sup.131I,
.sup.35S or .sup.3H.
Electronic Apparatus Readable Media and Arrays
[1414] Electronic apparatus readable media comprising a 16319
modulator of the present invention is also provided. As used
herein, "electronic apparatus readable media" refers to any
suitable medium for storing, holding or containing data or
information that can be read and accessed directly by an electronic
apparatus. Such media can include, but are not limited to: magnetic
storage media, such as floppy discs, hard disc storage medium, and
magnetic tape; optical storage media such as compact disc;
electronic storage media such as RAM, ROM, EPROM, EEPROM and the
like; general hard disks and hybrids of these categories such as
magnetic/optical storage media. The medium is adapted or configured
for having recorded thereon a marker of the present invention.
[1415] As used herein, the term "electronic apparatus" is intended
to include any suitable computing or processing apparatus or other
device configured or adapted for storing data or information.
Examples of electronic apparatus suitable for use with the present
invention include stand-alone computing apparatus; networks,
including a local area network (LAN), a wide area network (WAN)
Internet, Intranet, and Extranet; electronic appliances such as a
personal digital assistants (PDAs), cellular phone, pager and the
like; and local and distributed processing systems.
[1416] As used herein, "recorded" refers to a process for storing
or encoding information on the electronic apparatus readable
medium. Those skilled in the art can readily adopt any of the
presently known methods for recording information on known media to
generate manufactures comprising the 16319 modulators of the
present invention.
[1417] A variety of software programs and formats can be used to
store the marker information of the present invention on the
electronic apparatus readable medium. For example, the nucleic acid
sequence corresponding to the 16319 modulators can be represented
in a word processing text file, formatted in commercially-available
software such as WordPerfect and MicroSoft Word, or represented in
the form of an ASCII file, stored in a database application, such
as DB2, Sybase, Oracle, or the like, as well as in other forms. Any
number of data processor structuring formats (e.g., text file or
database) may be employed in order to obtain or create a medium
having recorded thereon the 16319 modulators of the present
invention.
[1418] By providing the 16319 modulators of the invention in
readable form, one can routinely access the marker sequence
information for a variety of purposes. For example, one skilled in
the art can use the nucleotide or amino acid sequences of the
present invention in readable form to compare a target sequence or
target structural motif with the sequence information stored within
the data storage means. Search means are used to identify fragments
or regions of the sequences of the invention which match a
particular target sequence or target motif.
[1419] The present invention therefore provides a medium for
holding instructions for performing a method for determining
whether a subject has a hematological disorder or a pre-disposition
to a hematological disorder, wherein the method comprises the steps
of determining the presence or absence of a 16319 modulator and
based on the presence or absence of the 16319 modulator,
determining whether the subject has a hematological disorder or a
pre-disposition to a hematological disorder and/or recommending a
particular treatment for the hematological disorder or
pre-hematological disorder condition.
[1420] The present invention further provides in an electronic
system and/or in a network, a method for determining whether a
subject has a hematological disorder or a pre-disposition to a
hematological disorder associated with a 16319 modulator wherein
the method comprises the steps of determining the presence or
absence of the 16319 modulator, and based on the presence or
absence of the 16319 modulator, determining whether the subject has
a hematological disorder or a pre-disposition to a hematological
disorder, and/or recommending a particular treatment for the
hematological disorder or pre-hematological disorder condition. The
method may further comprise the step of receiving phenotypic
information associated with the subject and/or acquiring from a
network phenotypic information associated with the subject.
[1421] The present invention also provides in a network, a method
for determining whether a subject has a hematological disorder or a
pre-disposition to a hematological disorder associated with a 16319
modulator, said method comprising the steps of receiving
information associated with the 16319 modulator receiving
phenotypic information associated with the subject, acquiring
information from the network corresponding to the 16319 modulator
and/or hematological disorder, and based on one or more of the
phenotypic information, the 16319 modulator, and the acquired
information, determining whether the subject has a hematological
disorder or a pre-disposition to a hematological disorder. The
method may further comprise the step of recommending a particular
treatment for the hematological disorder or pre-hematological
disorder condition.
[1422] The present invention also provides a business method for
determining whether a subject has a hematological disorder or a
pre-disposition to a hematological disorder, said method comprising
the steps of receiving information associated with the 16319
modulator, receiving phenotypic information associated with the
subject, acquiring information from the network corresponding to
the 16319 modulator and/or hematological disorder, and based on one
or more of the phenotypic information, the 16319 modulator, and the
acquired information, determining whether the subject has a
hematological disorder or a pre-disposition to a hematological
disorder. The method may further comprise the step of recommending
a particular treatment for the hematological disorder or
pre-hematological disorder condition.
[1423] The invention also includes an array comprising a 16319
modulator of the present invention. The array can be used to assay
expression of one or more genes in the array. In one embodiment,
the array can be used to assay gene expression in a tissue to
ascertain tissue specificity of genes in the array. In this manner,
up to about 7600 genes can be simultaneously assayed for
expression. This allows a profile to be developed showing a battery
of genes specifically expressed in one or more tissues.
[1424] In addition to such qualitative determination, the invention
allows the quantitation of gene expression. Thus, not only tissue
specificity, but also the level of expression of a battery of genes
in the tissue is ascertainable. Thus, genes can be grouped on the
basis of their tissue expression per se and level of expression in
that tissue. This is useful, for example, in ascertaining the
relationship of gene expression between or among tissues. Thus, one
tissue can be perturbed and the effect on gene expression in a
second tissue can be determined. In this context, the effect of one
cell type on another cell type in response to a biological stimulus
can be determined. Such a determination is useful, for example, to
know the effect of cell-cell interaction at the level of gene
expression. If an agent is administered therapeutically to treat
one cell type but has an undesirable effect on another cell type,
the invention provides an assay to determine the molecular basis of
the undesirable effect and thus provides the opportunity to
co-administer a counteracting agent or otherwise treat the
undesired effect. Similarly, even within a single cell type,
undesirable biological effects can be determined at the molecular
level. Thus, the effects of an agent on expression of other than
the target gene can be ascertained and counteracted.
[1425] In another embodiment, the array can be used to monitor the
time course of expression of one or more genes in the array. This
can occur in various biological contexts, as disclosed herein, for
example development of hematological disorder, progression of
hematological disorder, and processes, such a cellular
transformation associated with hematological disorder.
[1426] The array is also useful for ascertaining the effect of the
expression of a gene on the expression of other genes in the same
cell or in different cells. This provides, for example, for a
selection of alternate molecular targets for therapeutic
intervention if the ultimate or downstream target cannot be
regulated.
[1427] The array is also useful for ascertaining differential
expression patterns of one or more genes in normal and abnormal
cells. This provides a battery of genes that could serve as a
molecular target for diagnosis or therapeutic intervention.
[1428] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the Sequence Listing is
incorporated herein by reference.
EXAMPLES
Example 1
Identification of 16319 as a Modulator of Hematological
Disorders
[1429] In order to determine whether the 16319 molecules of the
present invention are involved in hematological disorders, 16319
gene expression during various points of hematopoietic cell
differentiation of different hematopoetic cell lineages (e.g.,
erythroid, myeloid and megakaryocyte lineages) was measured.
Materials and Methods
[1430] For analysis of human and murine 16319 expression in
hematopoietic cells and tissue, the following methods were
used:
[1431] Tissues were collected from 7 week old female C57/Bl6J mice.
Total RNA was prepared using the trizol method and treated with
DNAse to remove contaminating genomic DNA. cDNA was synthesized
using standard techniques. Mock cDNA synthesis in the absence of
reverse transcriptase resulted in samples with no detectable PCR
amplification of the control RNA gene confirming efficient removal
of genomic DNA contamination. 16319 expression was measured by
TaqMan.RTM. quantitative PCR analysis, performed according to the
manufacturer's directions (Perkin Elmer Applied Biosystems, Foster
City, Calif.).
[1432] The samples included the following normal cells and tissues:
lung, heart, spleen, kidney, liver, fetal liver, brain, colon,
muscle, bone marrow, cord blood, GPA hi, GPA lo, stromal, CD14 (B
cells), CD11b+, erythrocytes, BFU, mast cells, megakaryocytes,
neutrophils, CD3 (T cells), peripheral blood, K562 (leukemia
cells), HL60 (human peripheral blood leukemia promyelocytic cells),
MF11, MF12, HUVEC (human endothelial cells), HCEAC, platelets pool,
erythrleukemia, premyelocytic leukemia, thrombosis and virology
samples.
[1433] PCR probes were designed by PrimerExpress software (PE
Biosystems) based on the sequence of human 16319 (SEQ ID
NO:12).
[1434] To standardize the results between different tissues, two
probes, distinguished by different fluorescent labels, were added
to each sample. The differential labeling of the probe for the
16319 gene and the probe for control RNA as an internal control
thus enabled their simultaneous measurement in the same well.
Forward and reverse primers and the probes for both control RNA and
human 16319 were added to the TaqMan Universal PCR Master Mix (PE
Applied Biosystems). Although the final concentration of primer and
probe could vary, each was internally consistent within a given
experiment. A typical experiment contained 200 nM of forward and
reverse primers, plus 100 nM of the probe for the control RNA, and
4500 nM of each of the forward and reverse primers, plus 150 nM of
the probe for 16319. TaqMan matrix experiments were carried out
using an ABI PRISM 770 Sequence Detection System (PE Applied
Biosystems). The thermal cycler conditions were as follows: hold
for 2 minutes at 50.degree. C. and 10 minutes at 95.degree. C.,
followed by two-step PCR for 40 cycles of 95.degree. C. for 15
seconds, followed by 60.degree. C. for 1 minute.
[1435] The following method was used to quantitatively calculate
16319 gene expression in the tissue samples, relative to the
control RNA expression in the same tissue. The threshold values at
which the PCR amplification started were determined using the
manufacturer's software. PCR cycle number at threshold value was
designated as CT. Relative expression was calculated as:
2.sup.-((CTtest-CT18S)tissue of interest-(CTtest-CT18S)lowest
expressing tissue in panel).
[1436] Samples were run in duplicate and the averages of 2 relative
expression determinations are shown. All probes were tested on
serial dilutions of RNA from a tissue with high expression levels
and only probes which gave relative expression levels that were
linear to the amount of template cDNA with a slope similar to the
slope for the internal control 18S were used.
[1437] For Northern Blotting, human mRNA blots (Clontech) were
probed with a 520 nucleotide SacI fragment containing 420
nucleotides of the 5' coding sequence and 100 nucleotides of the 5'
UTR of human 16319. Probes were labeled with .sup.32P and
hybridized using the Rapid-Hyb buffer (Amersham).
Results
[1438] The expression of 16319 was examined during various points
of hematopoietic cell differentiation of different hematopoetic
cell lineages (e.g., erythroid, myeloid and megakaryocyte lineages)
using Taqman analysis. The results indicate that 16319 was most
highly expressed in CD34+ progenitor cells, and this high level of
expression was maintained in erythroid and Glycophorin A positive
cells. 16319 was also highly expressed in fetal liver cells. To
verify expression of 16319, Northern Blotting was performed using
commercially available Clontech Blots.
[1439] The results described above demonstrate that 16319 is
expressed in hematopoietic cells during different stages of
differentiation. Thus, 16319 is an important gene which is
expressed in early progenitor and committed cells and ultimately
plays a determinative role in hematopoietic cell proliferation.
VI. METHODS AND COMPOSITIONS FOR THE DIAGNOSIS AND TREATMENT OF
VIRAL DISEASE USING 55092
Background of the Invention
[1440] Phospholipases are involved in the signal transduction
pathway in which a cell response such as proliferation or secretion
is produced in response to an extracellular stimulus. The
interaction of extracellular signals (e.g., hormones, growth
factors, cytokines, neurotransmitters, and physical stimuli) with
cell surface receptors (e.g., G protein-coupled receptors and
receptor tyrosine kinases) often activates a phospholipase D
(PLD)-mediated signal transduction pathway that is important in the
regulation of cell function and cell fate. Phospholipase D
catalyzes the hydrolysis of phosphatidylcholine and other
phospholipids yielding phosphatidic acid and is, thus, able to
modify various lipid constituents of the plasma membrane and
generate intracellular messengers that act to recruit and/or
modulate specific target proteins. For example, addition of short
chain analogues of phosphatidic acid to intact cells has been shown
to regulate membrane transport, e.g., secretion of viral
glycoproteins and matrix metalloproteinase proteins (Bi, K et al.
(1997) Curr. Biol. 7:301-7; Williger, B T et al. (1999) J. Biol.
Chem. 74:735-8). Moreover, phosphatidic acid is further metabolized
to form diacylglycerol, a potent activator of protein kinase C, and
lysophosphatidic acid (Exton, J H (2000) Ann. NY Acad. Sci.
905:61-8; Ktistakis N T et al. (1999) Biochem. Soc. Trans.
27:634-637). PLD is also able to catalyze a transesterification
reaction (transphosphatidylation) utilizing short-chain primary
alcohols as phosphatidyl group acceptors and producing
phosphatidylalcohols. PLD activity is regulated by factors such as
small GTP binding proteins of the ADP-ribosylation factor (ARF) and
Rho families, and protein kinase C. PLD activities have been
identified in multiple cellular membranes including the nuclear
envelope, endoplasmic reticulum, Golgi apparatus,
transport/secretory vesicles, and the plasma membrane (Ktistakis N
T et al. (1999) Biochem. Soc. Trans. 27:634-637). Different PLD
isoforms are localized in distinct cellular organelles, and serve
diverse functions in signal transduction, membrane homeostasis,
membrane vesicle trafficking and cytoskeletal dynamics (Singer W D
et al. (1997) Ann. Rev. Biochem. 66:475-509; Exton, J H (2000) Ann.
NY Acad. Sci. 905:61-8).
[1441] The phospholipase D gene superfamily, as defined by
structural domains and sequence motifs, includes PLDs,
phosphatidyltransferases, phospholipid synthases,
phosphodiesterases, endonucleases, and viral envelope proteins
(Cao, J-X et al. (1997) Virus Research 48:11-18; Pedersen K M et
al. (1998) J. Biol. Chem. 273:31494-31504; Barcena J (2000) J. Gen.
Virol. 81:1073-1085; Liscovitch, M et al. (2000) Biochem. J.
345:401-415). PLD superfamily members share conserved motifs,
including the HKD motif (HXKX.sub.4D) (SEQ ID NO:19) which has been
implicated in catalytic activity (Ponting C P et al. (1996) Protein
Science 5:914-922; Koonin, E V (1996) TIBS 21:242-243; Sung T-C et
al. (1997) EMBO J. 16:4519-4530).
[1442] Vaccinia virus produces two different infectious forms,
intracellular mature virus (IMV) which are infectious when released
by cell lysis, and extracellular enveloped virus (EEV) which is
important in long-distance spread of infectious virus in vitro and
in vivo. Acquisition of the EEV envelope occurs by the wrapping of
IMV with vesicles derived from the trans-Golgi network. Two genes
encoding proteins with homology to PLD are present in vaccinia
virus and other poxviruses. The K4 protein contains two HKD motifs
and adjacent conserved sequences, and P37 contains a partially
conserved motif (Sung T-C et al. (1997) EMBO J. 16:4519-4530). P37,
a 37 kDa palmitylated protein encoded by the F13L gene, is the
major protein in the external envelope of EEV, and within infected
cells is localized in the Golgi region associated with vesicles
which form double-walled envelopes around IMV. P37 has been shown
to play an important role in the viral envelopment process and
subsequent release of enveloped virus (Borrego B et al. (1999) J.
Gen. Virol. 80:425-432). Viral mutants lacking P37 are severely
compromised, as trans-Golgi envelopment does not occur, thus,
blocking viral particle egress and cell-cell virus transmission
(Blasco R and Moss B (1991) J. Virol. 65:5910-5920; Blasco R and
Moss B (1995) Gene 158:157-162). Similarly, mutation of the P37 HKD
motif results in viruses that are unable to produce EEV and which
fail to mediate low-pH-induced fusion of infected cells (Roper R L
and Moss B (1999) J. Virol. 73:1108-1117).
[1443] Viruses are ubiquitous pathogens capable of producing
primary, latent, and recurrent infections which contribute to a
variety of clinical illnesses. Viruses may cause infected cells to
produce specific proteins that interact with each other and with
cellular proteins and viral nucleic acids to cause viral progeny to
be made, to destroy the infected cell, and to spread infection.
Thus, there is a vital need for antiviral drug development and
rapid diagnostic methods in order to achieve efficient management
strategies for viral infections.
Summary of the Invention
[1444] The present invention provides methods and compositions for
the diagnosis and treatment of viral disease, including but not
limited to, herpes simplex virus, hepatitis B virus, and hepatitis
C virus infection. The present invention is based, at least in
part, on the discovery that the PLD 55092 gene is differentially
expressed in cells infected with herpes simplex virus, hepatitis B
virus, and hepatitis C virus relative to their expression in
non-infected cells.
[1445] In one aspect, the invention provides a method for
identifying the presence of a nucleic acid molecule associated with
a viral disease, in a sample by contacting a sample comprising
nucleic acid molecules with a hybridization probe comprising at
least 25 contiguous nucleotides of SEQ ID NO:14, and detecting the
presence of a nucleic acid molecule associated with a viral
disease, when the sample contains a nucleic acid molecule that
hybridizes to the nucleic acid probe. In one embodiment, the
hybridization probe is detectably labeled. In another embodiment
the sample comprising nucleic acid molecules is subjected to
agarose gel electrophoresis and southern blotting prior to
contacting with the hybridization probe. In a further embodiment,
the sample comprising nucleic acid molecules is subjected to
agarose gel electrophoresis and northern blotting prior to
contacting with the hybridization probe. In yet another embodiment,
the detecting is by in situ hybridization. In other embodiments,
the method is used to detect mRNA or genomic DNA in the sample.
[1446] The invention also provides a method for identifying a
nucleic acid associated with a viral disease, in a sample, by
contacting a sample comprising nucleic acid molecules with a first
and a second amplification primer, the first primer comprising at
least 25 contiguous nucleotides of SEQ ID NO:14 and the second
primer comprising at least 25 contiguous nucleotides from the
complement of SEQ ID NO:14, incubating the sample under conditions
that allow for nucleic acid amplification, and detecting the
presence of a nucleic acid molecule associated with a viral
disease, when the sample contains a nucleic acid molecule that is
amplified. In one embodiment, the sample comprising nucleic acid
molecules is subjected to agarose gel electrophoresis after the
incubation step.
[1447] In addition, the invention provides a method for identifying
a polypeptide associated with a viral disease, in a sample by
contacting a sample comprising polypeptide molecules with a binding
substance specific for a PLD 55092 polypeptide, and detecting the
presence of a polypeptide associated with a viral disease, when the
sample contains a polypeptide molecule that binds to the binding
substance. In one embodiment the binding substance is an antibody.
In another embodiment, the binding substance is detectably
labeled.
[1448] In another aspect, the invention provides a method of
identifying a subject having or at risk for developing a viral
disease, by contacting a sample obtained from the subject
comprising nucleic acid molecules with a hybridization probe
comprising at least 25 contiguous nucleotides of SEQ ID NO:14, and
detecting the presence of a nucleic acid molecule when the sample
contains a nucleic acid molecule that hybridizes to the nucleic
acid probe, thereby identifying a subject having or at risk for
developing a viral disease.
[1449] In a further aspect, the invention provides a method for
identifying a subject having or at risk for developing a viral
disease, by contacting a sample obtained from a subject comprising
nucleic acid molecules with a first and a second amplification
primer, the first primer comprising at least 25 contiguous
nucleotides of SEQ ID NO:14 and the second primer comprising at
least 25 contiguous nucleotides from the complement of SEQ ID
NO:14, incubating the sample under conditions that allow for
nucleic acid amplification, and detecting a nucleic acid molecule
when the sample contains a nucleic acid molecule that is amplified,
thereby identifying a subject having or at risk for developing a
viral disease.
[1450] In yet another aspect, the invention provides a method of
identifying a subject having or at risk for developing a viral
disease, by contacting a sample obtained from the subject
comprising polypeptide molecules with a binding substance specific
for a PLD 55092 polypeptide by detecting the presence of a
polypeptide molecule in the sample that binds to the binding
substance, thereby identifying a subject having or at risk for
developing a viral disease.
[1451] In another aspect, the invention provides a method for
identifying a compound capable of treating a viral disease,
characterized by aberrant PLD 55092 nucleic acid expression or PLD
55092 protein activity, by assaying the ability of the compound to
modulate the expression of a PLD 55092 nucleic acid or the activity
of a PLD 55092 protein. In one embodiment, the disease is a disease
associated with herpes simplex virus infection. In another
embodiment, the disease is a disease associated with hepatitis B
virus infection. In yet another embodiment, the disease is a
disease associated with hepatitis C virus infection. In a further
embodiment, the ability of the compound to modulate the activity of
the PLD 55092 protein is determined by detecting the induction of
an intracellular second messenger, e.g., phosphatidic acid.
[1452] In yet another aspect, the invention provides a method for
treating a subject having a viral disease characterized by aberrant
PLD 55092 protein activity or aberrant PLD 55092 nucleic acid
expression by administering to the subject a PLD 55092 modulator.
The PLD 55092 modulator may be administered in a pharmaceutically
acceptable formulation, or using a gene therapy vector.
[1453] In one embodiment, a PLD 55092 modulator is capable of
modulating PLD 55092 polypeptide activity. For example, the PLD
55092 modulator may be a small molecule; an anti-PLD 55092
antibody; a PLD 55092 polypeptide comprising the amino acid
sequence of SEQ ID NO:15, or a fragment thereof; a PLD 55092
polypeptide comprising an amino acid sequence which is at least 90
percent identical to the amino acid sequence of SEQ ID NO:15,
wherein the percent identity is calculated using the ALIGN program
for comparing amino acid sequences, a PAM120 weight residue table,
a gap length penalty of 12, and a gap penalty of 4; or an isolated
naturally occurring allelic variant of a polypeptide consisting of
the amino acid sequence of SEQ ID NO:15, wherein the polypeptide is
encoded by a nucleic acid molecule which hybridizes to a complement
of a nucleic acid molecule consisting of SEQ ID NO:14 at
4.times.SSC at 65-70.degree. C. followed by one or more washes in
1.times.SSC, at 65-70.degree. C.
[1454] In another embodiment, the PLD 55092 modulator is capable of
modulating PLD 55092 nucleic acid expression. For example, the PLD
55092 modulator may be a small molecule; an antisense PLD 55092
nucleic acid molecule; a ribozyme; a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:14, or a fragment
thereof; a nucleic acid molecule encoding a polypeptide comprising
an amino acid sequence which is at least 90 percent identical to
the amino acid sequence of SEQ ID NO:15, wherein the percent
identity is calculated using the ALIGN program for comparing amino
acid sequences, a PAM120 weight residue table, a gap length penalty
of 12, and a gap penalty of 4; or a nucleic acid molecule encoding
a naturally occurring allelic variant of a polypeptide comprising
the amino acid sequence of SEQ ID NO:15, wherein the nucleic acid
molecule which hybridizes to a complement of a nucleic acid
molecule consisting of SEQ ID NO:14 at 4.times.SSC at 65-70.degree.
C. followed by one or more washes in 1.times.SSC, at 65-70.degree.
C.
[1455] In another aspect, the invention provides a method for
identifying a compound capable of modulating a virus activity,
e.g., virus replication, virus envelopment, extracellular virion
formation and/or cell-cell virus transmission. The method includes
contacting a virus or a virus infected cell with a test compound
and assaying the ability of the test compound to modulate the
expression of a PLD 55092 nucleic acid or the activity of a PLD
55092 protein.
[1456] Furthermore, the invention provides a method for modulating
a virus activity by contacting a virus or a virus infected cell
with a PLD 55092 modulator.
[1457] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
Detailed Description of the Invention
[1458] The present invention provides methods and compositions for
the diagnosis and treatment of viral disease, including but not
limited to herpes simplex virus infection, hepatitis B virus
infection and hepatitis C virus infection, and the clinical
sequelae associated with viral infection. The present invention is
based, at least in part, on the discovery that phospholipase D
(PLD) superfamily genes, referred to herein as "phospholipase D
55092" or "PLD 55092" nucleic acid and protein molecules, are
differentially expressed in viral disease states, e.g., viral
infection, relative to their expression in normal, or non-viral
disease states.
[1459] Without intending to be limited by mechanism, it is believed
that the PLD 55092 molecules of the present invention are involved
in signal transduction and membrane biogenesis events regulating
viral vesicular secretion and viral membrane biogenesis. The PLD
55092 molecules of the present invention may also mediate signal
transduction events necessary for viral replication. Moreover,
since the PLD 55092 molecules of the present invention are mostly
neuron specific (see Examples infra) it is believed that PLD 55092
function may regulate viral transport and/or secretion in neurons
and other infected cell types.
[1460] "Differential expression", as used herein, includes both
quantitative as well as qualitative differences in the temporal
and/or tissue expression pattern of a gene. Thus, a differentially
expressed gene may have its expression activated or inactivated in
normal versus viral disease conditions (for example, in virally
infected cells and/or tissues). The degree to which expression
differs in normal versus viral disease or control versus
experimental states need only be large enough to be visualized via
standard characterization techniques, e.g., quantitative PCR,
Northern analysis, subtractive hybridization. The expression
pattern of a differentially expressed gene may be used as part of a
prognostic or diagnostic viral disease evaluation, or may be used
in methods for identifying compounds useful for the treatment of
viral disease. In addition, a differentially expressed gene
involved in viral disease may represent a target gene such that
modulation of the level of target gene expression or of target gene
product activity may act to ameliorate a viral disease condition.
Compounds that modulate target gene expression or activity of the
target gene product can be used in the treatment of viral
disease.
[1461] Viral diseases include, but are not limited to, infection
with herpes simplex virus (type 1 and type 2), varicella zoster
virus, poliomyelitis virus, cytomegalovirus, influenza virus (A and
B), respiratory syncytial virus, coxsackie virus, ebola virus,
hantavirus, human papilloma virus, rotavirus, west nile virus,
Epstein-Barr virus, human immunodeficiency virus, and hepatitis
virus (A, B and C). The clinical sequelae of viral infection
include herpes, AIDS, lassa fever, kaposi's sarcoma, meningitis,
mumps, polio, chicken pox, colds and flu, dengue fever,
encephalitis, Fifth disease, shingles, genital warts, rubella,
yellow fever, hepatitis A, B and C, measles, rabies, and
smallpox.
[1462] Although the PLD 55092 genes described herein may be
differentially expressed with respect to viral disease, and/or
their products may interact with gene products important to viral
disease, the genes may also be involved in mechanisms important to
additional viral and cellular regulatory processes, e.g., lipid
metabolism, membrane homeostasis, vesicular trafficking and signal
transduction.
[1463] Accordingly, the PLD 55092 molecules of the present
invention may be involved in processes that modulate virus
activity. As used herein, a "virus activity" or "virus function"
includes virus replication, assembly, maturation, envelopment,
extracellular virus formation, virus egress, and virus
transmission.
[1464] The PLD 55092 molecules of the present invention may also
mediate signal transduction events involved in oncogenesis and/or
generation of pain signals. Thus, the PLD molecules of the present
invention may also act as novel diagnostic targets and therapeutic
agents for proliferative disorders, e.g., cancer, or pain
disorders.
[1465] The present invention provides methods for identifying the
presence of a PLD 55092 nucleic acid or polypeptide molecule
associated with viral disease. In addition, the invention provides
methods for identifying a subject having or at risk for developing
a viral disease, by detecting the presence of a PLD 55092 nucleic
acid or polypeptide molecule, within the subject or a sample, e.g.,
a tissue sample, obtained from the subject.
[1466] The invention also provides a method for identifying a
compound capable of treating a viral disease, characterized by
aberrant PLD 55092 nucleic acid expression or PLD 55092 protein
activity by assaying the ability of the compound to modulate the
expression of a PLD 55092 nucleic acid or the activity of a PLD
55092 protein. Furthermore, the invention provides a method for
treating a subject having a viral disease characterized by aberrant
PLD 55092 protein activity or aberrant PLD 55092 nucleic acid
expression by administering to the subject a PLD 55092 modulator
which is capable of modulating PLD 55092 protein activity or PLD
55092 nucleic acid expression.
[1467] Moreover, the invention provides a method for identifying a
compound capable of modulating a virus activity by modulating the
expression of a PLD 55092 nucleic acid or the activity of a PLD
55092 protein. The invention further provides a method for
modulating a virus activity by contacting a virus with a PLD 55092
modulator.
[1468] Various aspects of the invention are described in further
detail in the following subsections.
Screening Assays
[1469] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules (organic or inorganic) or other drugs) which bind to PLD
55092 proteins, have a stimulatory or inhibitory effect on, for
example, PLD 55092 expression or PLD 55092 activity, or have a
stimulatory or inhibitory effect on, for example, the expression or
activity of a PLD 55092 substrate.
[1470] These assays are designed to identify compounds that bind to
a PLD 55092 protein, bind to other intracellular or extracellular
proteins that interact with a PLD 55092 protein, and interfere with
the interaction of the PLD 55092 protein with other cellular or
extracellular proteins. For example, in the case of the PLD 55092
protein, which is a phospholipase D type protein, such techniques
can identify substrates and/or effectors for such a protein. A PLD
55092 protein substrate and/or effector can, for example, be used
to ameliorate viral diseases. Such compounds may include, but are
not limited to peptides, antibodies, or small organic or inorganic
compounds. Such compounds may also include other cellular
proteins.
[1471] Compounds identified via assays such as those described
herein may be useful, for example, for ameliorating viral disease.
In instances whereby a viral disease or condition associated with
viral infection results from an overall lower level of PLD 55092
gene expression and/or PLD 55092 protein in a cell or tissue,
compounds that interact with the PLD 55092 protein may include
compounds which accentuate or amplify the activity of the bound PLD
55092 protein. Such compounds would bring about an effective
increase in the level of PLD 55092 protein activity, thus,
ameliorating symptoms.
[1472] In other instances, mutations within the PLD 55092 gene may
cause aberrant types or excessive amounts of PLD 55092 proteins to
be made which have a deleterious effect that leads to a viral
disease. Similarly, physiological conditions may cause an excessive
increase in PLD 55092 gene expression leading to a viral disease.
In such cases, compounds that bind to a PLD 55092 protein may be
identified that inhibit the activity of the PLD 55092 protein.
Assays for testing the effectiveness of compounds identified by
techniques such as those described in this section are discussed
herein.
[1473] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a PLD
55092 protein or polypeptide or biologically active portion
thereof. In another embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of a PLD 55092 protein or polypeptide or biologically
active portion thereof. The test compounds of the present invention
can be obtained using any of the numerous approaches in
combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the `one-bead one-compound` library method; and
synthetic library methods using affinity chromatography selection.
The biological library approach is limited to peptide libraries,
while the other four approaches are applicable to peptide,
non-peptide oligomer or small molecule libraries of compounds (Lam,
K. S. (1997) Anticancer Drug Des. 12:145).
[1474] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med.
Chem. 37:1233.
[1475] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra.).
[1476] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a PLD 55092 protein or biologically active
portion thereof is contacted with a test compound and the ability
of the test compound to modulate PLD 55092 activity is determined.
Determining the ability of the test compound to modulate PLD 55092
activity can be accomplished by monitoring, for example,
intracellular phosphatidic acid, PIP.sub.2, diacylglycerol, or
phosphatidylalcohol concentration, cell proliferation and/or
migration, vesicle transport, or the activity of a PLD
55092-regulated transcription factor. The cell can be of mammalian
origin, e.g., a neuronal cell. In one embodiment, the cell is a
virally infected cell, and the ability of the test compound to
modulate PLD 55092 activity can be accomplished by monitoring
plaque formation and/or low pH fusion of infected cells. In another
embodiment, compounds that interact with a PLD 55092 protein can be
screened for their ability to function as substrates and/or
effectors, i.e., to bind to the PLD 55092 protein and modulate a
PLD 55092-mediated signal transduction pathway. Identification of
PLD 55092 substrates and/or effectors, and measuring the activity
of the substrate-protein and/or effector-protein complex, leads to
the identification of modulators (e.g., antagonists) of this
interaction. Such modulators may be useful in the treatment of
viral disease.
[1477] The ability of the test compound to modulate PLD 55092
binding to a substrate or to bind to PLD 55092 can also be
determined. Determining the ability of the test compound to
modulate PLD 55092 binding to a substrate can be accomplished, for
example, by coupling the PLD 55092 substrate with a radioisotope or
enzymatic label such that binding of the PLD 55092 substrate to PLD
55092 can be determined by detecting the labeled PLD 55092
substrate in a complex. PLD 55092 could also be coupled with a
radioisotope or enzymatic label to monitor the ability of a test
compound to modulate PLD 55092 binding to a PLD 55092 substrate in
a complex. Determining the ability of the test compound to bind PLD
55092 can be accomplished, for example, by coupling the compound
with a radioisotope or enzymatic label such that binding of the
compound to PLD 55092 can be determined by detecting the labeled
PLD 55092 compound in a complex. For example, compounds (e.g., PLD
55092 ligands or substrates) can be labeled with .sup.125I,
.sup.35S, .sup.14C, or .sup.3H, either directly or indirectly, and
the radioisotope detected by direct counting of radioemmission or
by scintillation counting. Compounds can further be enzymatically
labeled with, for example, horseradish peroxidase, alkaline
phosphatase, or luciferase, and the enzymatic label detected by
determination of conversion of an appropriate substrate to
product.
[1478] It is also within the scope of this invention to determine
the ability of a compound (e.g., a PLD 55092 ligand or substrate)
to interact with PLD 55092 without the labeling of any of the
interactants. For example, a microphysiometer can be used to detect
the interaction of a compound with PLD 55092 without the labeling
of either the compound or the PLD 55092 (McConnell, H. M. et al.
(1992) Science 257:1906-1912. As used herein, a "microphysiometer"
(e.g., Cytosensor) is an analytical instrument that measures the
rate at which a cell acidifies its environment using a
light-addressable potentiometric sensor (LAPS). Changes in this
acidification rate can be used as an indicator of the interaction
between a compound and PLD 55092.
[1479] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a PLD 55092 target molecule
(e.g., a PLD 55092 substrate) with a test compound and determining
the ability of the test compound to modulate (e.g., stimulate or
inhibit) the activity of the PLD 55092 target molecule. Determining
the ability of the test compound to modulate the activity of a PLD
55092 target molecule can be accomplished, for example, by
determining the ability of the PLD 55092 protein to bind to or
interact with the PLD 55092 target molecule.
[1480] Determining the ability of the PLD 55092 protein or a
biologically active fragment thereof, to bind to or interact with a
PLD 55092 target molecule can be accomplished by one of the methods
described above for determining direct binding. In a preferred
embodiment, determining the ability of the PLD 55092 protein to
bind to or interact with a PLD 55092 target molecule can be
accomplished by determining the activity of the target molecule.
For example, the activity of the target molecule can be determined
by detecting induction of a cellular second messenger of the target
(i.e., intracellular phosphatidic acid, diacylglycerol, PIP.sub.2),
detecting catalytic/enzymatic activity of the target on an
appropriate substrate, detecting the induction of a reporter gene
(comprising a target-responsive regulatory element operatively
linked to a nucleic acid encoding a detectable marker, e.g.,
luciferase), or detecting a target-regulated cellular response
(e.g., gene expression, cell proliferation or migration).
[1481] In yet another embodiment, an assay of the present invention
is a cell-free assay in which a PLD 55092 protein or biologically
active portion thereof, is contacted with a test compound and the
ability of the test compound to bind to the PLD 55092 protein or
biologically active portion thereof is determined. Preferred
biologically active portions of the PLD 55092 proteins to be used
in assays of the present invention include fragments which
participate in interactions with non-PLD 55092 molecules. Binding
of the test compound to the PLD 55092 protein can be determined
either directly or indirectly as described above. In a preferred
embodiment, the assay includes contacting the PLD 55092 protein or
biologically active portion thereof with a known compound which
binds PLD 55092 to form an assay mixture, contacting the assay
mixture with a test compound, and determining the ability of the
test compound to interact with a PLD 55092 protein, wherein
determining the ability of the test compound to interact with a PLD
55092 protein comprises determining the ability of the test
compound to preferentially bind to PLD 55092 or biologically active
portion thereof as compared to the known compound. Compounds that
modulate the interaction of PLD 55092 with a known target protein
may be useful in regulating the activity of a PLD 55092 protein,
especially a mutant PLD 55092 protein.
[1482] In another embodiment, the assay is a cell-free assay in
which a PLD 55092 protein or biologically active portion thereof is
contacted with a test compound and the ability of the test compound
to modulate (e.g., stimulate or inhibit) the activity of the PLD
55092 protein or biologically active portion thereof is determined.
Determining the ability of the test compound to modulate the
activity of a PLD 55092 protein can be accomplished, for example,
by determining the ability of the PLD 55092 protein to bind to a
PLD 55092 target molecule by one of the methods described above for
determining direct binding. Determining the ability of the PLD
55092 protein to bind to a PLD 55092 target molecule can also be
accomplished using a technology such as real-time Biomolecular
Interaction Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991)
Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin.
Struct. Biol. 5:699-705). As used herein, "BIA" is a technology for
studying biospecific interactions in real time, without labeling
any of the interactants (e.g., BIAcore). Changes in the optical
phenomenon of surface plasmon resonance (SPR) can be used as an
indication of real-time reactions between biological molecules.
Determining the ability of the test compound to modulate PLD 55092
activity can also be monitored using an assay for phospholipase D
activity, e.g., cleavage of a substrate,
transphosphatidylation.
[1483] In another embodiment, determining the ability of the test
compound to modulate the activity of a PLD 55092 protein can be
accomplished by determining the ability of the PLD 55092 protein to
further modulate the activity of a downstream effector of a PLD
55092 target molecule. For example, the activity of the effector
molecule on an appropriate target can be determined or the binding
of the effector to an appropriate target can be determined as
previously described.
[1484] In yet another embodiment, the cell-free assay involves
contacting a PLD 55092 protein or biologically active portion
thereof with a known compound which binds the PLD 55092 protein to
form an assay mixture, contacting the assay mixture with a test
compound, and determining the ability of the test compound to
interact with the PLD 55092 protein, wherein determining the
ability of the test compound to interact with the PLD 55092 protein
comprises determining the ability of the PLD 55092 protein to
preferentially bind to or modulate the activity of a PLD 55092
target molecule.
[1485] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either PLD
55092 or its target molecule to facilitate separation of complexed
from uncomplexed forms of one or both of the proteins, as well as
to accommodate automation of the assay. Binding of a test compound
to a PLD 55092 protein, or interaction of a PLD 55092 protein with
a target molecule in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtitre plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/PLD 55092 fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or PLD 55092 protein, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtitre plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of PLD 55092 binding or activity
determined using standard techniques.
[1486] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either a PLD 55092 protein or a PLD 55092 target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated PLD 55092 protein or target molecules can be prepared
from biotin-NHS (N-hydroxy-succinimide) using techniques known in
the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,
Ill.), and immobilized in the wells of streptavidin-coated 96 well
plates (Pierce Chemical). Alternatively, antibodies reactive with
PLD 55092 protein or target molecules but which do not interfere
with binding of the PLD 55092 protein to its target molecule can be
derivatized to the wells of the plate, and unbound target or PLD
55092 protein trapped in the wells by antibody conjugation. Methods
for detecting such complexes, in addition to those described above
for the GST-immobilized complexes, include immunodetection of
complexes using antibodies reactive with the PLD 55092 protein or
target molecule, as well as enzyme-linked assays which rely on
detecting an enzymatic activity associated with the PLD 55092
protein or target molecule.
[1487] In another embodiment, modulators of PLD 55092 expression
are identified in a method wherein a cell is contacted with a
candidate compound and the expression of PLD 55092 mRNA or protein
in the cell is determined. The level of expression of PLD 55092
mRNA or protein in the presence of the candidate compound is
compared to the level of expression of PLD 55092 mRNA or protein in
the absence of the candidate compound. The candidate compound can
then be identified as a modulator of PLD 55092 expression based on
this comparison. For example, when expression of PLD 55092 mRNA or
protein is greater (statistically significantly greater) in the
presence of the candidate compound than in its absence, the
candidate compound is identified as a stimulator of PLD 55092 mRNA
or protein expression. Alternatively, when expression of PLD 55092
mRNA or protein is less (statistically significantly less) in the
presence of the candidate compound than in its absence, the
candidate compound is identified as an inhibitor of PLD 55092 mRNA
or protein expression. The level of PLD 55092 mRNA or protein
expression in the cells can be determined by methods described
herein for detecting PLD 55092 mRNA or protein.
[1488] In yet another aspect of the invention, the PLD 55092
proteins can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with PLD
55092 ("PLD 55092-binding proteins" or "PLD 55092-bp") and are
involved in PLD 55092 activity. Such PLD 55092-binding proteins are
also likely to be involved in the propagation of signals by the PLD
55092 proteins or PLD 55092 targets as, for example, downstream
elements of a PLD 55092-mediated signaling pathway. Alternatively,
such PLD 55092-binding proteins are likely to be PLD 55092
inhibitors.
[1489] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a PLD 55092
protein, or a fragment thereof, is fused to a gene encoding the DNA
binding domain of a known transcription factor (e.g., GAL-4). In
the other construct, a DNA sequence, from a library of DNA
sequences, that encodes an unidentified protein ("prey" or
"sample") is fused to a gene that codes for the activation domain
of the known transcription factor. If the "bait" and the "prey"
proteins are able to interact, in vivo, forming a PLD
55092-dependent complex, the DNA-binding and activation domains of
the transcription factor are brought into close proximity. This
proximity allows transcription of a reporter gene (e.g., LacZ)
which is operably linked to a transcriptional regulatory site
responsive to the transcription factor. Expression of the reporter
gene can be detected and cell colonies containing the functional
transcription factor can be isolated and used to obtain the cloned
gene which encodes the protein which interacts with the PLD 55092
protein.
[1490] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a PLD 55092 protein can be confirmed in vivo, e.g., in an animal
such as an animal model for viral disease, as described herein.
[1491] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein (e.g., a PLD 55092 modulating
agent, an antisense PLD 55092 nucleic acid molecule, a PLD
55092-specific antibody, or a PLD 55092-binding partner) can be
used in an animal model to determine the efficacy, toxicity, or
side effects of treatment with such an agent. Alternatively, an
agent identified as described herein can be used in an animal model
to determine the mechanism of action of such an agent. Furthermore,
this invention pertains to uses of novel agents identified by the
above-described screening assays for treatments as described
herein.
[1492] Any of the compounds, including but not limited to compounds
such as those identified in the foregoing assay systems, may be
tested for the ability to ameliorate viral disease symptoms and/or
viral infection. Cell-based and animal model-based assays for the
identification of compounds exhibiting such an ability to
ameliorate viral disease systems are described herein.
[1493] In one aspect, cell-based systems, as described herein, may
be used to identify compounds which may act to ameliorate viral
disease symptoms, e.g., viral infection. For example, such cell
systems (e.g., cells infected with virus) may be exposed to a
compound, suspected of exhibiting an ability to ameliorate viral
disease symptoms, at a sufficient concentration and for a time
sufficient to elicit such an amelioration of viral disease symptoms
in the exposed cells. After exposure, the cells are examined to
determine whether one or more of the viral disease cellular
phenotypes has been altered to resemble a more normal or more wild
type, non-viral disease phenotype. Cellular phenotypes that are
associated with viral disease include viral infection (e.g., virus
burden), cell lysis, plaque formation, and low pH induced fusion of
infected cells.
[1494] In addition, animal-based viral disease systems, such as
those described herein, may be used to identify compounds capable
of ameliorating viral 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 viral disease. For example, animal models may
be exposed to a compound, suspected of exhibiting an ability to
ameliorate viral disease symptoms and/or viral infection, at a
sufficient concentration and for a time sufficient to elicit such
an amelioration of viral 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 viral disease,
for example, by monitoring viral burden before and after
treatment.
[1495] With regard to intervention, any treatments which reverse
any aspect of viral disease symptoms should be considered as
candidates for human viral disease therapeutic intervention.
Dosages of test agents may be determined by deriving dose-response
curves.
[1496] Additionally, gene expression patterns may be utilized to
assess the ability of a compound to ameliorate viral disease
symptoms. For example, the expression pattern of one or more genes
may form part of a "gene expression profile" or "transcriptional
profile" which may be then be used in such an assessment. "Gene
expression profile" or "transcriptional profile", as used herein,
includes 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, infection with herpes simplex
virus, hepatitis B virus or hepatitis C virus, including any of the
control or experimental conditions described herein. Gene
expression profiles may be generated, for example, by utilizing a
differential display procedure, Northern analysis and/or RT-PCR. In
one embodiment, PLD 55092 gene sequences may be used as probes
and/or PCR primers for the generation and corroboration of such
gene expression profiles.
[1497] Gene expression profiles may be characterized for known
states, either viral disease or normal, within the cell- and/or
animal-based model systems. Subsequently, these known gene
expression profiles may be compared to ascertain the effect a test
compound has to modify such gene expression profiles, and to cause
the profile to more closely resemble that of a more desirable
profile.
[1498] For example, administration of a compound may cause the gene
expression profile of a viral disease model system to more closely
resemble the control system. Administration of a compound may,
alternatively, cause the gene expression profile of a control
system to begin to mimic a viral 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.
Predictive Medicine
[1499] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to
diagnostic assays for determining PLD 55092 protein and/or nucleic
acid expression as well as PLD 55092 activity, in the context of a
biological sample (e.g., blood, serum, cells, tissue) to thereby
determine whether an individual is afflicted with a disease or
disorder, or is at risk of developing a viral disease, a pain
disorder, or a cellular proliferation, growth, differentiation, or
migration disorder, associated with aberrant or unwanted PLD 55092
expression or activity. The invention also provides for prognostic
(or predictive) assays for determining whether an individual is at
risk of developing a disorder associated with PLD 55092 protein,
nucleic acid expression or activity. For example, mutations in a
PLD 55092 gene can be assayed in a biological sample. Such assays
can be used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with PLD 55092 protein,
nucleic acid expression or activity.
[1500] Another aspect of the invention pertains to monitoring the
influence of agents (e.g., drugs, compounds) on the expression or
activity of PLD 55092 in clinical trials.
[1501] These and other agents are described in further detail in
the following sections.
Diagnostic Assays
[1502] The present invention encompasses methods for diagnostic and
prognostic evaluation of viral disease conditions, and for the
identification of subjects exhibiting a predisposition to such
conditions.
[1503] An exemplary method for detecting the presence or absence of
PLD 55092 protein or nucleic acid in a biological sample involves
obtaining a biological sample from a test subject and contacting
the biological sample with a compound or an agent capable of
detecting PLD 55092 protein or nucleic acid (e.g., mRNA, or genomic
DNA) that encodes PLD 55092 protein such that the presence of PLD
55092 protein or nucleic acid is detected in the biological sample.
A preferred agent for detecting PLD 55092 mRNA or genomic DNA is a
labeled nucleic acid probe capable of hybridizing to PLD 55092 mRNA
or genomic DNA. The nucleic acid probe can be, for example, the PLD
55092 nucleic acid set forth in SEQ ID NO:14, or a portion thereof,
such as an oligonucleotide of at least 15, 20, 25, 30, 35, 40, 45,
50, 100, 250 or 500 nucleotides in length and sufficient to
specifically hybridize under stringent conditions to PLD 55092 mRNA
or genomic DNA. Other suitable probes for use in the diagnostic
assays of the invention are described herein.
[1504] A preferred agent for detecting PLD 55092 protein is an
antibody capable of binding to PLD 55092 protein, preferably an
antibody with a detectable label. Antibodies can be polyclonal, or
more preferably, monoclonal. An intact antibody, or a fragment
thereof (e.g., Fab or F(ab')2) can be used. The term "labeled",
with regard to the probe or antibody, is intended to encompass
direct labeling of the probe or antibody by coupling (i.e.,
physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently labeled streptavidin. The term "biological sample" is
intended to include tissues, cells and biological fluids isolated
from a subject, as well as tissues, cells and fluids present within
a subject. That is, the detection method of the invention can be
used to detect PLD 55092 mRNA, protein, or genomic DNA in a
biological sample in vitro as well as in vivo. For example, in
vitro techniques for detection of PLD 55092 mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detection of PLD 55092 protein include enzyme linked immunosorbent
assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro techniques for detection of PLD 55092
genomic DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of PLD 55092 protein include introducing
into a subject a labeled anti-PLD 55092 antibody. For example, the
antibody can be labeled with a radioactive marker whose presence
and location in a subject can be detected by standard imaging
techniques.
[1505] In one embodiment, the biological sample contains protein
molecules from the test subject. Alternatively, the biological
sample can contain mRNA molecules from the test subject or genomic
DNA molecules from the test subject. A preferred biological sample
is a serum sample isolated by conventional means from a
subject.
[1506] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting PLD
55092 protein, mRNA, or genomic DNA, such that the presence of PLD
55092 protein, mRNA or genomic DNA is detected in the biological
sample, and comparing the presence of PLD 55092 protein, mRNA or
genomic DNA in the control sample with the presence of PLD 55092
protein, mRNA or genomic DNA in the test sample.
[1507] The invention also encompasses kits for detecting the
presence of PLD 55092 in a biological sample. For example, the kit
can comprise a labeled compound or agent capable of detecting PLD
55092 protein or mRNA in a biological sample; means for determining
the amount of PLD 55092 in the sample; and means for comparing the
amount of PLD 55092 in the sample with a standard. The compound or
agent can be packaged in a suitable container. The kit can further
comprise instructions for using the kit to detect PLD 55092 protein
or nucleic acid.
Prognostic Assays
[1508] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
viral disease or disorder associated with aberrant or unwanted PLD
55092 expression or activity. As used herein, the term "aberrant"
includes a PLD 55092 expression or activity which deviates from the
wild type PLD 55092 expression or activity. Aberrant expression or
activity includes increased or decreased expression or activity, as
well as expression or activity which does not follow the wild type
developmental pattern of expression or the subcellular pattern of
expression. For example, aberrant PLD 55092 expression or activity
is intended to include the cases in which a mutation in the PLD
55092 gene causes the PLD 55092 gene to be under-expressed or
over-expressed and situations in which such mutations result in a
non-functional PLD 55092 protein or a protein which does not
function in a wild-type fashion, e.g., a protein which does not
interact with a PLD 55092 ligand or substrate, or one which
interacts with a non-PLD 55092 ligand or substrate. As used herein,
the term "unwanted" includes an unwanted phenomenon involved in a
biological response such as viral replication and dissemination.
For example, the term unwanted includes a PLD 55092 expression
pattern or a PLD 55092 protein activity which is undesirable in a
subject.
[1509] The assays described herein, such as the preceding
diagnostic assays or the following assays, can be utilized to
identify a subject having or at risk of developing a disorder
associated with a misregulation in PLD 55092 protein activity or
nucleic acid expression, such as a viral disease, a pain disorder,
or a cellular proliferation, growth, differentiation, or migration
disorder. Alternatively, the prognostic assays can be utilized to
identify a subject having or at risk for developing a viral
disease, a pain disorder, or a cellular proliferation, growth,
differentiation, or migration disorder, associated with a
misregulation in PLD 55092 protein activity or nucleic acid
expression. Thus, the present invention provides a method for
identifying a disease or disorder associated with aberrant or
unwanted PLD 55092 expression or activity in which a test sample is
obtained from a subject and PLD 55092 protein or nucleic acid
(e.g., mRNA or genomic DNA) is detected, wherein the presence of
PLD 55092 protein or nucleic acid is diagnostic for a subject
having or at risk of developing a disease or disorder associated
with aberrant or unwanted PLD 55092 expression or activity. As used
herein, a "test sample" refers to a biological sample obtained from
a subject of interest. For example, a test sample can be a
biological fluid (e.g., serum), cell sample, or tissue.
[1510] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder associated with aberrant or unwanted PLD 55092
expression or activity. For example, such methods can be used to
determine whether a subject can be effectively treated with an
agent for a viral disease, a pain disorder, or a cellular
proliferation, growth, differentiation, or migration disorder.
Thus, the present invention provides methods for determining
whether a subject can be effectively treated with an agent for a
viral disease, a pain disorder, or a cellular proliferation,
growth, differentiation, or migration disorder associated with
aberrant or unwanted PLD 55092 expression or activity in which a
test sample is obtained and PLD 55092 protein or nucleic acid
expression or activity is detected (e.g., wherein the abundance of
PLD 55092 protein or nucleic acid expression or activity is
diagnostic for a subject that can be administered the agent to
treat a disorder associated with aberrant or unwanted PLD 55092
expression or activity).
[1511] The methods of the invention can also be used to detect
genetic alterations in a PLD 55092 gene, thereby determining if a
subject with the altered gene is at risk for a disorder
characterized by misregulation in PLD 55092 protein activity or
nucleic acid expression, such as a viral disease, a pain disorder,
or a cellular proliferation, growth, differentiation, or migration
disorder. In preferred embodiments, the methods include detecting,
in a sample of cells from the subject, the presence or absence of a
genetic alteration characterized by at least one of an alteration
affecting the integrity of a gene encoding a PLD 55092 protein, or
the mis-expression of the PLD 55092 gene. For example, such genetic
alterations can be detected by ascertaining the existence of at
least one of 1) a deletion of one or more nucleotides from a PLD
55092 gene; 2) an addition of one or more nucleotides to a PLD
55092 gene; 3) a substitution of one or more nucleotides of a PLD
55092 gene, 4) a chromosomal rearrangement of a PLD 55092 gene; 5)
an alteration in the level of a messenger RNA transcript of a PLD
55092 gene, 6) aberrant modification of a PLD 55092 gene, such as
of the methylation pattern of the genomic DNA, 7) the presence of a
non-wild type splicing pattern of a messenger RNA transcript of a
PLD 55092 gene, 8) a non-wild type level of a PLD 55092 protein, 9)
allelic loss of a PLD 55092 gene, and 10) inappropriate
post-translational modification of a PLD 55092 protein. As
described herein, there are a large number of assays known in the
art which can be used for detecting alterations in a PLD 55092
gene. A preferred biological sample is a tissue or serum sample
isolated by conventional means from a subject.
[1512] In certain embodiments, detection of the alteration involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364),
the latter of which can be particularly useful for detecting point
mutations in the PLD 55092 gene (see Abravaya et al. (1995) Nucleic
Acids Res. 23:675-682). This method can include the steps of
collecting a sample of cells from a subject, isolating nucleic acid
(e.g., genomic, mRNA or both) from the cells of the sample,
contacting the nucleic acid sample with one or more primers which
specifically hybridize to a PLD 55092 gene under conditions such
that hybridization and amplification of the PLD 55092 gene (if
present) occurs, and detecting the presence or absence of an
amplification product, or detecting the size of the amplification
product and comparing the length to a control sample. It is
anticipated that PCR and/or LCR may be desirable to use as a
preliminary amplification step in conjunction with any of the
techniques used for detecting mutations described herein.
[1513] Other amplification methods include: 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.
[1514] In an alternative embodiment, mutations in a PLD 55092 gene
from a sample cell can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
for example, U.S. Pat. No. 5,498,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[1515] In other embodiments, genetic mutations in PLD 55092 can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, to high density arrays containing hundreds or thousands
of oligonucleotides probes (Cronin, M. T. et al. (1996) Human
Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2:
753-759). For example, genetic mutations in PLD 55092 can be
identified in two dimensional arrays containing light-generated DNA
probes as described in Cronin, M. T. et al. supra. Briefly, a first
hybridization array of probes can be used to scan through long
stretches of DNA in a sample and control to identify base changes
between the sequences by making linear arrays of sequential
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene.
[1516] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the PLD
55092 gene and detect mutations by comparing the sequence of the
sample PLD 55092 with the corresponding wild-type (control)
sequence. Examples of sequencing reactions include those based on
techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad.
Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA
74:5463). It is also contemplated that any of a variety of
automated sequencing procedures can be utilized when performing the
diagnostic assays ((1995) Biotechniques 19:448), including
sequencing by mass spectrometry (see, e.g., PCT International
Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.
36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.
38:147-159).
[1517] Other methods for detecting mutations in the PLD 55092 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230:1242). In general, the art technique of
"mismatch cleavage" starts by providing heteroduplexes of formed by
hybridizing (labeled) RNA or DNA containing the wild-type PLD 55092
sequence with potentially mutant RNA or DNA obtained from a tissue
sample. The double-stranded duplexes are treated with an agent
which cleaves single-stranded regions of the duplex such as which
will exist due to basepair mismatches between the control and
sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically
digesting the mismatched regions. In other embodiments, either
DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or
osmium tetroxide and with piperidine in order to digest mismatched
regions. After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing polyacrylamide
gels to determine the site of mutation. See, for example, Cotton et
al. (1988) Proc. Nail Acad Sci USA 85:4397; Saleeba et al. (1992)
Methods Enzymol. 217:286-295. In a preferred embodiment, the
control DNA or RNA can be labeled for detection.
[1518] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in PLD
55092 cDNAs obtained from samples of cells. For example, the mutY
enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15:1657-1662). According to an exemplary
embodiment, a probe based on a PLD 55092 sequence, e.g., a
wild-type PLD 55092 sequence, is hybridized to a cDNA or other DNA
product from a test cell(s). The duplex is treated with a DNA
mismatch repair enzyme, and the cleavage products, if any, can be
detected from electrophoresis protocols or the like (described in,
for example, U.S. Pat. No. 5,459,039).
[1519] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in PLD 55092 genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144;
and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79).
Single-stranded DNA fragments of sample and control PLD 55092
nucleic acids will be denatured and allowed to renature. The
secondary structure of single-stranded nucleic acids varies
according to sequence, the resulting alteration in electrophoretic
mobility enables the detection of even a single base change. The
DNA fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al. (1991) Trends Genet 7:5).
[1520] In yet another embodiment the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265:12753).
[1521] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki
et al. (1989) Proc. Natl Acad. Sci USA 86:6230). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA or a number of different mutations when the oligonucleotides
are attached to the hybridizing membrane and hybridized with
labeled target DNA.
[1522] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner (1993) Tibtech 11:238). In
addition it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88:189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[1523] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a PLD 55092 gene.
[1524] Furthermore, any cell type or tissue in which PLD 55092 is
expressed may be utilized in the prognostic assays described
herein.
Monitoring of Effects During Clinical Trials
[1525] The present invention provides methods for evaluating the
efficacy of drugs and monitoring the progress of patients involved
in clinical trials for the treatment of viral disease.
[1526] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of a PLD 55092 protein (e.g., the modulation
of viral replication, assembly, maturation, and/or transmission;
lipid metabolism; vesicle trafficking; or cell proliferation,
differentiation and/or migration) can be applied not only in basic
drug screening, but also in clinical trials. For example, the
effectiveness of an agent determined by a screening assay as
described herein to increase PLD 55092 gene expression, protein
levels, or upregulate PLD 55092 activity, can be monitored in
clinical trials of subjects exhibiting decreased PLD 55092 gene
expression, protein levels, or downregulated PLD 55092 activity.
Alternatively, the effectiveness of an agent determined by a
screening assay to decrease PLD 55092 gene expression, protein
levels, or downregulate PLD 55092 activity, can be monitored in
clinical trials of subjects exhibiting increased PLD 55092 gene
expression, protein levels, or upregulated PLD 55092 activity. In
such clinical trials, the expression or activity of a PLD 55092
gene, and preferably, other genes that have been implicated in, for
example, a PLD 55092-associated disorder can be used as a "read
out" or markers of the phenotype a particular cell, e.g., a
neuronal cell. In addition, the expression of a PLD 55092 gene, or
the level of PLD 55092 protein activity may be used as a read out
of a particular drug or agent's effect on a viral disease
state.
[1527] For example, and not by way of limitation, genes, including
PLD 55092, that are modulated in cells by treatment with an agent
(e.g., compound, drug or small molecule) which modulates PLD 55092
activity (e.g., identified in a screening assay as described
herein) can be identified. Thus, to study the effect of agents on
PLD 55092-associated disorders (e.g., viral disease, pain
disorders, or cellular proliferation, growth, differentiation, or
migration disorders), for example, in a clinical trial, cells can
be isolated and RNA prepared and analyzed for the levels of
expression of PLD 55092 and other genes implicated in the PLD
55092-associated disorder, respectively. The levels of gene
expression (e.g., a gene expression pattern) can be quantified by
northern blot analysis or RT-PCR, as described herein, or
alternatively by measuring the amount of protein produced, by one
of the methods as described herein, or by measuring the levels of
activity of PLD 55092 or other genes. In this way, the gene
expression pattern can serve as a marker, indicative of the
physiological response of the cells to the agent. Accordingly, this
response state may be determined before, and at various points
during treatment of the individual with the agent.
[1528] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
including the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of a PLD 55092 protein, mRNA, or genomic
DNA in the preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the PLD 55092 protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the PLD 55092 protein, mRNA, or
genomic DNA in the pre-administration sample with the PLD 55092
protein, mRNA, or genomic DNA in the post administration sample or
samples; and (vi) altering the administration of the agent to the
subject accordingly. For example, increased administration of the
agent may be desirable to increase the expression or activity of
PLD 55092 to higher levels than detected, i.e., to increase the
effectiveness of the agent. Alternatively, decreased administration
of the agent may be desirable to decrease expression or activity of
PLD 55092 to lower levels than detected, i.e. to decrease the
effectiveness of the agent. According to such an embodiment, PLD
55092 expression or activity may be used as an indicator of the
effectiveness of an agent, even in the absence of an observable
phenotypic response.
Methods of Treatment
[1529] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted PLD 55092 expression or activity, e.g. a viral
disease, a pain disorder, or a cellular proliferation, growth,
differentiation, or migration disorder. With regards to both
prophylactic and therapeutic methods of treatment, such treatments
may be specifically tailored or modified, based on knowledge
obtained from the field of pharmacogenomics. "Pharmacogenomics", as
used herein, refers to the application of genomics technologies
such as gene sequencing, statistical genetics, and gene expression
analysis to drugs in clinical development and on the market. More
specifically, the term refers the study of how a patient's genes
determine his or her response to a drug (e.g., a patient's "drug
response phenotype", or "drug response genotype".) Thus, another
aspect of the invention provides methods for tailoring an
individual's prophylactic or therapeutic treatment with either the
PLD 55092 molecules of the present invention or PLD 55092
modulators according to that individual's drug response genotype.
Pharmacogenomics allows a clinician or physician to target
prophylactic or therapeutic treatments to patients who will most
benefit from the treatment and to avoid treatment of patients who
will experience toxic drug-related side effects.
[1530] Treatment is defined as the application or administration of
a therapeutic agent to a patient, or the application or
administration of a therapeutic agent to an isolated tissue or cell
line from a patient, who has a disease, a symptom of disease or a
predisposition toward a disease, with the purpose of curing,
healing, alleviating, relieving, altering, remedying, ameliorating,
improving or affecting the disease, the symptoms of disease or the
predisposition toward disease as described herein.
[1531] A therapeutic agent includes, but is not limited to, small
molecules, peptides, antibodies, ribozymes and antisense
oligonucleotides.
Prophylactic Methods
[1532] In one aspect, the invention provides a method for
preventing in a subject, a viral disease, a pain disorder, or a
cellular proliferation, growth, differentiation, or migration
disorder associated with an aberrant or unwanted PLD 55092
expression or activity, by administering to the subject a PLD 55092
or an agent which modulates PLD 55092 expression or at least one
PLD 55092 activity. Subjects at risk for a viral disease, a pain
disorder, or a cellular proliferation, growth, differentiation, or
migration disorder which is caused or contributed to by aberrant or
unwanted PLD 55092 expression or activity can be identified by, for
example, any or a combination of diagnostic or prognostic assays as
described herein. Administration of a prophylactic agent can occur
prior to the manifestation of symptoms characteristic of the PLD
55092 aberrancy, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on the type of
PLD 55092 aberrancy, for example, a PLD 55092, PLD 55092 agonist or
PLD 55092 antagonist agent can be used for treating the subject.
The appropriate agent can be determined based on screening assays
described herein.
Therapeutic Methods
[1533] Described herein are methods and compositions whereby viral
disease symptoms may be ameliorated. Certain viral diseases are
brought about, at least in part, by an excessive level of a 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 viral disease symptoms. Techniques for the
reduction of gene expression levels or the activity of a protein
are discussed below.
[1534] Alternatively, certain other viral 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 protein's
activity. As such, an increase in the level of gene expression
and/or the activity of such proteins would bring about the
amelioration of viral disease symptoms.
[1535] 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 gene's
expression, or the activity of the gene product, will reinforce the
protective effect it exerts. Some viral 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 viral disease symptoms. Techniques for increasing
target gene expression levels or target gene product activity
levels are discussed herein.
[1536] Accordingly, another aspect of the invention pertains to
methods of modulating PLD 55092 expression or activity for
therapeutic purposes. Accordingly, in an exemplary embodiment, the
modulatory method of the invention involves contacting a cell with
a PLD 55092 or agent that modulates one or more of the activities
of PLD 55092 protein activity associated with the cell. An agent
that modulates PLD 55092 protein activity can be an agent as
described herein, such as a nucleic acid or a protein, a
naturally-occurring target molecule of a PLD 55092 protein (e.g., a
PLD 55092 ligand or substrate), a PLD 55092 antibody, a PLD 55092
agonist or antagonist, a peptidomimetic of a PLD 55092 agonist or
antagonist, or other small molecule. In one embodiment, the agent
stimulates one or more PLD 55092 activities. Examples of such
stimulatory agents include active PLD 55092 protein and a nucleic
acid molecule encoding PLD 55092 that has been introduced into the
cell. In another embodiment, the agent inhibits one or more PLD
55092 activities. Examples of such inhibitory agents include
antisense PLD 55092 nucleic acid molecules, anti-PLD 55092
antibodies, and PLD 55092 inhibitors. These modulatory methods can
be performed in vitro (e.g., by culturing the cell with the agent)
or, alternatively, in vivo (e.g., by administering the agent to a
subject). As such, the present invention provides methods of
treating an individual afflicted with a disease or disorder
characterized by aberrant or unwanted expression or activity of a
PLD 55092 protein or nucleic acid molecule. In one embodiment, the
method involves administering an agent (e.g., an agent identified
by a screening assay described herein), or combination of agents
that modulates (e.g., upregulates or downregulates) PLD 55092
expression or activity. In another embodiment, the method involves
administering a PLD 55092 protein or nucleic acid molecule as
therapy to compensate for reduced, aberrant, or unwanted PLD 55092
expression or activity.
[1537] Stimulation of PLD 55092 activity is desirable in situations
in which PLD 55092 is abnormally downregulated and/or in which
increased PLD 55092 activity is likely to have a beneficial effect.
Likewise, inhibition of PLD 55092 activity is desirable in
situations in which PLD 55092 is abnormally upregulated and/or in
which decreased PLD 55092 activity is likely to have a beneficial
effect.
[1538] Methods for Inhibiting Target Gene Expression, Synthesis, or
Activity
[1539] As discussed above, genes involved in viral disease, pain
disorders, or cellular proliferation, growth, differentiation, or
migration disorders may cause such disorders via an increased level
of gene activity. In some cases, such up-regulation may have a
causative or exacerbating effect on the disease state. A variety of
techniques may be used to inhibit the expression, synthesis, or
activity of such genes and/or proteins.
[1540] For example, compounds such as those identified through
assays described above, which exhibit inhibitory activity, may be
used in accordance with the invention to ameliorate viral disease
symptoms. Such molecules may include, but are not limited to, small
organic molecules, peptides, antibodies, and the like.
[1541] For example, compounds can be administered that compete with
endogenous substrate and/or ligand for the PLD 55092 protein. The
resulting reduction in the amount of substrate-bound or
ligand-bound PLD 55092 protein will modulate virus and/or 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 biologically active domains,
or portions and/or analogs thereof, of the PLD 55092 protein,
including, for example, soluble fusion proteins such as Ig-tailed
fusion proteins. (For a discussion of the production of Ig-tailed
fusion proteins, see, for example, U.S. Pat. No. 5,116,964).
Alternatively, compounds, such as ligand analogs or antibodies,
that bind to the PLD 55092 catalytic site, but do not activate the
protein, (e.g., antagonists) can be effective in inhibiting PLD
55092 protein activity.
[1542] Further, antisense and ribozyme molecules which inhibit
expression of the PLD 55092 gene may also be used in accordance
with the invention to inhibit aberrant PLD 55092 gene activity.
Still further, triple helix molecules may be utilized in inhibiting
aberrant PLD 55092 gene activity.
[1543] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a PLD 55092 protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of
a route of administration of antisense nucleic acid molecules of
the invention include direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to
target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be
modified such that they specifically bind to receptors or antigens
expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies which
bind to cell surface receptors or antigens. The antisense nucleic
acid molecules can also be delivered to cells using the vectors
described herein. To achieve sufficient intracellular
concentrations of the antisense molecules, vector constructs in
which the antisense nucleic acid molecule is placed under the
control of a strong pol II or pol III promoter are preferred.
[1544] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[1545] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can
be used to catalytically cleave PLD 55092 mRNA transcripts to
thereby inhibit translation of PLD 55092 mRNA. A ribozyme having
specificity for a PLD 55092-encoding nucleic acid can be designed
based upon the nucleotide sequence of a PLD 55092 cDNA disclosed
herein (i.e., SEQ ID NO:14). For example, a derivative of a
Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide
sequence of the active site is complementary to the nucleotide
sequence to be cleaved in a PLD 55092-encoding mRNA (see, for
example, Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S.
Pat. No. 5,116,742). Alternatively, PLD 55092 mRNA can be used to
select a catalytic RNA having a specific ribonuclease activity from
a pool of RNA molecules (see, for example, Bartel, D. and Szostak,
J. W. (1993) Science 261:1411-1418).
[1546] PLD 55092 gene expression can also be inhibited by targeting
nucleotide sequences complementary to the regulatory region of the
PLD 55092 (e.g., the PLD 55092 promoter and/or enhancers) to form
triple helical structures that prevent transcription of the PLD
55092 gene in target cells (see, for example, 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).
[1547] Antibodies that are both specific for the PLD 55092 protein
and interfere with its activity may also be used to modulate or
inhibit PLD 55092 protein function. Such antibodies may be
generated using standard techniques described herein, against the
PLD 55092 protein itself or against peptides corresponding to
portions of the protein. Such antibodies include but are not
limited to polyclonal, monoclonal, Fab fragments, single chain
antibodies, or chimeric antibodies.
[1548] In instances where the target gene protein is intracellular
and whole antibodies are used, internalizing antibodies may be
preferred. Lipofectin liposomes may be used to deliver the antibody
or a fragment of the Fab region which binds to the target 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 (described in,
for example, Creighton (1983), supra; and Sambrook et al. (1989)
supra). 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. (1993) Proc.
Natl. Acad. Sci. USA 90:7889-7893).
[1549] In some instances, the target gene protein is extracellular,
or is a transmembrane protein. Antibodies that are specific for one
or more extracellular domains of the protein, for example, and that
interfere with its activity, are particularly useful in treating
disease. Such antibodies are especially efficient because they can
access the target domains directly from the bloodstream. Any of the
administration techniques described below which are appropriate for
peptide administration may be utilized to effectively administer
inhibitory target gene antibodies to their site of action.
Methods for Restoring or Enhancing Target Gene Activity
[1550] Genes that cause viral disease, a pain disorder, or a
cellular proliferation, growth, differentiation, or migration
disorder may be underexpressed within disease situations.
Alternatively, the activity of the protein products of such genes
may be decreased, leading to the development of symptoms of viral
disease, a pain disorder, or a cellular proliferation, growth,
differentiation, or migration disorder. Such down-regulation of
gene expression or decrease of protein activity might have a
causative or exacerbating effect on the disease state.
[1551] In some cases, genes that are up-regulated in the disease
state might be exerting a protective effect. A variety of
techniques may be used to increase the expression, synthesis, or
activity of genes and/or proteins that exert a protective effect in
response to viral disease conditions.
[1552] Described in this section are methods whereby the level PLD
55092 activity may be increased to levels wherein viral disease
symptoms are ameliorated. The level of PLD 55092 activity may be
increased, for example, by either increasing the level of PLD 55092
gene expression or by increasing the level of active PLD 55092
protein which is present.
[1553] For example, a PLD 55092 protein, at a level sufficient to
ameliorate viral disease symptoms may be administered to a patient
exhibiting such symptoms. Any of the techniques discussed below may
be used for such administration. One of skill in the art will
readily know how to determine the concentration of effective,
non-toxic doses of the PLD 55092 protein, utilizing techniques such
as those described below.
[1554] Additionally, RNA sequences encoding a PLD 55092 protein may
be directly administered to a patient exhibiting viral disease
symptoms, at a concentration sufficient to produce a level of PLD
55092 protein such that viral disease symptoms are ameliorated. Any
of the techniques discussed below, which achieve intracellular
administration of compounds, such as, for example, liposome
administration, may be used for the administration of such RNA
molecules. The RNA molecules may be produced, for example, by
recombinant techniques such as those described herein.
[1555] Further, subjects may be treated by gene replacement
therapy. One or more copies of a PLD 55092 gene, or a portion
thereof, that directs the production of a normal PLD 55092 protein
with PLD 55092 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 used for the
introduction of PLD 55092 gene sequences into human cells.
[1556] Cells, preferably, autologous cells, containing PLD 55092
expressing gene sequences may then be introduced or reintroduced
into the subject at positions which allow for the amelioration of
viral disease symptoms. Such cell replacement techniques may be
preferred, for example, when the gene product is a secreted,
extracellular gene product.
Pharmacogenomics
[1557] The PLD 55092 molecules of the present invention, as well as
agents, or modulators which have a stimulatory or inhibitory effect
on PLD 55092 activity (e.g., PLD 55092 gene expression) as
identified by a screening assay described herein can be
administered to individuals to treat (prophylactically or
therapeutically) PLD 55092-associated disorders (e.g., a viral
disease, a pain disorder, or a cellular proliferation, growth,
differentiation, or migration disorder) associated with aberrant or
unwanted PLD 55092 activity. In conjunction with such treatment,
pharmacogenomics (i.e., the study of the relationship between an
individual's genotype and that individual's response to a foreign
compound or drug) may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug. Thus, a physician or clinician may
consider applying knowledge obtained in relevant pharmacogenomics
studies in determining whether to administer a PLD 55092 molecule
or a PLD 55092 modulator as well as tailoring the dosage and/or
therapeutic regimen of treatment with a PLD 55092 molecule or PLD
55092 modulator.
[1558] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin.
Chem. 43(2):254-266. In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body (altered
drug action) or genetic conditions transmitted as single factors
altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare genetic
defects or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[1559] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[1560] Alternatively, a method termed the "candidate gene
approach", can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drugs
target is known (e.g., a PLD 55092 protein of the present
invention), all common variants of that gene can be fairly easily
identified in the population and it can be determined if having one
version of the gene versus another is associated with a particular
drug response.
[1561] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[1562] Alternatively, a method termed the "gene expression
profiling", can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a PLD 55092 molecule or PLD 55092 modulator of the
present invention) can give an indication whether gene pathways
related to toxicity have been turned on.
[1563] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment an individual. This knowledge, when applied to dosing or
drug selection, can avoid adverse reactions or therapeutic failure
and thus enhance therapeutic or prophylactic efficiency when
treating a subject with a PLD 55092 molecule or PLD 55092
modulator, such as a modulator identified by one of the exemplary
screening assays described herein.
Use of PLD 55092 Molecules as Surrogate Markers
[1564] The PLD 55092 molecules of the invention are also useful as
markers of disorders or disease states, as markers for precursors
of disease states, as markers for predisposition of disease states,
as markers of drug activity, or as markers of the pharmacogenomic
profile of a subject. Using the methods described herein, the
presence, absence and/or quantity of the PLD 55092 molecules of the
invention may be detected, and may be correlated with one or more
biological states in vivo. For example, the PLD 55092 molecules of
the invention may serve as surrogate markers for one or more
disorders or disease states or for conditions leading up to disease
states.
[1565] As used herein, a "surrogate marker" is an objective
biochemical marker which correlates with the absence or presence of
a disease or disorder, or with the progression of a disease or
disorder (e.g., with the presence or absence of a tumor). The
presence or quantity of such markers is independent of the
causation of the disease. Therefore, these markers may serve to
indicate whether a particular course of treatment is effective in
lessening a disease state or disorder. Surrogate markers are of
particular use when the presence or extent of a disease state or
disorder is difficult to assess through standard methodologies
(e.g., early stage tumors), or when an assessment of disease
progression is desired before a potentially dangerous clinical
endpoint is reached (e.g., an assessment of cardiovascular disease
may be made using cholesterol levels as a surrogate marker, and an
analysis of HIV infection may be made using HIV RNA levels as a
surrogate marker, well in advance of the undesirable clinical
outcomes of myocardial infarction or fully-developed AIDS).
Examples of the use of surrogate markers in the art include: Koomen
et al. (2000) J. Mass. Spectrom. 35:258-264; and James (1994) AIDS
Treatment News Archive 209.
[1566] The PLD 55092 molecules of the invention are also useful as
pharmacodynamic markers. As used herein, a "pharmacodynamic marker"
is an objective biochemical marker which correlates specifically
with drug effects. The presence or quantity of a pharmacodynamic
marker is not related to the disease state or disorder for which
the drug is being administered; therefore, the presence or quantity
of the marker is indicative of the presence or activity of the drug
in a subject. For example, a pharmacodynamic marker may be
indicative of the concentration of the drug in a biological tissue,
in that the marker is either expressed or transcribed or not
expressed or transcribed in that tissue in relationship to the
level of the drug. In this fashion, the distribution or uptake of
the drug may be monitored by the pharmacodynamic marker. Similarly,
the presence or quantity of the pharmacodynamic marker may be
related to the presence or quantity of the metabolic product of a
drug, such that the presence or quantity of the marker is
indicative of the relative breakdown rate of the drug in vivo.
Pharmacodynamic markers are of particular use in increasing the
sensitivity of detection of drug effects, particularly when the
drug is administered in low doses. Since even a small amount of a
drug may be sufficient to activate multiple rounds of marker (e.g.,
a PLD 55092 marker) transcription or expression, the amplified
marker may be in a quantity which is more readily detectable than
the drug itself. Also, the marker may be more easily detected due
to the nature of the marker itself; for example, using the methods
described herein, anti-PLD 55092 antibodies may be employed in an
immune-based detection system for a PLD 55092 protein marker, or
PLD 55092-specific radiolabeled probes may be used to detect a PLD
55092 mRNA marker. Furthermore, the use of a pharmacodynamic marker
may offer mechanism-based prediction of risk due to drug treatment
beyond the range of possible direct observations. Examples of the
use of pharmacodynamic markers in the art include: Matsuda et al.
U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect.
90:229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl.
3:S21-S24; and Nicolau (1999) Am. J. Health-Syst. Pharm. 56 Suppl.
3:S16-S20.
[1567] The PLD 55092 molecules of the invention are also useful as
pharmacogenomic markers. As used herein, a "pharmacogenomic marker"
is an objective biochemical marker which correlates with a specific
clinical drug response or susceptibility in a subject (see, e.g.,
McLeod et al. (1999) Eur. J. Cancer 35(12):1650-1652). The presence
or quantity of the pharmacogenomic marker is related to the
predicted response of the subject to a specific drug or class of
drugs prior to administration of the drug. By assessing the
presence or quantity of one or more pharmacogenomic markers in a
subject, a drug therapy which is most appropriate for the subject,
or which is predicted to have a greater degree of success, may be
selected. For example, based on the presence or quantity of RNA, or
protein (e.g., PLD 55092 protein or RNA) for specific tumor markers
in a subject, a drug or course of treatment may be selected that is
optimized for the treatment of the specific tumor likely to be
present in the subject. Similarly, the presence or absence of a
specific sequence mutation in PLD 55092 DNA may correlate PLD 55092
drug response. The use of pharmacogenomic markers therefore permits
the application of the most appropriate treatment for each subject
without having to administer the therapy.
Detection Assays
[1568] Portions or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. For example, these
sequences can be used to: (i) map their respective genes on a
chromosome; and, thus, locate gene regions associated with genetic
disease; (ii) identify an individual from a minute biological
sample (tissue typing); and (iii) aid in forensic identification of
a biological sample. These applications are described in the
subsections below.
Chromosome Mapping
[1569] Once the sequence (or a portion of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. This process is called chromosome
mapping. Accordingly, portions or fragments of the PLD 55092
nucleotide sequences, described herein, can be used to map the
location of the PLD 55092 genes on a chromosome. The mapping of the
PLD 55092 sequences to chromosomes is an important first step in
correlating these sequences with genes associated with disease.
[1570] Briefly, PLD 55092 genes can be mapped to chromosomes by
preparing PCR primers (preferably 15-25 bp in length) from the PLD
55092 nucleotide sequences. Computer analysis of the PLD 55092
sequences can be used to predict primers that do not span more than
one exon in the genomic DNA, thus complicating the amplification
process. These primers can then be used for PCR screening of
somatic cell hybrids containing individual human chromosomes. Only
those hybrids containing the human gene corresponding to the PLD
55092 sequences will yield an amplified fragment.
[1571] Somatic cell hybrids are prepared by fusing somatic cells
from different mammals (e.g., human and mouse cells). As hybrids of
human and mouse cells grow and divide, they gradually lose human
chromosomes in random order, but retain the mouse chromosomes. By
using media in which mouse cells cannot grow, because they lack a
particular enzyme, but human cells can, the one human chromosome
that contains the gene encoding the needed enzyme, will be
retained. By using various media, panels of hybrid cell lines can
be established. Each cell line in a panel contains either a single
human chromosome or a small number of human chromosomes, and a full
set of mouse chromosomes, allowing easy mapping of individual genes
to specific human chromosomes. (D'Eustachio P. et al. (1983)
Science 220:919-924). Somatic cell hybrids containing only
fragments of human chromosomes can also be produced by using human
chromosomes with translocations and deletions.
[1572] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the PLD 55092 nucleotide sequences to design
oligonucleotide primers, sublocalization can be achieved with
panels of fragments from specific chromosomes. Other mapping
strategies which can similarly be used to map a PLD 55092 sequence
to its chromosome include in situ hybridization (described in Fan,
Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),
pre-screening with labeled flow-sorted chromosomes, and
pre-selection by hybridization to chromosome specific cDNA
libraries.
[1573] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. Chromosome spreads can be
made using cells whose division has been blocked in metaphase by a
chemical such as colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then stained
with Giemsa. A pattern of light and dark bands develops on each
chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500
or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location with
sufficient signal intensity for simple detection. Preferably 1,000
bases, and more preferably 2,000 bases will suffice to get good
results at a reasonable amount of time. For a review of this
technique, see Verma et al., Human Chromosomes: A Manual of Basic
Techniques (Pergamon Press, New York 1988).
[1574] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[1575] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between a gene and a disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland, J. et al. (1987) Nature, 325:783-787.
[1576] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the PLD 55092 gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
Tissue Typing
[1577] The PLD 55092 sequences of the present invention can also be
used to identify individuals from minute biological samples. The
United States military, for example, is considering the use of
restriction fragment length polymorphism (RFLP) for identification
of its personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identification. This method
does not suffer from the current limitations of "Dog Tags" which
can be lost, switched, or stolen, making positive identification
difficult. The sequences of the present invention are useful as
additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[1578] Furthermore, the sequences of the present invention can be
used to provide an alternative technique which determines the
actual base-by-base DNA sequence of selected portions of an
individual's genome. Thus, the PLD 55092 nucleotide sequences
described herein can be used to prepare two PCR primers from the 5'
and 3' ends of the sequences. These primers can then be used to
amplify an individual's DNA and subsequently sequence it.
[1579] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
present invention can be used to obtain such identification
sequences from individuals and from tissue. The PLD 55092
nucleotide sequences of the invention uniquely represent portions
of the human genome. Allelic variation occurs to some degree in the
coding regions of these sequences, and to a greater degree in the
noncoding regions. It is estimated that allelic variation between
individual humans occurs with a frequency of about once per each
500 bases. Each of the sequences described herein can, to some
degree, be used as a standard against which DNA from an individual
can be compared for identification purposes. Because greater
numbers of polymorphisms occur in the noncoding regions, fewer
sequences are necessary to differentiate individuals. The noncoding
sequences of PLD 55092 gene sequences can comfortably provide
positive individual identification with a panel of perhaps 10 to
1,000 primers which each yield a noncoding amplified sequence of
100 bases. If predicted coding sequences are used, a more
appropriate number of primers for positive individual
identification would be 500-2,000.
[1580] If a panel of reagents from PLD 55092 nucleotide sequences
described herein is used to generate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
Use of Partial PLD 55092 Sequences in Forensic Biology
[1581] DNA-based identification techniques can also be used in
forensic biology. Forensic biology is a scientific field employing
genetic typing of biological evidence found at a crime scene as a
means for positively identifying, for example, a perpetrator of a
crime. To make such an identification, PCR technology can be used
to amplify DNA sequences taken from very small biological samples
such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, or semen found at a crime scene. The amplified sequence can
then be compared to a standard, thereby allowing identification of
the origin of the biological sample.
[1582] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As mentioned
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
noncoding regions of PLD 55092 gene sequences are particularly
appropriate for this use as greater numbers of polymorphisms occur
in the noncoding regions, making it easier to differentiate
individuals using this technique. Examples of polynucleotide
reagents include the PLD 55092 nucleotide sequences or portions
thereof, e.g., fragments derived from the noncoding regions having
a length of at least 20 bases, preferably at least 30 bases.
[1583] The PLD 55092 nucleotide sequences described herein can
further be used to provide polynucleotide reagents, e.g., labeled
or labelable probes which can be used in, for example, an in situ
hybridization technique, to identify a specific tissue, e.g., brain
tissue. This can be very useful in cases where a forensic
pathologist is presented with a tissue of unknown origin. Panels of
such PLD 55092 probes can be used to identify tissue by species
and/or by organ type.
[1584] In a similar fashion, these reagents, e.g., PLD 55092
primers or probes can be used to screen tissue culture for
contamination (i.e. screen for the presence of a mixture of
different types of cells in a culture).
Recombinant Expression Vectors and Host Cells
[1585] The methods of the invention include the use of vectors,
preferably expression vectors, containing a nucleic acid encoding a
PLD 55092 protein (or a portion thereof). As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. One type of
vector is a "plasmid", which refers to a circular double stranded
DNA loop into which additional DNA segments can be ligated. Another
type of vector is a viral vector, wherein additional DNA segments
can be ligated into the viral genome. Certain vectors are capable
of autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the methods of the invention may include other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[1586] The recombinant expression vectors used in the methods of
the invention comprise a nucleic acid of the invention in a form
suitable for expression of the nucleic acid in a host cell, which
means that the recombinant expression vectors include one or more
regulatory sequences, selected on the basis of the host cells to be
used for expression, which is operatively linked to the nucleic
acid sequence to be expressed. Within a recombinant expression
vector, "operably linked" is intended to mean that the nucleotide
sequence of interest is linked to the regulatory sequence(s) in a
manner which allows for expression of the nucleotide sequence
(e.g., in an in vitro transcription/translation system or in a host
cell when the vector is introduced into the host cell). The term
"regulatory sequence" is intended to include promoters, enhancers
and other expression control elements (e.g., polyadenylation
signals). Such regulatory sequences are described, for example, in
Goeddel; Gene Expression Technology Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990). Regulatory sequences
include those which direct constitutive expression of a nucleotide
sequence in many types of host cells and those which direct
expression of the nucleotide sequence only in certain host cells
(e.g., tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of protein
desired, and the like. The expression vectors of the invention can
be introduced into host cells to thereby produce proteins or
peptides, including fusion proteins or peptides, encoded by nucleic
acids as described herein (e.g., PLD 55092 proteins, mutant forms
of PLD 55092 proteins, fusion proteins, and the like).
[1587] The recombinant expression vectors used in the methods of
the invention can be designed for expression of PLD 55092 proteins
in prokaryotic or eukaryotic cells, e.g., for use in the cell-based
assays of the invention. For example, PLD 55092 proteins can be
expressed in bacterial cells such as E. coli, insect cells (using
baculovirus expression vectors) yeast cells or mammalian cells.
Suitable host cells are discussed further in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[1588] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein.
[1589] Purified fusion proteins can be utilized in PLD 55092
activity assays, (e.g., direct assays or competitive assays
described in detail below), or to generate antibodies specific for
PLD 55092 proteins, for example. In a preferred embodiment, a PLD
55092 fusion protein expressed in a retroviral expression vector of
the present invention can be utilized to infect bone marrow cells
which are subsequently transplanted into irradiated recipients. The
pathology of the subject recipient is then examined after
sufficient time has passed (e.g., six (6) weeks).
[1590] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
prophage harboring a T7 gn1 gene under the transcriptional control
of the lacUV 5 promoter.
[1591] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid
to be inserted into an expression vector so that the individual
codons for each amino acid are those preferentially utilized in E.
coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
[1592] In another embodiment, the PLD 55092 expression vector is a
yeast expression vector. Examples of vectors for expression in
yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO
J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell
30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2
(Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen
Corp, San Diego, Calif.).
[1593] Alternatively, PLD 55092 proteins can be expressed in insect
cells using baculovirus expression vectors. Baculovirus vectors
available for expression of proteins in cultured insect cells
(e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol.
Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers
(1989) Virology 170:31-39).
[1594] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)
EMBO J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd,
ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989.
[1595] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), endothelial cell-specific promoters
(e.g., KDR/flk promoter; U.S. Pat. No. 5,888,765),
pancreas-specific promoters (Edlund et al. (1985) Science
230:912-916), and mammary gland-specific promoters (e.g., milk whey
promoter; U.S. Pat. No. 4,873,316 and European Application
Publication No. 264,166). Developmentally-regulated promoters are
also encompassed, for example the murine hox promoters (Kessel and
Gruss (1990) Science 249:374-379) and the .alpha.-fetoprotein
promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
[1596] The expression characteristics of an endogenous PLD 55092
gene within a cell line or microorganism may be modified by
inserting a heterologous DNA regulatory element into the genome of
a stable cell line or cloned microorganism such that the inserted
regulatory element is operatively linked with the endogenous PLD
55092 gene. For example, an endogenous PLD 55092 gene which is
normally "transcriptionally silent", i.e., a PLD 55092 gene which
is normally not expressed, or is expressed only at very low levels
in a cell line or microorganism, may be activated by inserting a
regulatory element which is capable of promoting the expression of
a normally expressed gene product in that cell line or
microorganism. Alternatively, a transcriptionally silent,
endogenous PLD 55092 gene may be activated by insertion of a
promiscuous regulatory element that works across cell types.
[1597] A heterologous regulatory element may be inserted into a
stable cell line or cloned microorganism, such that it is
operatively linked with an endogenous PLD 55092 gene, using
techniques, such as targeted homologous recombination, which are
well known to those of skill in the art, and described, e.g., in
Chappel, U.S. Pat. No. 5,272,071; PCT publication No. WO 91/06667,
published May 16, 1991.
[1598] The methods of the invention use a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to PLD 55092 mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[1599] Another aspect the methods of the invention pertains to the
use of host cells into which a PLD 55092 nucleic acid molecule of
the invention is introduced, e.g., a PLD 55092 nucleic acid
molecule within a recombinant expression vector or a PLD 55092
nucleic acid molecule containing sequences which allow it to
homologously recombine into a specific site of the host cell's
genome. The terms "host cell" and "recombinant host cell" are used
interchangeably herein. It is understood that such terms refer not
only to the particular subject cell but to the progeny or potential
progeny of such a cell. Because certain modifications may occur in
succeeding generations due to either mutation or environmental
influences, such progeny may not, in fact, be identical to the
parent cell, but are still included within the scope of the term as
used herein.
[1600] A host cell can be any prokaryotic or eukaryotic cell. For
example, a PLD 55092 protein can be expressed in bacterial cells
such as E. coli, insect cells, yeast or mammalian cells (such as
Chinese hamster ovary cells (CHO) HEPG2 cells, NT2 cells, MRC5
cells, or COS cells). Other suitable host cells are known to those
skilled in the art.
[1601] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[1602] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin, puromycin, zeomycin and methotrexate. Nucleic acid
encoding a selectable marker can be introduced into a host cell on
the same vector as that encoding a PLD 55092 protein or can be
introduced on a separate vector. Cells stably transfected with the
introduced nucleic acid can be identified by drug selection (e.g.,
cells that have incorporated the selectable marker gene will
survive, while the other cells die).
[1603] A host cell used in the methods of the invention, such as a
prokaryotic or eukaryotic host cell in culture, can be used to
produce (i.e., express) a PLD 55092 protein. Accordingly, the
invention further provides methods for producing a PLD 55092
protein using the host cells of the invention. In one embodiment,
the method comprises culturing the host cell of the invention (into
which a recombinant expression vector encoding a PLD 55092 protein
has been introduced) in a suitable medium such that a PLD 55092
protein is produced. In another embodiment, the method further
comprises isolating a PLD 55092 protein from the medium or the host
cell.
Cell- and Animal-Based Model Systems
[1604] Described herein are cell- and animal-based systems which
act as models for viral 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 genes associated with viral disease, e.g., PLD 55092. In
addition, animal- and cell-based assays may be used as part of
screening strategies designed to identify compounds which are
capable of ameliorating viral disease symptoms, as described,
below. Thus, the animal- and cell-based models may be used to
identify drugs, pharmaceuticals, therapies and interventions which
may be effective in treating viral disease. Furthermore, such
animal models may be used to determine the LD50 and the ED50 in
animal subjects, and such data can be used to determine the in vivo
efficacy of potential viral disease treatments.
Animal-Based Systems
[1605] Animal-based model systems of viral disease may include, but
are not limited to, non-recombinant and engineered transgenic
animals.
[1606] Non-recombinant animal models for viral disease may include,
for example, genetic models. Transgenic mouse models for viral
disease are reviewed in Rall G F et al. (Virol. (2000)
271:220-226), Eckert R L et al. (Int. J. Oncol. (2000) 16:853-70),
and Morrey J D et al. (Antiviral Ther. (1998) 3:59-68).
[1607] Non-recombinant, non-genetic animal models of viral disease
may include, for example, animal models in which the animal has
been exposed to viral infection, as described in, for example,
Mosier, D (2000), Virol. 271:215-219; Lavi, E et al. (1999) J.
Neuropathol. Exp. Neurol. 58:1197-1206; Briese, T et al. (1999) J.
Neurovirol. 5:604-612; Johannessen, I et al. (1999) Rev. Med.
Virol. 9:263-277; Hayashi, K et al. (2000) Pathol. Int. 50:85-97;
Michalak, T I (2000) Immunol. Rev. 174:98-111; McSharry, J J (1999)
Antiviral Res. 43:1-21; Bernstein, D I et al. (2000) Antiviral Res.
47:159-169; Thackray, A M et al. (2000) J. Gen. Virol.
81:2385-2396; Nakazato, I et al. (2000) Pathol. Res. Pract.
196:635-645; and Takasaki, I et al. (2000) Jpn. J. Pharmacol.
83:319-326.
[1608] Additionally, animal models exhibiting viral disease
symptoms may be engineered by using, for example, PLD 55092 gene
sequences described above, in conjunction with techniques for
producing transgenic animals that are well known to those of skill
in the art. For example, PLD 55092 gene sequences may be introduced
into, and overexpressed in, the genome of the animal of interest,
or, if endogenous PLD 55092 gene sequences are present, they may
either be overexpressed or, alternatively, be disrupted in order to
underexpress or inactivate PLD 55092 gene expression, such as
described for the disruption of apoE in mice (Plump et al., 1992,
Cell 71: 343-353).
[1609] The host cells of the invention can also be used to produce
non-human transgenic animals. For example, in one embodiment, a
host cell of the invention is a fertilized oocyte or an embryonic
stem cell into which PLD 55092-coding sequences have been
introduced. Such host cells can then be used to create non-human
transgenic animals in which exogenous PLD 55092 sequences have been
introduced into their genome or homologous recombinant animals in
which endogenous PLD 55092 sequences have been altered. Such
animals are useful for studying the function and/or activity of a
PLD 55092 and for identifying and/or evaluating modulators of PLD
55092 activity. As used herein, a "transgenic animal" is a
non-human animal, preferably a mammal, more preferably a rodent
such as a rat or mouse, in which one or more of the cells of the
animal includes a transgene. Other examples of transgenic animals
include non-human primates, sheep, dogs, cows, goats, chickens,
amphibians, and the like. A transgene is exogenous DNA which is
integrated into the genome of a cell from which a transgenic animal
develops and which remains in the genome of the mature animal,
thereby directing the expression of an encoded gene product in one
or more cell types or tissues of the transgenic animal. As used
herein, a "homologous recombinant animal" is a non-human animal,
preferably a mammal, more preferably a mouse, in which an
endogenous PLD 55092 gene has been altered by homologous
recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal, e.g., an embryonic
cell of the animal, prior to development of the animal.
[1610] A transgenic animal used in the methods of the invention can
be created by introducing a PLD 55092-encoding nucleic acid into
the male pronuclei of a fertilized oocyte, e.g., by microinjection,
retroviral infection, and allowing the oocyte to develop in a
pseudopregnant female foster animal. The PLD 55092 cDNA sequence of
SEQ ID NO:14 can be introduced as a transgene into the genome of a
non-human animal. Alternatively, a nonhuman homologue of a human
PLD 55092 gene, such as a mouse or rat PLD 55092 gene, can be used
as a transgene. Alternatively, a PLD 55092 gene homologue, such as
another PLD 55092 family member, can be isolated based on
hybridization to the PLD 55092 cDNA sequences of SEQ ID NO:14 and
used as a transgene. Intronic sequences and polyadenylation signals
can also be included in the transgene to increase the efficiency of
expression of the transgene. A tissue-specific regulatory
sequence(s) can be operably linked to a PLD 55092 transgene to
direct expression of a PLD 55092 protein to particular cells.
Methods for generating transgenic animals via embryo manipulation
and microinjection, particularly animals such as mice, have become
conventional in the art and are described, for example, in U.S.
Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat.
No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the
Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1986). Similar methods are used for production of
other transgenic animals. A transgenic founder animal can be
identified based upon the presence of a PLD 55092 transgene in its
genome and/or expression of PLD 55092 mRNA in tissues or cells of
the animals. A transgenic founder animal can then be used to breed
additional animals carrying the transgene. Moreover, transgenic
animals carrying a transgene encoding a PLD 55092 protein can
further be bred to other transgenic animals carrying other
transgenes.
[1611] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of a PLD 55092 gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the PLD 55092 gene. The
PLD 55092 gene can be a human gene (e.g., the cDNA of SEQ ID
NO:14), but more preferably, is a non-human homologue of a human
PLD 55092 gene (e.g., a cDNA isolated by stringent hybridization
with the nucleotide sequence of SEQ ID NO:14). For example, a mouse
PLD 55092 gene can be used to construct a homologous recombination
nucleic acid molecule, e.g., a vector, suitable for altering an
endogenous PLD 55092 gene in the mouse genome. In a preferred
embodiment, the homologous recombination nucleic acid molecule is
designed such that, upon homologous recombination, the endogenous
PLD 55092 gene is functionally disrupted (i.e., no longer encodes a
functional protein; also referred to as a "knock out" vector).
Alternatively, the homologous recombination nucleic acid molecule
can be designed such that, upon homologous recombination, the
endogenous PLD 55092 gene is mutated or otherwise altered but still
encodes functional protein (e.g., the upstream regulatory region
can be altered to thereby alter the expression of the endogenous
PLD 55092 protein). In the homologous recombination nucleic acid
molecule, the altered portion of the PLD 55092 gene is flanked at
its 5' and 3' ends by additional nucleic acid sequence of the PLD
55092 gene to allow for homologous recombination to occur between
the exogenous PLD 55092 gene carried by the homologous
recombination nucleic acid molecule and an endogenous PLD 55092
gene in a cell, e.g., an embryonic stem cell. The additional
flanking PLD 55092 nucleic acid sequence is of sufficient length
for successful homologous recombination with the endogenous gene.
Typically, several kilobases of flanking DNA (both at the 5' and 3'
ends) are included in the homologous recombination nucleic acid
molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell
51:503 for a description of homologous recombination vectors). The
homologous recombination nucleic acid molecule is introduced into a
cell, e.g., an embryonic stem cell line (e.g., by electroporation)
and cells in which the introduced PLD 55092 gene has homologously
recombined with the endogenous PLD 55092 gene are selected (see
e.g., Li, E. et al. (1992) Cell 69:915). The selected cells can
then injected into a blastocyst of an animal (e.g., a mouse) to
form aggregation chimeras (see e.g., Bradley, A. in
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric
embryo can then be implanted into a suitable pseudopregnant female
foster animal and the embryo brought to term. Progeny harboring the
homologously recombined DNA in their germ cells can be used to
breed animals in which all cells of the animal contain the
homologously recombined DNA by germline transmission of the
transgene. Methods for constructing homologous recombination
nucleic acid molecules, e.g., vectors, or homologous recombinant
animals are described further in Bradley, A. (1991) Current Opinion
in Biotechnology 2:823-829 and in PCT International Publication
Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et
al.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et
al.
[1612] In another embodiment, transgenic non-human animals for use
in the methods of the invention can be produced which contain
selected systems which allow for regulated expression of the
transgene. One example of such a system is the cre/loxP recombinase
system of bacteriophage P1. For a description of the cre/loxP
recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl.
Acad. Sci. USA 89:6232-6236. Another example of a recombinase
system is the FLP recombinase system of Saccharomyces cerevisiae
(O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP
recombinase system is used to regulate expression of the transgene,
animals containing transgenes encoding both the Cre recombinase and
a selected protein are required. Such animals can be provided
through the construction of "double" transgenic animals, e.g., by
mating two transgenic animals, one containing a transgene encoding
a selected protein and the other containing a transgene encoding a
recombinase.
[1613] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. (1997) Nature 385:810-813 and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.o phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyte and then transferred to pseudopregnant female
foster animal. The offspring borne of this female foster animal
will be a clone of the animal from which the cell, e.g., the
somatic cell, is isolated.
[1614] The PLD 55092 transgenic animals that express PLD 55092 mRNA
or a PLD 55092 peptide (detected immunocytochemically, using
antibodies directed against PLD 55092 epitopes) at easily
detectable levels should then be further evaluated to identify
those animals which display characteristic viral disease symptoms.
Such viral disease symptoms may include, for example, viremia.
[1615] Additionally, specific cell types (e.g., neuronal cells)
within the transgenic animals may be analyzed and assayed for
cellular phenotypes characteristic of viral disease. Cellular
phenotypes may include a particular cell type's pattern of
expression of genes associated with viral disease as compared to
known expression profiles of the particular cell type in animals
exhibiting viral disease symptoms.
Cell-Based Systems
[1616] Cells that contain and express PLD 55092 gene sequences
which encode a PLD 55092 protein, and, further, exhibit cellular
phenotypes associated with viral disease, may be used to identify
compounds that exhibit anti-viral disease activity. Such cells may
include 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 viral
disease animal models of the invention, discussed above, may be
used to generate cell lines, containing one or more cell types
involved in viral disease, that can be used as cell culture models
for this disorder. While primary cultures derived from the viral
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.
[1617] Alternatively, cells of a cell type known to be involved in
viral disease and/or susceptible to viral infection may be
transfected with sequences capable of increasing or decreasing the
amount of PLD 55092 gene expression within the cell. For example,
PLD 55092 gene sequences may be introduced into, and overexpressed
in, the genome of the cell of interest, or, if endogenous PLD 55092
gene sequences are present, they may be either overexpressed or,
alternatively disrupted in order to underexpress or inactivate PLD
55092 gene expression.
[1618] In order to overexpress a PLD 55092 gene, the coding portion
of the PLD 55092 gene may be ligated to a regulatory sequence which
is capable of driving gene expression in the cell type of interest,
e.g., a neuronal cell or a liver cell. 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 above.
[1619] For underexpression of an endogenous PLD 55092 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 PLD 55092 alleles will be inactivated. Preferably, the
engineered PLD 55092 sequence is introduced via gene targeting such
that the endogenous PLD 55092 sequence is disrupted upon
integration of the engineered PLD 55092 sequence into the cell's
genome. Transfection of host cells with PLD 55092 genes is
discussed, above.
[1620] Cells (e.g., virally infected cells) treated with compounds
or transfected with PLD 55092 genes can be examined for phenotypes
associated with viral infection and/or disease, e.g., plaque
formation or low pH induced fusion of infected cells (Sung T-C et
al. (1997) EMBO J. 16:4519-4530; Roper R L and Moss B (1999) J.
Virol. 73:1108-1117; Blasco R and Moss B (1991) J. Virol.
65:5910-5920). Moreover, cells treated with compounds or
transfected with PLD 55092 genes can be examined for phenotypes,
including, but not limited to changes in cellular morphology, cell
proliferation, cell differentiation, cell migration, and vesicular
trafficking.
[1621] Transfection of PLD 55092 nucleic acid may be accomplished
by using standard techniques (described in, for example, Ausubel
(1989) supra). Transfected cells should be evaluated for the
presence of the recombinant PLD 55092 gene sequences, for
expression and accumulation of PLD 55092 mRNA, and for the presence
of recombinant PLD 55092 protein production. In instances wherein a
decrease in PLD 55092 gene expression is desired, standard
techniques may be used to demonstrate whether a decrease in
endogenous PLD 55092 gene expression and/or in PLD 55092 protein
production is achieved.
[1622] Cellular models for the study of viral disease include
models of cell infection with virus, e.g., herpes simplex virus,
Epstein Barr virus, hepatitis virus, human papilloma virus.
Pharmaceutical Compositions
[1623] Active compounds for use in the methods of the invention can
be incorporated into pharmaceutical compositions suitable for
administration. As used herein, the language "active compounds"
includes PLD 55092 nucleic acid molecules, fragments of PLD 55092
proteins, and anti-PLD 55092 antibodies, as well as identified
compounds that modulate PLD 55092 gene expression, synthesis,
and/or activity. Such compositions typically comprise the compound,
nucleic acid molecule, protein, or antibody and a pharmaceutically
acceptable carrier. As used herein the language "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[1624] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[1625] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[1626] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a fragment of a PLD 55092
protein or a PLD 55092 substrate) in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[1627] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[1628] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[1629] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[1630] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[1631] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[1632] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals. In one embodiment, a therapeutically effective dose
refers to that amount of an active compound sufficient to result in
amelioration of symptoms of viral disease or infection. In other
embodiments, a therapeutically effective dose refers to that amount
of an active compound sufficient to suppress disease recurrence,
reduce and/or delay disease onset, reduce viremia, and protect
against viral infection.
[1633] 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 LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. 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.
[1634] 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 ED50 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
IC50 (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.
[1635] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
skilled artisan will appreciate that certain factors may influence
the dosage required to effectively treat a subject, including but
not limited to the severity of the disease or disorder, previous
treatments, the general health and/or age of the subject, and other
diseases present. Moreover, treatment of a subject with a
therapeutically effective amount of a protein, polypeptide, or
antibody can include a single treatment or, preferably, can include
a series of treatments.
[1636] In a preferred example, a subject is treated with antibody,
protein, or polypeptide in the range of between about 0.1 to 20
mg/kg body weight, one time per week for between about 1 to 10
weeks, preferably between 2 to 8 weeks, more preferably between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. It will also be appreciated that the effective dosage of
antibody, protein, or polypeptide used for treatment may increase
or decrease over the course of a particular treatment. Changes in
dosage may result and become apparent from the results of
diagnostic assays as described herein.
[1637] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds. It is understood that appropriate doses of small
molecule agents depends upon a number of factors within the ken of
the ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention.
[1638] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram. It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. Such appropriate doses may be determined using the
assays described herein. When one or more of these small molecules
is to be administered to an animal (e.g., a human) in order to
modulate expression or activity of a polypeptide or nucleic acid of
the invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[1639] In certain embodiments of the invention, a modulator of PLD
55092 activity is administered in combination with other agents
(e.g., a small molecule), or in conjunction with another,
complementary treatment regime. For example, in one embodiment, a
modulator of PLD 55092 activity is used to treat a viral disease,
e.g., a disease associated with Herpes simplex virus infection.
Accordingly, modulation of PLD 55092 activity may be used in
conjunction with, for example, antiviral agents, e.g., acyclovir,
valaciclovir, famciclovir.
[1640] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (CDDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[1641] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
.alpha.-interferon, .beta.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophase colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors.
[1642] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2.sup.nd Ed.),
Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);
Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
[1643] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[1644] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
Isolated Nucleic Acid Molecules
[1645] The nucleotide sequence of the isolated human PLD 55092 cDNA
and the predicted amino acid sequence of the human PLD 55092
polypeptide are shown in SEQ ID NOs:14, respectively.
[1646] The human PLD 55092 gene, which is approximately 1917
nucleotides in length, encodes a protein having a molecular weight
of approximately 55 kD and which is approximately 506 amino acid
residues in length.
[1647] The methods of the invention include the use of isolated
nucleic acid molecules that encode PLD 55092 proteins or
biologically active portions thereof, as well as nucleic acid
fragments sufficient for use as hybridization probes to identify
PLD 55092-encoding nucleic acid molecules (e.g., PLD 55092 mRNA)
and fragments for use as PCR primers for the amplification or
mutation of PLD 55092 nucleic acid molecules. As used herein, the
term "nucleic acid molecule" is intended to include DNA molecules
(e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and
analogs of the DNA or RNA generated using nucleotide analogs. The
nucleic acid molecule can be single-stranded or double-stranded,
but preferably is double-stranded DNA.
[1648] The term "isolated nucleic acid molecule" includes nucleic
acid molecules which are separated from other nucleic acid
molecules which are present in the natural source of the nucleic
acid. For example, with regards to genomic DNA, the term "isolated"
includes nucleic acid molecules which are separated from the
chromosome with which the genomic DNA is naturally associated.
Preferably, an "isolated" nucleic acid is free of sequences which
naturally flank the nucleic acid (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. For example, in various
embodiments, the isolated PLD 55092 nucleic acid molecule can
contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1
kb of nucleotide sequences which naturally flank the nucleic acid
molecule in genomic DNA of the cell from which the nucleic acid is
derived. Moreover, an "isolated" nucleic acid molecule, such as a
cDNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized.
[1649] A nucleic acid molecule used in the methods of the present
invention, e.g., a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:14, or a portion thereof, can be isolated
using standard molecular biology techniques and the sequence
information provided herein. Using all or portion of the nucleic
acid sequence of SEQ ID NO:14, as a hybridization probe, PLD 55092
nucleic acid molecules can be isolated using standard hybridization
and cloning techniques (e.g., as described in Sambrook, J., Fritsh,
E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.
2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989).
[1650] Moreover, a nucleic acid molecule encompassing all or a
portion of SEQ ID NO:14 or 16 can be isolated by the polymerase
chain reaction (PCR) using synthetic oligonucleotide primers
designed based upon the sequence of SEQ ID NO:14.
[1651] A nucleic acid used in the methods of the invention can be
amplified using cDNA, mRNA or alternatively, genomic DNA, as a
template and appropriate oligonucleotide primers according to
standard PCR amplification techniques. The nucleic acid so
amplified can be cloned into an appropriate vector and
characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to PLD 55092 nucleotide sequences
can be prepared by standard synthetic techniques, e.g., using an
automated DNA synthesizer.
[1652] In a preferred embodiment, an isolated nucleic acid molecule
used in the methods of the invention comprises the nucleotide
sequence shown in SEQ ID NO:14. This cDNA may comprise sequences
encoding the human PLD 55092 protein (i.e., "the coding region",
from nucleotides 122-1642), as well as 5' untranslated sequences
(nucleotides 1-121) and 3' untranslated sequences (nucleotides
1643-1917) of SEQ ID NO:14. Alternatively, the nucleic acid
molecule can comprise only the coding region of SEQ ID NO:14 (e.g.,
nucleotides 122-1642 of SEQ ID NO:14).
[1653] In another preferred embodiment, an isolated nucleic acid
molecule used in the methods of the invention comprises a nucleic
acid molecule which is a complement of the nucleotide sequence
shown in SEQ ID NO:14, or a portion of any of this nucleotide
sequence. A nucleic acid molecule which is complementary to the
nucleotide sequence shown in SEQ ID NO:14 is one which is
sufficiently complementary to the nucleotide sequence shown in SEQ
ID NO:14 such that it can hybridize to the nucleotide sequence
shown in SEQ ID NO:14, thereby forming a stable duplex.
[1654] In still another preferred embodiment, an isolated nucleic
acid molecule used in the methods of the present invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the entire
length of the nucleotide sequence shown in SEQ ID NO:14, or a
portion of any of this nucleotide sequence.
[1655] Moreover, a nucleic acid molecule used in the methods of the
invention can comprise only a portion of the nucleic acid sequence
of SEQ ID NO:14, for example, a fragment which can be used as a
probe or primer or a fragment encoding a portion of a PLD 55092
protein, e.g., a biologically active portion of a PLD 55092
protein. The nucleotide sequence determined from the cloning of the
PLD 55092 gene allows for the generation of probes and primers
designed for use in identifying and/or cloning other PLD 55092
family members, as well as PLD 55092 homologues from other species.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12 or 15, preferably about 20 or 25, more
preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive
nucleotides of a sense sequence of SEQ ID NO:14, of an anti-sense
sequence of SEQ ID NO:14, or of a naturally occurring allelic
variant or mutant of SEQ ID NO:14. In one embodiment, a nucleic
acid molecule of the present invention comprises a nucleotide
sequence which is greater than 100, 100-200, 200-300, 300-400,
400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100,
1100-120, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700,
1700-1800, 1800 or more nucleotides in length and hybridizes under
stringent hybridization conditions to a nucleic acid molecule of
SEQ ID NO:14.
[1656] Probes based on the PLD 55092 nucleotide sequence can be
used to detect transcripts or genomic sequences encoding the same
or homologous proteins. In preferred embodiments, the probe further
comprises a label group attached thereto, e.g., the label group can
be a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells or tissue which misexpress a PLD 55092
protein, such as by measuring a level of a PLD 55092-encoding
nucleic acid in a sample of cells from a subject e.g., detecting
PLD 55092 mRNA levels or determining whether a genomic PLD 55092
gene has been mutated or deleted.
[1657] A nucleic acid fragment encoding a "biologically active
portion of a PLD 55092 protein" can be prepared by isolating a
portion of the nucleotide sequence of SEQ ID NO:14 which encodes a
polypeptide having a PLD 55092 biological activity (the biological
activities of the PLD 55092 protein is described herein),
expressing the encoded portion of the PLD 55092 protein (e.g., by
recombinant expression in vitro) and assessing the activity of the
encoded portion of the PLD 55092 protein.
[1658] The methods of the invention further encompass the use of
nucleic acid molecules that differ from the nucleotide sequence
shown in SEQ ID NO:14, due to degeneracy of the genetic code and
thus encode the same PLD 55092 protein as those encoded by the
nucleotide sequence shown in SEQ ID NO:14. In another embodiment,
an isolated nucleic acid molecule of the invention has a nucleotide
sequence encoding a protein having an amino acid sequence shown in
SEQ ID NO:15.
[1659] In addition to the PLD 55092 nucleotide sequence shown in
SEQ ID NO:14, it will be appreciated by those skilled in the art
that DNA sequence polymorphisms that lead to changes in the amino
acid sequences of the PLD 55092 protein may exist within a
population (e.g., the human population). Such genetic polymorphism
in the PLD 55092 gene may exist among individuals within a
population due to natural allelic variation. As used herein, the
terms "gene" and "recombinant gene" refer to nucleic acid molecules
which include an open reading frame encoding a PLD 55092 protein,
preferably a mammalian PLD 55092 protein, and can further include
non-coding regulatory sequences, and introns.
[1660] Allelic variants of human PLD 55092 include both functional
and non-functional PLD 55092 proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the human
PLD 55092 protein that maintain the ability to bind a PLD 55092
ligand or substrate and/or modulate signal transduction, lipid
metabolism, and/or vesicle trafficking mechanisms. Functional
allelic variants will typically contain only conservative
substitution of one or more amino acids of SEQ ID NO:15, or
substitution, deletion or insertion of non-critical residues in
non-critical regions of the protein.
[1661] Non-functional allelic variants are naturally occurring
amino acid sequence variants of the human PLD 55092 protein that do
not have the ability to either bind a PLD 55092 ligand or substrate
and/or modulate signal transduction, lipid metabolism, and/or
vesicle trafficking mechanisms. Non-functional allelic variants
will typically contain a non-conservative substitution, a deletion,
or insertion or premature truncation of the amino acid sequence of
SEQ ID NO:15, or a substitution, insertion or deletion in critical
residues or critical regions.
[1662] The methods of the present invention may further use
non-human orthologues of the human PLD 55092 protein. Orthologues
of the human PLD 55092 protein are proteins that are isolated from
non-human organisms and possess the same PLD 55092 ligand binding
and/or modulation of signal transduction, lipid metabolism, and/or
vesicle trafficking mechanisms of the human PLD 55092 protein.
Orthologues of the human PLD 55092 protein can readily be
identified as comprising an amino acid sequence that is
substantially identical to SEQ ID NO:15.
[1663] Moreover, nucleic acid molecules encoding other PLD 55092
family members and, thus, which have a nucleotide sequence which
differs from the PLD 55092 sequence of SEQ ID NO:14 are intended to
be within the scope of the invention. For example, another PLD
55092 cDNA can be identified based on the nucleotide sequence of
human PLD 55092. Moreover, nucleic acid molecules encoding PLD
55092 proteins from different species, and which, thus, have a
nucleotide sequence which differs from the PLD 55092 sequence of
SEQ ID NO:14 are intended to be within the scope of the invention.
For example, a mouse PLD 55092 cDNA can be identified based on the
nucleotide sequence of human PLD 55092.
[1664] Nucleic acid molecules corresponding to natural allelic
variants and homologues of the PLD 55092 cDNA of the invention can
be isolated based on their homology to the PLD 55092 nucleic acid
disclosed herein using the cDNAs disclosed herein, or a portion
thereof, as a hybridization probe according to standard
hybridization techniques under stringent hybridization conditions.
Nucleic acid molecules corresponding to natural allelic variants
and homologues of the PLD 55092 cDNA of the invention can further
be isolated by mapping to the same chromosome or locus as the PLD
55092 gene.
[1665] Accordingly, in another embodiment, an isolated nucleic acid
molecule used in the methods of the invention is at least 15, 20,
25, 30 or more nucleotides in length and hybridizes under stringent
conditions to the nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:14. In other embodiment, the nucleic acid is
at least 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 1000, 1200, 1400, 1600 or more nucleotides
in length.
[1666] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences that are significantly
identical or homologous to each other remain hybridized to each
other. Preferably, the conditions are such that sequences at least
about 70%, more preferably at least about 80%, even more preferably
at least about 85% or 90% identical to each other remain hybridized
to each other. Such stringent conditions are known to those skilled
in the art and can be found in Current Protocols in Molecular
Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995),
sections 2, 4 and 6. Additional stringent conditions can be found
in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9
and 11. A preferred, non-limiting example of stringent
hybridization conditions includes hybridization in 4.times. sodium
chloride/sodium citrate (SSC), at about 65-70.degree. C. (or
hybridization in 4.times.SSC plus 50% formamide at about
42-50.degree. C.) followed by one or more washes in 1.times.SSC, at
about 65-70.degree. C. A preferred, non-limiting example of highly
stringent hybridization conditions includes hybridization in
1.times.SSC, at about 65-70.degree. C. (or hybridization in
1.times.SSC plus 50% formamide at about 42-50.degree. C.) followed
by one or more washes in 0.3.times.SSC, at about 65-70.degree. C. A
preferred, non-limiting example of reduced stringency hybridization
conditions includes hybridization in 4.times.SSC, at about
50-60.degree. C. (or hybridization in 6.times.SSC plus 50%
formamide at about 40-45.degree. C.) followed by one or more washes
in 2.times.SSC, at about 50-60.degree. C. Ranges intermediate to
the above-recited values, e.g., at 65-70.degree. C. or at
42-50.degree. C. are also intended to be encompassed by the present
invention. SSPE (1.times.SSPE is 0.15M NaCl, 10 mM
NaH.sub.2PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be substituted for
SSC (1.times.SSC is 0.15M NaCl and 15 mM sodium citrate) in the
hybridization and wash buffers; washes are performed for 15 minutes
each after hybridization is complete. The hybridization temperature
for hybrids anticipated to be less than 50 base pairs in length
should be 5-10.degree. C. less than the melting temperature
(T.sub.m) of the hybrid, where T.sub.m is determined according to
the following equations. For hybrids less than 18 base pairs in
length, T.sub.m(.degree. C.)=2(# of A+T bases)+4(# of G+C bases).
For hybrids between 18 and 49 base pairs in length,
T.sub.m(.degree. C.)=81.5+16.6(log.sub.10[Na.sup.+])+0.41(%
G+C)-(600/N), where N is the number of bases in the hybrid, and
[Na.sup.+] is the concentration of sodium ions in the hybridization
buffer ([Na.sup.+] for 1.times.SSC=0.165 M). It will also be
recognized by the skilled practitioner that additional reagents may
be added to hybridization and/or wash buffers to decrease
non-specific hybridization of nucleic acid molecules to membranes,
for example, nitrocellulose or nylon membranes, including but not
limited to blocking agents (e.g., BSA or salmon or herring sperm
carrier DNA), detergents (e.g., SDS), chelating agents (e.g.,
EDTA), Ficoll, PVP and the like. When using nylon membranes, in
particular, an additional preferred, non-limiting example of
stringent hybridization conditions is hybridization in 0.25-0.5M
NaH.sub.2PO.sub.4, 7% SDS at about 65.degree. C., followed by one
or more washes at 0.02M NaH.sub.2PO.sub.4, 1% SDS at 65.degree. C.,
see, e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA
81:1991-1995, (or alternatively 0.2.times.SSC, 1% SDS).
[1667] Preferably, an isolated nucleic acid molecule of the
invention that hybridizes under stringent conditions to the
sequence of SEQ ID NO:14 corresponds to a naturally-occurring
nucleic acid molecule. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein).
[1668] In addition to naturally-occurring allelic variants of the
PLD 55092 sequences that may exist in the population, the skilled
artisan will further appreciate that changes can be introduced by
mutation into the nucleotide sequence of SEQ ID NO:14, thereby
leading to changes in the amino acid sequence of the encoded PLD
55092 protein, without altering the functional ability of the PLD
55092 protein. For example, nucleotide substitutions leading to
amino acid substitutions at "non-essential" amino acid residues can
be made in the sequence of SEQ ID NO:14. A "non-essential" amino
acid residue is a residue that can be altered from the wild-type
sequence of PLD 55092 (e.g., the sequence of SEQ ID NO:15) without
altering the biological activity, whereas an "essential" amino acid
residue is required for biological activity. For example, amino
acid residues that are conserved among the PLD 55092 proteins of
the present invention, e.g., those present in a HKD motif, are
predicted to be particularly unamenable to alteration. Furthermore,
additional amino acid residues that are conserved between the PLD
55092 proteins of the present invention and other members of the
PLD gene superfamily (Koonin, E V (1996) TIBS 21:242-243; Ponting,
C P et al. (1996) Protein Sci. 5:914-922; Liscovitch, M et al.
(2000) Biochem. J. 345:401-415) are not likely to be amenable to
alteration.
[1669] Accordingly, the methods of the invention may include the
use of nucleic acid molecules encoding PLD 55092 proteins that
contain changes in amino acid residues that are not essential for
activity. Such PLD 55092 proteins differ in amino acid sequence
from SEQ ID NO:15, yet retain biological activity. In one
embodiment, the isolated nucleic acid molecule comprises a
nucleotide sequence encoding a protein, wherein the protein
comprises an amino acid sequence at least about 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:15.
[1670] An isolated nucleic acid molecule encoding a PLD 55092
protein identical to the protein of SEQ ID NO:15 can be created by
introducing one or more nucleotide substitutions, additions or
deletions into the nucleotide sequence of SEQ ID NO:14 such that
one or more amino acid substitutions, additions or deletions are
introduced into the encoded protein. Mutations can be introduced
into SEQ ID NO:14 by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Preferably, conservative
amino acid substitutions are made at one or more predicted
non-essential amino acid residues. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in
the art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a predicted nonessential amino acid residue in a
PLD 55092 protein is preferably replaced with another amino acid
residue from the same side chain family. Alternatively, in another
embodiment, mutations can be introduced randomly along all or part
of a PLD 55092 coding sequence, such as by saturation mutagenesis,
and the resultant mutants can be screened for PLD 55092 biological
activity to identify mutants that retain activity. Following
mutagenesis of SEQ ID NO:14, the encoded protein can be expressed
recombinantly and the activity of the protein can be
determined.
[1671] In a preferred embodiment, a mutant PLD 55092 protein can be
assayed for the ability to (1) interact with a non-PLD 55092
protein molecule, e.g., a PLD 55092 ligand or substrate; (2)
activate a PLD 55092-dependent signal transduction pathway; (3)
modulate lipid metabolism; (4) modulate membrane vesicular
trafficking; (5) modulate membrane homeostasis; or (6) modulate
cell proliferation, differentiation and/or migration
mechanisms.
[1672] In addition to the nucleic acid molecules encoding PLD 55092
proteins described herein, another aspect of the invention pertains
to isolated nucleic acid molecules which are antisense thereto. An
"antisense" nucleic acid comprises a nucleotide sequence which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. Accordingly, an
antisense nucleic acid can hydrogen bond to a sense nucleic acid.
The antisense nucleic acid can be complementary to an entire PLD
55092 coding strand, or to only a portion thereof. In one
embodiment, an antisense nucleic acid molecule is antisense to a
"coding region" of the coding strand of a nucleotide sequence
encoding PLD 55092. The term "coding region" refers to the region
of the nucleotide sequence comprising codons which are translated
into amino acid residues (e.g., the coding region of human PLD
55092 corresponds to nucleotides 122-1642 of SEQ ID NO:14). In
another embodiment, the antisense nucleic acid molecule is
antisense to a "noncoding region" of the coding strand of a
nucleotide sequence encoding PLD 55092. The term "noncoding region"
refers to 5' and 3' sequences which flank the coding region that
are not translated into amino acids (i.e., also referred to as 5'
and 3' untranslated regions).
[1673] Given the coding strand sequences encoding PLD 55092
disclosed herein (e.g., nucleotides 122-1642 of SEQ ID NO:14),
antisense nucleic acids of the invention can be designed according
to the rules of Watson and Crick base pairing. The antisense
nucleic acid molecule can be complementary to the entire coding
region of PLD 55092 mRNA, but more preferably is an oligonucleotide
which is antisense to only a portion of the coding or noncoding
region of PLD 55092 mRNA. For example, the antisense
oligonucleotide can be complementary to the region surrounding the
translation start site of PLD 55092 mRNA. An antisense
oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30,
35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid
of the invention can be constructed using chemical synthesis and
enzymatic ligation reactions using procedures known in the art. For
example, an antisense nucleic acid (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense
and sense nucleic acids, e.g., phosphorothioate derivatives and
acridine substituted nucleotides can be used. Examples of modified
nucleotides which can be used to generate the antisense nucleic
acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, 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-isopentenyladenine,
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. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest).
[1674] In yet another embodiment, the PLD 55092 nucleic acid
molecules of the present invention can be modified at the base
moiety, sugar moiety or phosphate backbone to improve, e.g., the
stability, hybridization, or solubility of the molecule. For
example, the deoxyribose phosphate backbone of the nucleic acid
molecules can be modified to generate peptide nucleic acids (see
Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1):
5-23). As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics, e.g., DNA mimics, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl.
Acad. Sci. 93: 14670-675.
[1675] PNAs of PLD 55092 nucleic acid molecules can be used in
therapeutic and diagnostic applications. For example, PNAs can be
used as antisense or antigene agents for sequence-specific
modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs
of PLD 55092 nucleic acid molecules can also be used in the
analysis of single base pair mutations in a gene, (e.g., by
PNA-directed PCR clamping); as `artificial restriction enzymes`
when used in combination with other enzymes, (e.g., S1 nucleases
(Hyrup B. (1996) supra)); or as probes or primers for DNA
sequencing or hybridization (Hyrup B. et al. (1996) supra;
Perry-O'Keefe supra).
[1676] In another embodiment, PNAs of PLD 55092 can be modified,
(e.g., to enhance their stability or cellular uptake), by attaching
lipophilic or other helper groups to PNA, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of
drug delivery known in the art. For example, PNA-DNA chimeras of
PLD 55092 nucleic acid molecules can be generated which may combine
the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, (e.g., RNase H and DNA polymerases), to
interact with the DNA portion while the PNA portion would provide
high binding affinity and specificity. PNA-DNA chimeras can be
linked using linkers of appropriate lengths selected in terms of
base stacking, number of bonds between the nucleobases, and
orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA
chimeras can be performed as described in Hyrup B. (1996) supra and
Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For
example, a DNA chain can be synthesized on a solid support using
standard phosphoramidite coupling chemistry and modified nucleoside
analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite, can be used as a between the PNA and the 5' end of
DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA
monomers are then coupled in a stepwise manner to produce a
chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn
P. J. et al. (1996) supra). Alternatively, chimeric molecules can
be synthesized with a 5' DNA segment and a 3' PNA segment
(Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Let. 5:
1119-11124).
[1677] In other embodiments, 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. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In
addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (See, e.g., Krol et al.
(1988) Bio-Techniques 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, or hybridization-triggered cleavage agent).
Isolated PLD 55092 Proteins and Anti-PLD 55092 Antibodies
[1678] The methods of the invention include the use of isolated PLD
55092 proteins, and biologically active portions thereof, as well
as polypeptide fragments suitable for use as immunogens to raise
anti-PLD 55092 antibodies.
[1679] Isolated proteins used in the methods of the present
invention, preferably PLD 55092 proteins, have an amino acid
sequence sufficiently identical to the amino acid sequence of SEQ
ID NO:15, or are encoded by a nucleotide sequence sufficiently
identical to SEQ ID NO:14. As used herein, the term "sufficiently
identical" refers to a first amino acid or nucleotide sequence
which contains a sufficient or minimum number of identical or
equivalent (e.g., an amino acid residue which has a similar side
chain) amino acid residues or nucleotides to a second amino acid or
nucleotide sequence such that the first and second amino acid or
nucleotide sequences share common structural domains or motifs
and/or a common functional activity. For example, amino acid or
nucleotide sequences which share common structural domains have at
least 30%, 40%, or 50% homology, preferably 60% homology, more
preferably 70%-80%, and even more preferably 90-95% homology across
the amino acid sequences of the domains and contain at least one
and preferably two structural domains or motifs, are defined herein
as sufficiently identical. Furthermore, amino acid or nucleotide
sequences which share at least 30%, 40%, or 50%, preferably 60%,
more preferably 70-80%, or 90-95% homology and share a common
functional activity are defined herein as sufficiently
identical.
[1680] As used interchangeably herein, a "PLD 55092 activity",
"biological activity of PLD 55092" or "functional activity of PLD
55092", refers to an activity exerted by a PLD 55092 protein,
polypeptide or nucleic acid molecule on a PLD 55092 responsive cell
(e.g., a neuronal cell) or tissue (e.g., brain), or on a PLD 55092
substrate, as determined in vivo, or in vitro, according to
standard techniques. In one embodiment, a PLD 55092 activity is a
direct activity, such as an association with a PLD 55092 target
molecule. As used herein, a "target molecule" or "binding partner"
is a molecule with which a PLD 55092 protein binds or interacts in
nature, such that PLD 55092-mediated function is achieved. A PLD
55092 target molecule can be a non-PLD 55092 molecule or a PLD
55092 protein or polypeptide of the present invention. In an
exemplary embodiment, a PLD 55092 target molecule is a PLD 55092
substrate (e.g., a phospholipid). Alternatively, a PLD 55092
activity is an indirect activity, such as a cellular signaling
activity mediated by interaction of the PLD 55092 protein with a
PLD 55092 substrate. Preferably, a PLD 55092 activity is the
ability to act as a signal transduction molecule and to modulate
cellular proliferation, differentiation and/or migration
mechanisms. In another embodiment, a PLD 55092 activity is the
ability to modulate lipid metabolism, membrane vesicular
trafficking and/or membrane homeostasis. In yet another embodiment,
a PLD 55092 activity is the ability to modulate virus replication,
assembly, maturation and transmission. Accordingly, another
embodiment of the invention features isolated PLD 55092 proteins
and polypeptides having a PLD 55092 activity.
[1681] In one embodiment, native PLD 55092 proteins can be isolated
from cells or tissue sources by an appropriate purification scheme
using standard protein purification techniques. In another
embodiment, PLD 55092 proteins are produced by recombinant DNA
techniques. Alternative to recombinant expression, a PLD 55092
protein or polypeptide can be synthesized chemically using standard
peptide synthesis techniques.
[1682] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the PLD 55092 protein is derived, or substantially free from
chemical precursors or other chemicals when chemically synthesized.
The language "substantially free of cellular material" includes
preparations of PLD 55092 protein in which the protein is separated
from cellular components of the cells from which it is isolated or
recombinantly produced. In one embodiment, the language
"substantially free of cellular material" includes preparations of
PLD 55092 protein having less than about 30% (by dry weight) of
non-PLD 55092 protein (also referred to herein as a "contaminating
protein"), more preferably less than about 20% of non-PLD 55092
protein, still more preferably less than about 10% of non-PLD 55092
protein, and most preferably less than about 5% non-PLD 55092
protein. When the PLD 55092 protein or biologically active portion
thereof is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
protein preparation.
[1683] The language "substantially free of chemical precursors or
other chemicals" includes preparations of PLD 55092 protein in
which the protein is separated from chemical precursors or other
chemicals which are involved in the synthesis of the protein. In
one embodiment, the language "substantially free of chemical
precursors or other chemicals" includes preparations of PLD 55092
protein having less than about 30% (by dry weight) of chemical
precursors or non-PLD 55092 chemicals, more preferably less than
about 20% chemical precursors or non-PLD 55092 chemicals, still
more preferably less than about 10% chemical precursors or non-PLD
55092 chemicals, and most preferably less than about 5% chemical
precursors or non-PLD 55092 chemicals.
[1684] As used herein, a "biologically active portion" of a PLD
55092 protein includes a fragment of a PLD 55092 protein which
participates in an interaction between a PLD 55092 molecule and a
non-PLD 55092 molecule. Biologically active portions of a PLD 55092
protein include peptides comprising amino acid sequences
sufficiently identical to or derived from the amino acid sequence
of the PLD 55092 protein, e.g., the amino acid sequence shown in
SEQ ID NO:15, which include less amino acids than the full length
PLD 55092 protein, and exhibit at least one activity of a PLD 55092
protein. Typically, biologically active portions comprise a domain
or motif with at least one activity of the PLD 55092 protein, e.g.,
modulating cell signaling mechanisms, lipid homeostasis, vesicle
trafficking, and/or cell proliferation, differentiation and
migration mechanisms. A biologically active portion of a PLD 55092
protein can be a polypeptide which is, for example, 10, 25, 50,
100, 200, or more amino acids in length. Biologically active
portions of a PLD 55092 protein can be used as targets for
developing agents which modulate a PLD 55092 mediated activity,
e.g., a cell signaling mechanism, lipid homeostasis mechanism,
vesicle trafficking mechanism, and/or a cell proliferation,
differentiation and migration mechanism. A biologically active
portion of a PLD 55092 protein comprises a protein in which regions
of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of a native PLD 55092 protein.
[1685] In a preferred embodiment, the PLD 55092 protein has an
amino acid sequence shown in SEQ ID NO:15. In other embodiments,
the PLD 55092 protein is substantially identical to SEQ ID NO:15,
and retains the functional activity of the protein of SEQ ID NO:15,
yet differs in amino acid sequence due to natural allelic variation
or mutagenesis, as described in detail in subsection I above.
Accordingly, in another embodiment, the PLD 55092 protein is a
protein which comprises an amino acid sequence at least about 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID
NO:15.
[1686] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-identical
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence (e.g., when aligning a second sequence to
the PLD 55092 amino acid sequence of SEQ ID NO:15 having 506 amino
acid residues, at least 152, preferably at least 202, more
preferably at least 253, even more preferably at least 304, and
even more preferably at least 354, 405 or 455 amino acid residues
are aligned). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position (as used herein amino acid or nucleic acid "identity" is
equivalent to amino acid or nucleic acid "homology"). The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences, taking into account
the number of gaps, and the length of each gap, which need to be
introduced for optimal alignment of the two sequences.
[1687] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm
which has been incorporated into the GAP program in the GCG
software package, using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package, using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. A preferred, non-limiting
example of parameters to be used in conjunction with the GAP
program include a Blosum 62 scoring matrix with a gap penalty of
12, a gap extend penalty of 4, and a frameshift gap penalty of
5.
[1688] In another embodiment, the percent identity between two
amino acid or nucleotide sequences is determined using the
algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,
4:11-17 (1988)) which has been incorporated into the ALIGN program
(version 2.0 or version 2.0U), using a PAM120 weight residue table,
a gap length penalty of 12 and a gap penalty of 4.
[1689] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against public databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can
be performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to PLD 55092 nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=100, wordlength=3 to obtain amino
acid sequences homologous to PLD 55092 protein molecules of the
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) can be used.
[1690] The methods of the invention may also use PLD 55092 chimeric
or fusion proteins. As used herein, a PLD 55092 "chimeric protein"
or "fusion protein" comprises a PLD 55092 polypeptide operatively
linked to a non-PLD 55092 polypeptide. A "PLD 55092 polypeptide"
refers to a polypeptide having an amino acid sequence corresponding
to PLD 55092, whereas a "non-PLD 55092 polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous to the PLD 55092
protein, e.g., a protein which is different from the PLD 55092
protein and which is derived from the same or a different organism.
Within a PLD 55092 fusion protein the PLD 55092 polypeptide can
correspond to all or a portion of a PLD 55092 protein. In a
preferred embodiment, a PLD 55092 fusion protein comprises at least
one biologically active portion of a PLD 55092 protein. In another
preferred embodiment, a PLD 55092 fusion protein comprises at least
two biologically active portions of a PLD 55092 protein. Within the
fusion protein, the term "operatively linked" is intended to
indicate that the PLD 55092 polypeptide and the non-PLD 55092
polypeptide are fused in-frame to each other. The non-PLD 55092
polypeptide can be fused to the N-terminus or C-terminus of the PLD
55092 polypeptide.
[1691] For example, in one embodiment, the fusion protein is a
GST-PLD 55092 fusion protein in which the PLD 55092 sequences are
fused to the C-terminus of the GST sequences. Such fusion proteins
can facilitate the purification of recombinant PLD 55092.
[1692] In another embodiment, the fusion protein is a PLD 55092
protein containing a heterologous signal sequence at its
N-terminus. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of PLD 55092 can be increased through
use of a heterologous signal sequence.
[1693] The PLD 55092 fusion proteins used in the methods of the
invention can be incorporated into pharmaceutical compositions and
administered to a subject in vivo. The PLD 55092 fusion proteins
can be used to affect the bioavailability of a PLD 55092 substrate.
Use of PLD 55092 fusion proteins may be useful therapeutically for
the treatment of disorders caused by, for example, (i) aberrant
modification or mutation of a gene encoding a PLD 55092 protein;
(ii) mis-regulation of the PLD 55092 gene; and (iii) aberrant
post-translational modification of a PLD 55092 protein. In one
embodiment, a PLD 55092 fusion protein may be used to treat a viral
disease. In another embodiment, a PLD 55092 fusion protein may be
used to treat a pain disorder. In a further embodiment, a PLD 55092
fusion protein may be used to treat a cellular proliferation,
growth, differentiation, or migration disorder.
[1694] Moreover, the PLD 55092-fusion proteins of the invention can
be used as immunogens to produce anti-PLD 55092 antibodies in a
subject, to purify PLD 55092 ligands and in screening assays to
identify molecules which inhibit the interaction of PLD 55092 with
a PLD 55092 substrate.
[1695] Preferably, a PLD 55092 chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A PLD 55092-encoding nucleic acid can be cloned
into such an expression vector such that the fusion moiety is
linked in-frame to the PLD 55092 protein.
[1696] The methods of the present invention may also include the
use of variants of the PLD 55092 protein which function as either
PLD 55092 agonists (mimetics) or as PLD 55092 antagonists. Variants
of the PLD 55092 protein can be generated by mutagenesis, e.g.,
discrete point mutation or truncation of a PLD 55092 protein. An
agonist of the PLD 55092 protein can retain substantially the same,
or a subset, of the biological activities of the naturally
occurring form of a PLD 55092 protein. An antagonist of a PLD 55092
protein can inhibit one or more of the activities of the naturally
occurring form of the PLD 55092 protein by, for example,
competitively modulating a PLD 55092-mediated activity of a PLD
55092 protein. Thus, specific biological effects can be elicited by
treatment with a variant of limited function. In one embodiment,
treatment of a subject with a variant having a subset of the
biological activities of the naturally occurring form of the
protein has fewer side effects in a subject relative to treatment
with the naturally occurring form of the PLD 55092 protein.
[1697] In one embodiment, variants of a PLD 55092 protein which
function as either PLD 55092 agonists (mimetics) or as PLD 55092
antagonists can be identified by screening combinatorial libraries
of mutants, e.g., truncation mutants, of a PLD 55092 protein for
PLD 55092 protein agonist or antagonist activity. In one
embodiment, a variegated library of PLD 55092 variants is generated
by combinatorial mutagenesis at the nucleic acid level and is
encoded by a variegated gene library. A variegated library of PLD
55092 variants can be produced by, for example, enzymatically
ligating a mixture of synthetic oligonucleotides into gene
sequences such that a degenerate set of potential PLD 55092
sequences is expressible as individual polypeptides, or
alternatively, as a set of larger fusion proteins (e.g., for phage
display) containing the set of PLD 55092 sequences therein. There
are a variety of methods which can be used to produce libraries of
potential PLD 55092 variants from a degenerate oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be
performed in an automatic DNA synthesizer, and the synthetic gene
then ligated into an appropriate expression vector. Use of a
degenerate set of genes allows for the provision, in one mixture,
of all of the sequences encoding the desired set of potential PLD
55092 sequences. Methods for synthesizing degenerate
oligonucleotides are known in the art (see, e.g., Narang, S. A.
(1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.
53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)
Nucleic Acid Res. 11:477.
[1698] In addition, libraries of fragments of a PLD 55092 protein
coding sequence can be used to generate a variegated population of
PLD 55092 fragments for screening and subsequent selection of
variants of a PLD 55092 protein. In one embodiment, a library of
coding sequence fragments can be generated by treating a double
stranded PCR fragment of a PLD 55092 coding sequence with a
nuclease under conditions wherein nicking occurs only about once
per molecule, denaturing the double stranded DNA, renaturing the
DNA to form double stranded DNA which can include sense/antisense
pairs from different nicked products, removing single stranded
portions from reformed duplexes by treatment with S1 nuclease, and
ligating the resulting fragment library into an expression vector.
By this method, an expression library can be derived which encodes
N-terminal, C-terminal and internal fragments of various sizes of
the PLD 55092 protein.
[1699] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of PLD 55092 proteins. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recrusive ensemble mutagenesis
(REM), a new technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify PLD 55092 variants (Arkin and Yourvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.
(1993) Protein Engineering 6(3):327-331).
[1700] In one embodiment, cell based assays can be exploited to
analyze a variegated PLD 55092 library. For example, a library of
expression vectors can be transfected into a cell line, e.g., a
neuronal cell line, which ordinarily responds to a ligand in a
particular PLD 55092-dependent manner. The transfected cells are
then contacted with a ligand and the effect of expression of the
mutant on signaling by PLD 55092 can be detected, e.g., by
monitoring the generation of an intracellular second messenger
(e.g., phosphatidic acid, PIP.sub.2, or diacylglycerol), vesicle
trafficking, cell proliferation, differentiation and/or migration,
or the activity of a PLD 55092-regulated transcription factor.
Plasmid DNA can then be recovered from the cells which score for
inhibition, or alternatively, potentiation of signaling by PLD
55092, and the individual clones further characterized.
[1701] An isolated PLD 55092 protein, or a portion or fragment
thereof, can be used as an immunogen to generate antibodies that
bind PLD 55092 using standard techniques for polyclonal and
monoclonal antibody preparation. A full-length PLD 55092 protein
can be used or, alternatively, the invention provides antigenic
peptide fragments of PLD 55092 for use as immunogens. The antigenic
peptide of PLD 55092 comprises at least 8 amino acid residues of
the amino acid sequence shown in SEQ ID NO:15 and encompasses an
epitope of PLD 55092 such that an antibody raised against the
peptide forms a specific immune complex with PLD 55092. Preferably,
the antigenic peptide comprises at least 10 amino acid residues,
more preferably at least 15 amino acid residues, even more
preferably at least 20 amino acid residues, and most preferably at
least 30 amino acid residues. Preferred epitopes encompassed by the
antigenic peptide are regions of PLD 55092 that are located on the
surface of the protein, e.g., hydrophilic regions, as well as
regions with high antigenicity.
[1702] A PLD 55092 immunogen typically is used to prepare
antibodies by immunizing a suitable subject, (e.g., rabbit, goat,
mouse or other mammal) with the immunogen. An appropriate
immunogenic preparation can contain, for example, recombinantly
expressed PLD 55092 protein or a chemically synthesized PLD 55092
polypeptide. The preparation can further include an adjuvant, such
as Freund's complete or incomplete adjuvant, or similar
immunostimulatory agent. Immunization of a suitable subject with an
immunogenic PLD 55092 preparation induces a polyclonal anti-PLD
55092 antibody response.
[1703] Accordingly, another aspect of the invention pertains to the
use of anti-PLD 55092 antibodies. The term "antibody" as used
herein refers to immunoglobulin molecules and immunologically
active portions of immunoglobulin molecules, i.e., molecules that
contain an antigen binding site which specifically binds
(immunoreacts with) an antigen, such as PLD 55092. Examples of
immunologically active portions of immunoglobulin molecules include
F(ab) and F(ab').sub.2 fragments which can be generated by treating
the antibody with an enzyme such as pepsin. The invention provides
polyclonal and monoclonal antibodies that bind PLD 55092. The term
"monoclonal antibody" or "monoclonal antibody composition", as used
herein, refers to a population of antibody molecules that contain
only one species of an antigen binding site capable of
immunoreacting with a particular epitope of PLD 55092. A monoclonal
antibody composition thus typically displays a single binding
affinity for a particular PLD 55092 protein with which it
immunoreacts.
[1704] Polyclonal anti-PLD 55092 antibodies can be prepared as
described above by immunizing a suitable subject with a PLD 55092
immunogen. The anti-PLD 55092 antibody titer in the immunized
subject can be monitored over time by standard techniques, such as
with an enzyme linked immunosorbent assay (ELISA) using immobilized
PLD 55092. If desired, the antibody molecules directed against PLD
55092 can be isolated from the mammal (e.g., from the blood) and
further purified by well known techniques, such as protein A
chromatography to obtain the IgG fraction. At an appropriate time
after immunization, e.g., when the anti-PLD 55092 antibody titers
are highest, antibody-producing cells can be obtained from the
subject and used to prepare monoclonal antibodies by standard
techniques, such as the hybridoma technique originally described by
Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et
al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol.
Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA
76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the
more recent human B cell hybridoma technique (Kozbor et al. (1983)
Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.
(1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
monoclonal antibody hybridomas is well known (see generally R. H.
Kenneth, in Monoclonal Antibodies: A New Dimension In Biological
Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A.
Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al.
(1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell
line (typically a myeloma) is fused to lymphocytes (typically
splenocytes) from a mammal immunized with a PLD 55092 immunogen as
described above, and the culture supernatants of the resulting
hybridoma cells are screened to identify a hybridoma producing a
monoclonal antibody that binds PLD 55092.
[1705] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-PLD 55092 monoclonal antibody (see,
e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al.
Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited
supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the
ordinarily skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines can be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from ATCC. Typically, HAT-sensitive mouse myeloma cells
are fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using
HAT medium, which kills unfused and unproductively fused myeloma
cells (unfused splenocytes die after several days because they are
not transformed). Hybridoma cells producing a monoclonal antibody
of the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind PLD 55092, e.g., using a
standard ELISA assay.
[1706] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-PLD 55092 antibody can be identified
and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with PLD 55092 to thereby isolate immunoglobulin library members
that bind PLD 55092. Kits for generating and screening phage
display libraries are commercially available (e.g., the Pharmacia
Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene SurfZAP.TM. Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
library can be found in, for example, Ladner et al. U.S. Pat. No.
5,223,409; Kang et al. PCT International Publication No. WO
92/18619; Dower et al. PCT International Publication No. WO
91/17271; Winter et al. PCT International Publication WO 92/20791;
Markland et al. PCT International Publication No. WO 92/15679;
Breitling et al. PCT International Publication WO 93/01288;
McCafferty et al. PCT International Publication No. WO 92/01047;
Garrard et al. PCT International Publication No. WO 92/09690;
Ladner et al. PCT International Publication No. WO 90/02809; Fuchs
et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.
Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins
et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991)
Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA
89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377;
Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al.
(1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et
al. Nature (1990) 348:552-554.
[1707] Additionally, recombinant anti-PLD 55092 antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, can also be used in the methods of the
present invention. Such chimeric and humanized monoclonal
antibodies can be produced by recombinant DNA techniques known in
the art, for example using methods described in Robinson et al.
International Application No. PCT/US86/02269; Akira, et al.
European Patent Application 184,187; Taniguchi, M., European Patent
Application 171,496; Morrison et al. European Patent Application
173,494; Neuberger et al. PCT International Publication No. WO
86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.
European Patent Application 125,023; Better et al. (1988) Science
240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA
84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et
al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.
(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature
314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.
80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et
al. (1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539;
Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[1708] An anti-PLD 55092 antibody (e.g., monoclonal antibody) can
be used to isolate PLD 55092 by standard techniques, such as
affinity chromatography or immunoprecipitation. An anti-PLD 55092
antibody can facilitate the purification of natural PLD 55092 from
cells and of recombinantly produced PLD 55092 expressed in host
cells. Moreover, an anti-PLD 55092 antibody can be used to detect
PLD 55092 protein (e.g., in a cellular lysate or cell supernatant)
in order to evaluate the abundance and pattern of expression of the
PLD 55092 protein. Anti-PLD 55092 antibodies can be used
diagnostically to monitor protein levels in tissue as part of a
clinical testing procedure, e.g., to, for example, determine the
efficacy of a given treatment regimen. Detection can be facilitated
by coupling (i.e., physically linking) the antibody to a detectable
substance. Examples of detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[1709] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the Sequence Listing, are
incorporated herein by reference.
EXAMPLES
Example 1
Regulation of PLD 55092 Expression in Virus Infected Tissues
[1710] The expression of PLD 55092 in virus infected human tissues
was analyzed by TaqMan.RTM. Quantitative Polymerase Chain
Reaction.
[1711] Probes were designed by PrimerExpress software (PE
Biosystems) based on the sequence of the PLD 55092 gene. Each PLD
55092 gene probe was labeled using FAM (6-carboxyfluorescein), and
the .beta.2-microglobulin reference probe was labeled with a
different fluorescent dye, VIC. The differential labeling of the
target gene and internal reference gene, thus, enabled measurement
in the same well. Forward and reverse primers and probes for both
the .beta.2-microglobulin and the target gene were added to the
TaqMan.RTM. Universal PCR Master Mix (PE Applied Biosystems).
Although the final concentration of primer and probe could vary,
each was internally consistent within a given experiment. A typical
experiment contained 200 nM of forward and reverse primers plus 100
nM of probe for .beta.-2 microglobulin and 600 nM of forward and
reverse primers plus 200 nM of probe for the target gene. TaqMan
matrix experiments were carried out using an ABI PRISM 7700
Sequence Detection System (PE Applied Biosystems). The thermal
cycler conditions were as follows: hold for 2 minutes at 50.degree.
C. and 10 minutes at 95.degree. C., followed by two-step PCR for 40
cycles of 95.degree. C. for 15 seconds followed by 60.degree. C.
for 1 minute.
[1712] A comparative Ct method was used for the relative
quantitation of gene expression. The following method was used to
quantitatively calculate PLD 55092 gene expression in the various
samples relative to .beta.-2 microglobulin expression in the same
sample. The threshold cycle (Ct) value was defined as the cycle at
which a statistically significant increase in fluorescence is
detected. A lower Ct value was indicative of a higher mRNA
concentration. The Ct value of the PLD 55092 gene was normalized by
subtracting the Ct value of the .beta.-2 microglobulin gene to
obtain a .DELTA.Ct value using the following formula:
.DELTA.Ct=Ct.sub.55092-Ct.sub..beta.-2 microglobulin
[1713] Expression was then calibrated against a cDNA control sample
containing no template. The .DELTA.Ct value for the calibrator
sample was then subtracted from .DELTA.Ct for each tissue sample
according to the following formula:
.DELTA..DELTA.Ct=.DELTA.Ct-.sub.sample-.DELTA.Ct-.sub.calibrator
[1714] Relative expression was then calculated using the arithmetic
formula given by 2.sup.-.DELTA..DELTA.Ct.
[1715] As demonstrated using this TaqMan technology, PLD 55092 gene
expression was up-regulated in hepatitis B and C virus infected
human livers as compared to control normal human liver samples, in
hepatitis B virus infected tissue culture cells, and in herpes
simplex virus infected human ganglia, but not in herpes simplex
virus infected human neuroblastoma cells. There was no induction in
resting or activated T cells suggesting that induction is not an
immune response.
[1716] Thus, modulation of PLD 55092 activity and/or PLD 55092
mediated signal transduction may be of therapeutic importance in
viral infection.
Example 2
PLD 55092 Expression in Human and Mouse Tissues
[1717] The expression of PLD 55092 in normal or uninfected human
tissues obtained from pathology phase I of human biopsy and autopsy
materials was analyzed by TaqMan.RTM. Quantitative Polymerase Chain
Reaction, as described above.
[1718] PLD 55092 was strongly expressed in the brain cortex and
hypothalamus, as well as in glioblastoma cells. PLD 55092 was also
expressed in dorsal root ganglia, the spinal cord, and tonsil
cells, and expressed at lower levels in prostate, lymph node, and
bone marrow mononuclear cells. There was no induction in resting or
activated T cells.
VII. METHODS AND COMPOSITIONS FOR TREATING CARDIOVASCULAR DISEASE
USING 10218
Background of the Invention
[1719] 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 peripheral vascular disease resulting in significant
disability and limb loss, and thereby the principle cause of death
in the United States.
[1720] Atherosclerosis is a complex disease involving many cell
types and molecular factors (described in, for example, 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. Injury or dysfunction of the vascular
endothelium is a common feature of many conditions that predispose
an individual to accelerated development of atherosclerotic
cardiovascular disease. 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.
[1721] 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 LDLs
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.
[1722] 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 smooth muscle cells 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.
[1723] Such plaque may totally or partially block blood flow
through a blood vessel leading to a heart attack or stroke. Plaque
can also weaken the arterial wall, resulting in an aneurysm.
Moreover, occlusion of the blood vessels caused by plaques restrict
the flow of blood, resulting in ischemia. Ischemia is a condition
characterized by a lack of oxygen supply in tissues of organs due
to inadequate perfusion. Such inadequate perfusion can have a
number of natural causes, including atherosclerotic or restenotic
lesions, anemia, or stroke. 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.
[1724] The P2X receptors are a family of ligand-gated membrane ion
channels activated by the binding of extracellular adenosine
5'-triphosphate (ATP). Seven different P2X receptor subunit cDNAs
have been identified (P2X.sub.1, P2X.sub.2, P2X.sub.3, P2.times.4,
P2X.sub.5, P2.times.6, and P2X.sub.7) (MacKenzie, et al. (1999)
Ann. N.Y. Acad. Sci. 868:716-729). They are characterized by two
transmembrane domains with a large extracellular loop where 10
cysteine residues are preserved; and by intracellular N- and
C-terminals (Burnstock (2000) British Journal of Anesthesia
84:476-880). P2X receptors are widely distributed in various
tissues of mammals, including smooth muscle of the urinary bladder
and arteries, kidney, pancreas, lung, cardiac myocytes, sensory and
sympathetic ganglia, brain and spinal cord, and each subtype seems
to be preferentially expressed in different tissue (Yamamoto, et
al. (2000) Am. J. Physiol. Heart Circ. Physiol. 279:H285-H292).
[1725] The human P2X.sub.4 gene was cloned from the brain and forms
functional homomeric ATP-activated channels when expressed in
heterologous cellular systems (Garcia-Guzman, et al. (1997)
Molecular Pharmacology 51:109-118). This receptor has been found to
be expressed in human endothelial cells, and is involved in
ATP-induced Ca.sup.2+ influx in endothelial cells (Yamamoto, et al.
(2000) Am. J. Physiol. Heart Circ. Physiol. 279:H285-H292).
[1726] Calcium concentration plays a role in cardiovascular
diseases, including atherosclerosis. Calcium channel blockers (CCB)
have been used to effectively modulate high blood pressure. It has
been postulated that CCB's could also be used to avoid calcium
deposits in arterial walls, which is one of the main components of
atherosclerotic plaques (Perez (2000) J. Hum. Hypertens. 14 Suppl
1:S96-9). Intracellular calcium levels have also been correlated
with late phase platelet aggregation and formation of a hemostaic
plug, which has been implicated in the pathogenesis of
atherosclerosis (Covic, et al. (2000) Biochemistry 39:5458-5467).
Recent studies also have focused on the role of free radicals on
calcium signaling. Vascular calcium signaling is altered by oxidant
stress in ischemia-related disease states (Lounsbury et al. (2000)
Free Radical. Biol. Med. 28:1362). Extracellular calcium has been
shown to function as an ionic chemokinetic agent capable of
modulating the innate immune response in vivo and in vitro by
direct and indirect actions on monocytic cells. Therefore, calcium
deposition may be both a consequence of and/or a cause of chronic
inflammatory changes at sites of injury, infection, and
atherosclerosis (Olszak, et al. (2000) J. Clin. Invest.
105:1299-305).
Summary of the Invention
[1727] The present invention provides methods and compositions for
the diagnosis and treatment of cardiovascular disease, including,
but not limited to, atherosclerosis. The present invention is
based, at least in part, on the discovery that the P2X4 gene
(referred to herein as "10218"), is differentially expressed in
macrophages stimulated by highly atherosclerotic agents, e.g.,
interferon gamma (IFN.gamma.) and CD40L, and in atherosclerotic
lesions as compared to non-lesioned vessels in an animal model of
atherosclerosis and normal vessels in wild-type animals. Moreover,
10218 is expressed in highly vascularized organs and blood vessels.
Accordingly, the present invention provides methods for the
diagnosis and treatment of cardiovascular disease including, but
not limited to, atherosclerosis.
[1728] In one aspect, the present invention provides methods for
identifying a compound capable of treating a cardiovascular
disease, e.g., atherosclerosis, characterized by aberrant 10218
nucleic acid expression or 10218 polypeptide activity by assaying
the ability of the compound or agent to modulate 10218 expression
or activity. In one embodiment, the identified compound inhibits
10218 expression or activity.
[1729] In another aspect, the present invention provides methods
for identifying a subject suffering from a cardiovascular disease,
e.g., atherosclerosis, comprising obtaining a biological sample
from the subject, and detecting in the sample aberrant or abnormal
10218 expression or activity, thereby identifying a subject
suffering from a cardiovascular disease.
[1730] In yet another embodiment, the present invention provides
methods for identifying a subject having a cardiovascular disease,
e.g., atherosclerosis, or at risk for developing a cardiovascular
disease comprising contacting a sample obtained from the subject
containing nucleic acid molecules with a hybridization probe
comprising at least 25 contiguous nucleotides of SEQ ID NO:16 and
detecting the presence of a nucleic acid molecule in the sample
that hybridizes to the probe. In one embodiment, the hybridization
probe is detectably labeled. In another embodiment, the sample is
subjected to agarose gel electrophoresis and southern blotting
prior to contacting with the hybridization probe. In yet another
embodiment, the sample is subjected to agarose gel electrophoresis
and northern blotting prior to contacting with the hybridization
probe. In a further embodiment, the detecting is by in situ
hybridization.
[1731] In yet another aspect, the present invention provides
methods for treating a subject having a cardiovascular disease,
e.g., atherosclerosis, characterized by aberrant 10281 polypeptide
activity or aberrant 10281 nucleic acid expression by administering
to the subject a 10281 modulator, for example, a small molecule, an
antibody specific for 10281, a 10281 polypeptide, a fragment of a
10281 polypeptide, a 10281 nucleic acid molecule, a fragment of a
10281 nucleic acid molecule, an antisense 10281 nucleic acid
molecule, and a ribozyme. In one embodiment, the 10281 modulator is
administered in a pharmaceutically acceptable formulation. In a
further embodiment, the 10281 modulator is administered using a
gene therapy vector. In another embodiment, the 10281 polypeptide
comprises the amino acid sequence of SEQ ID NO:17, or a fragment
thereof or an amino acid sequence which is at least 90 percent
identical to the amino acid sequence of SEQ ID NO:17, where the
percent identity is calculated using the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4. In yet another
embodiment, the 10281 polypeptide is an isolated naturally
occurring allelic variant of a polypeptide consisting of the amino
acid sequence of SEQ ID NO:17, wherein the polypeptide is encoded
by a nucleic acid molecule which hybridizes to a complement of a
nucleic acid molecule consisting of SEQ ID NO:16 at 6.times.SSC at
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 65.degree. C. In still another embodiment, the 10281
nucleic acid molecule comprises the nucleotide sequence of SEQ ID
NO:16, or a fragment thereof.
[1732] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
Detailed Description of the Invention
[1733] The present invention provides methods and compositions for
the diagnosis and treatment of cardiovascular disease, including
but not limited to, atherosclerosis, ischemia/reperfusion injury,
hypertension, restenosis, arterial inflammation, and endothelial
cell disorders. "Treatment", as used herein, is defined as the
application or administration of a therapeutic agent to a patient,
or application or administration of a therapeutic agent to an
isolated tissue or cell line from a patient, who has a disease or
disorder, a symptom of disease or disorder or a predisposition
toward a disease or disorder, with the purpose of curing, healing,
alleviating, relieving, altering, remedying, ameliorating,
improving or affecting the disease or disorder, the symptoms of
disease or disorder or the predisposition toward a disease or
disorder. A therapeutic agent includes, but is not limited to, the
small molecules, peptides, antibodies, ribozymes and antisense
oligonucleotides described herein.
[1734] The present invention is based, at least in part, on the
discovery that the P2X.sub.4 nucleic acid and protein molecules
(referred to herein as "10218" nucleic acid and protein molecules),
are differentially expressed in cardiovascular disease states
relative to their expression in normal, or non-cardiovascular
disease states, as well as in macrophages stimulated with highly
atherogenic cytokines, e.g., interferon gamma (IFN.gamma.) and
CD40L. 10218 nucleic acid and protein molecules are also expressed
in highly vascularized organs, e.g., heart, kidney, liver, and
skeletal muscle, and blood vessels, e.g., arteries and veins. The
10281 modulators identified according to the methods of the
invention can be used to modulate (e.g., inhibit, treat, or
prevent) or diagnose cardiovascular disease, including, but not
limited to, atherosclerosis.
[1735] "Differential expression", as used herein, includes both
quantitative as well as qualitative differences in the temporal
and/or tissue expression pattern of a gene. Thus, a differentially
expressed gene may have its expression activated or inactivated in
normal versus cardiovascular disease conditions (for example, in an
experimental cardiovascular disease system such as in an animal
model for atherosclerosis). 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, e.g., quantitative PCR,
Northern analysis, subtractive hybridization. The expression
pattern of a differentially expressed gene may be used as part of a
prognostic or diagnostic cardiovascular disease, e.g.,
artherosclerosis, evaluation, or may be used in methods for
identifying compounds useful for the treatment of cardiovascular
disease, e.g., atherosclerosis. In addition, a differentially
expressed gene involved in cardiovascular disease may represent a
target gene such that modulation of the level of target gene
expression or of target gene product activity may act to ameliorate
a cardiovascular disease condition, e.g., atherosclerosis.
Compounds that modulate target gene expression or activity of the
target gene product can be used in the treatment of cardiovascular
disease. Although the 10218 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 cell
processes.
[1736] The 10218 molecules used in the methods of the invention are
ligand-gated membrane ion channels which are activated by the
binding of extracellular adenosine 5'-triphosphate (ATP). They are
involved in ATP-induced Ca.sup.2+ influx in endothelial cells
(Yamamoto, et al. (2000) Am. J. Physiol. Heart Circ. Physiol.
279:H285-H292). Calcium concentration is postulated to be involved
in cardiovascular disease, including, but not limited to
atherosclerosis. For example, calcium is a major component of
atherosclerotic plaques and is also implicated in high blood
pressure (Perez (2000) J. Hum. Hypertens. 14 Suppl 1:S96-9).
Calcium is also involved in late phase platelet aggregation and
formation (Covic, et al. (2000) Biochemistry 39:5458-5467) and
calcium deposition may be both a consequence and/or a cause of
chronic inflammatory changes at atherosclerotic sites (Olszak, et
al. (2000) J. Clin. Invest. 105:1299-305). Therefore, given the
differential expression of the 10218 molecules in cardiovascular
disease states and in macrophages stimulated with highly
atherogenic cytokines, as well as their expression in vessels and
arteries, modulation of the 10218 molecules may modulate, e.g.,
inhibit, treat, or prevent, cardiovascular disease, and, in
particular, atherosclerosis.
[1737] As used herein, "cardiovascular disease" or a
"cardiovascular disorder" includes a disease or disorder which
affects the cardiovascular system, e.g., the heart or the blood
vessels. A cardiovascular disease includes disorders such as
atherosclerosis, ischemia reperfusion injury, restenosis, arterial
inflammation, vascular wall remodeling, ventricular remodeling,
rapid ventricular pacing, coronary microembolism, tachycardia,
bradycardia, pressure overload, aortic bending, coronary artery
ligation, vascular heart disease, atrial fibrillation, long-QT
syndrome, congestive heart failure, sinus node dysfunction, angina,
heart failure, hypertension, atrial fibrillation, atrial flutter,
dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial
infarction, coronary artery disease, coronary artery spasm,
ischemic disease, arrhythmia, and cardiovascular developmental
disorders (e.g., arteriovenous malformations, arteriovenous
fistulae, raynaud's syndrome, neurogenic thoracic outlet syndrome,
causalgia/reflex sympathetic dystrophy, hemangioma, aneurysm,
cavernous angioma, aortic valve stenosis, atrial septal defects,
atrioventricular canal, coarctation of the aorta, ebsteins anomaly,
hypoplastic left heart syndrome, interruption of the aortic arch,
mitral valve prolapse, ductus arteriosus, patent foramen ovale,
partial anomalous pulmonary venous return, pulmonary atresia with
ventricular septal defect, pulmonary atresia without ventricular
septal defect, persistance of the fetal circulation, pulmonary
valve stenosis, single ventricle, total anomalous pulmonary venous
return, transposition of the great vessels, tricuspid atresia,
truncus arteriosus, ventricular septal defects). In a preferred
embodiment, a cardiovascular disease is atherosclerosis. A
cardiovascular disease or disorder also includes an endothelial
cell disorder.
[1738] As used herein, an "endothelial cell disorder" includes a
disorder characterized by aberrant, unregulated, or unwanted
endothelial cell activity, e.g., proliferation, migration,
angiogenesis, or vascularization; or aberrant expression of cell
surface adhesion molecules or genes associated with angiogenesis,
e.g., TIE-2, FLT and FLK. Endothelial cell disorders include
tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy,
endometriosis, Grave's disease, ischemic disease (e.g.,
atherosclerosis), and chronic inflammatory diseases (e.g.,
rheumatoid arthritis).
[1739] As used interchangeably herein, "10218 activity,"
"biological activity of 10218" or "functional activity of 10218,"
includes an activity exerted by a 10218 protein, polypeptide or
nucleic acid molecule on a 10218 responsive cell or tissue, e.g.,
endothelial cells or vascular tissue, or on a 10218 protein
substrate, as determined in vivo, or in vitro, according to
standard techniques. 10218 activity can be a direct activity, such
as an association with a 10218-target molecule. As used herein, a
"substrate" or "target molecule" or "binding partner" is a molecule
with which a 10218 protein binds or interacts in nature, e.g. ATP,
such that 10218-mediated function, e.g., modulation of calcium
concentration, is achieved. A 10218 target molecule can be a
non-10218 molecule or a 10218 protein or polypeptide. Examples of
such target molecules include proteins in the same signaling path
as the 10218 protein, e.g., proteins which may function upstream
(including both stimulators and inhibitors of activity) or
downstream of the 10218 protein in a pathway involving regulation
of intercellular or extracellular calcium concentration, e.g.,
calcium influx modulated by ATP binding. Alternatively, a 10218
activity is an indirect activity, such as a cellular signaling
activity mediated by interaction of the 10218 protein with a 10218
target molecule. The biological activities of 10218 are described
herein. For example, the 10218 proteins can have one or more of the
following activities: 1) they bind ATP; 2) they bind calcium; 3)
they modulate intercellular calcium infux in cells, e.g.,
endothelial cells; 4) they modulate cellular migration, e.g.,
monocyte or platelet migration; and 5) they modulate
atherosclerotic lesion formation.
[1740] Various aspects of the invention are described in further
detail in the following subsections:
Screening Assays
[1741] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules (organic or inorganic) or other drugs) which bind to
10218 proteins, have a stimulatory or inhibitory effect on, for
example, 10218 expression or 10218 activity, or have a stimulatory
or inhibitory effect on, for example, the expression or activity of
a 10218 substrate. Compounds identified using the assays described
herein may be useful for treating cardiovascular diseases, e.g.,
atherosclerosis.
[1742] These assays are designed to identify compounds that bind to
a 10218 protein, bind to other intracellular or extracellular
proteins that interact with a 10218 protein, and interfere with the
interaction of the 10218 protein with other intercellular or
extracellular proteins. For example, in the case of the 10218
protein, which is a transmembrane receptor-type protein, such
techniques can identify ligands for such a receptor. A 10218
protein ligand can, for example, be used to ameliorate
cardiovascular diseases, e.g., atherosclerosis,
ischemia/reperfusion, hypertension, restenosis, arterial
inflammation, and endothelial cell disorders. Such compounds may
include, but are not limited to peptides, antibodies, or small
organic or inorganic compounds. Such compounds may also include
other cellular proteins.
[1743] Compounds identified via assays such as those described
herein may be useful, for example, for ameliorating cardiovascular
disease, e.g., athersclerosis. In instances whereby a
cardiovascular disease condition results from an overall lower
level of 10218 gene expression and/or 10218 protein in a cell or
tissue, compounds that interact with the 10218 protein may include
compounds which accentuate or amplify the activity of the bound
10218 protein. Such compounds would bring about an effective
increase in the level of 10218 protein activity, thus ameliorating
symptoms.
[1744] In other instances, mutations within the 10218 gene may
cause aberrant types or excessive amounts of 10218 proteins to be
made which have a deleterious effect that leads to a cardiovascular
disease. Similarly, physiological conditions may cause an excessive
increase in 10218 gene expression leading to a cardiovascular
disease. In such cases, compounds that bind to a 10218 protein may
be identified that inhibit the activity of the 10218 protein.
Assays for testing the effectiveness of compounds identified by
techniques such as those described in this section are discussed
herein.
[1745] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of a
10218 protein or polypeptide or biologically active portion
thereof. In another embodiment, the invention provides assays for
screening candidate or test compounds which bind to or modulate the
activity of a 10218 protein or polypeptide or biologically active
portion thereof. The test compounds of the present invention can be
obtained using any of the numerous approaches in combinatorial
library methods known in the art, including: biological libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
four approaches are applicable to peptide, non-peptide oligomer or
small molecule libraries of compounds (Lam, K. S. (1997) Anticancer
Drug Des. 12:145).
[1746] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med.
Chem. 37:1233.
[1747] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra.).
[1748] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a 10218 protein or biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to modulate 10218 activity is determined. Determining
the ability of the test compound to modulate 10218 activity can be
accomplished by monitoring, for example, intracellular calcium,
IP.sub.3, cAMP, or diacylglycerol concentration, the
phosphorylation profile of intracellular proteins, cell
proliferation and/or migration, gene expression of, for example,
cell surface adhesion molecules or genes associated with
angiogenesis, or the activity of a 10218-regulated transcription
factor. The cell can be of mammalian origin, e.g., an endothelial
cell. In one embodiment, compounds that interact with a 10218
receptor domain can be screened for their ability to function as
ligands, i.e., to bind to the 10218 receptor and modulate a signal
transduction pathway. Identification of 10218 ligands, and
measuring the activity of the ligand-receptor complex, leads to the
identification of modulators (e.g., antagonists) of this
interaction. Such modulators may be useful in the treatment of
cardiovascular disease.
[1749] The ability of the test compound to modulate 10218 binding
to a substrate or to bind to 10218 can also be determined.
Determining the ability of the test compound to modulate 10218
binding to a substrate can be accomplished, for example, by
coupling the 10218 substrate with a radioisotope or enzymatic label
such that binding of the 10218 substrate to 10218 can be determined
by detecting the labeled 10218 substrate in a complex. 10218 could
also be coupled with a radioisotope or enzymatic label to monitor
the ability of a test compound to modulate 10218 binding to a 10218
substrate in a complex. Determining the ability of the test
compound to bind 10218 can be accomplished, for example, by
coupling the compound with a radioisotope or enzymatic label such
that binding of the compound to 10218 can be determined by
detecting the labeled 10218 compound in a complex. For example,
compounds (e.g., 10218 ligands or substrates) can be labeled with
.sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Compounds can further
be enzymatically labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product.
[1750] The ability of a test compound to modulate the 10218
receptor's ability to associate with (e.g., bind) calcium can
tested for using the assays described in, for example, Liu L.
(1999) Cell Signal. 11(5):317-24 and Kawai T. et al. (1999)
Oncogene 18(23):3471-80, the contents of which are incorporated
herein by reference.
[1751] It is also within the scope of this invention to determine
the ability of a compound (e.g., a 10218 ligand or substrate) to
interact with 10218 without the labeling of any of the
interactants. For example, a microphysiometer can be used to detect
the interaction of a compound with 10218 without the labeling of
either the compound or the 10218 (McConnell, H. M. et al. (1992)
Science 257:1906-1912. As used herein, a "microphysiometer" (e.g.,
Cytosensor) is an analytical instrument that measures the rate at
which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate
can be used as an indicator of the interaction between a compound
and 10218.
[1752] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a 10218 target molecule
(e.g., a 10218 substrate) with a test compound and determining the
ability of the test compound to modulate (e.g., stimulate or
inhibit) the activity of the 10218 target molecule. Determining the
ability of the test compound to modulate the activity of a 10218
target molecule can be accomplished, for example, by determining
the ability of the 10218 protein to bind to or interact with the
10218 target molecule.
[1753] Determining the ability of the 10218 protein or a
biologically active fragment thereof, to bind to or interact with a
10218 target molecule can be accomplished by one of the methods
described above for determining direct binding. In a preferred
embodiment, determining the ability of the 10218 protein to bind to
or interact with a 10218 target molecule can be accomplished by
determining the activity of the target molecule. For example, the
activity of the target molecule can be determined by detecting
induction of a cellular second messenger of the target (i.e.,
intracellular Ca.sup.2+, diacylglycerol, IP.sub.3, cAMP), detecting
catalytic/enzymatic activity of the target on an appropriate
substrate, detecting the induction of a reporter gene (comprising a
target-responsive regulatory element operatively linked to a
nucleic acid encoding a detectable marker, e.g., luciferase), or
detecting a target-regulated cellular response (e.g., gene
expression).
[1754] In yet another embodiment, an assay of the present invention
is a cell-free assay in which a 10218 protein or biologically
active portion thereof, is contacted with a test compound and the
ability of the test compound to bind to the 10218 protein or
biologically active portion thereof is determined. Preferred
biologically active portions of the 10218 proteins to be used in
assays of the present invention include fragments which participate
in interactions with non-10218 molecules, e.g., fragments with high
surface probability scores. Binding of the test compound to the
10218 protein can be determined either directly or indirectly as
described above. In a preferred embodiment, the assay includes
contacting the 10218 protein or biologically active portion thereof
with a known compound which binds 10218 to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with a 10218 protein,
wherein determining the ability of the test compound to interact
with a 10218 protein comprises determining the ability of the test
compound to preferentially bind to 10218 or biologically active
portion thereof as compared to the known compound. Compounds that
modulate the interaction of 10218 with a known target protein may
be useful in regulating the activity of a 10218 protein, especially
a mutant 10218 protein.
[1755] In another embodiment, the assay is a cell-free assay in
which a 10218 protein or biologically active portion thereof is
contacted with a test compound and the ability of the test compound
to modulate (e.g., stimulate or inhibit) the activity of the 10218
protein or biologically active portion thereof is determined.
Determining the ability of the test compound to modulate the
activity of a 10218 protein can be accomplished, for example, by
determining the ability of the 10218 protein to bind to a 10218
target molecule by one of the methods described above for
determining direct binding. Determining the ability of the 10218
protein to bind to a 10218 target molecule can also be accomplished
using a technology such as real-time Biomolecular Interaction
Analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem.
63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol.
5:699-705). As used herein, "BIA" is a technology for studying
biospecific interactions in real time, without labeling any of the
interactants (e.g., BIAcore). Changes in the optical phenomenon of
surface plasmon resonance (SPR) can be used as an indication of
real-time reactions between biological molecules.
[1756] In another embodiment, determining the ability of the test
compound to modulate the activity of a 10218 protein can be
accomplished by determining the ability of the 10218 protein to
further modulate the activity of a downstream effector of a 10218
target molecule. For example, the activity of the effector molecule
on an appropriate target can be determined or the binding of the
effector to an appropriate target can be determined as previously
described.
[1757] In yet another embodiment, the cell-free assay involves
contacting a 10218 protein or biologically active portion thereof
with a known compound which binds the 10218 protein to form an
assay mixture, contacting the assay mixture with a test compound,
and determining the ability of the test compound to interact with
the 10218 protein, wherein determining the ability of the test
compound to interact with the 10218 protein comprises determining
the ability of the 10218 protein to preferentially bind to or
modulate the activity of a 10218 target molecule.
[1758] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either
10218 or its target molecule to facilitate separation of complexed
from uncomplexed forms of one or both of the proteins, as well as
to accommodate automation of the assay. Binding of a test compound
to a 10218 protein, or interaction of a 10218 protein with a target
molecule in the presence and absence of a candidate compound, can
be accomplished in any vessel suitable for containing the
reactants. Examples of such vessels include microtitre plates, test
tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows one or both
of the proteins to be bound to a matrix. For example,
glutathione-S-transferase/10218 fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or 10218 protein, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtitre plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of 10218 binding or activity
determined using standard techniques.
[1759] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either a 10218 protein or a 10218 target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated 10218 protein or target molecules can be prepared from
biotin-NHS (N-hydroxy-succinimide) using techniques known in the
art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),
and immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with 10218
protein or target molecules but which do not interfere with binding
of the 10218 protein to its target molecule can be derivatized to
the wells of the plate, and unbound target or 10218 protein trapped
in the wells by antibody conjugation. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the 10218 protein or target
molecule, as well as enzyme-linked assays which rely on detecting
an enzymatic activity associated with the 10218 protein or target
molecule.
[1760] In another embodiment, modulators of 10218 expression are
identified in a method wherein a cell is contacted with a candidate
compound and the expression of 10218 mRNA or protein in the cell is
determined. The level of expression of 10218 mRNA or protein in the
presence of the candidate compound is compared to the level of
expression of 10218 mRNA or protein in the absence of the candidate
compound. The candidate compound can then be identified as a
modulator of 10218 expression based on this comparison. For
example, when expression of 10218 mRNA or protein is greater
(statistically significantly greater) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of 10218 mRNA or protein expression.
Alternatively, when expression of 10218 mRNA or protein is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of 10218 mRNA or protein expression. The level of
10218 mRNA or protein expression in the cells can be determined by
methods described herein for detecting 10218 mRNA or protein.
[1761] In yet another aspect of the invention, the 10218 proteins
can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with 10218
("10218-binding proteins" or "10218-bp") and are involved in 10218
activity. Such 10218-binding proteins are also likely to be
involved in the propagation of signals by the 10218 proteins or
10218 targets as, for example, downstream elements of a
10218-mediated signaling pathway. Alternatively, such 10218-binding
proteins are likely to be 10218 inhibitors.
[1762] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a 10218
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a 10218-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the 10218 protein.
[1763] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of a 10218 protein can be confirmed in vivo, e.g., in an animal
such as an animal model for cardiovascular disease, e.g.,
atherosclerosis, as described herein.
[1764] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein (e.g., a 10218 modulating
agent, an antisense 10218 nucleic acid molecule, a 10218-specific
antibody, or a 10218-binding partner) can be used in an animal
model to determine the efficacy, toxicity, or side effects of
treatment with such an agent. Alternatively, an agent identified as
described herein can be used in an animal model to determine the
mechanism of action of such an agent. Furthermore, this invention
pertains to uses of novel agents identified by the above-described
screening assays for treatments as described herein.
[1765] Any of the 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 systems are described herein.
[1766] In one aspect, cell-based systems, as described herein, 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. Cellular phenotypes that are
associated with cardiovascular disease states include aberrant
proliferation and migration, angiogenesis, deposition of
extracellular matrix components, accumulation of intracellular
lipids, and expression of growth factors, cytokines, and other
inflammatory mediators.
[1767] In addition, animal-based cardiovascular disease systems,
such as those described herein, 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.
[1768] 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.
[1769] 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 genes may form part of a "gene expression profile" or
"transcriptional profile" which may be then be used in such an
assessment. "Gene expression profile" or "transcriptional profile",
as used herein, includes 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 herein, for example, atherogenic
cytokine stimulation of macrophages. Gene expression profiles may
be generated, for example, by utilizing a differential display
procedure, Northern analysis and/or RT-PCR. In one embodiment,
10218 gene sequences may be used as probes and/or PCR primers for
the generation and corroboration of such gene expression
profiles.
[1770] Gene expression profiles may be characterized for known
states, either cardiovascular disease or normal, within the cell-
and/or animal-based model systems. Subsequently, these known gene
expression profiles may be compared to ascertain the effect a test
compound has to modify such gene expression profiles, and to cause
the profile to more closely resemble that of a more desirable
profile.
[1771] For example, administration of a compound may cause the gene
expression profile of a cardiovascular disease model system to more
closely resemble the control system. Administration of a compound
may, alternatively, cause the gene expression 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.
Cell- and Animal-Based Model Systems
[1772] 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 genes associated with cardiovascular
disease, e.g., 10218. In addition, animal- and cell-based assays
may be used as part of screening strategies designed to identify
compounds which are capable of ameliorating cardiovascular disease
symptoms, as described, below. 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. Furthermore, such animal models may be used to determine
the LD50 and the ED50 in animal subjects, and such data can be used
to determine the in vivo efficacy of potential cardiovascular
disease treatments.
Animal-Based Systems
[1773] Animal-based model systems of cardiovascular disease may
include, but are not limited to, non-recombinant and engineered
transgenic animals.
[1774] 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). Transgenic mouse models in
cardiovascular disease and angiogenesis are reviewed in Carmeliet,
P. and Collen, D. (2000) J. Pathol. 190:387-405.
[1775] 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. Animal models of
cardiovascular disease also include rat myocardial infarction
models (described in, for example, Schwarz, E R et al. (2000) J.
Am. Coll. Cardiol. 35:1323-1330) and models of chromic cardiac
ischemia in rabbits (described in, for example, Operschall, C et
al. (2000) J. Appl. Physiol. 88:1438-1445).
[1776] Additionally, animal models exhibiting cardiovascular
disease symptoms may be engineered by using, for example, 10218
gene sequences described above, in conjunction with techniques for
producing transgenic animals that are well known to those of skill
in the art. For example, 10218 gene sequences may be introduced
into, and overexpressed in, the genome of the animal of interest,
or, if endogenous 10218 gene sequences are present, they may either
be overexpressed or, alternatively, be disrupted in order to
underexpress or inactivate 10218 gene expression, such as described
for the disruption of ApoE in mice (Plump et al., 1992, Cell 71:
343-353).
[1777] The host cells of the invention can also be used to produce
non-human transgenic animals. For example, in one embodiment, a
host cell of the invention is a fertilized oocyte or an embryonic
stem cell into which 10218-coding sequences have been introduced.
Such host cells can then be used to create non-human transgenic
animals in which exogenous 10218 sequences have been introduced
into their genome or homologous recombinant animals in which
endogenous 10218 sequences have been altered. Such animals are
useful for studying the function and/or activity of a 10218 and for
identifying and/or evaluating modulators of 10218 activity. As used
herein, a "transgenic animal" is a non-human animal, preferably a
mammal, more preferably a rodent such as a rat or mouse, in which
one or more of the cells of the animal includes a transgene. Other
examples of transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, amphibians, and the like. A transgene
is exogenous DNA which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal, thereby directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal. As used herein, a "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably
a mouse, in which an endogenous 10218 gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[1778] A transgenic animal used in the methods of the invention can
be created by introducing a 10218-encoding nucleic acid into the
male pronuclei of a fertilized oocyte, e.g., by microinjection,
retroviral infection, and allowing the oocyte to develop in a
pseudopregnant female foster animal. The 10218 cDNA sequence of SEQ
ID NO:16 or 18 can be introduced as a transgene into the genome of
a non-human animal. Alternatively, a nonhuman homologue of a human
10218 gene, such as a mouse or rat 10218 gene, can be used as a
transgene. Alternatively, a 10218 gene homologue, such as another
10218 family member, can be isolated based on hybridization to the
10218 cDNA sequences of SEQ ID NO:16 or 18 and used as a transgene.
Intronic sequences and polyadenylation signals can also be included
in the transgene to increase the efficiency of expression of the
transgene. A tissue-specific regulatory sequence(s) can be operably
linked to a 10218 transgene to direct expression of a 10218 protein
to particular cells. Methods for generating transgenic animals via
embryo manipulation and microinjection, particularly animals such
as mice, have become conventional in the art and are described, for
example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder
et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of a 10218
transgene in its genome and/or expression of 10218 mRNA in tissues
or cells of the animals. A transgenic founder animal can then be
used to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene encoding a 10218 protein
can further be bred to other transgenic animals carrying other
transgenes.
[1779] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of a 10218 gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the 10218 gene. The
10218 gene can be a human gene (e.g., the cDNA of SEQ ID NO:16 or
18), but more preferably, is a non-human homologue of a human 10218
gene (e.g., a cDNA isolated by stringent hybridization with the
nucleotide sequence of SEQ ID NO:16 or 18). For example, a rat
10218 gene can be used to construct a homologous recombination
nucleic acid molecule, e.g., a vector, suitable for altering an
endogenous 10218 gene in the mouse genome. In a preferred
embodiment, the homologous recombination nucleic acid molecule is
designed such that, upon homologous recombination, the endogenous
10218 gene is functionally disrupted (i.e., no longer encodes a
functional protein; also referred to as a "knock out" vector).
Alternatively, the homologous recombination nucleic acid molecule
can be designed such that, upon homologous recombination, the
endogenous 10218 gene is mutated or otherwise altered but still
encodes functional protein (e.g., the upstream regulatory region
can be altered to thereby alter the expression of the endogenous
10218 protein). In the homologous recombination nucleic acid
molecule, the altered portion of the 10218 gene is flanked at its
5' and 3' ends by additional nucleic acid sequence of the 10218
gene to allow for homologous recombination to occur between the
exogenous 10218 gene carried by the homologous recombination
nucleic acid molecule and an endogenous 10218 gene in a cell, e.g.,
an embryonic stem cell. The additional flanking 10218 nucleic acid
sequence is of sufficient length for successful homologous
recombination with the endogenous gene. Typically, several
kilobases of flanking DNA (both at the 5' and 3' ends) are included
in the homologous recombination nucleic acid molecule (see, e.g.,
Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a
description of homologous recombination vectors). The homologous
recombination nucleic acid molecule is introduced into a cell,
e.g., an embryonic stem cell line (e.g., by electroporation) and
cells in which the introduced 10218 gene has homologously
recombined with the endogenous 10218 gene are selected (see e.g.,
Li, E. et al. (1992) Cell 69:915). The selected cells can then
injected into a blastocyst of an animal (e.g., a mouse) to form
aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and
Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.
(IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be
implanted into a suitable pseudopregnant female foster animal and
the embryo brought to term. Progeny harboring the homologously
recombined DNA in their germ cells can be used to breed animals in
which all cells of the animal contain the homologously recombined
DNA by germline transmission of the transgene. Methods for
constructing homologous recombination nucleic acid molecules, e.g.,
vectors, or homologous recombinant animals are described further in
Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and
in PCT International Publication Nos.: WO 90/11354 by Le Mouellec
et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et
al.; and WO 93/04169 by Berns et al.
[1780] In another embodiment, transgenic non-human animals for use
in the methods of the invention can be produced which contain
selected systems which allow for regulated expression of the
transgene. One example of such a system is the cre/loxP recombinase
system of bacteriophage P1. For a description of the cre/loxP
recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl.
Acad. Sci. USA 89:6232-6236. Another example of a recombinase
system is the FLP recombinase system of Saccharomyces cerevisiae
(O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP
recombinase system is used to regulate expression of the transgene,
animals containing transgenes encoding both the Cre recombinase and
a selected protein are required. Such animals can be provided
through the construction of "double" transgenic animals, e.g., by
mating two transgenic animals, one containing a transgene encoding
a selected protein and the other containing a transgene encoding a
recombinase.
[1781] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. (1997) Nature 385:810-813 and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.o phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyte and then transferred to pseudopregnant female
foster animal. The offspring borne of this female foster animal
will be a clone of the animal from which the cell, e.g., the
somatic cell, is isolated.
[1782] The 10218 transgenic animals that express 10218 mRNA or a
10218 peptide (detected immunocytochemically, using antibodies
directed against 10218 epitopes) at easily detectable levels should
then be further evaluated to identify those animals which display
characteristic cardiovascular disease symptoms. Such cardiovascular
disease symptoms may include, for example, increased prevalence and
size of fatty streaks and/or cardiovascular disease plaques.
[1783] Additionally, specific cell types (e.g., endothelial cells)
within the transgenic animals may be analyzed and assayed for
cellular phenotypes characteristic of cardiovascular disease. In
the case of endothelial cells, such phenotypes include, but are not
limited to cell proliferation, migration, angiogenesis, production
of proinflammatory growth factors and cytokines, and adhesion to
inflammatory cells. 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 phenotypes may include a particular cell type's
pattern of expression of genes associated with cardiovascular
disease as compared to known expression profiles of the particular
cell type in animals exhibiting cardiovascular disease
symptoms.
Cell-Based Systems
[1784] Cells that contain and express 10218 gene sequences which
encode a 10218 protein, and, further, exhibit cellular phenotypes
associated with cardiovascular disease, may be used to identify
compounds that exhibit anti-cardiovascular disease activity. Such
cells may include non-recombinant monocyte cell lines, such as U937
(ATCC# CRL-1593), THP-1 (ATCC#TIB-202), and P388D1 (ATCC# TIB-63);
endothelial cells such as human umbilical vein endothelial cells
(HUVECs), human microvascular endothelial cells (HMVEC), and bovine
aortic endothelial cells (BAECs); 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, 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.
[1785] 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 10218 gene expression within
the cell. For example, 10218 gene sequences may be introduced into,
and overexpressed in, the genome of the cell of interest, or, if
endogenous 10218 gene sequences are present, they may be either
overexpressed or, alternatively disrupted in order to underexpress
or inactivate 10218 gene expression.
[1786] In order to overexpress a 10218 gene, the coding portion of
the 10218 gene may be ligated to a regulatory sequence which is
capable of driving gene expression in the cell type of interest,
e.g., an endothelial cell. 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 above.
[1787] For underexpression of an endogenous 10218 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 10218 alleles will be inactivated. Preferably, the
engineered 10218 sequence is introduced via gene targeting such
that the endogenous 10218 sequence is disrupted upon integration of
the engineered 10218 sequence into the cell's genome. Transfection
of host cells with 10218 genes is discussed, above.
[1788] Cells treated with compounds or transfected with 10218 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. (1988) J. Clin. Invest. 82:1853-1863, by quantifying
the number of monocytes that migrate across the endothelial
monolayer and into the collagen layer of the subendothelial
space.
[1789] Similarly, endothelial cells can be treated with test
compounds or transfected with genetically engineered 10218 genes.
The endothelial cells 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 adhesion molecules (e.g., ICAM, VCAM, E-selectin), growth
factors and cytokines (e.g., PDGF, IL-1.beta., TNF.alpha., MCF),
and proteins involved in angiogenesis (e.g., FLK, FLT).
[1790] Transfection of 10218 nucleic acid may be accomplished by
using standard techniques (described in, for example, Ausubel
(1989) supra). Transfected cells should be evaluated for the
presence of the recombinant 10218 gene sequences, for expression
and accumulation of 10218 mRNA, and for the presence of recombinant
10218 protein production. In instances wherein a decrease in 10218
gene expression is desired, standard techniques may be used to
demonstrate whether a decrease in endogenous 10218 gene expression
and/or in 10218 protein production is achieved.
[1791] Cellular models for the study of cardiovascular disease and
angiogenesis include models of endothelial cell differentiation on
Matrigel (Baatout, S. et al. (1996) Rom. J. Intern. Med.
34:263-269; Benelli, R et al. (1999) Int. J. Biol. Markers
14:243-246), embryonic stem cell models of vascular morphogenesis
(Doetschman, T. et al. (1993) Hypertension 22:618-629), the culture
of microvessel fragments in physiological gels (Hoying, J B et al.
(1996) In Vitro Cell Dev. Biol. Anim. 32: 409-419; U.S. Pat. No.
5,976,782), and the treatment of endothelial cells and smooth
muscle cells with atherogenic and angiogenic factors including
growth factors and cytokines (e.g., IL-1.beta., PDGF, TNF.alpha.,
VEGF), homocysteine, and LDL. In vitro angiogenesis models are
described in, for example, Black, A F et al. (1999) Cell Biol.
Toxicol. 15:81-90.
Predictive Medicine
[1792] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to
diagnostic assays for determining 10218 protein and/or nucleic acid
expression as well as 10218 activity, in the context of a
biological sample (e.g., blood, serum, cells, e.g., endothelial
cells, or tissue, e.g., vascular tissue) to thereby determine
whether an individual is afflicted with a cardiovascular disease.
The invention also provides for prognostic (or predictive) assays
for determining whether an individual is at risk of developing a
cardiovascular disorder. For example, mutations in a 10218 gene can
be assayed for in a biological sample. Such assays can be used for
prognostic or predictive purpose to thereby prophylactically treat
an individual prior to the onset of a cardiovascular disorder,
e.g., atherosclerosis.
[1793] Another aspect of the invention pertains to monitoring the
influence of 10218 modulators (e.g., anti-10218 antibodies or 10218
ribozymes) on the expression or activity of 10218 in clinical
trials.
[1794] These and other agents are described in further detail in
the following sections.
Diagnostic Assays For Cardiovascular Disease
[1795] To determine whether a subject is afflicted with a
cardiovascular disease, a biological sample may be obtained from a
subject and the biological sample may be contacted with a compound
or an agent capable of detecting a 10218 protein or nucleic acid
(e.g., mRNA or genomic DNA) that encodes a 10218 protein, in the
biological sample. A preferred agent for detecting 10218 mRNA or
genomic DNA is a labeled nucleic acid probe capable of hybridizing
to 10218 mRNA or genomic DNA. The nucleic acid probe can be, for
example, the 10218 nucleic acid set forth in SEQ ID NO:16, or a
portion thereof, such as an oligonucleotide of at least 15, 20, 25,
30, 25, 40, 45, 50, 100, 250 or 500 nucleotides in length and
sufficient to specifically hybridize under stringent conditions to
10218 mRNA or genomic DNA. Other suitable probes for use in the
diagnostic assays of the invention are described herein.
[1796] A preferred agent for detecting 10218 protein in a sample is
an antibody capable of binding to 10218 protein, preferably an
antibody with a detectable label. Antibodies can be polyclonal, or
more preferably, monoclonal. An intact antibody, or a fragment
thereof (e.g., Fab or F(ab')2) can be used. The term "labeled",
with regard to the probe or antibody, is intended to encompass
direct labeling of the probe or antibody by coupling (i.e.,
physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently labeled streptavidin.
[1797] The term "biological sample" is intended to include tissues,
cells, and biological fluids isolated from a subject, as well as
tissues, cells, and fluids present within a subject. That is, the
detection method of the invention can be used to detect 10218 mRNA,
protein, or genomic DNA in a biological sample in vitro as well as
in vivo. For example, in vitro techniques for detection of 10218
mRNA include Northern hybridizations and in situ hybridizations. In
vitro techniques for detection of 10218 protein include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. In vitro techniques
for detection of 10218 genomic DNA include Southern hybridizations.
Furthermore, in vivo techniques for detection of 10218 protein
include introducing into a subject a labeled anti-10218 antibody.
For example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[1798] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting 10218
protein, mRNA, or genomic DNA, such that the presence of 10218
protein, mRNA or genomic DNA is detected in the biological sample,
and comparing the presence of 10218 protein, mRNA or genomic DNA in
the control sample with the presence of 10218 protein, mRNA or
genomic DNA in the test sample.
Prognostic Assays for Cardiovascular Disease
[1799] The present invention further pertains to methods for
identifying subjects having or at risk of developing a
cardiovascular disease associated with aberrant 10218 expression or
activity.
[1800] As used herein, the term "aberrant" includes a 10218
expression or activity which deviates from the wild type 10218
expression or activity. Aberrant expression or activity includes
increased or decreased expression or activity, as well as
expression or activity which does not follow the wild type
developmental pattern of expression or the subcellular pattern of
expression. For example, aberrant 10218 expression or activity is
intended to include the cases in which a mutation in the 10218 gene
causes the 10218 gene to be under-expressed or over-expressed and
situations in which such mutations result in a non-functional 10218
protein or a protein which does not function in a wild-type
fashion, e.g., a protein which does not interact with a 10218
substrate, or one which interacts with a non-10218 substrate.
[1801] The assays described herein, such as the preceding
diagnostic assays or the following assays, can be used to identify
a subject having or at risk of developing a cardiovascular disease,
e.g., including but not limited to, atherosclerosis,
ischemia/reperfusion injury, hypertension, restenosis, arterial
inflammation, and endothelial cell disorders. A biological sample
may be obtained from a subject and tested for the presence or
absence of a genetic alteration. For example, such genetic
alterations can be detected by ascertaining the existence of at
least one of 1) a deletion of one or more nucleotides from a 10218
gene, 2) an addition of one or more nucleotides to a 10218 gene, 3)
a substitution of one or more nucleotides of a 10218 gene, 4) a
chromosomal rearrangement of a 10218 gene, 5) an alteration in the
level of a messenger RNA transcript of a 10218 gene, 6) aberrant
modification of a 10218 gene, such as of the methylation pattern of
the genomic DNA, 7) the presence of a non-wild type splicing
pattern of a messenger RNA transcript of a 10218 gene, 8) a
non-wild type level of a 10218-protein, 9) allelic loss of a 10218
gene, and 10) inappropriate post-translational modification of a
10218-protein.
[1802] As described herein, there are a large number of assays
known in the art which can be used for detecting genetic
alterations in a 10218 gene. For example, a genetic alteration in a
10218 gene may be detected using a probe/primer in a polymerase
chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and
4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a
ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988)
Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad.
Sci. USA 91:360-364), the latter of which can be particularly
useful for detecting point mutations in a 10218 gene (see Abravaya
et al. (1995) Nucleic Acids Res. 23:675-682). This method includes
collecting a biological sample from a subject, isolating nucleic
acid (e.g., genomic DNA, mRNA or both) from the sample, contacting
the nucleic acid sample with one or more primers which specifically
hybridize to a 10218 gene under conditions such that hybridization
and amplification of the 10218 gene (if present) occurs, and
detecting the presence or absence of an amplification product, or
detecting the size of the amplification product and comparing the
length to a control sample. It is anticipated that PCR and/or LCR
may be desirable to use as a preliminary amplification step in
conjunction with any of the techniques used for detecting mutations
described herein.
[1803] Alternative amplification methods include: 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.
[1804] In an alternative embodiment, mutations in a 10218 gene from
a biological sample can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
for example, U.S. Pat. No. 5,498,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[1805] In other embodiments, genetic mutations in 10218 can be
identified by hybridizing biological sample derived and control
nucleic acids, e.g., DNA or RNA, to high density arrays containing
hundreds or thousands of oligonucleotide probes (Cronin, M. T. et
al. (1996) Human Mutation 7:244-255; Kozal, M. J. et al. (1996)
Nature Medicine 2:753-759). For example, genetic mutations in 10218
can be identified in two dimensional arrays containing
light-generated DNA probes as described in Cronin, M. T. et al.
(1996) supra. Briefly, a first hybridization array of probes can be
used to scan through long stretches of DNA in a sample and control
to identify base changes between the sequences by making linear
arrays of sequential, overlapping probes. This step allows for the
identification of point mutations. This step is followed by a
second hybridization array that allows for the characterization of
specific mutations by using smaller, specialized probe arrays
complementary to all variants or mutations detected. Each mutation
array is composed of parallel probe sets, one complementary to the
wild-type gene and the other complementary to the mutant gene.
[1806] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
10218 gene in a biological sample and detect mutations by comparing
the sequence of the 10218 in the biological sample with the
corresponding wild-type (control) sequence. Examples of sequencing
reactions include those based on techniques developed by Maxam and
Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger (1977)
Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that
any of a variety of automated sequencing procedures can be utilized
when performing the diagnostic assays (Naeve, C. W. (1995)
Biotechniques 19:448-53), including sequencing by mass spectrometry
(see, e.g., PCT International Publication No. WO 94/16101; Cohen et
al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993)
Appl. Biochem. Biotechnol. 38:147-159).
[1807] Other methods for detecting mutations in the 10218 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230:1242). In general, the art technique of
"mismatch cleavage" starts by providing heteroduplexes formed by
hybridizing (labeled) RNA or DNA containing the wild-type 10218
sequence with potentially mutant RNA or DNA obtained from a tissue
sample. The double-stranded duplexes are treated with an agent
which cleaves single-stranded regions of the duplex such as which
will exist due to basepair mismatches between the control and
sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically
digest the mismatched regions. In other embodiments, either DNA/DNA
or RNA/DNA duplexes can be treated with hydroxylamine or osmium
tetroxide and with piperidine in order to digest mismatched
regions. After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing polyacrylamide
gels to determine the site of mutation. See, for example, Cotton et
al. (1988) Proc. Natl. Acad Sci USA 85:4397 and Saleeba et al.
(1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the
control DNA or RNA can be labeled for detection.
[1808] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in 10218
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15:1657-1662). According to an exemplary
embodiment, a probe based on a 10218 sequence, e.g., a wild-type
10218 sequence, is hybridized to a cDNA or other DNA product from a
test cell(s). The duplex is treated with a DNA mismatch repair
enzyme, and the cleavage products, if any, can be detected from
electrophoresis protocols or the like. See, for example, U.S. Pat.
No. 5,459,039.
[1809] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in 10218 genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA: 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144
and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79).
Single-stranded DNA fragments of sample and control 10218 nucleic
acids will be denatured and allowed to renature. The secondary
structure of single-stranded nucleic acids varies according to
sequence, the resulting alteration in electrophoretic mobility
enables the detection of even a single base change. The DNA
fragments may be labeled or detected with labeled probes. The
sensitivity of the assay may be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al. (1991) Trends Genet 7:5).
[1810] In yet another embodiment the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to ensure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265:12753).
[1811] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki
et al. (1989) Proc. Natl Acad. Sci USA 86:6230). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA or a number of different mutations when the oligonucleotides
are attached to the hybridizing membrane and hybridized with
labeled target DNA.
[1812] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner (1993) Tibtech 11:238). In
addition it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88:189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[1813] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered a 10218
modulator (e.g., an agonist, antagonist, peptidomimetic, protein,
peptide, nucleic acid, or small molecule) to effectively treat a
cardiovascular disease, e.g., atherosclerosis.
Monitoring of Effects During Clinical Trials
[1814] The present invention further provides methods for
determining the effectiveness of a 10218 modulator (e.g., a 10218
modulator identified herein) in treating a cardiovascular disease,
e.g., atherosclerosis, in a subject. For example, the effectiveness
of a 10218 modulator in increasing 10218 gene expression, protein
levels, or in upregulating 10218 activity, can be monitored in
clinical trials of subjects exhibiting decreased 10218 gene
expression, protein levels, or down-regulated 10218 activity.
Alternatively, the effectiveness of a 10218 modulator in decreasing
10218 gene expression, protein levels, or in downregulating 10218
activity, can be monitored in clinical trials of subjects
exhibiting increased 10218 gene expression, protein levels, or
10218 activity. In such clinical trials, the expression or activity
of a 10218 gene, and preferably, other genes that have been
implicated in, for example, atherosclerosis can be used as a "read
out" or marker of the phenotype of a particular cell, e.g., a
vascular endothelial cell.
[1815] For example, and not by way of limitation, genes, including
10218, that are modulated in cells by treatment with an agent which
modulates 10218 activity (e.g., identified in a screening assay as
described herein) can be identified. Thus, to study the effect of
agents which modulate 10218 activity on subjects suffering from a
cardiovascular disease in, for example, a clinical trial, cells can
be isolated and RNA prepared and analyzed for the levels of
expression of 10218 and other genes implicated in the
cardiovascular disease. The levels of gene expression (e.g., a gene
expression pattern) can be quantified by Northern blot analysis or
RT-PCR, as described herein, or alternatively by measuring the
amount of protein produced, by one of the methods described herein,
or by measuring the levels of activity of 10218 or other genes. In
this way, the gene expression pattern can serve as a marker,
indicative of the physiological response of the cells to the agent
which modulates 10218 activity. This response state may be
determined before, and at various points during treatment of the
individual with the agent which modulates 10218 activity.
[1816] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent which modulates 10218 activity (e.g., an agonist,
antagonist, peptidomimetic, protein, peptide, nucleic acid, or
small molecule identified by the screening assays described herein)
including the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of a 10218 protein, mRNA, or genomic DNA in
the pre-administration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the 10218 protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the 10218 protein, mRNA, or
genomic DNA in the pre-administration sample with the 10218
protein, mRNA, or genomic DNA in the post administration sample or
samples; and (vi) altering the administration of the agent to the
subject accordingly. For example, increased administration of the
agent may be desirable to increase the expression or activity of
10218 to higher levels than detected, i.e., to increase the
effectiveness of the agent. Alternatively, decreased administration
of the agent may be desirable to decrease expression or activity of
10218 to lower levels than detected, i.e. to decrease the
effectiveness of the agent. According to such an embodiment, 10218
expression or activity may be used as an indicator of the
effectiveness of an agent, even in the absence of an observable
phenotypic response.
Methods of Treatment of Subjects Suffering from Cardiovascular
Disease
[1817] The present invention provides for both prophylactic and
therapeutic methods of treating a subject, e.g., a human, at risk
of (or susceptible to) a cardiovascular disease such as
atherosclerosis, ischemia/reperfusion injury, hypertension,
restenosis, arterial inflammation, and endothelial cell disorders.
With regard to both prophylactic and therapeutic methods of
treatment, such treatments may be specifically tailored or
modified, based on knowledge obtained from the field of
pharmacogenomics. "Pharmacogenomics," as used herein, refers to the
application of genomics technologies such as gene sequencing,
statistical genetics, and gene expression analysis to drugs in
clinical development and on the market. More specifically, the term
refers to the study of how a patient's genes determine his or her
response to a drug (e.g., a patient's "drug response phenotype", or
"drug response genotype").
[1818] Thus, another aspect of the invention provides methods for
tailoring an subject's prophylactic or therapeutic treatment with
either the 10218 molecules of the present invention or 10218
modulators according to that individual's drug response genotype.
Pharmacogenomics allows a clinician or physician to target
prophylactic or therapeutic treatments to patients who will most
benefit from the treatment and to avoid treatment of patients who
will experience toxic drug-related side effects.
Prophylactic Methods
[1819] In one aspect, the invention provides a method for
preventing in a subject, a cardiovascular disease by administering
to the subject an agent which modulates 10218 expression or 10218
activity, e.g., modulation of calcium influx, cellular migration,
or formation of atherosclerotic lesions. Subjects at risk for a
cardiovascular disease, e.g., atherosclerosis, can be identified
by, for example, any or a combination of the diagnostic or
prognostic assays described herein. Administration of a
prophylactic agent can occur prior to the manifestation of symptoms
characteristic of aberrant 10218 expression or activity, such that
a cardiovascular disease is prevented or, alternatively, delayed in
its progression. Depending on the type of 10218 aberrancy, for
example, a 10218, 10218 agonist or 10218 antagonist agent can be
used for treating the subject. The appropriate agent can be
determined based on screening assays described herein.
Therapeutic Methods
[1820] Described herein are methods and compositions whereby
cardiovascular disease symptoms may be ameliorated. Certain
cardiovascular diseases are brought about, at least in part, by an
excessive level of a 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 gene expression levels or the
activity of a protein are discussed below.
[1821] 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
protein's activity. As such, an increase in the level of gene
expression and/or the activity of such proteins would bring about
the amelioration of cardiovascular disease symptoms.
[1822] 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 gene's
expression, or the activity of the 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 herein.
[1823] Accordingly, another aspect of the invention pertains to
methods of modulating 10281 expression or activity for therapeutic
purposes. Accordingly, in an exemplary embodiment, the modulatory
method of the invention involves contacting a cell with a 10281 or
agent that modulates one or more of the activities of 10281 protein
activity associated with the cell (e.g., an endothelial cell or an
ovarian cell). An agent that modulates 10281 protein activity can
be an agent as described herein, such as a nucleic acid or a
protein, a naturally-occurring target molecule of a 10281 protein
(e.g., a 10281 ligand or substrate), a 10281 antibody, a 10281
agonist or antagonist, a peptidomimetic of a 10281 agonist or
antagonist, or other small molecule. In one embodiment, the agent
stimulates one or more 10281 activities. Examples of such
stimulatory agents include active 10281 protein and a nucleic acid
molecule encoding 10281 that has been introduced into the cell. In
another embodiment, the agent inhibits one or more 10281
activities. Examples of such inhibitory agents include antisense
10281 nucleic acid molecules, anti-10281 antibodies, and 10281
inhibitors. These modulatory methods can be performed in vitro
(e.g., by culturing the cell with the agent) or, alternatively, in
vivo (e.g., by administering the agent to a subject). As such, the
present invention provides methods of treating an individual
afflicted with a disease or disorder characterized by aberrant or
unwanted expression or activity of a 10281 protein or nucleic acid
molecule. In one embodiment, the method involves administering an
agent (e.g., an agent identified by a screening assay described
herein), or combination of agents that modulates (e.g., upregulates
or down-regulates) 10281 expression or activity. In another
embodiment, the method involves administering a 10281 protein or
nucleic acid molecule as therapy to compensate for reduced,
aberrant, or unwanted 10281 expression or activity.
[1824] Stimulation of 10281 activity is desirable in situations in
which 10281 is abnormally downregulated and/or in which increased
10281 activity is likely to have a beneficial effect. Likewise,
inhibition of 10281 activity is desirable in situations in which
10281 is abnormally upregulated and/or in which decreased 10281
activity is likely to have a beneficial effect.
Methods for Inhibiting Target Gene Expression, Synthesis, or
Activity
[1825] As discussed above, genes involved in cardiovascular
disorders may cause such disorders via an increased level of gene
activity. In some cases, such up-regulation may have a causative or
exacerbating effect on the disease state. A variety of techniques
may be used to inhibit the expression, synthesis, or activity of
such genes and/or proteins.
[1826] For example, compounds such as those identified through
assays described above, which exhibit inhibitory activity, may be
used in accordance with the invention to ameliorate cardiovascular
disease symptoms. Such molecules may include, but are not limited
to, small organic molecules, peptides, antibodies, and the
like.
[1827] For example, compounds can be administered that compete with
endogenous ligand for the 10281 protein. The resulting reduction in
the amount of ligand-bound 10281 protein will modulate endothelial
cell physiology. Compounds that can be particularly useful for this
purpose include, for example, soluble proteins or peptides, such as
peptides comprising one or more of the extracellular domains, or
portions and/or analogs thereof, of the 10281 protein, including,
for example, soluble fusion proteins such as Ig-tailed fusion
proteins. (For a discussion of the production of Ig-tailed fusion
proteins, see, for example, U.S. Pat. No. 5,116,964).
Alternatively, compounds, such as ligand analogs or antibodies,
that bind to the 10281 receptor site, but do not activate the
protein, (e.g., receptor-ligand antagonists) can be effective in
inhibiting 10281 protein activity.
[1828] Further, antisense and ribozyme molecules which inhibit
expression of the 10281 gene may also be used in accordance with
the invention to inhibit aberrant 10281 gene activity. Still
further, triple helix molecules may be utilized in inhibiting
aberrant 10281 gene activity.
[1829] The antisense nucleic acid molecules used in the methods of
the invention are typically administered to a subject or generated
in situ such that they hybridize with or bind to cellular mRNA
and/or genomic DNA encoding a 10281 protein to thereby inhibit
expression of the protein, e.g., by inhibiting transcription and/or
translation. The hybridization can be by conventional nucleotide
complementarity to form a stable duplex, or, for example, in the
case of an antisense nucleic acid molecule which binds to DNA
duplexes, through specific interactions in the major groove of the
double helix. An example of a route of administration of antisense
nucleic acid molecules of the invention include direct injection at
a tissue site. Alternatively, antisense nucleic acid molecules can
be modified to target selected cells and then administered
systemically. For example, for systemic administration, antisense
molecules can be modified such that they specifically bind to
receptors or antigens expressed on a selected cell surface, e.g.,
by linking the antisense nucleic acid molecules to peptides or
antibodies which bind to cell surface receptors or antigens. The
antisense nucleic acid molecules can also be delivered to cells
using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which the antisense nucleic acid molecule is placed
under the control of a strong pol II or pol III promoter are
preferred.
[1830] In yet another embodiment, an antisense nucleic acid
molecule used in the methods of the invention is an
.alpha.-anomeric nucleic acid molecule. An .alpha.-anomeric nucleic
acid molecule forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gaultier et al. (1987) Nucleic
Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can
also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987)
Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue
(Inoue et al. (1987) FEBS Lett. 215:327-330).
[1831] In still another embodiment, an antisense nucleic acid used
in the methods of the invention is a ribozyme. Ribozymes are
catalytic RNA molecules with ribonuclease activity which are
capable of cleaving a single-stranded nucleic acid, such as an
mRNA, to which they have a complementary region. Thus, ribozymes
(e.g., hammerhead ribozymes (described in Haselhoff and Gerlach
(1988) Nature 334:585-591)) can be used to catalytically cleave
10281 mRNA transcripts to thereby inhibit translation of 10281
mRNA. A ribozyme having specificity for a 10281-encoding nucleic
acid can be designed based upon the nucleotide sequence of a 10281
cDNA disclosed herein (i.e., SEQ ID NO:16 or 18). For example, a
derivative of a Tetrahymena L-19 IVS RNA can be constructed in
which the nucleotide sequence of the active site is complementary
to the nucleotide sequence to be cleaved in a 10281-encoding mRNA
(see, for example, Cech et al. U.S. Pat. No. 4,987,071; and Cech et
al. U.S. Pat. No. 5,116,742). Alternatively, 10281 mRNA can be used
to select a catalytic RNA having a specific ribonuclease activity
from a pool of RNA molecules (see, for example, Bartel, D. and
Szostak, J. W. (1993) Science 261:1411-1418).
[1832] 10281 gene expression can also be inhibited by targeting
nucleotide sequences complementary to the regulatory region of the
10281 (e.g., the 10281 promoter and/or enhancers) to form triple
helical structures that prevent transcription of the 10281 gene in
target cells (see, for example, 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).
[1833] Antibodies that are both specific for the 10281 protein and
interfere with its activity may also be used to modulate or inhibit
10281 protein function. Such antibodies may be generated using
standard techniques described herein, against the 10281 protein
itself or against peptides corresponding to portions of the
protein. Such antibodies include but are not limited to polyclonal,
monoclonal, Fab fragments, single chain antibodies, or chimeric
antibodies.
[1834] In instances where the target gene protein is intracellular
and whole antibodies are used, internalizing antibodies may be
preferred. Lipofectin liposomes may be used to deliver the antibody
or a fragment of the Fab region which binds to the target 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 (described in,
for example, Creighton (1983), supra; and Sambrook et al. (1989)
supra). 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. (1993) Proc.
Natl. Acad. Sci. USA 90:7889-7893).
[1835] In some instances, the target gene protein is extracellular,
or is a transmembrane protein, such as the 10281 protein.
Antibodies that are specific for one or more extracellular domains
of the 10281 protein, 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 which are appropriate for
peptide administration may be utilized to effectively administer
inhibitory target gene antibodies to their site of action.
Methods for Restoring or Enhancing Target Gene Activity
[1836] Genes that cause cardiovascular disease may be
underexpressed within cardiovascular disease situations.
Alternatively, the activity of the protein products of such genes
may be decreased, leading to the development of cardiovascular
disease symptoms. Such down-regulation of gene expression or
decrease of protein activity might have a causative or exacerbating
effect on the disease state.
[1837] In some cases, genes that are up-regulated in the disease
state might be exerting a protective effect. A variety of
techniques may be used to increase the expression, synthesis, or
activity of genes and/or proteins that exert a protective effect in
response to cardiovascular disease conditions.
[1838] Described in this section are methods whereby the level
10281 activity may be increased to levels wherein cardiovascular
disease symptoms are ameliorated. The level of 10281 activity may
be increased, for example, by either increasing the level of 10281
gene expression or by increasing the level of active 10281 protein
which is present.
[1839] For example, a 10281 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 may be used for such administration. One of skill in the art
will readily know how to determine the concentration of effective,
non-toxic doses of the 10281 protein, utilizing techniques such as
those described below.
[1840] Additionally, RNA sequences encoding a 10281 protein may be
directly administered to a patient exhibiting cardiovascular
disease symptoms, at a concentration sufficient to produce a level
of 10281 protein such that cardiovascular disease symptoms are
ameliorated. Any of the techniques discussed below, which achieve
intracellular administration of compounds, such as, for example,
liposome administration, may be used for the administration of such
RNA molecules. The RNA molecules may be produced, for example, by
recombinant techniques such as those described herein.
[1841] Further, subjects may be treated by gene replacement
therapy. One or more copies of a 10281 gene, or a portion thereof,
that directs the production of a normal 10281 protein with 10281
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 used for the introduction of 10281
gene sequences into human cells.
[1842] Cells, preferably, autologous cells, containing 10281
expressing gene sequences may then be introduced or reintroduced
into the subject at positions which allow for the amelioration of
cardiovascular disease symptoms. Such cell replacement techniques
may be preferred, for example, when the gene product is a secreted,
extracellular gene product.
Pharmaceutical Compositions
[1843] Another aspect of the invention pertains to methods for
treating a subject suffering from a cardiovascular disease, e.g.,
atherosclerosis. These methods involve administering to a subject
an agent which modulates 10218 expression or activity (e.g., an
agent identified by a screening assay described herein), or a
combination of such agents. In another embodiment, the method
involves administering to a subject a 10218 protein or nucleic acid
molecule as therapy to compensate for reduced, aberrant, or
unwanted 10218 expression or activity.
[1844] Stimulation of 10218 activity is desirable in situations in
which 10218 is abnormally downregulated and/or in which increased
10218 activity is likely to have a beneficial effect. Likewise,
inhibition of 10218 activity is desirable in situations in which
10218 is abnormally upregulated and/or in which decreased 10218
activity is likely to have a beneficial effect, e.g., inhibition of
atherosclerotic lesion formation.
[1845] The agents which modulate 10218 activity can be administered
to a subject using pharmaceutical compositions suitable for such
administration. Such compositions typically comprise the agent
(e.g., nucleic acid molecule, protein, or antibody) and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[1846] A pharmaceutical composition used in the therapeutic methods
of the invention is formulated to be compatible with its intended
route of administration. Examples of routes of administration
include parenteral, e.g., intravenous, intradermal, subcutaneous,
oral (e.g., inhalation), transdermal (topical), transmucosal, and
rectal administration. Solutions or suspensions used for
parenteral, intradermal, or subcutaneous application can include
the following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[1847] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, and sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[1848] Sterile injectable solutions can be prepared by
incorporating the agent that modulates 10218 activity (e.g., a
fragment of a 10218 protein or an anti-10218 antibody) in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[1849] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[1850] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[1851] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[1852] The agents that modulate 10218 activity can also be prepared
in the form of suppositories (e.g., with conventional suppository
bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
[1853] In one embodiment, the agents that modulate 10218 activity
are prepared with carriers that will protect the compound against
rapid elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811.
[1854] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the agent that modulates 10218 activity
and the particular therapeutic effect to be achieved, and the
limitations inherent in the art of compounding such an agent for
the treatment of subjects.
[1855] Toxicity and therapeutic efficacy of such agents can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
can be expressed as the ratio LD50/ED50. Agents which exhibit large
therapeutic indices are preferred. While agents that exhibit toxic
side effects may be used, care should be taken to design a delivery
system that targets such agents to the site of affected tissue in
order to minimize potential damage to uninfected cells and,
thereby, reduce side effects.
[1856] 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 10218 modulating agents lies preferably
within a range of circulating concentrations that include the ED50
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 agent used in the therapeutic
methods 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 IC50 (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.
[1857] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The
skilled artisan will appreciate that certain factors may influence
the dosage required to effectively treat a subject, including but
not limited to the severity of the disease or disorder, previous
treatments, the general health and/or age of the subject, and other
diseases present. Moreover, treatment of a subject with a
therapeutically effective amount of a protein, polypeptide, or
antibody can include a single treatment or, preferably, can include
a series of treatments.
[1858] In a preferred example, a subject is treated with antibody,
protein, or polypeptide in the range of between about 0.1 to 20
mg/kg body weight, one time per week for between about 1 to 10
weeks, preferably between 2 to 8 weeks, more preferably between
about 3 to 7 weeks, and even more preferably for about 4, 5, or 6
weeks. It will also be appreciated that the effective dosage of
antibody, protein, or polypeptide used for treatment may increase
or decrease over the course of a particular treatment. Changes in
dosage may result and become apparent from the results of
diagnostic assays as described herein.
[1859] The present invention encompasses agents which modulate
expression or activity. An agent may, for example, be a small
molecule. For example, such small molecules include, but are not
limited to, peptides, peptidomimetics, amino acids, amino acid
analogs, polynucleotides, polynucleotide analogs, nucleotides,
nucleotide analogs, organic or inorganic compounds (i.e., including
heteroorganic and organometallic compounds) having a molecular
weight less than about 10,000 grams per mole, organic or inorganic
compounds having a molecular weight less than about 5,000 grams per
mole, organic or inorganic compounds having a molecular weight less
than about 1,000 grams per mole, organic or inorganic compounds
having a molecular weight less than about 500 grams per mole, and
salts, esters, and other pharmaceutically acceptable forms of such
compounds. It is understood that appropriate doses of small
molecule agents depends upon a number of factors within the ken of
the ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the small molecule will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the small molecule to have upon the
nucleic acid or polypeptide of the invention.
[1860] Exemplary doses include milligram or microgram amounts of
the small molecule per kilogram of subject or sample weight (e.g.,
about 1 microgram per kilogram to about 500 milligrams per
kilogram, about 100 micrograms per kilogram to about 5 milligrams
per kilogram, or about 1 microgram per kilogram to about 50
micrograms per kilogram). It is furthermore understood that
appropriate doses of a small molecule depend upon the potency of
the small molecule with respect to the expression or activity to be
modulated. Such appropriate doses may be determined using the
assays described herein. When one or more of these small molecules
is to be administered to an animal (e.g., a human) in order to
modulate expression or activity of a polypeptide or nucleic acid of
the invention, a physician, veterinarian, or researcher may, for
example, prescribe a relatively low dose at first, subsequently
increasing the dose until an appropriate response is obtained. In
addition, it is understood that the specific dose level for any
particular animal subject will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[1861] Further, an antibody (or fragment thereof) may be conjugated
to a therapeutic moiety such as a cytotoxin, a therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes
any agent that is detrimental to cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[1862] The conjugates of the invention can be used for modifying a
given biological response, the drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a
desired biological activity. Such proteins may include, for
example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor,
alpha-interferon, beta-interferon, nerve growth factor, platelet
derived growth factor, tissue plasminogen activator; or biological
response modifiers such as, for example, lymphokines, interleukin-1
("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte macrophase colony stimulating factor ("GM-CSF"),
granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
[1863] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be
conjugated to a second antibody to form an antibody heteroconjugate
as described by Segal in U.S. Pat. No. 4,676,980.
[1864] The nucleic acid molecules used in the methods of the
invention can be inserted into vectors and used as gene therapy
vectors. Gene therapy vectors can be delivered to a subject by, for
example, intravenous injection, local administration (see U.S. Pat.
No. 5,328,470) or by stereotactic injection (see, e.g., Chen et al.
(1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system.
Pharmacogenomics
[1865] In conjunction with the therapeutic methods of the
invention, pharmacogenomics (i.e., the study of the relationship
between a subject's genotype and that subject's response to a
foreign compound or drug) may be considered. Differences in
metabolism of therapeutics can lead to severe toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus, a
physician or clinician may consider applying knowledge obtained in
relevant pharmacogenomics studies in determining whether to
administer an agent which modulates 10218 activity, as well as
tailoring the dosage and/or therapeutic regimen of treatment with
an agent which modulates 10218 activity.
[1866] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol.
Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin.
Chem. 43(2):254-266. In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body (altered
drug action) or genetic conditions transmitted as single factors
altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare genetic
defects or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate aminopeptidase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[1867] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association", relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants). Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP may occur once per every 1000
bases of DNA. A SNP may be involved in a disease process, however,
the vast majority may not be disease-associated. Given a genetic
map based on the occurrence of such SNPs, individuals can be
grouped into genetic categories depending on a particular pattern
of SNPs in their individual genome. In such a manner, treatment
regimens can be tailored to groups of genetically similar
individuals, taking into account traits that may be common among
such genetically similar individuals.
[1868] Alternatively, a method termed the "candidate gene approach"
can be utilized to identify genes that predict drug response.
According to this method, if a gene that encodes a drug target is
known (e.g., a 10218 protein used in the methods of the present
invention), all common variants of that gene can be fairly
easily-identified in the population and it can be determined if
having one version of the gene versus another is associated with a
particular drug response.
[1869] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and the cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[1870] Alternatively, a method termed the "gene expression
profiling" can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., a 10218 molecule or 10218 modulator used in the
methods of the present invention) can give an indication whether
gene pathways related to toxicity have been turned on.
[1871] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of a subject. This knowledge, when applied to dosing or
drug selection, can avoid adverse reactions or therapeutic failure
and, thus, enhance therapeutic or prophylactic efficiency when
treating a subject suffering from a cardiovascular disease, e.g.,
atherosclerosis, with an agent which modulates 10218 activity.
Recombinant Expression Vectors and Host Cells Used in the Methods
of the Invention
[1872] The methods of the invention (e.g., the screening assays
described herein) include the use of vectors, preferably expression
vectors, containing a nucleic acid encoding a 10218 protein (or a
portion thereof). As used herein, the term "vector" refers to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments can be ligated. Another type of vector is a
viral vector, wherein additional DNA segments can be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) are integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "expression
vectors". In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" can be used interchangeably
as the plasmid is the most commonly used form of vector. However,
the invention is intended to include such other forms of expression
vectors, such as viral vectors (e.g., replication defective
retroviruses, adenoviruses and adeno-associated viruses), which
serve equivalent functions.
[1873] The recombinant expression vectors to be used in the methods
of the invention comprise a nucleic acid of the invention in a form
suitable for expression of the nucleic acid in a host cell, which
means that the recombinant expression vectors include one or more
regulatory sequences, selected on the basis of the host cells to be
used for expression, which is operatively linked to the nucleic
acid sequence to be expressed. Within a recombinant expression
vector, "operably linked" is intended to mean that the nucleotide
sequence of interest is linked to the regulatory sequence(s) in a
manner which allows for expression of the nucleotide sequence
(e.g., in an in vitro transcription/translation system or in a host
cell when the vector is introduced into the host cell). The term
"regulatory sequence" is intended to include promoters, enhancers
and other expression control elements (e.g., polyadenylation
signals). Such regulatory sequences are described, for example, in
Goeddel (1990) Methods Enzymol. 185:3-7. Regulatory sequences
include those which direct constitutive expression of a nucleotide
sequence in many types of host cells and those which direct
expression of the nucleotide sequence only in certain host cells
(e.g., tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of protein
desired, and the like. The expression vectors of the invention can
be introduced into host cells to thereby produce proteins or
peptides, including fusion proteins or peptides, encoded by nucleic
acids as described herein (e.g., 10218 proteins, mutant forms of
10218 proteins, fusion proteins, and the like).
[1874] The recombinant expression vectors to be used in the methods
of the invention can be designed for expression of 10218 proteins
in prokaryotic or eukaryotic cells. For example, 10218 proteins can
be expressed in bacterial cells such as E. coli, insect cells
(using baculovirus expression vectors), yeast cells, or mammalian
cells. Suitable host cells are discussed further in Goeddel (1990)
supra. Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[1875] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein.
[1876] Purified fusion proteins can be utilized in 10218 activity
assays, (e.g., direct assays or competitive assays described in
detail below), or to generate antibodies specific for 10218
proteins. In a preferred embodiment, a 10218 fusion protein
expressed in a retroviral expression vector of the present
invention can be utilized to infect bone marrow cells which are
subsequently transplanted into irradiated recipients. The pathology
of the subject recipient is then examined after sufficient time has
passed (e.g., six weeks).
[1877] In another embodiment, a nucleic acid of the invention is
expressed in mammalian cells using a mammalian expression vector.
Examples of mammalian expression vectors include pCDM8 (Seed, B.
(1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.
6:187-195). When used in mammalian cells, the expression vector's
control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40. For other
suitable expression systems for both prokaryotic and eukaryotic
cells see chapters 16 and 17 of Sambrook, J. et al., Molecular
Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[1878] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
[1879] The methods of the invention may further use a recombinant
expression vector comprising a DNA molecule of the invention cloned
into the expression vector in an antisense orientation. That is,
the DNA molecule is operatively linked to a regulatory sequence in
a manner which allows for expression (by transcription of the DNA
molecule) of an RNA molecule which is antisense to 10218 mRNA.
Regulatory sequences operatively linked to a nucleic acid cloned in
the antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific,
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid, or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes, see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[1880] Another aspect of the invention pertains to the use of host
cells into which a 10218 nucleic acid molecule of the invention is
introduced, e.g., a 10218 nucleic acid molecule within a
recombinant expression vector or a 10218 nucleic acid molecule
containing sequences which allow it to homologously recombine into
a specific site of the host cell's genome. The terms "host cell"
and "recombinant host cell" are used interchangeably herein. It is
understood that such terms refer not only to the particular subject
cell but to the progeny or potential progeny of such a cell.
Because certain modifications may occur in succeeding generations
due to either mutation or environmental influences, such progeny
may not, in fact, be identical to the parent cell, but are still
included within the scope of the term as used herein.
[1881] A host cell can be any prokaryotic or eukaryotic cell. For
example, a 10218 protein can be expressed in bacterial cells such
as E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[1882] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[1883] A host cell used in the methods of the invention, such as a
prokaryotic or eukaryotic host cell in culture, can be used to
produce (i.e., express) a 10218 protein. Accordingly, the invention
further provides methods for producing a 10218 protein using the
host cells of the invention. In one embodiment, the method
comprises culturing the host cell of the invention (into which a
recombinant expression vector encoding a 10218 protein has been
introduced) in a suitable medium such that a 10218 protein is
produced. In another embodiment, the method further comprises
isolating a 10218 protein from the medium or the host cell.
Isolated Nucleic Acid Molecules Used in the Methods of the
Invention
[1884] The coding sequence of the isolated human 10218 cDNA (also
referred to herein as P2X.sub.4) and the predicted amino acid
sequence of the human 10218 polypeptide are shown in SEQ ID NOs:18
and 17, respectively. The 10218 amino acid sequence is also
described in Garcia-Guzman, et al. (1997) Molecular Pharmacology
51:109 (the contents of which are incorporated herein by
reference). The nucleotide sequence of 10218 is also described in
GenBank Accession Nos. NM.sub.--002560 (SEQ ID NO:16) and Y07684
(the contents of which are included herein by reference).
[1885] The methods of the invention include the use of isolated
nucleic acid molecules that encode 10218 proteins or biologically
active portions thereof, as well as nucleic acid fragments
sufficient for use as hybridization probes to identify
10218-encoding nucleic acid molecules (e.g., 10218 mRNA) and
fragments for use as PCR primers for the amplification or mutation
of 10218 nucleic acid molecules. As used herein, the term "nucleic
acid molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA
or RNA generated using nucleotide analogs. The nucleic acid
molecule can be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[1886] A nucleic acid molecule used in the methods of the present
invention, e.g., a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:16, or a portion thereof, can be isolated
using standard molecular biology techniques and the sequence
information provided herein. Using all or portion of the nucleic
acid sequence of SEQ ID NO:16 as a hybridization probe, 10218
nucleic acid molecules can be isolated using standard hybridization
and cloning techniques (e.g., as described in Sambrook, J., Fritsh,
E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.
2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989).
[1887] Moreover, a nucleic acid molecule encompassing all or a
portion of SEQ ID NO:16 can be isolated by the polymerase chain
reaction (PCR) using synthetic oligonucleotide primers designed
based upon the sequence of SEQ ID NO:16.
[1888] A nucleic acid used in the methods of the invention can be
amplified using cDNA, mRNA or, alternatively, genomic DNA as a
template and appropriate oligonucleotide primers according to
standard PCR amplification techniques. Furthermore,
oligonucleotides corresponding to 10218 nucleotide sequences can be
prepared by standard synthetic techniques, e.g., using an automated
DNA synthesizer.
[1889] In a preferred embodiment, the isolated nucleic acid
molecules used in the methods of the invention comprise the
nucleotide sequence shown in SEQ ID NO:16, a complement of the
nucleotide sequence shown in SEQ ID NO:16, or a portion of any of
these nucleotide sequences. A nucleic acid molecule which is
complementary to the nucleotide sequence shown in SEQ ID NO:16, is
one which is sufficiently complementary to the nucleotide sequence
shown in SEQ ID NO:16 such that it can hybridize to the nucleotide
sequence shown in SEQ ID NO:16 thereby forming a stable duplex.
[1890] In still another preferred embodiment, an isolated nucleic
acid molecule used in the methods of the present invention
comprises a nucleotide sequence which is at least about 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more
identical to the entire length of the nucleotide sequence shown in
SEQ ID NO:16 or a portion of any of this nucleotide sequence.
[1891] Moreover, the nucleic acid molecules used in the methods of
the invention can comprise only a portion of the nucleic acid
sequence of SEQ ID NO:16, for example, a fragment which can be used
as a probe or primer or a fragment encoding a portion of a 10218
protein, e.g., a biologically active portion of a 10218 protein.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12 or 15, preferably about 20 or 25, more
preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive
nucleotides of a sense sequence of SEQ ID NO:16 of an anti-sense
sequence of SEQ ID NO:16 or of a naturally occurring allelic
variant or mutant of SEQ ID NO:16. In one embodiment, a nucleic
acid molecule used in the methods of the present invention
comprises a nucleotide sequence which is greater than 100, 100-200,
200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,
900-1000, 1000-1100, 1100-1200, 1200-1300, or more nucleotides in
length and hybridizes under stringent hybridization conditions to a
nucleic acid molecule of SEQ ID NO:16.
[1892] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences that are significantly
identical or homologous to each other remain hybridized to each
other. Preferably, the conditions are such that sequences at least
about 70%, more preferably at least about 80%, even more preferably
at least about 85% or 90% identical to each other remain hybridized
to each other. Such stringent conditions are known to those skilled
in the art and can be found in Current Protocols in Molecular
Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995),
sections 2, 4 and 6. Additional stringent conditions can be found
in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9
and 11. A preferred, non-limiting example of stringent
hybridization conditions includes hybridization in 4.times. sodium
chloride/sodium citrate (SSC), at about 65-70.degree. C. (or
hybridization in 4.times.SSC plus 50% formamide at about
42-50.degree. C.) followed by one or more washes in 1.times.SSC, at
about 65-70.degree. C. A preferred, non-limiting example of highly
stringent hybridization conditions includes hybridization in
1.times.SSC, at about 65-70.degree. C. (or hybridization in
1.times.SSC plus 50% formamide at about 42-50.degree. C.) followed
by one or more washes in 0.3.times.SSC, at about 65-70.degree. C. A
preferred, non-limiting example of reduced stringency hybridization
conditions includes hybridization in 4.times.SSC, at about
50-60.degree. C. (or alternatively hybridization in 6.times.SSC
plus 50% formamide at about 40-45.degree. C.) followed by one or
more washes in 2.times.SSC, at about 50-60.degree. C. Ranges
intermediate to the above-recited values, e.g., at 65-70.degree. C.
or at 42-50.degree. C. are also intended to be encompassed by the
present invention. SSPE (1.times.SSPE is 0.15M NaCl, 10 mM
NaH.sub.2PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be substituted for
SSC (1.times.SSC is 0.15M NaCl and 15 mM sodium citrate) in the
hybridization and wash buffers; washes are performed for 15 minutes
each after hybridization is complete. The hybridization temperature
for hybrids anticipated to be less than 50 base pairs in length
should be 5-10.degree. C. less than the melting temperature
(T.sub.m) of the hybrid, where T.sub.m is determined according to
the following equations. For hybrids less than 18 base pairs in
length, T.sub.m(.degree. C.)=2(# of A+T bases)+4(# of G+C bases).
For hybrids between 18 and 49 base pairs in length,
T.sub.m(.degree. C.)=81.5+16.6(log.sub.10[Na.sup.+])+0.41(%
G+C)-(600/N), where N is the number of bases in the hybrid, and
[Na.sup.+] is the concentration of sodium ions in the hybridization
buffer ([Na.sup.+] for 1.times.SSC=0.165 M). It will also be
recognized by the skilled practitioner that additional reagents may
be added to hybridization and/or wash buffers to decrease
non-specific hybridization of nucleic acid molecules to membranes,
for example, nitrocellulose or nylon membranes, including but not
limited to blocking agents (e.g., BSA or salmon or herring sperm
carrier DNA), detergents (e.g., SDS), chelating agents (e.g.,
EDTA), Ficoll, PVP and the like. When using nylon membranes, in
particular, an additional preferred, non-limiting example of
stringent hybridization conditions is hybridization in 0.25-0.5M
NaH.sub.2PO.sub.4, 7% SDS at about 65.degree. C., followed by one
or more washes at 0.02M NaH.sub.2PO.sub.4, 1% SDS at 65.degree. C.,
see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA
81:1991-1995, (or alternatively 0.2.times.SSC, 1% SDS).
[1893] In preferred embodiments, the probe further comprises a
label group attached thereto, e.g., the label group can be a
radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells or tissue which misexpress a 10218
protein, such as by measuring a level of a 10218-encoding nucleic
acid in a sample of cells from a subject e.g., detecting 10218 mRNA
levels or determining whether a genomic 10218 gene has been mutated
or deleted.
[1894] The methods of the invention further encompass the use of
nucleic acid molecules that differ from the nucleotide sequence
shown in SEQ ID NO:16 due to degeneracy of the genetic code and
thus encode the same 10218 proteins as those encoded by the
nucleotide sequence shown in SEQ ID NO:16. In another embodiment,
an isolated nucleic acid molecule included in the methods of the
invention has a nucleotide sequence encoding a protein having an
amino acid sequence shown in SEQ ID NO:17.
[1895] The methods of the invention further include the use of
allelic variants of human 10218, e.g., functional and
non-functional allelic variants. Functional allelic variants are
naturally occurring amino acid sequence variants of the human 10218
protein that maintain a 10218 activity. Functional allelic variants
will typically contain only conservative substitution of one or
more amino acids of SEQ ID NO:17, or substitution, deletion or
insertion of non-critical residues in non-critical regions of the
protein.
[1896] Non-functional allelic variants are naturally occurring
amino acid sequence variants of the human 10218 protein that do not
have a 10218 activity. Non-functional allelic variants will
typically contain a non-conservative substitution, deletion, or
insertion or premature truncation of the amino acid sequence of SEQ
ID NO:17, or a substitution, insertion or deletion in critical
residues or critical regions of the protein.
[1897] The methods of the present invention may further use
non-human orthologues of the human 10218 protein. Orthologues of
the human 10218 protein are proteins that are isolated from
non-human organisms and possess the same 10218 activity.
[1898] The methods of the present invention further include the use
of nucleic acid molecules comprising the nucleotide sequence of SEQ
ID NO:16 or a portion thereof, in which a mutation has been
introduced. The mutation may lead to amino acid substitutions at
"non-essential" amino acid residues or at "essential" amino acid
residues. A "non-essential" amino acid residue is a residue that
can be altered from the wild-type sequence of 10218 (e.g., the
sequence of SEQ ID NO:17) without altering the biological activity,
whereas an "essential" amino acid residue is required for
biological activity. For example, amino acid residues that are
conserved among the 10218 proteins of the present invention and
other members of the P2X family (e.g., P2X.sub.1, P2X.sub.2,
P2X.sub.3, P2X.sub.5, P2X.sub.6, and P2X.sub.7) are not likely to
be amenable to alteration.
[1899] Mutations can be introduced into SEQ ID NO:16 by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Preferably, conservative amino acid substitutions are
made at one or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., asparagine, glutamine,
serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
glycine, alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a 10218 protein is
preferably replaced with another amino acid residue from the same
side chain family. Alternatively, in another embodiment, mutations
can be introduced randomly along all or part of a 10218 coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for 10218 biological activity to identify
mutants that retain activity. Following mutagenesis of SEQ ID NO:16
the encoded protein can be expressed recombinantly and the activity
of the protein can be determined using the assay described
herein.
[1900] Another aspect of the invention pertains to the use of
isolated nucleic acid molecules which are antisense to the
nucleotide sequence of SEQ ID NO:16. An "antisense" nucleic acid
comprises a nucleotide sequence which is complementary to a "sense"
nucleic acid encoding a protein, e.g., complementary to the coding
strand of a double-stranded cDNA molecule or complementary to an
mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen
bond to a sense nucleic acid. The antisense nucleic acid can be
complementary to an entire 10218 coding strand, or to only a
portion thereof. In one embodiment, an antisense nucleic acid
molecule is antisense to a "coding region" of the coding strand of
a nucleotide sequence encoding a 10218. The term "coding region"
refers to the region of the nucleotide sequence comprising codons
which are translated into amino acid residues. In another
embodiment, the antisense nucleic acid molecule is antisense to a
"noncoding region" of the coding strand of a nucleotide sequence
encoding 10218. The term "noncoding region" refers to 5' and 3'
sequences which flank the coding region that are not translated
into amino acids (also referred to as 5' and 3' untranslated
regions).
[1901] Given the coding strand sequences encoding 10218 disclosed
herein, antisense nucleic acids of the invention can be designed
according to the rules of Watson and Crick base pairing. The
antisense nucleic acid molecule can be complementary to the entire
coding region of 10218 mRNA, but more preferably is an
oligonucleotide which is antisense to only a portion of the coding
or noncoding region of 10218 mRNA. For example, the antisense
oligonucleotide can be complementary to the region surrounding the
translation start site of 10218 mRNA. An antisense oligonucleotide
can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50
nucleotides in length. An antisense nucleic acid of the invention
can be constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used. Examples of modified nucleotides which can
be used to generate the antisense nucleic acid include
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, 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-isopentenyladenine,
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. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest).
Antisense nucleic acid molecules used in the methods of the
invention are further described above, in section IV.
[1902] In yet another embodiment, the 10218 nucleic acid molecules
used in the methods of the present invention can be modified at the
base moiety, sugar moiety or phosphate backbone to improve, e.g.,
the stability, hybridization, or solubility of the molecule. For
example, the deoxyribose phosphate backbone of the nucleic acid
molecules can be modified to generate peptide nucleic acids (see
Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1):
5-23). As used herein, the terms "peptide nucleic acids" or "PNAs"
refer to nucleic acid mimics, e.g., DNA mimics, in which the
deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The
neutral backbone of PNAs has been shown to allow for specific
hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. (1996) Proc.
Natl. Acad. Sci. 93:14670-675.
[1903] PNAs of 10218 nucleic acid molecules can be used in the
therapeutic and diagnostic applications described herein. For
example, PNAs can be used as antisense or antigene agents for
sequence-specific modulation of gene expression by, for example,
inducing transcription or translation arrest or inhibiting
replication. PNAs of 10218 nucleic acid molecules can also be used
in the analysis of single base pair mutations in a gene, (e.g., by
PNA-directed PCR clamping); as `artificial restriction enzymes`
when used in combination with other enzymes, (e.g., S1 nucleases
(Hyrup B. et al. (1996) supra)); or as probes or primers for DNA
sequencing or hybridization (Hyrup B. et al. (1996) supra;
Perry-O'Keefe et al. (1996) supra).
[1904] In another embodiment, PNAs of 10218 can be modified, (e.g.,
to enhance their stability or cellular uptake), by attaching
lipophilic or other helper groups to PNA, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of
drug delivery known in the art. For example, PNA-DNA chimeras of
10218 nucleic acid molecules can be generated which may combine the
advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, (e.g., RNAse H and DNA polymerases), to
interact with the DNA portion while the PNA portion would provide
high binding affinity and specificity. PNA-DNA chimeras can be
linked using linkers of appropriate lengths selected in terms of
base stacking, number of bonds between the nucleobases, and
orientation (Hyrup B. et al. (1996) supra). The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup B. et al.
(1996) supra and Finn P. J. et al. (1996) Nucleic Acids Res. 24
(17): 3357-63. For example, a DNA chain can be synthesized on a
solid support using standard phosphoramidite coupling chemistry and
modified nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can
be used as a between the PNA and the 5' end of DNA (Mag, M. et al.
(1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then
coupled in a stepwise manner to produce a chimeric molecule with a
5' PNA segment and a 3' DNA segment (Finn P. J. et al. (1996)
supra). Alternatively, chimeric molecules can be synthesized with a
5' DNA segment and a 3' PNA segment (Peterser, K. H. et al. (1975)
Bioorganic Med. Chem. Lett. 5: 1119-11124).
[1905] In other embodiments, the oligonucleotide used in the
methods of the invention 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. USA 86:6553-6556;
Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT
Publication No. WO88/09810) or the blood-brain barrier (see, e.g.,
PCT Publication No. WO89/10134). In addition, oligonucleotides can
be modified with hybridization-triggered cleavage agents (See,
e.g., Krol et al. (1988) Bio-Techniques 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, or hybridization-triggered cleavage agent).
Isolated 10218 Proteins and Anti-10218 Antibodies Used in the
Methods of the Invention
[1906] The methods of the invention include the use of isolated
10218 proteins, and biologically active portions thereof, as well
as polypeptide fragments suitable for use as immunogens to raise
anti-10218 antibodies. In one embodiment, native 10218 proteins can
be isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques.
In another embodiment, 10218 proteins are produced by recombinant
DNA techniques. Alternative to recombinant expression, a 10218
protein or polypeptide can be synthesized chemically using standard
peptide synthesis techniques.
[1907] As used herein, a "biologically active portion" of a 10218
protein includes a fragment of a 10218 protein having a 10218
activity. Biologically active portions of a 10218 protein include
peptides comprising amino acid sequences sufficiently identical to
or derived from the amino acid sequence of the 10218 protein, e.g.,
the amino acid sequence shown in SEQ ID NO:17, which include fewer
amino acids than the full length 10218 proteins, and exhibit at
least one activity of a 10218 protein. Typically, biologically
active portions comprise a domain or motif with at least one
activity of the 10218 protein (e.g., the N-terminal region of the
10218 protein that is believed to be involved in the regulation of
apoptotic activity). A biologically active portion of a 10218
protein can be a polypeptide which is, for example, 25, 50, 75,
100, 125, 150, 175, 200, 250, 300 or more amino acids in length.
Biologically active portions of a 10218 protein can be used as
targets for developing agents which modulate a 10218 activity.
[1908] In a preferred embodiment, the 10218 protein used in the
methods of the invention has an amino acid sequence shown in SEQ ID
NO:17. In other embodiments, the 10218 protein is substantially
identical to SEQ ID NO:17, and retains the functional activity of
the protein of SEQ ID NO:17, yet differs in amino acid sequence due
to natural allelic variation or mutagenesis, as described in detail
in subsection V above. Accordingly, in another embodiment, the
10218 protein used in the methods of the invention is a protein
which comprises an amino acid sequence at least about 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more
identical to SEQ ID NO:17.
[1909] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-identical
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference sequence aligned
for comparison purposes is at least 30%, preferably at least 40%,
more preferably at least 50%, even more preferably at least 60%,
and even more preferably at least 70%, 80%, or 90% of the length of
the reference sequence (e.g., when aligning a second sequence to
the 10218 amino acid sequence of SEQ ID NO:17 having 500 amino acid
residues, at least 75, preferably at least 150, more preferably at
least 225, even more preferably at least 300, and even more
preferably at least 400 or more amino acid residues are aligned).
The amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position (as
used herein amino acid or nucleic acid "identity" is equivalent to
amino acid or nucleic acid "homology"). The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which need to be introduced
for optimal alignment of the two sequences.
[1910] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two amino acid sequences is determined using the
Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm
which has been incorporated into the GAP program in the GCG
software package, using either a Blosum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package, using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and
a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the
percent identity between two amino acid or nucleotide sequences is
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci. 4:11-17 (1988)) which has been incorporated into the
ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4.
[1911] The methods of the invention may also use 10218 chimeric or
fusion proteins. As used herein, a 10218 "chimeric protein" or
"fusion protein" comprises a 10218 polypeptide operatively linked
to a non-10218 polypeptide. An "10218 polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a 10218
molecule, whereas a "non-10218 polypeptide" refers to a polypeptide
having an amino acid sequence corresponding to a protein which is
not substantially homologous to the 10218 protein, e.g., a protein
which is different from the 10218 protein and which is derived from
the same or a different organism. Within a 10218 fusion protein the
10218 polypeptide can correspond to all or a portion of a 10218
protein. In a preferred embodiment, a 10218 fusion protein
comprises at least one biologically active portion of a 10218
protein. In another preferred embodiment, a 10218 fusion protein
comprises at least two biologically active portions of a 10218
protein. Within the fusion protein, the term "operatively linked"
is intended to indicate that the 10218 polypeptide and the
non-10218 polypeptide are fused in-frame to each other. The
non-10218 polypeptide can be fused to the N-terminus or C-terminus
of the 10218 polypeptide.
[1912] For example, in one embodiment, the fusion protein is a
GST-10218 fusion protein in which the 10218 sequences are fused to
the C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant 10218.
[1913] In another embodiment, this fusion protein is a 10218
protein containing a heterologous signal sequence at its
N-terminus. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of 10218 can be increased through use
of a heterologous signal sequence.
[1914] The 10218 fusion proteins used in the methods of the
invention can be incorporated into pharmaceutical compositions and
administered to a subject in vivo. The 10218 fusion proteins can be
used to affect the bioavailability of a 10218 substrate. Use of
10218 fusion proteins may be useful therapeutically for the
treatment of disorders caused by, for example, (i) aberrant
modification or mutation of a gene encoding a 10218 protein; (ii)
mis-regulation of the 10218 gene; and (iii) aberrant
post-translational modification of a 10218 protein.
[1915] Moreover, the 10218-fusion proteins used in the methods of
the invention can be used as immunogens to produce anti-10218
antibodies in a subject, to purify 10218 ligands and in screening
assays to identify molecules which inhibit the interaction of 10218
with a 10218 substrate.
[1916] Preferably, a 10218 chimeric or fusion protein used in the
methods of the invention is produced by standard recombinant DNA
techniques. For example, DNA fragments coding for the different
polypeptide sequences are ligated together in-frame in accordance
with conventional techniques, for example by employing blunt-ended
or stagger-ended termini for ligation, restriction enzyme digestion
to provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). A 10218-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked
in-frame to the 10218 protein.
[1917] The present invention also pertains to the use of variants
of the 10218 proteins which function as either 10218 agonists
(mimetics) or as 10218 antagonists. Variants of the 10218 proteins
can be generated by mutagenesis, e.g., discrete point mutation or
truncation of a 10218 protein. An agonist of the 10218 proteins can
retain substantially the same, or a subset, of the biological
activities of the naturally occurring form of a 10218 protein. An
antagonist of a 10218 protein can inhibit one or more of the
activities of the naturally occurring form of the 10218 protein by,
for example, competitively modulating a 10218-mediated activity of
a 10218 protein. Thus, specific biological effects can be elicited
by treatment with a variant of limited function. In one embodiment,
treatment of a subject with a variant having a subset of the
biological activities of the naturally occurring form of the
protein has fewer side effects in a subject relative to treatment
with the naturally occurring form of the 10218 protein.
[1918] In one embodiment, variants of a 10218 protein which
function as either 10218 agonists (mimetics) or as 10218
antagonists can be identified by screening combinatorial libraries
of mutants, e.g., truncation mutants, of a 10218 protein for 10218
protein agonist or antagonist activity. In one embodiment, a
variegated library of 10218 variants is generated by combinatorial
mutagenesis at the nucleic acid level and is encoded by a
variegated gene library. A variegated library of 10218 variants can
be produced by, for example, enzymatically ligating a mixture of
synthetic oligonucleotides into gene sequences such that a
degenerate set of potential 10218 sequences is expressible as
individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display) containing the set of
10218 sequences therein. There are a variety of methods which can
be used to produce libraries of potential 10218 variants from a
degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an
appropriate expression vector. Use of a degenerate set of genes
allows for the provision, in one mixture, of all of the sequences
encoding the desired set of potential 10218 sequences. Methods for
synthesizing degenerate oligonucleotides are known in the art (see,
e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984)
Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056;
Ike et al. (1983) Nucleic Acid Res. 11:477).
[1919] In addition, libraries of fragments of a 10218 protein
coding sequence can be used to generate a variegated population of
10218 fragments for screening and subsequent selection of variants
of a 10218 protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of a 10218 coding sequence with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form
double stranded DNA which can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. By this
method, an expression library can be derived which encodes
N-terminal, C-terminal and internal fragments of various sizes of
the 10218 protein.
[1920] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of 10218 proteins. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recursive ensemble mutagenesis
(REM), a new technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify 10218 variants (Arkin and Yourvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.
(1993) Protein Engineering 6(3):327-331).
[1921] The methods of the present invention further include the use
of anti-10218 antibodies. An isolated 10218 protein, or a portion
or fragment thereof, can be used as an immunogen to generate
antibodies that bind 10218 using standard techniques for polyclonal
and monoclonal antibody preparation. A full-length 10218 protein
can be used or, alternatively, antigenic peptide fragments of 10218
can be used as immunogens. The antigenic peptide of 10218 comprises
at least 8 amino acid residues of the amino acid sequence shown in
SEQ ID NO:17 and encompasses an epitope of 10218 such that an
antibody raised against the peptide forms a specific immune complex
with the 10218 protein. Preferably, the antigenic peptide comprises
at least 10 amino acid residues, more preferably at least 15 amino
acid residues, even more preferably at least 20 amino acid
residues, and most preferably at least 30 amino acid residues.
[1922] Preferred epitopes encompassed by the antigenic peptide are
regions of 10218 that are located on the surface of the protein,
e.g., hydrophilic regions, as well as regions with high
antigenicity.
[1923] A 10218 immunogen is typically used to prepare antibodies by
immunizing a suitable subject, (e.g., rabbit, goat, mouse, or other
mammal) with the immunogen. An appropriate immunogenic preparation
can contain, for example, recombinantly expressed 10218 protein or
a chemically synthesized 10218 polypeptide. The preparation can
further include an adjuvant, such as Freund's complete or
incomplete adjuvant, or similar immunostimulatory agent.
Immunization of a suitable subject with an immunogenic 10218
preparation induces a polyclonal anti-10218 antibody response.
[1924] The term "antibody" as used herein refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
which specifically binds (immunoreacts with) an antigen, such as a
10218. Examples of immunologically active portions of
immunoglobulin molecules include F(ab) and F(ab').sub.2 fragments
which can be generated by treating the antibody with an enzyme such
as pepsin. The invention provides polyclonal and monoclonal
antibodies that bind 10218 molecules. The term "monoclonal
antibody" or "monoclonal antibody composition", as used herein,
refers to a population of antibody molecules that contain only one
species of an antigen binding site capable of immunoreacting with a
particular epitope of 10218. A monoclonal antibody composition thus
typically displays a single binding affinity for a particular 10218
protein with which it immunoreacts.
[1925] Polyclonal anti-10218 antibodies can be prepared as
described above by immunizing a suitable subject with a 10218
immunogen. The anti-10218 antibody titer in the immunized subject
can be monitored over time by standard techniques, such as with an
enzyme linked immunosorbent assay (ELISA) using immobilized 10218.
If desired, the antibody molecules directed against 10218 can be
isolated from the mammal (e.g., from the blood) and further
purified by well known techniques, such as protein A chromatography
to obtain the IgG fraction. At an appropriate time after
immunization, e.g., when the anti-10218 antibody titers are
highest, antibody-producing cells can be obtained from the subject
and used to prepare monoclonal antibodies by standard techniques,
such as the hybridoma technique originally described by Kohler and
Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981)
J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.
255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA
76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the
more recent human B cell hybridoma technique (Kozbor et al. (1983)
Immunol Today 4:72), the EBV-hybridoma technique (Cole et al.
(1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
monoclonal antibody hybridomas is well known (see generally
Kenneth, R. H. in Monoclonal Antibodies: A New Dimension In
Biological Analyses, Plenum Publishing Corp., New York, N.Y.
(1980); Lerner, E. A. (1981) Yale J. Biol. Med. 54:387-402; Gefter,
M. L. et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an
immortal cell line (typically a myeloma) is fused to lymphocytes
(typically splenocytes) from a mammal immunized with a 10218
immunogen as described above, and the culture supernatants of the
resulting hybridoma cells are screened to identify a hybridoma
producing a monoclonal antibody that binds 10218.
[1926] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-10218 monoclonal antibody (see, e.g.,
G. Galfre et al. (1977) Nature 266:55052; Gefter et al. (1977)
supra; Lerner (1981) supra; and Kenneth (1980) supra). Moreover,
the ordinarily skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines can be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from ATCC. Typically, HAT-sensitive mouse myeloma cells
are fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using
HAT medium, which kills unfused and unproductively fused myeloma
cells (unfused splenocytes die after several days because they are
not transformed). Hybridoma cells producing a monoclonal antibody
of the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind 10218, e.g., using a standard
ELISA assay.
[1927] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-10218 antibody can be identified and
isolated by screening a recombinant combinatorial immunoglobulin
library (e.g., an antibody phage display library) with 10218 to
thereby isolate immunoglobulin library members that bind 10218.
Kits for generating and screening phage display libraries are
commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the Stratagene
SurfZAP.TM. Phage Display Kit, Catalog No. 240612). Additionally,
examples of methods and reagents particularly amenable for use in
generating and screening antibody display library can be found in,
for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT
International Publication No. WO 92/18619; Dower et al. PCT
International Publication No. WO 91/17271; Winter et al. PCT
International Publication WO 92/20791; Markland et al. PCT
International Publication No. WO 92/15679; Breitling et al. PCT
International Publication WO 93/01288; McCafferty et al. PCT
International Publication No. WO 92/01047; Garrard et al. PCT
International Publication No. WO 92/09690; Ladner et al. PCT
International Publication No. WO 90/02809; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J.
Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628;
Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad
et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991)
Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad.
Sci. USA 88:7978-7982; and McCafferty et al. (1990) Nature
348:552-554.
[1928] Additionally, recombinant anti-10218 antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the methods of
the invention. Such chimeric and humanized monoclonal antibodies
can be produced by recombinant DNA techniques known in the art, for
example using methods described in Robinson et al. International
Application No. PCT/US86/02269; Akira, et al. European Patent
Application 184,187; Taniguchi, M., European Patent Application
171,496; Morrison et al. European Patent Application 173,494;
Neuberger et al. PCT International Publication No. WO 86/01533;
Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European
Patent Application 125,023; Better et al. (1988) Science
240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA
84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et
al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.
(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature
314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559;
Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986)
BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.
(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science
239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.
[1929] An anti-10218 antibody can be used to detect 10218 protein
(e.g., in a cellular lysate or cell supernatant) in order to
evaluate the abundance and pattern of expression of the 10218
protein. Anti-10218 antibodies can be used diagnostically to
monitor protein levels in tissue as part of a clinical testing
procedure, e.g., to, for example, determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling (i.e.,
physically linking) the antibody to a detectable substance.
Examples of detectable substances include various enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, and radioactive materials. Examples of
suitable enzymes include horseradish peroxidase, alkaline
phosphatase, .beta.-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin, and examples
of suitable radioactive material include .sup.125I, .sup.131I,
.sup.35S or .sup.3H.
[1930] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the Sequence Listing is
incorporated herein by reference.
EXAMPLES
Example 1
Analysis of Expression of Human P2X.sub.4 (10218) in
Macrophages
[1931] This experiment describes the expression of 10218 in
macrophages stimulated with interferon gamma (IFN.gamma.) and
CD40L, cytokines which are known to be highly atherogenic, in order
to mimic the physiologic conditions involved in the atherosclerotic
state.
[1932] Macrophages were treated with IFN.gamma. and CD40L and
expression of 10218 mRNA was assessed by Taqman.TM. analysis.
[1933] Macrophages treated with IFN.gamma. and CD40L show increased
expression of 10218 at 4 hours and at 18 hours after treatment.
This data indicates a role of 10218 in the formation of
atherosclerotic lesions.
Example 2
Analysis of Expression of Human P2X.sub.4 (10218) mRNA in
Atherosclerotic Lesions in ApoE Knockout Mice
[1934] This experiment describes the use of ApoE knockout mice to
study the regulation of 10218 in atherosclerotic lesions at various
stages of lesion development and as compared to normal vessels.
[1935] The ApoE knockout mouse was created by gene targeting in
embryonic stem cells to disrupt the ApoE gene. The homozygous
inactivation of the ApoE gene results in animals that are devoid of
ApoE in their sera. These mice exhibit five times the normal serum
plasma cholesterol and spontaneous atherosclerotic lesions. This is
similar to a disease in humans who have a variant form of the ApoE
gene that is defective in binding to the LDL receptor and are at
risk for early development of atherosclerosis, and increased plasma
triglyceride and cholesterol levels. ApoE knockout mice are
routinely used to study modulators of atherosclerosis and the
pathogenesis of atherosclerosis.
[1936] In the ApoE knockout animals, the aortic arch region is
prone to formation of atherosclerotic lesions, whereas the
abdominal aorta is typically free of such lesions. At 5 weeks of
age lesion development is minimal, whereas by 18 weeks of age
complex lesion formation is observed, which persists at 33 weeks of
age.
[1937] In this experiment, the expression of 10218 was assessed in
C57 ApoE knockout animals at 8, 12, 17, 20, 22, 25, and 30 weeks of
age. Non-lesioned and lesioned tissue sections were dissected from
either the abdominal aorta (non-lesioned) or the aortic arch
(lesioned) from ApoE knockout animals at each of the above ages.
Vessels from wild-type mice were used as a control. 10218 is
upregulated in lesioned vessels as compared to non-lesioned vessels
and vessels obtained from normal animals at 17, 20, 22, 25, and 30
weeks of age indicating a correlation between 10218 expression and
the pathogenesis of atherosclerosis.
Example 3
Tissue Distribution of Human P2X.sub.4 (10218) mRNA Using
TaqMan.TM. Analysis
[1938] This example describes the tissue distribution of human
10218 mRNA in a variety of cells and tissues, as determined using
the TaqMan.TM. procedure. The Taqman.TM. procedure is a
quantitative, reverse transcription PCR-based approach for
detecting mRNA. The RT-PCR reaction exploits the 5' nuclease
activity of AmpliTaq Gold.TM. DNA Polymerase to cleave a TaqMan.TM.
probe during PCR. Briefly, cDNA was generated from the samples of
interest, e.g., heart, kidney, liver, skeletal muscle, and various
vessels, and used as the starting material for PCR amplification.
In addition to the 5' and 3' gene-specific primers, a gene-specific
oligonucleotide probe (complementary to the region being amplified)
was included in the reaction (i.e., the Taqman.TM. probe). The
TaqMan.TM. probe includes the oligonucleotide with a fluorescent
reporter dye covalently linked to the 5' end of the probe (such as
FAM (6-carboxyfluorescein), TET
(6-carboxy-4,7,2',7'-tetrachlorofluorescein), JOE
(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and a
quencher dye (TAMRA (6-carboxy-N,N,N',N'-tetramethylrhodamine) at
the 3' end of the probe.
[1939] During the PCR reaction, cleavage of the probe separates the
reporter dye and the quencher dye, resulting in increased
fluorescence of the reporter. Accumulation of PCR products is
detected directly by monitoring the increase in fluorescence of the
reporter dye. When the probe is intact, the proximity of the
reporter dye to the quencher dye results in suppression of the
reporter fluorescence. During PCR, if the target of interest is
present, the probe specifically anneals between the forward and
reverse primer sites. The 5'-3' nucleolytic activity of the
AmpliTaq.TM. Gold DNA Polymerase cleaves the probe between the
reporter and the quencher only if the probe hybridizes to the
target. The probe fragments are then displaced from the target, and
polymerization of the strand continues. The 3' end of the probe is
blocked to prevent extension of the probe during PCR. This process
occurs in every cycle and does not interfere with the exponential
accumulation of product. RNA was prepared using the trizol method
and treated with DNase to remove contaminating genomic DNA. cDNA
was synthesized using standard techniques. Mock cDNA synthesis in
the absence of reverse transcriptase resulted in samples with no
detectable PCR amplification of the control gene confirms efficient
removal of genomic DNA contamination.
[1940] A phase 1.3.4 panel including human normal and tumor tissue
indicated highest expression of 10218 mRNA in the pancreas, static
and shear HUVEC, and the brain. Expression of 10219 was also
detected in the kidney, heart, skeletal muscle, and liver, which
are all vascular rich organs. A cardiovascular vessel panel
indicated expression in various human vessels, including aortic
smooth muscle cells (SMC), coronary SMC, carotid artery, muscular
artery, diseased aorta, and normal vein. Highest expression was
detected in LSS HUVEC and static HUVEC. These expression data
indicate expression of 10218 across various vessels and in highly
vascularized organs, indicating a role of 10218 in the modulation
of cardiovascular disease, e.g., atherosclerosis.
Sequence CWU 1
1
28 1 5353 DNA Homo Sapiens 1 aaagggaata agcttgcggc cgcccggttc
ctgccatgcc cggcggcccc agtccccgca 60 gccccgcgcc tttgctgcgc
cccctcctcc tgctcctctg cgctctggct cccggcgccc 120 ccggacccgc
accaggacgt gcaaccgagg gccgggcggc actggacatc gtgcacccgg 180
ttcgagtcga cgcggggggc tccttcctgt cctacgagct gtggccccgc gcactgcgca
240 agcgggatgt atctgtgcgc cgagacgcgc ccgccttcta cgagctacaa
taccgcgggc 300 gcgagctgcg cttcaacctg accgccaatc agcacctgct
ggcgcccggc tttgtgagcg 360 agacgcggcg gcgcggcggc ctgggccgcg
cgcacatccg ggcccacacc ccggcctgcc 420 acctgcttgg cgaggtgcag
gaccctgagc tcgagggtgg cctggcggcc atcagcgcct 480 gcgacggcct
gaaaggtgtg ttccaactct ccaacgagga ctacttcatt gagcccctgg 540
acagtgcccc ggcccggcct ggccacgccc agccccatgt ggtgtacaag cgtcaggccc
600 cggagaggct ggcacagcgg ggtgattcca gtgctccaag cacctgtgga
gtgcaagtgt 660 acccagagct ggagtctcga cgggagcgtt gggagcagcg
gcagcagtgg cggcggccac 720 ggctgaggcg tctacaccag cggtcggtca
gcaaagagaa gtgggtggag accctggtag 780 tagctgatgc caaaatggtg
gagtaccacg gacagccgca ggttgagagc tatgtgctga 840 ccatcatgaa
catggtggct ggcctgtttc atgaccccag cattgggaac cccatccaca 900
tcaccattgt gcgcctggtc ctgctggaag atgaggagga ggacctaaag atcacgcacc
960 atgcagacaa caccctgaag agcttctgca agtggcagaa aagcatcaac
atgaaggggg 1020 atgcccatcc cctgcaccat gacactgcca tcctgctcac
cagaaaggac ctgtgtgcag 1080 ccatgaaccg gccctgtgag accctgggac
tgtcccatgt ggcgggcatg tgccagccgc 1140 accgcagctg cagcatcaac
gaggacacgg gcctgccgct ggccttcact gtagcccacg 1200 agctcgggca
cagttttggc attcagcatg acggaagcgg caatgactgt gagcccgttg 1260
ggaaacgacc tttcatcatg tctccacagc tcctgtacga cgccgctccc ctcacctggt
1320 cccgctgcag ccgccagtat atcaccaggt tccttgaccg tgggtggggc
ctgtgcctgg 1380 acgaccctcc tgccaaggac attatcgact tcccctcggt
gccacctggc gtcctctatg 1440 atgtaagcca ccagtgccgc ctccagtacg
gggcctactc tgccttctgc gaggacatgg 1500 ataatgtctg ccacacactc
tggtgctctg tggggaccac ctgtcactcc aagctggatg 1560 cagccgtgga
cggcacccgg tgtggggaga ataagtggtg tctcagtggg gagtgcgtac 1620
ccgtgggctt ccggcccgag gccgtggatg gtggctggtc tggctggagc gcctggtcca
1680 tctgctcacg gagctgtggc atgggcgtac agagcgccga gcggcagtgc
acgcagccta 1740 cgcccaaata caaaggcaga tactgtgtgg gtgagcgcaa
gcgcttccgc ctctgcaacc 1800 tgcaggcctg ccctgctggc crcccctcct
tccgccacgt ccagtgcagc cactttgacg 1860 ctatgctcta caagggccag
ctgcacacat gggtgcccgt ggtcaatgac gtgaacccct 1920 gcgagctgca
ctgccggccc gcgaatgagt actttgccga gaagctgcgg gacgccgtgg 1980
tcgatggcac cccctgctac caggtccgag ccagccggga cctctgcatc aacggcatct
2040 gtaagaacgt gggctgtgac ttcgagattg actccggtgc tatggaggac
cgctgtggtg 2100 tgtgccacgg caacggctcc acctgccaca ccgtgagcgg
gaccttcgag gaggccgagg 2160 gcctggggta tgtggatgtg gggctgatcc
cagccggcgc acgcgagatc cgcatccaag 2220 aggttgccga ggctgccaac
ttcctggcac tgcggagtga ggacccggag aagtacttcc 2280 tcaatggtgg
ctggaccatc cagtggaacg gggactacca ggtggcaggg accaccttca 2340
catacgcacg caggggcaac tgggagaacc tcacgtcccc gggtcccacc aaggagcctg
2400 tctggatcca gctgctgttc caggagagca accctggggt gcactacgag
tacaccatcc 2460 acagggaggc aggtggccac gacgaggtcc cgccgcccgt
gttctcctgg cattatgggc 2520 cctggaccaa gtgcacagtc acctgcggca
gaggtgtgca gaggcagaat gtgtactgct 2580 tggagcggca ggcagggccc
gtggacgagg agcactgtga ccccctgggc cggcctgatg 2640 accaacagag
gaagtgcagc gagcagccct gccctgccag gtggtgggca ggtgagtggc 2700
agctgtgctc cagctcctgc gggcctgggg gcctctcccg ccgggccgtg ctctgcatcc
2760 gcagcgtggg gctggatgag cagagcgccc tggagccacc cgcctgtgaa
caccttcccc 2820 ggccccctac tgaaacccct tgcaaccgcc atgtaccctg
tccggccacc tgggctgtgg 2880 ggaactggtc tcagtgctca gtgacatgtg
gggagggcac tcagcgccga aatgtcctct 2940 gcaccaatga caccggtgtc
ccctgtgacg aggcccagca gccagccagc gaagtcacct 3000 gctctctgcc
actctgtcgg tggcccctgg gcacactggg ccctgaaggc tcaggcagcg 3060
gctcctccag ccacgagctc ttcaacgagg ctgacttcat cccgcaccac ctggccccac
3120 gcccttcacc cgcctcatca cccaagccag gcaccatggg caacgccatt
gaggaggagg 3180 ctccagagct ggacctgccg gggcccgtgt ttgtggacga
cttctactac gactacaatt 3240 tcatcaattt ccacgaggat ctgtcctacg
ggccctctga ggagcccgat ctagacctgg 3300 cggggacagg ggaccggaca
cccccaccac acagccgtcc tgctgcgccc tccacgggta 3360 gccctgtgcc
tgccacagag cctcctgcag ccaaggagga gggggtactg ggaccttggt 3420
ccccgagccc ttggcctagc caggccggcc gctccccacc cccaccctca gagcagaccc
3480 ctgggaaccc tttgatcaat ttcctgcctg aggaagacac ccccataggg
gccccagatc 3540 ttgggctccc cagcctgtcc tggcccaggg tttccactga
tggcctgcag acacctgcca 3600 cccctgagag ccaaaatgat ttcccagttg
gcaaggacag ccagagccag ctgccccctc 3660 catggcggga caggaccaat
gaggttttca aggatgatga ggaacccaag ggccgcggag 3720 caccccacct
gcccccgaga cccagctcca cgctgccccc tttgtcccct gttggcagca 3780
cccactcctc tcctagtcct gacgtggcgg agctgtggac aggaggcaca gtggcctggg
3840 agccagctct ggagggtggc ctggggcctg tggacagtga actgtggccc
actgttgggg 3900 tggcttctct ccttcctcct cccatagccc ctctgccaga
gatgaaggtc agggacagtt 3960 ccctggagcc ggggactccc tccttcccag
ccccaggacc aggctcatgg gacctgcaga 4020 ctgtggcagt gtgggggacc
ttcctcccca caaccctgac tggcctcggg cacatgcctg 4080 agcctgccct
gaacccagga cccaagggtc agcctgagtc cctcacccct gaggtgcccc 4140
tgagctctag gctgctgtcc acaccagctt gggacagccc cgccaacagc cacagagtcc
4200 ctgagaccca gccgctggct cccagcctgg ctgaagcggg gccccccgcg
gacccgttgg 4260 ttgtcaggaa cgccagctgg caagcgggaa actggagcga
gtgctctacc acctgtggcc 4320 tgggtgcggt ctggaggccg gtgcgctgta
gctccggccg ggatgaggac tgcgcccccg 4380 ctggccggcc ccagcctgcc
cgccgctgcc acctacggcc ctgtgccacc tggcactcag 4440 gcaactggag
taagtgctcc cgcagctgcg acggaggttc ctcagtgcgg gacgtgcagt 4500
gtgtggacac acgggacctc cggccactgc ggcccttcca ttgtcagccc gggcctgcca
4560 agccgcatgc gcaccggccc tgcggggccc agccctgcct cagctggtac
acatcttcct 4620 ggagggagtg ctccgaggcc tgtggcggtg gtgagcagca
gcgtctagtg acctgcccgg 4680 agccaggcct ctgcgaggag gcgctgagac
ccaacaccac ccggccctgc aacacccacc 4740 cctgcacgca gtgggtggtg
gggccctggg gccagtgctc agccccctgt ggtggtggtg 4800 tccagcggcg
cctggtcaag tgtgtcaaca cccagacagg gctgcccgag gaagacagtg 4860
accagtgtgg ccacgaggcc tggcctgaga gctcccggcc gtgtggcacc gaggattgtg
4920 agcccgtcga gcctccccgc tgtgagcggg accgcctgtc cttcgggttc
tgcgagacgc 4980 tgcgcctact gggccgctgc cagctgccca ccatccgcac
ccagtgctgc cgctcgtgct 5040 ctccgcccag ccacggcgcc ccctcccgag
gccatcagcg ggttgcccgc cgctgactgt 5100 gccaggatgc acagaccgac
cgacagacct cagtgcccac cacgggctgt ggcggagctc 5160 ccgccccctg
cgccctaatg gtgctaaccc cctctcacta cccagcagca ggctggggac 5220
ctcctccccc tcaaaaaagg tattttttta ttctaacagt ttgtgtaaca tttattatga
5280 ttttacataa atgagcatct accaaaaaaa aaaaaaaagg gcggccgcta
gactagtcta 5340 gagaaaaaac ctc 5353 2 1686 PRT Homo Sapiens 2 Met
Pro Gly Gly Pro Ser Pro Arg Ser Pro Ala Pro Leu Leu Arg Pro 1 5 10
15 Leu Leu Leu Leu Leu Cys Ala Leu Ala Pro Gly Ala Pro Gly Pro Ala
20 25 30 Pro Gly Arg Ala Thr Glu Gly Arg Ala Ala Leu Asp Ile Val
His Pro 35 40 45 Val Arg Val Asp Ala Gly Gly Ser Phe Leu Ser Tyr
Glu Leu Trp Pro 50 55 60 Arg Ala Leu Arg Lys Arg Asp Val Ser Val
Arg Arg Asp Ala Pro Ala 65 70 75 80 Phe Tyr Glu Leu Gln Tyr Arg Gly
Arg Glu Leu Arg Phe Asn Leu Thr 85 90 95 Ala Asn Gln His Leu Leu
Ala Pro Gly Phe Val Ser Glu Thr Arg Arg 100 105 110 Arg Gly Gly Leu
Gly Arg Ala His Ile Arg Ala His Thr Pro Ala Cys 115 120 125 His Leu
Leu Gly Glu Val Gln Asp Pro Glu Leu Glu Gly Gly Leu Ala 130 135 140
Ala Ile Ser Ala Cys Asp Gly Leu Lys Gly Val Phe Gln Leu Ser Asn 145
150 155 160 Glu Asp Tyr Phe Ile Glu Pro Leu Asp Ser Ala Pro Ala Arg
Pro Gly 165 170 175 His Ala Gln Pro His Val Val Tyr Lys Arg Gln Ala
Pro Glu Arg Leu 180 185 190 Ala Gln Arg Gly Asp Ser Ser Ala Pro Ser
Thr Cys Gly Val Gln Val 195 200 205 Tyr Pro Glu Leu Glu Ser Arg Arg
Glu Arg Trp Glu Gln Arg Gln Gln 210 215 220 Trp Arg Arg Pro Arg Leu
Arg Arg Leu His Gln Arg Ser Val Ser Lys 225 230 235 240 Glu Lys Trp
Val Glu Thr Leu Val Val Ala Asp Ala Lys Met Val Glu 245 250 255 Tyr
His Gly Gln Pro Gln Val Glu Ser Tyr Val Leu Thr Ile Met Asn 260 265
270 Met Val Ala Gly Leu Phe His Asp Pro Ser Ile Gly Asn Pro Ile His
275 280 285 Ile Thr Ile Val Arg Leu Val Leu Leu Glu Asp Glu Glu Glu
Asp Leu 290 295 300 Lys Ile Thr His His Ala Asp Asn Thr Leu Lys Ser
Phe Cys Lys Trp 305 310 315 320 Gln Lys Ser Ile Asn Met Lys Gly Asp
Ala His Pro Leu His His Asp 325 330 335 Thr Ala Ile Leu Leu Thr Arg
Lys Asp Leu Cys Ala Ala Met Asn Arg 340 345 350 Pro Cys Glu Thr Leu
Gly Leu Ser His Val Ala Gly Met Cys Gln Pro 355 360 365 His Arg Ser
Cys Ser Ile Asn Glu Asp Thr Gly Leu Pro Leu Ala Phe 370 375 380 Thr
Val Ala His Glu Leu Gly His Ser Phe Gly Ile Gln His Asp Gly 385 390
395 400 Ser Gly Asn Asp Cys Glu Pro Val Gly Lys Arg Pro Phe Ile Met
Ser 405 410 415 Pro Gln Leu Leu Tyr Asp Ala Ala Pro Leu Thr Trp Ser
Arg Cys Ser 420 425 430 Arg Gln Tyr Ile Thr Arg Phe Leu Asp Arg Gly
Trp Gly Leu Cys Leu 435 440 445 Asp Asp Pro Pro Ala Lys Asp Ile Ile
Asp Phe Pro Ser Val Pro Pro 450 455 460 Gly Val Leu Tyr Asp Val Ser
His Gln Cys Arg Leu Gln Tyr Gly Ala 465 470 475 480 Tyr Ser Ala Phe
Cys Glu Asp Met Asp Asn Val Cys His Thr Leu Trp 485 490 495 Cys Ser
Val Gly Thr Thr Cys His Ser Lys Leu Asp Ala Ala Val Asp 500 505 510
Gly Thr Arg Cys Gly Glu Asn Lys Trp Cys Leu Ser Gly Glu Cys Val 515
520 525 Pro Val Gly Phe Arg Pro Glu Ala Val Asp Gly Gly Trp Ser Gly
Trp 530 535 540 Ser Ala Trp Ser Ile Cys Ser Arg Ser Cys Gly Met Gly
Val Gln Ser 545 550 555 560 Ala Glu Arg Gln Cys Thr Gln Pro Thr Pro
Lys Tyr Lys Gly Arg Tyr 565 570 575 Cys Val Gly Glu Arg Lys Arg Phe
Arg Leu Cys Asn Leu Gln Ala Cys 580 585 590 Pro Ala Gly Arg Pro Ser
Phe Arg His Val Gln Cys Ser His Phe Asp 595 600 605 Ala Met Leu Tyr
Lys Gly Gln Leu His Thr Trp Val Pro Val Val Asn 610 615 620 Asp Val
Asn Pro Cys Glu Leu His Cys Arg Pro Ala Asn Glu Tyr Phe 625 630 635
640 Ala Glu Lys Leu Arg Asp Ala Val Val Asp Gly Thr Pro Cys Tyr Gln
645 650 655 Val Arg Ala Ser Arg Asp Leu Cys Ile Asn Gly Ile Cys Lys
Asn Val 660 665 670 Gly Cys Asp Phe Glu Ile Asp Ser Gly Ala Met Glu
Asp Arg Cys Gly 675 680 685 Val Cys His Gly Asn Gly Ser Thr Cys His
Thr Val Ser Gly Thr Phe 690 695 700 Glu Glu Ala Glu Gly Leu Gly Tyr
Val Asp Val Gly Leu Ile Pro Ala 705 710 715 720 Gly Ala Arg Glu Ile
Arg Ile Gln Glu Val Ala Glu Ala Ala Asn Phe 725 730 735 Leu Ala Leu
Arg Ser Glu Asp Pro Glu Lys Tyr Phe Leu Asn Gly Gly 740 745 750 Trp
Thr Ile Gln Trp Asn Gly Asp Tyr Gln Val Ala Gly Thr Thr Phe 755 760
765 Thr Tyr Ala Arg Arg Gly Asn Trp Glu Asn Leu Thr Ser Pro Gly Pro
770 775 780 Thr Lys Glu Pro Val Trp Ile Gln Leu Leu Phe Gln Glu Ser
Asn Pro 785 790 795 800 Gly Val His Tyr Glu Tyr Thr Ile His Arg Glu
Ala Gly Gly His Asp 805 810 815 Glu Val Pro Pro Pro Val Phe Ser Trp
His Tyr Gly Pro Trp Thr Lys 820 825 830 Cys Thr Val Thr Cys Gly Arg
Gly Val Gln Arg Gln Asn Val Tyr Cys 835 840 845 Leu Glu Arg Gln Ala
Gly Pro Val Asp Glu Glu His Cys Asp Pro Leu 850 855 860 Gly Arg Pro
Asp Asp Gln Gln Arg Lys Cys Ser Glu Gln Pro Cys Pro 865 870 875 880
Ala Arg Trp Trp Ala Gly Glu Trp Gln Leu Cys Ser Ser Ser Cys Gly 885
890 895 Pro Gly Gly Leu Ser Arg Arg Ala Val Leu Cys Ile Arg Ser Val
Gly 900 905 910 Leu Asp Glu Gln Ser Ala Leu Glu Pro Pro Ala Cys Glu
His Leu Pro 915 920 925 Arg Pro Pro Thr Glu Thr Pro Cys Asn Arg His
Val Pro Cys Pro Ala 930 935 940 Thr Trp Ala Val Gly Asn Trp Ser Gln
Cys Ser Val Thr Cys Gly Glu 945 950 955 960 Gly Thr Gln Arg Arg Asn
Val Leu Cys Thr Asn Asp Thr Gly Val Pro 965 970 975 Cys Asp Glu Ala
Gln Gln Pro Ala Ser Glu Val Thr Cys Ser Leu Pro 980 985 990 Leu Cys
Arg Trp Pro Leu Gly Thr Leu Gly Pro Glu Gly Ser Gly Ser 995 1000
1005 Gly Ser Ser Ser His Glu Leu Phe Asn Glu Ala Asp Phe Ile Pro
His 1010 1015 1020 His Leu Ala Pro Arg Pro Ser Pro Ala Ser Ser Pro
Lys Pro Gly Thr 1025 1030 1035 1040 Met Gly Asn Ala Ile Glu Glu Glu
Ala Pro Glu Leu Asp Leu Pro Gly 1045 1050 1055 Pro Val Phe Val Asp
Asp Phe Tyr Tyr Asp Tyr Asn Phe Ile Asn Phe 1060 1065 1070 His Glu
Asp Leu Ser Tyr Gly Pro Ser Glu Glu Pro Asp Leu Asp Leu 1075 1080
1085 Ala Gly Thr Gly Asp Arg Thr Pro Pro Pro His Ser Arg Pro Ala
Ala 1090 1095 1100 Pro Ser Thr Gly Ser Pro Val Pro Ala Thr Glu Pro
Pro Ala Ala Lys 1105 1110 1115 1120 Glu Glu Gly Val Leu Gly Pro Trp
Ser Pro Ser Pro Trp Pro Ser Gln 1125 1130 1135 Ala Gly Arg Ser Pro
Pro Pro Pro Ser Glu Gln Thr Pro Gly Asn Pro 1140 1145 1150 Leu Ile
Asn Phe Leu Pro Glu Glu Asp Thr Pro Ile Gly Ala Pro Asp 1155 1160
1165 Leu Gly Leu Pro Ser Leu Ser Trp Pro Arg Val Ser Thr Asp Gly
Leu 1170 1175 1180 Gln Thr Pro Ala Thr Pro Glu Ser Gln Asn Asp Phe
Pro Val Gly Lys 1185 1190 1195 1200 Asp Ser Gln Ser Gln Leu Pro Pro
Pro Trp Arg Asp Arg Thr Asn Glu 1205 1210 1215 Val Phe Lys Asp Asp
Glu Glu Pro Lys Gly Arg Gly Ala Pro His Leu 1220 1225 1230 Pro Pro
Arg Pro Ser Ser Thr Leu Pro Pro Leu Ser Pro Val Gly Ser 1235 1240
1245 Thr His Ser Ser Pro Ser Pro Asp Val Ala Glu Leu Trp Thr Gly
Gly 1250 1255 1260 Thr Val Ala Trp Glu Pro Ala Leu Glu Gly Gly Leu
Gly Pro Val Asp 1265 1270 1275 1280 Ser Glu Leu Trp Pro Thr Val Gly
Val Ala Ser Leu Leu Pro Pro Pro 1285 1290 1295 Ile Ala Pro Leu Pro
Glu Met Lys Val Arg Asp Ser Ser Leu Glu Pro 1300 1305 1310 Gly Thr
Pro Ser Phe Pro Ala Pro Gly Pro Gly Ser Trp Asp Leu Gln 1315 1320
1325 Thr Val Ala Val Trp Gly Thr Phe Leu Pro Thr Thr Leu Thr Gly
Leu 1330 1335 1340 Gly His Met Pro Glu Pro Ala Leu Asn Pro Gly Pro
Lys Gly Gln Pro 1345 1350 1355 1360 Glu Ser Leu Thr Pro Glu Val Pro
Leu Ser Ser Arg Leu Leu Ser Thr 1365 1370 1375 Pro Ala Trp Asp Ser
Pro Ala Asn Ser His Arg Val Pro Glu Thr Gln 1380 1385 1390 Pro Leu
Ala Pro Ser Leu Ala Glu Ala Gly Pro Pro Ala Asp Pro Leu 1395 1400
1405 Val Val Arg Asn Ala Ser Trp Gln Ala Gly Asn Trp Ser Glu Cys
Ser 1410 1415 1420 Thr Thr Cys Gly Leu Gly Ala Val Trp Arg Pro Val
Arg Cys Ser Ser 1425 1430 1435 1440 Gly Arg Asp Glu Asp Cys Ala Pro
Ala Gly Arg Pro Gln Pro Ala Arg 1445 1450 1455 Arg Cys His Leu Arg
Pro Cys Ala Thr Trp His Ser Gly Asn Trp Ser 1460 1465 1470 Lys Cys
Ser Arg Ser Cys Gly Gly Gly Ser Ser Val Arg Asp Val Gln 1475 1480
1485 Cys Val Asp Thr Arg Asp Leu Arg Pro Leu Arg Pro Phe His Cys
Gln 1490 1495 1500 Pro Gly Pro Ala Lys Pro Pro Ala His Arg Pro Cys
Gly Ala Gln Pro 1505 1510 1515 1520 Cys Leu Ser Trp Tyr Thr Ser Ser
Trp Arg Glu Cys Ser Glu Ala Cys 1525 1530 1535 Gly Gly Gly Glu Gln
Gln Arg Leu Val Thr Cys Pro Glu Pro Gly Leu 1540 1545 1550 Cys Glu
Glu Ala Leu
Arg Pro Asn Thr Thr Arg Pro Cys Asn Thr His 1555 1560 1565 Pro Cys
Thr Gln Trp Val Val Gly Pro Trp Gly Gln Cys Ser Ala Pro 1570 1575
1580 Cys Gly Gly Gly Val Gln Arg Arg Leu Val Lys Cys Val Asn Thr
Gln 1585 1590 1595 1600 Thr Gly Leu Pro Glu Glu Asp Ser Asp Gln Cys
Gly His Glu Ala Trp 1605 1610 1615 Pro Glu Ser Ser Arg Pro Cys Gly
Thr Glu Asp Cys Glu Pro Val Glu 1620 1625 1630 Pro Pro Arg Cys Glu
Arg Asp Arg Leu Ser Phe Gly Phe Cys Glu Thr 1635 1640 1645 Leu Arg
Leu Leu Gly Arg Cys Gln Leu Pro Thr Ile Arg Thr Gln Cys 1650 1655
1660 Cys Arg Ser Cys Ser Pro Pro Ser His Gly Ala Pro Ser Arg Gly
His 1665 1670 1675 1680 Gln Arg Val Ala Arg Arg 1685 3 2662 DNA
Homo Sapiens misc_feature (1)...(2662) n = A,T,C or G 3 ggagggcctg
aagagacagg gaggttgtgc caggctggag gaggcttgtc tttccgaagc 60
tggagaggat cttacggggg ttcgcttttc cctgcctggg aagaatttcc cctgtggtag
120 cagcagcagc agcagcagaa gcagaaacag cagcagcagc aacagcagca
gcagcagcag 180 caccaccacc accactacct cctcttctgg ggcacaagac
agaatgcctg tgctagagcg 240 atatttccac ccagcagagc taggcaggag
gtggacaggc ccagaaggtg tgctgccctc 300 ctccccggga agccggccgg
ggtgccagca ggggccgctg ccctgggact tgccagagat 360 gatcaggatg
gtaaagctgg tttggaaatc caaaagtgag ctgcaggcga ccaaacagag 420
aggcattctg gacaatgaag atgctctccg cagctttcca ggagatatac gactaagggg
480 tcagacgggg gttcgtgctg aacgccgtgg ctcctaccca ttcattgact
tccgcctact 540 taacagtaca acatactcag gggagattgg caccaagaaa
aaggtgaaaa gactattaag 600 ctttcaaaga tacttccatg catcaaggct
gcttcgtgga attataccac aagcccctct 660 gcacctgctg gatgaagact
accttggaca agcaaggcat atgctctcca aagtgggaat 720 gtgggatttt
gacattttct tgtttgatcg cttgacaaat ggaaacagcc tggtaacact 780
gttgtgccac ctcttcaata cccatggact cattcaccat ttcaagttag atatggtgac
840 cttacaccga tttttagtca tggttcaaga agattaccac agccaaaacc
cgtatcacaa 900 tgctgttcac gcagccgacg tcacccaggc catgcactgc
tacctgaaag agccaaagct 960 tgccagcttc ctcacgcctc tggacatcat
gcttggactg ctggctgcag cagcacacga 1020 tgtggaccac ccaggggtga
accagccatt tttgataaaa actaaccacc atcttgcaaa 1080 cctatatcag
aatatgtctg tgctggagaa tcatcactgg cgatctacaa ttggcatgct 1140
tcgagaatca aggcttcttg ctcatttgcc aaaggaaatg acacaggata ttgaacagca
1200 gctgggctcc ttgatcttgg caacagacat caacaggcag aatgaatttt
tgaccagatt 1260 gaaagctcac ctccacaata aagacttaag actggaggat
gcacaggaca ggcactttat 1320 gcttcagatc gccttgaagt gtgctgacat
ttgcaatcct tgtagaatct gggagatgag 1380 caagcagtgg agtgaaaggg
tctgtgaaga attctacagg caaggtgaac ttgaacagaa 1440 atttgaactg
gaaatcagtc ctctttgtaa tcaacagaaa gattccatcc ctagtataca 1500
aattggtttc atgagctaca tcgtggagcc gctcttccgg gaatgggccc atttcacggg
1560 taacagcacc ctgtcggaga acatgctggg ccacctcgca cacaacaagg
cccagtggaa 1620 gagcctgttg cccaggcagc acagaagcag gggcagcagt
ggcagcgggc ctgaccacga 1680 ccacgcaggc caagggactg agagcgagga
gcaggaaggc gacagcccct aggggccggc 1740 ccaacttaga cgcggctctc
ctccggcagg gcccccagag ggcagaagca gcgtggaggg 1800 gccctcacgc
agcagcccag ccactttctg agtgttgtcc tggggctctt tggaacgcca 1860
tcttcctccc acttacctgc ctcccctcct tttcgcaaat gtacagaagc catttgtcac
1920 ctcagcattc gctgccgaaa tgagcaactc cattcagtaa cgtgggagct
gatcccacgg 1980 gcaggctctc cctgctccag gagaagacta ggaggaagaa
tgaggtgctc ctgccgtgtc 2040 cgccttgttc cgggtcgcac tggaacaggc
agcaattcct aagtccggag cgtttgagcg 2100 tttgctatct gactgctgat
ctgcgtgaca gaaacaccag catatttgca acgccaagga 2160 tattggtctt
aagtgcaaga gcacaaatga gagtgtgaga gaaagkacct tctattttaa 2220
taataatatt attataaaaa taataaatct ttttaacttt tatattttat gcactagnca
2280 atggatctgc aactttggac taaggtcatt caatgtaccc aaacttgaac
agggggttca 2340 ttgttttgct attgacttta ttatgccact ttggggcaga
gacttggcat cttcgcagtt 2400 taagaaacca cgtttcctat ccaatccgaa
gggaaggtgc tgtacagttc attcctttgc 2460 accattagcc aatctgtctt
ttatggattc tgtgacatgt ttatattcac ccatgtacat 2520 tttctgtaaa
taccaaacgg ctactgattc ccatgccaaa atacatgagt attatgggat 2580
tgctacctgt ataaacaatg gcactgtgaa aatactgtta gttttaatac aanagaatgc
2640 atttgtaaaa aaaaaaaaaa aa 2662 4 502 PRT Homo Sapiens 4 Met Pro
Val Leu Glu Arg Tyr Phe His Pro Ala Glu Leu Gly Arg Arg 1 5 10 15
Trp Thr Gly Pro Glu Gly Val Leu Pro Ser Ser Pro Gly Ser Arg Pro 20
25 30 Gly Cys Gln Gln Gly Pro Leu Pro Trp Asp Leu Pro Glu Met Ile
Arg 35 40 45 Met Val Lys Leu Val Trp Lys Ser Lys Ser Glu Leu Gln
Ala Thr Lys 50 55 60 Gln Arg Gly Ile Leu Asp Asn Glu Asp Ala Leu
Arg Ser Phe Pro Gly 65 70 75 80 Asp Ile Arg Leu Arg Gly Gln Thr Gly
Val Arg Ala Glu Arg Arg Gly 85 90 95 Ser Tyr Pro Phe Ile Asp Phe
Arg Leu Leu Asn Ser Thr Thr Tyr Ser 100 105 110 Gly Glu Ile Gly Thr
Lys Lys Lys Val Lys Arg Leu Leu Ser Phe Gln 115 120 125 Arg Tyr Phe
His Ala Ser Arg Leu Leu Arg Gly Ile Ile Pro Gln Ala 130 135 140 Pro
Leu His Leu Leu Asp Glu Asp Tyr Leu Gly Gln Ala Arg His Met 145 150
155 160 Leu Ser Lys Val Gly Met Trp Asp Phe Asp Ile Phe Leu Phe Asp
Arg 165 170 175 Leu Thr Asn Gly Asn Ser Leu Val Thr Leu Leu Cys His
Leu Phe Asn 180 185 190 Thr His Gly Leu Ile His His Phe Lys Leu Asp
Met Val Thr Leu His 195 200 205 Arg Phe Leu Val Met Val Gln Glu Asp
Tyr His Ser Gln Asn Pro Tyr 210 215 220 His Asn Ala Val His Ala Ala
Asp Val Thr Gln Ala Met His Cys Tyr 225 230 235 240 Leu Lys Glu Pro
Lys Leu Ala Ser Phe Leu Thr Pro Leu Asp Ile Met 245 250 255 Leu Gly
Leu Leu Ala Ala Ala Ala His Asp Val Asp His Pro Gly Val 260 265 270
Asn Gln Pro Phe Leu Ile Lys Thr Asn His His Leu Ala Asn Leu Tyr 275
280 285 Gln Asn Met Ser Val Leu Glu Asn His His Trp Arg Ser Thr Ile
Gly 290 295 300 Met Leu Arg Glu Ser Arg Leu Leu Ala His Leu Pro Lys
Glu Met Thr 305 310 315 320 Gln Asp Ile Glu Gln Gln Leu Gly Ser Leu
Ile Leu Ala Thr Asp Ile 325 330 335 Asn Arg Gln Asn Glu Phe Leu Thr
Arg Leu Lys Ala His Leu His Asn 340 345 350 Lys Asp Leu Arg Leu Glu
Asp Ala Gln Asp Arg His Phe Met Leu Gln 355 360 365 Ile Ala Leu Lys
Cys Ala Asp Ile Cys Asn Pro Cys Arg Ile Trp Glu 370 375 380 Met Ser
Lys Gln Trp Ser Glu Arg Val Cys Glu Glu Phe Tyr Arg Gln 385 390 395
400 Gly Glu Leu Glu Gln Lys Phe Glu Leu Glu Ile Ser Pro Leu Cys Asn
405 410 415 Gln Gln Lys Asp Ser Ile Pro Ser Ile Gln Ile Gly Phe Met
Ser Tyr 420 425 430 Ile Val Glu Pro Leu Phe Arg Glu Trp Ala His Phe
Thr Gly Asn Ser 435 440 445 Thr Leu Ser Glu Asn Met Leu Gly His Leu
Ala His Asn Lys Ala Gln 450 455 460 Trp Lys Ser Leu Leu Pro Arg Gln
His Arg Ser Arg Gly Ser Ser Gly 465 470 475 480 Ser Gly Pro Asp His
Asp His Ala Gly Gln Gly Thr Glu Ser Glu Glu 485 490 495 Gln Glu Gly
Asp Ser Pro 500 5 3336 DNA Homo Sapiens misc_feature (1)...(3336) n
= A,T,C or G 5 gagggcctga agagacaggg aggttgtgcc aggctggagg
aggcttgtct ttccgaagct 60 ggagaggatc ttacgggggt tcgcttttcc
ctgcctggga agaatttccc ctgtggtagc 120 agcagcagca gcagcagaag
cagaaacagc agcagcagca acagcagcag cagcagcagc 180 accaccacca
ccactacctc ctcttctggg gcacaagaca gaatgcctgt gctagagcgc 240
tatttccacc cagcagagct aggcaggagg tggacaggcc cagaaggtgt gctgccctcc
300 tccccgggaa gccggccggg gtgccagcag gggccgctgc cctgggactt
gccagagatg 360 atcaggatgg taaagctggt ttggaaatcc aaaagtgagc
tgcaggcgac caaacagaga 420 ggcattctgg acaatgaaga tgctctccgc
agctttccag gagatatacg actaaggggt 480 cagacggggg ttcgtgctga
acgccgtggc tcctacccat tcattgactt ccgcctactt 540 aacagtacaa
catactcagg ggagattggc accaagaaaa aggtgaaaag actattaagc 600
tttcaaagat acttccatgc atcaaggctg cttcgtggaa ttataccaca agcccctctg
660 cacctgctgg atgaagacta ccttggacaa gcaaggcata tgctctccaa
agtgggaatg 720 tgggattttg acattttctt gtttgatcgc ttgacaaatg
gaaacagcct ggtaacactg 780 ttgtgccacc tcttcaatac ccatggactc
attcaccatt tcaagttaga tatggtgacc 840 ttacaccgat ttttagtcat
ggttcaagaa gattaccaca gccaaaaccc gtatcacaat 900 gctgttcacg
cagccgacgt cacccaggcc atgcactgct acctgaaaga gccaaagctt 960
gccagcttcc tcacgcctct ggacatcatg cttggactgc tggctgcagc agcacacgat
1020 gtggaccacc caggggtgaa ccagccattt ttgataaaaa ctaaccacca
tcttgcaaac 1080 ctatatcaga atatgtctgt gctggagaat catcactggc
gatctacaat tggcatgctt 1140 cgagaatcaa ggcttcttgc tcatttgcca
aaggaaatga cgtaagtgct gccgagatga 1200 aacatactga tgtgcatgca
gtaaagataa gccactttct ctagggcagg cttgggacct 1260 tttgcgtgaa
tggcagagag ccccccggct gtacttcctg cctgcactga gctgtctatc 1320
agaggagatt tggtgtcagt tacagcaacc cagaaaccaa aatctctctg tgtgctttga
1380 aagggccttg cagagtcaat gacctacagt caggaaaagg gataataaac
agctctcagt 1440 tttcacacgc ttcagtatca gtgctcgact ttgccaaatt
cccgaccttt agtttagcaa 1500 aattgtcctt ccatgtagct ccaaatagta
aatatttatc aagaaggaac ccaggcattc 1560 taaagctaga gttcaaaaaa
gtatattttg taattgctag tctcagcaaa aatagaagtc 1620 agaaattctt
ttctaaaatg tcttttgcta agtaattgaa atggccctag catttttttc 1680
accaattaat ttaccttacg tctcttgcac tttaaacaga aggggagaca ctcattttct
1740 ggttcactat ttgatagcca tggtatgtag gctgagtccc actaaatctg
aggccattgt 1800 ttcattttcc tggtggcccc aagttagctg ctaatactgt
cttccaaggc caccattaat 1860 tctgatctgt ttaatgaaca cgtgcagaac
ccaagaaacc taggtgaaaa gagtacatag 1920 attgctgtac ccttcttcaa
gacaagcaca taacttgagg tcaaggacca agtgctgtct 1980 cccaactgaa
caagcagtat actctgggtt gtggattgat tcctggccct ctgatttgat 2040
ctcatgctgt ttcctagcac ccagaggaat gtgaaatttg caggaggaat ttcagttctg
2100 ataaattttt actccctgga actaaataaa accagttctc gtgcatggaa
taaaaactta 2160 tgcctcttac tagaataata aattgcaaag attgaaagaa
ttaaatgcaa aaagaactaa 2220 aaactagagc aaaagatcaa gtgagaagaa
gaaaagagga ggtaaggaga gagacaagga 2280 agaaagaagg agaaggaaag
gaagaatagt gaggacagga aagaagaaaa tgcaagggaa 2340 atgggaaagg
actctggggt gaccagactt ctcctggtca gtacctgcat tcatcctgtt 2400
tgttactcaa tatttctttc ctaaaatatt catttcacat ctatggattc caatgaaaaa
2460 tatattttta tgtgtctttg tggaacacag tgttataaat tgtttttgcc
agaagaataa 2520 ttgttataca ataatatatg tgaaaacttt attacaaaag
ccattatcat aatcattatt 2580 attccttcta tcacaggtaa atgctttaat
gtcatttttc tgattttaaa agtagggcag 2640 gttaattgta gaaagtaagg
aaaattcagg aaagtgttag tttgaactat gtgaagttgc 2700 tctttttaag
ggccaaaaac aggagacttt tagcactttc atatgtttca gcttgatatg 2760
aaagagaaaa ctgaaactgc tagtaatcct gccatccagg tatagttcat gttaacctgg
2820 ctagtttatt ttcttttagt cttttttcaa tacaaactta ttttaacaaa
atatgattan 2880 atttggggaa cttattttac agtttacgtc ctgaaatttt
ttatttacaa taaagacttt 2940 tttccaaatc attaaacctg ttaaattaaa
atgattttgt cagccgtatg gcattattgt 3000 ataccactac tgcctttcat
ttggaattca aatggtttcc aatatcccaa actttgatac 3060 tctgttttct
caggaagtat ttgtagataa aaattattgg tcagaaaggt ctgaactttt 3120
aagtttcttg tatattatcc agttgttctt ctaaaaggct gtatctacct gtattccaac
3180 tgatggattg taagaaaatg taccaatgta ccatcaccaa aattgagttt
atttttatct 3240 ttttaaaata tttgcaaatt tgacatatat gtatgtatat
acacaaatat atatgtaaag 3300 tggttttcat taaattagta tgcatccttt acttac
3336 6 320 PRT Homo Sapiens 6 Met Pro Val Leu Glu Arg Tyr Phe His
Pro Ala Glu Leu Gly Arg Arg 1 5 10 15 Trp Thr Gly Pro Glu Gly Val
Leu Pro Ser Ser Pro Gly Ser Arg Pro 20 25 30 Gly Cys Gln Gln Gly
Pro Leu Pro Trp Asp Leu Pro Glu Met Ile Arg 35 40 45 Met Val Lys
Leu Val Trp Lys Ser Lys Ser Glu Leu Gln Ala Thr Lys 50 55 60 Gln
Arg Gly Ile Leu Asp Asn Glu Asp Ala Leu Arg Ser Phe Pro Gly 65 70
75 80 Asp Ile Arg Leu Arg Gly Gln Thr Gly Val Arg Ala Glu Arg Arg
Gly 85 90 95 Ser Tyr Pro Phe Ile Asp Phe Arg Leu Leu Asn Ser Thr
Thr Tyr Ser 100 105 110 Gly Glu Ile Gly Thr Lys Lys Lys Val Lys Arg
Leu Leu Ser Phe Gln 115 120 125 Arg Tyr Phe His Ala Ser Arg Leu Leu
Arg Gly Ile Ile Pro Gln Ala 130 135 140 Pro Leu His Leu Leu Asp Glu
Asp Tyr Leu Gly Gln Ala Arg His Met 145 150 155 160 Leu Ser Lys Val
Gly Met Trp Asp Phe Asp Ile Phe Leu Phe Asp Arg 165 170 175 Leu Thr
Asn Gly Asn Ser Leu Val Thr Leu Leu Cys His Leu Phe Asn 180 185 190
Thr His Gly Leu Ile His His Phe Lys Leu Asp Met Val Thr Leu His 195
200 205 Arg Phe Leu Val Met Val Gln Glu Asp Tyr His Ser Gln Asn Pro
Tyr 210 215 220 His Asn Ala Val His Ala Ala Asp Val Thr Gln Ala Met
His Cys Tyr 225 230 235 240 Leu Lys Glu Pro Lys Leu Ala Ser Phe Leu
Thr Pro Leu Asp Ile Met 245 250 255 Leu Gly Leu Leu Ala Ala Ala Ala
His Asp Val Asp His Pro Gly Val 260 265 270 Asn Gln Pro Phe Leu Ile
Lys Thr Asn His His Leu Ala Asn Leu Tyr 275 280 285 Gln Asn Met Ser
Val Leu Glu Asn His His Trp Arg Ser Thr Ile Gly 290 295 300 Met Leu
Arg Glu Ser Arg Leu Leu Ala His Leu Pro Lys Glu Met Thr 305 310 315
320 7 2898 DNA Homo Sapiens 7 gcagcggcgg cggcggcggg gcctggagcc
ggatctaaga tggcagcggc ggcagcggcg 60 gtggggccgg gcgcgggggg
cgcggggtcg gcggtcccgg gcggcgcggg gccctgcgct 120 accgtgtcgg
tgttccccgg cgcccgcctc ctcaccatcg gcgacgcgaa cggcgagatc 180
cagcggcacg cggagcagca ggcgctgcgc ctcgaggtgc gcgccggccc ggactcggcg
240 ggcatcgccc tctacagcca tgaagatgtg tgtgtcttta agtgctcagt
gtcccgagag 300 acagagtgca gccgtgtggg caagcagtcc ttcatcatca
ccctgggctg caacagcgtc 360 ctcatccagt tcgccacacc caacgatttc
tgttccttct acaacatcct gaaaacctgc 420 cggggccaca ccctggagcg
gtctgtgttc agcgagcgga cggaggagtc ttctgccgtg 480 cagtacttcc
agttttatgg ctacctgtcc cagcagcaga acatgatgca ggactacgtg 540
cggacaggca cctaccagcg cgccatcctg caaaaccaca ccgacttcaa ggacaagatc
600 gttcttgatg ttggctgtgg ctctgggatc ctgtcgtttt ttgccgccca
agctggagca 660 cggaaaatct acgcggtgga ggccagcacc atggcccagc
acgctgaggt cttggtgaag 720 agtaacaacc tgacggaccg catcgtggtc
atcccgggca aggtggagga ggtgtcactc 780 cccgagcagg tggacatcat
catctcggag cccatgggct acatgctctt caacgagcgc 840 atgctggaga
gctacctcca cgccaagaag tacctgaagc ccagcggaaa catgtttcct 900
accattggtg acgtccacct tgcacccttc acggatgaac agctctacat ggagcagttc
960 accaaggcca acttctggta ccagccatct ttccatggag tggacctgtc
ggccctccga 1020 ggtgccgcgg tggatgagta tttccggcag cctgtggtgg
acacatttga catccggatc 1080 ctgatggcca agtctgtcaa gtacacggtg
aacttcttag aagccaaaga aggagatttg 1140 cacaggatag aaatcccatt
caaattccac atgctgcatt cagggctggt ccacggcctg 1200 gctttctggt
ttgacgttgc tttcatcggc tccataatga ccgtgtggct gtccacagcc 1260
ccgacagagc ccctgaccca ctggtaccag gtgcggtgcc tgttccagtc accactgttc
1320 gccaaggcag gggacacgct ctcagggaca tgtctgctta ttgccaacaa
aagacagagc 1380 tacgacatca gtattgtggc ccaggtggac cagaccggct
ccaagtccag taacctcctg 1440 gatctgaaaa accccttctt tagatacacg
ggcacaacgc cctcaccccc acccggctcc 1500 cactacacat ctccctcgga
aaacatgtgg aacacgggca gcacctacaa cctcagcagc 1560 gggatggccg
tggcagggat gccgaccgcc tatgacttga gcagtgttat tgccagtggc 1620
tccagcgtgg gccacaacaa cctgattcct ttagccaaca cggggattgt caatcacacc
1680 cactcccgga tgggctccat aatgagcacg gggattgtcc aagggtcctc
cggcgcccag 1740 ggcagtggtg gtggcagcac gagtgcccac tatgcagtca
acagccagtt caccatgggc 1800 ggccccgcca tctccwtggc gtcgcccatg
tccatcccga ccaacaccat gcactacggg 1860 agctaggggc ccgccccgcg
gactgacagc accaggaaac caaatgatgt ccctgcccgc 1920 cgcccccgcc
gggcggcttt cccccttgta ctggagaagc tcgaacaccc ggtcacagct 1980
ctctttgcta tgggaactgg gacacttttt tacacgatgt tgccgccgtc cccaccctaa
2040 cccccacctc ccggccctga gcgtgtgtcg ctgccatatt ttacacaaaa
tcatgttgtg 2100 ggagccctcg tcccccctcc tgcccgctct accctgacct
gggcttgtca tctgctggaa 2160 caggcgccat ggggcctgcc agccctgcct
gccaggtccc ttagcacctg tccccctgcc 2220 tgtctccagt gggaaggtag
cctggccagg cggggcctcc ccttcgacga ccaggcctcg 2280 gtcacaacgg
acgtgacatg ctgctttttt taattttatt tttttatgaa aagaaccagt 2340
gtcaatccgc agaccctctg tgaagccagg ccggccgggc cgagccagca gcccctctcc
2400 ctagactcag aggcgccgcg gggaggggtg gccccgccga ggcttcaggg
gccccctccc 2460 caccaaaggg ttcacctcac acttgaatgt acaacccacc
ccactgtcgg gaaggcctcc 2520 gtcctcggcc cctgcctctt gctgctgtcc
tgtccccgag cccctgcagg tccccccccg 2580 cccccccact caagagttag
agcaggtggc tgcaggcctt gggcccggag ggaaggccac 2640 tgccggccac
ttggggcaga cacagacacc tcaaggatct gtcacggaag gcgtcctttt 2700
tccttgtagc taacgttagg cctgagtagc tcccctccat ccttgtagac gctccagtcc
2760 ctactactgt gacggcattt ccatccctcc cctgcccggg aagggacctt
gcagggacct 2820 ctccctccaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaagggsr aacgtgttgc 2880 aaaaaaaaaa aaaaaaaa 2898 8 608 PRT
Homo Sapiens VARIANT (1)...(608) Xaa = Any Amino Acid 8 Met Ala Ala
Ala Ala Ala Ala Val Gly Pro Gly Ala Gly Gly Ala Gly 1 5 10 15 Ser
Ala Val Pro Gly Gly Ala Gly Pro Cys Ala Thr Val Ser Val Phe 20 25
30 Pro Gly Ala Arg Leu Leu Thr Ile Gly Asp Ala Asn Gly Glu Ile Gln
35 40 45 Arg His Ala Glu Gln Gln Ala Leu Arg Leu Glu Val Arg Ala
Gly Pro 50 55 60 Asp Ser Ala Gly Ile Ala Leu Tyr Ser His Glu Asp
Val Cys Val Phe 65 70 75 80 Lys Cys Ser Val Ser Arg Glu Thr Glu Cys
Ser Arg Val Gly Lys Gln 85 90 95 Ser Phe Ile Ile Thr Leu Gly Cys
Asn Ser Val Leu Ile Gln Phe Ala 100 105 110 Thr Pro Asn Asp Phe Cys
Ser Phe Tyr Asn Ile Leu Lys Thr Cys Arg 115 120 125 Gly His Thr Leu
Glu Arg Ser Val Phe Ser Glu Arg Thr Glu Glu Ser 130 135 140 Ser Ala
Val Gln Tyr Phe Gln Phe Tyr Gly Tyr Leu Ser Gln Gln Gln 145 150 155
160 Asn Met Met Gln Asp Tyr Val Arg Thr Gly Thr Tyr Gln Arg Ala Ile
165 170 175 Leu Gln Asn His Thr Asp Phe Lys Asp Lys Ile Val Leu Asp
Val Gly 180 185 190 Cys Gly Ser Gly Ile Leu Ser Phe Phe Ala Ala Gln
Ala Gly Ala Arg 195 200 205 Lys Ile Tyr Ala Val Glu Ala Ser Thr Met
Ala Gln His Ala Glu Val 210 215 220 Leu Val Lys Ser Asn Asn Leu Thr
Asp Arg Ile Val Val Ile Pro Gly 225 230 235 240 Lys Val Glu Glu Val
Ser Leu Pro Glu Gln Val Asp Ile Ile Ile Ser 245 250 255 Glu Pro Met
Gly Tyr Met Leu Phe Asn Glu Arg Met Leu Glu Ser Tyr 260 265 270 Leu
His Ala Lys Lys Tyr Leu Lys Pro Ser Gly Asn Met Phe Pro Thr 275 280
285 Ile Gly Asp Val His Leu Ala Pro Phe Thr Asp Glu Gln Leu Tyr Met
290 295 300 Glu Gln Phe Thr Lys Ala Asn Phe Trp Tyr Gln Pro Ser Phe
His Gly 305 310 315 320 Val Asp Leu Ser Ala Leu Arg Gly Ala Ala Val
Asp Glu Tyr Phe Arg 325 330 335 Gln Pro Val Val Asp Thr Phe Asp Ile
Arg Ile Leu Met Ala Lys Ser 340 345 350 Val Lys Tyr Thr Val Asn Phe
Leu Glu Ala Lys Glu Gly Asp Leu His 355 360 365 Arg Ile Glu Ile Pro
Phe Lys Phe His Met Leu His Ser Gly Leu Val 370 375 380 His Gly Leu
Ala Phe Trp Phe Asp Val Ala Phe Ile Gly Ser Ile Met 385 390 395 400
Thr Val Trp Leu Ser Thr Ala Pro Thr Glu Pro Leu Thr His Trp Tyr 405
410 415 Gln Val Arg Cys Leu Phe Gln Ser Pro Leu Phe Ala Lys Ala Gly
Asp 420 425 430 Thr Leu Ser Gly Thr Cys Leu Leu Ile Ala Asn Lys Arg
Gln Ser Tyr 435 440 445 Asp Ile Ser Ile Val Ala Gln Val Asp Gln Thr
Gly Ser Lys Ser Ser 450 455 460 Asn Leu Leu Asp Leu Lys Asn Pro Phe
Phe Arg Tyr Thr Gly Thr Thr 465 470 475 480 Pro Ser Pro Pro Pro Gly
Ser His Tyr Thr Ser Pro Ser Glu Asn Met 485 490 495 Trp Asn Thr Gly
Ser Thr Tyr Asn Leu Ser Ser Gly Met Ala Val Ala 500 505 510 Gly Met
Pro Thr Ala Tyr Asp Leu Ser Ser Val Ile Ala Ser Gly Ser 515 520 525
Ser Val Gly His Asn Asn Leu Ile Pro Leu Ala Asn Thr Gly Ile Val 530
535 540 Asn His Thr His Ser Arg Met Gly Ser Ile Met Ser Thr Gly Ile
Val 545 550 555 560 Gln Gly Ser Ser Gly Ala Gln Gly Ser Gly Gly Gly
Ser Thr Ser Ala 565 570 575 His Tyr Ala Val Asn Ser Gln Phe Thr Met
Gly Gly Pro Ala Ile Ser 580 585 590 Xaa Ala Ser Pro Met Ser Ile Pro
Thr Asn Thr Met His Tyr Gly Ser 595 600 605 9 1824 DNA Homo Sapiens
9 atggcagcgg cggcagcggc ggtggggccg ggcgcggggg gcgcggggtc ggcggtcccg
60 ggcggcgcgg ggccctgcgc taccgtgtcg gtgttccccg gcgcccgcct
cctcaccatc 120 ggcgacgcga acggcgagat ccagcggcac gcggagcagc
aggcgctgcg cctcgaggtg 180 cgcgccggcc cggactcggc gggcatcgcc
ctctacagcc atgaagatgt gtgtgtcttt 240 aagtgctcag tgtcccgaga
gacagagtgc agccgtgtgg gcaagcagtc cttcatcatc 300 accctgggct
gcaacagcgt cctcatccag ttcgccacac ccaacgattt ctgttccttc 360
tacaacatcc tgaaaacctg ccggggccac accctggagc ggtctgtgtt cagcgagcgg
420 acggaggagt cttctgccgt gcagtacttc cagttttatg gctacctgtc
ccagcagcag 480 aacatgatgc aggactacgt gcggacaggc acctaccagc
gcgccatcct gcaaaaccac 540 accgacttca aggacaagat cgttcttgat
gttggctgtg gctctgggat cctgtcgttt 600 tttgccgccc aagctggagc
acggaaaatc tacgcggtgg aggccagcac catggcccag 660 cacgctgagg
tcttggtgaa gagtaacaac ctgacggacc gcatcgtggt catcccgggc 720
aaggtggagg aggtgtcact ccccgagcag gtggacatca tcatctcgga gcccatgggc
780 tacatgctct tcaacgagcg catgctggag agctacctcc acgccaagaa
gtacctgaag 840 cccagcggaa acatgtttcc taccattggt gacgtccacc
ttgcaccctt cacggatgaa 900 cagctctaca tggagcagtt caccaaggcc
aacttctggt accagccatc tttccatgga 960 gtggacctgt cggccctccg
aggtgccgcg gtggatgagt atttccggca gcctgtggtg 1020 gacacatttg
acatccggat cctgatggcc aagtctgtca agtacacggt gaacttctta 1080
gaagccaaag aaggagattt gcacaggata gaaatcccat tcaaattcca catgctgcat
1140 tcagggctgg tccacggcct ggctttctgg tttgacgttg ctttcatcgg
ctccataatg 1200 accgtgtggc tgtccacagc cccgacagag cccctgaccc
actggtacca ggtgcggtgc 1260 ctgttccagt caccactgtt cgccaaggca
ggggacacgc tctcagggac atgtctgctt 1320 attgccaaca aaagacagag
ctacgacatc agtattgtgg cccaggtgga ccagaccggc 1380 tccaagtcca
gtaacctcct ggatctgaaa aaccccttct ttagatacac gggcacaacg 1440
ccctcacccc cacccggctc ccactacaca tctccctcgg aaaacatgtg gaacacgggc
1500 agcacctaca acctcagcag cgggatggcc gtggcaggga tgccgaccgc
ctatgacttg 1560 agcagtgtta ttgccagtgg ctccagcgtg ggccacaaca
acctgattcc tttagccaac 1620 acggggattg tcaatcacac ccactcccgg
atgggctcca taatgagcac ggggattgtc 1680 caagggtcct ccggcgccca
gggcagtggt ggtggcagca cgagtgccca ctatgcagtc 1740 aacagccagt
tcaccatggg cggccccgcc atctccwtgg cgtcgcccat gtccatcccg 1800
accaacacca tgcactacgg gagc 1824 10 2795 DNA Homo Sapiens 10
cggggacatg aggtggatac tgttcattgg ggcccttatt gggtccagca tctgtggccg
60 agaaaaattt tttggggacc aagttttgag gattaatgtc agaaatggag
acgagatcag 120 caaattgagt caactagtga attcaaacaa cttgaagctc
aatttctgga aatctccctc 180 ctccttcaat cggcctgtgg atgtcctggt
cccatctgtc agtctgcagg catttaaatc 240 cttcctgaga tcccagggct
tagagtacgc agtgacaatt gaggacctgc aggccctttt 300 agacaatgaa
gatgatgaaa tgcaacacaa tgaagggcaa gaacggagca gtaataactt 360
caactacggg gcttaccatt ccctggaagc tatttaccac gagatggaca acattgccgc
420 agactttcct gacctggcga ggagggtgaa gattggacat tcgtttgaaa
accggccgat 480 gtatgtactg aagttcagca ctgggaaagg cgtgaggcgg
ccggccgttt ggctgaatgc 540 aggcatccat tcccgagagt ggatctccca
ggccactgca atctggacgg caaggaagat 600 tgtatctgat taccagaggg
atccagctat cacctccatc ttggagaaaa tggatatttt 660 cttgttgcct
gtggccaatc ctgatggata tgtgtatact caaactcaaa accgattatg 720
gaggaagacg cggtcccgaa atcctggaag ctcctgcatt ggtgctgacc caaatagaaa
780 ctggaacgct agttttgcag gaaagggagc cagcgacaac ccttgctccg
aagtgtacca 840 tggaccccac gccaattcgg aagtggaggt gaaatcagtg
gtagatttca tccaaaaaca 900 tgggaatttc aagggcttca tcgacctgca
cagctactcg cagctgctga tgtatccata 960 tgggtactca gtcaaaaagg
ccccagatgc cgaggaactc gacaaggtgg cgaggcttgc 1020 ggccaaagct
ctggcttctg tgtcgggcac tgagtaccaa gtgggtccca cctgcaccac 1080
tgtctatcca gctagcggga gcagcatcga ctgggcgtat gacaacggca tcaaatttgc
1140 attcacattt gagttgagag ataccgggac ctatggcttc ctcctgccag
ctaaccagat 1200 catccccact gcagaggaga cgtggctggg gctgaagacc
atcatggagc atgtgcggga 1260 caacctctac taggcgatgg ctctgctctg
tctacattta tttgtaccca cacgtgcacg 1320 cactgaggcc attgttaaag
gagctctttc ctacctgtgt gagtcagagc cctctgggtt 1380 tgtggagcac
acaggcctgc ccctctccag ccagctccct ggagtcgtgt gtcctggcgg 1440
tgtccctgca agaactggtt ctgccagcct gctcaatttt ggtcctgctg tttttgatga
1500 gccttttgtc tgtttctcct tccaccctgc tggctgggcg gctgcactca
gcatcacccc 1560 ttcctgggtg gcatgtctct ctctacctca tttttagaac
caaagaacat ctgagatgat 1620 tctctaccct cattcacatc tagccaagcc
agtgaccttt gctctggtgg cactgtggga 1680 gacaccactt gtctttaggt
gggtctcaaa gatgatgtag aatttccttt aatttctcgc 1740 agtcttcctg
gaaaatattt tcctttgagc agcaaatctt gtagggatat cagtgaaggt 1800
ctctccctcc ctcctctcct gttttttttt tttgaggcag agttttgctc ttgttgccca
1860 ggctggagtg tgatgggctc gatcttggct caccacaacc tctgcctcct
gggttcaagc 1920 aattctcctg cctcagcctc ttgagtagct tggtttatag
gcgcatgcca ccatgcctgg 1980 ctaattttgt gtttttagta gagacagggt
ttctccatgt tggtcaggct ggtctcaaac 2040 tcccaacctc aggtgatctg
ccctccttgg cctcccagag tgctgggatt acagggggag 2100 ccactgtgcc
ggtcccgtcc cctccttttt taggcctgaa tacaaagtag aagatcactt 2160
tccttcactg tgctgagaat ttctagatac tacagttctt actcctctct tccctttgtt
2220 attcagtgtg accaggatgg gcgggagggg atctgtgtca ctgtaggtac
tgtgcccagg 2280 aaggctgggt gaagtcccca tctaaattgc aggatggcga
aattatcccc atctgtccta 2340 atgggcttcc ctcctctttg ccttttgaac
tcacttcaaa gatgtaggcc tcatcttaca 2400 ggtcctaaat cactcatctg
gcctggataa tctcactgcc ctggcacatt cccatttgtg 2460 ctggggtatc
ctgtgtttcc ttgtcctggt ttgtgtgtgt gtgtgtgtgt gtgtgtgtgt 2520
gtgtgtgtgt ttgtgtgtgt gtgtctgtct attttgatcc ggcccaagtt cctaagtaga
2580 gcaagaattc atcaaccagc tgcctttgtt tcatttcacc tcagcacgta
ccatcgtcct 2640 ttggggggtt gtttgttttt gttttttgct ttaaccaaaa
tgtttgtaaa tcttaacctc 2700 ctgcctagga tttgtacagc atttggtgtg
tgcttataag ccaataaata ttcaatgtga 2760 gttccaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaa 2795 11 421 PRT Homo Sapiens 11 Met Arg Trp Ile
Leu Phe Ile Gly Ala Leu Ile Gly Ser Ser Ile Cys 1 5 10 15 Gly Arg
Glu Lys Phe Phe Gly Asp Gln Val Leu Arg Ile Asn Val Arg 20 25 30
Asn Gly Asp Glu Ile Ser Lys Leu Ser Gln Leu Val Asn Ser Asn Asn 35
40 45 Leu Lys Leu Asn Phe Trp Lys Ser Pro Ser Ser Phe Asn Arg Pro
Val 50 55 60 Asp Val Leu Val Pro Ser Val Ser Leu Gln Ala Phe Lys
Ser Phe Leu 65 70 75 80 Arg Ser Gln Gly Leu Glu Tyr Ala Val Thr Ile
Glu Asp Leu Gln Ala 85 90 95 Leu Leu Asp Asn Glu Asp Asp Glu Met
Gln His Asn Glu Gly Gln Glu 100 105 110 Arg Ser Ser Asn Asn Phe Asn
Tyr Gly Ala Tyr His Ser Leu Glu Ala 115 120 125 Ile Tyr His Glu Met
Asp Asn Ile Ala Ala Asp Phe Pro Asp Leu Ala 130 135 140 Arg Arg Val
Lys Ile Gly His Ser Phe Glu Asn Arg Pro Met Tyr Val 145 150 155 160
Leu Lys Phe Ser Thr Gly Lys Gly Val Arg Arg Pro Ala Val Trp Leu 165
170 175 Asn Ala Gly Ile His Ser Arg Glu Trp Ile Ser Gln Ala Thr Ala
Ile 180 185 190 Trp Thr Ala Arg Lys Ile Val Ser Asp Tyr Gln Arg Asp
Pro Ala Ile 195 200 205 Thr Ser Ile Leu Glu Lys Met Asp Ile Phe Leu
Leu Pro Val Ala Asn 210 215 220 Pro Asp Gly Tyr Val Tyr Thr Gln Thr
Gln Asn Arg Leu Trp Arg Lys 225 230 235 240 Thr Arg Ser Arg Asn Pro
Gly Ser Ser Cys Ile Gly Ala Asp Pro Asn 245 250 255 Arg Asn Trp Asn
Ala Ser Phe Ala Gly Lys Gly Ala Ser Asp Asn Pro 260 265 270 Cys Ser
Glu Val Tyr His Gly Pro His Ala Asn Ser Glu Val Glu Val 275 280 285
Lys Ser Val Val Asp Phe Ile Gln Lys His Gly Asn Phe Lys Gly Phe 290
295 300 Ile Asp Leu His Ser Tyr Ser Gln Leu Leu Met Tyr Pro Tyr Gly
Tyr 305 310 315 320 Ser Val Lys Lys Ala Pro Asp Ala Glu Glu Leu Asp
Lys Val Ala Arg 325 330 335 Leu Ala Ala Lys Ala Leu Ala Ser Val Ser
Gly Thr Glu Tyr Gln Val 340 345 350 Gly Pro Thr Cys Thr Thr Val Tyr
Pro Ala Ser Gly Ser Ser Ile Asp 355 360 365 Trp Ala Tyr Asp Asn Gly
Ile Lys Phe Ala Phe Thr Phe Glu Leu Arg 370 375 380 Asp Thr Gly Thr
Tyr Gly Phe Leu Leu Pro Ala Asn Gln Ile Ile Pro 385 390 395 400 Thr
Ala Glu Glu Thr Trp Leu Gly Leu Lys Thr Ile Met Glu His Val 405 410
415 Arg Asp Asn Leu Tyr 420 12 1705 DNA Homo Sapiens 12 atgtctacag
cctctgccgc ctcctcctcc tcctcgtctt cggccggtga gatgatcgaa 60
gccccttccc aggtcctcaa ctttgaagag atcgactaca aggagatcga ggtggaagag
120 gttgttggaa gaggagcctt tggagttgtt tgcaaagcta agtggagagc
aaaagatgtt 180 gctattaaac aaatagaaag tgaatctgag aggaaagcgt
ttattgtaga gcttcggcag 240 ttatcccgtg tgaaccatcc taatattgta
aagctttatg gagcctgctt gaatccagtg 300 tgtcttgtga tggaatatgc
tgaagggggc tctttatata atgtgctgca tggtgctgaa 360 ccattgccat
attatactgc tgcccacgca atgagttggt gtttacagtg ttcccaagga 420
gtggcttatc ttcacagcat gcaacccaaa gcgctaattc acagggacct gaaaccacca
480 aacttactgc tggttgcagg ggggacagtt ctaaaaattt gtgattttgg
tacagcctgt 540 gacattcaga cacacatgac caataacaag gggagtgctg
cttggatggc acctgaagtt 600 tttgaaggta gtaattacag tgaaaaatgt
gacgtcttca gctggggtat tattctttgg 660 gaagtgataa cgcgtcggaa
accctttgat gagattggtg gcccagcttt ccgaatcatg 720 tgggctgttc
ataatggtac tcgaccacca ctgataaaaa atttacctaa gcccattgag 780
agcctgatga ctcgttgttg gtctaaagat ccttcccagc gcccttcaat ggaggaaatt
840 gtgaaaataa tgactcactt gatgcggtac tttccaggag cagatgagcc
attacagtat 900 ccttgtcagt attcagatga aggacagagc aactctgcca
ccagtacagg ctcattcatg 960 gacattgctt ctacaaatac gagtaacaaa
agtgacacta atatggagca agttcctgcc 1020 acaaatgata ctattaagcg
cttagaatca aaattgttga aaaatcaggc aaagcaacag 1080 agtgaatctg
gacgtttaag cttgggagcc tcccgtggga gcagtgtgga gagcttgccc 1140
ccaacctctg agggcaagag gatgagtgct gacatgtctg aaatagaagc taggatcgcc
1200 gcaaccacag cctattccaa gcctaaacgg ggccaccgta aaactgcttc
atttggcaac 1260 attctggatg tccctgagat cgtcatatca ggcaacggac
agccaagacg tagatccatc 1320 caagacttga ctgtaactgg aacagaacct
ggtcaggtga gcagtaggtc atccagtccc 1380 agtgtcagaa tgattactac
ctcaggacca acctcagaaa agccaactcg aagtcatcca 1440 tggacccctg
atgattccac agataccaat ggatcagata actccatccc aatggcttat 1500
cttacactgg atcaccaact acaggcaaga actagttgca gaactggacc aggatgaaaa
1560 ggaccagcaa aatacatctc gcctggtaca ggaacataaa aagcttttag
atgaaaacaa 1620 aggcctttct acttactacc agcaatgcaa aaaacaacta
gaggtcatca gaagtcagca 1680 gcagaaacga caaggcactt catga 1705 13 518
PRT Homo Sapiens 13 Met Ser Thr Ala Ser Ala Ala Ser Ser Ser Ser Ser
Ser Ser Ala Gly 1 5 10 15 Glu Met Ile Glu Ala Pro Ser Gln Val Leu
Asn Phe Glu Glu Ile Asp 20 25 30 Tyr Lys Glu Ile Glu Val Glu Glu
Val Val Gly Arg Gly Ala Phe Gly 35 40 45 Val Val Cys Lys Ala Lys
Trp Arg Ala Lys Asp Val Ala Ile Lys Gln 50 55 60 Ile Glu Ser Glu
Ser Glu Arg Lys Ala Phe Ile Val Glu Leu Arg Gln 65 70 75 80 Leu Ser
Arg Val Asn His Pro Asn Ile Val Lys Leu Tyr Gly Ala Cys 85 90 95
Leu Asn Pro Val Cys Leu Val Met Glu Tyr Ala Glu Gly Gly Ser Leu 100
105 110 Tyr Asn Val Leu His Gly Ala Glu Pro Leu Pro Tyr Tyr Thr Ala
Ala 115 120 125 His Ala Met Ser Trp Cys Leu Gln Cys Ser Gln Gly Val
Ala Tyr Leu 130 135 140 His Ser Met Gln Pro Lys Ala Leu Ile His Arg
Asp Leu Lys Pro Pro 145 150 155 160 Asn Leu Leu Leu Val Ala Gly Gly
Thr Val Leu Lys Ile Cys Asp Phe 165 170 175 Gly Thr Ala Cys Asp Ile
Gln Thr His Met Thr Asn Asn Lys Gly Ser 180 185 190 Ala Ala Trp Met
Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr Ser Glu 195 200 205 Lys Cys
Asp Val Phe Ser Trp Gly Ile Ile Leu Trp Glu Val Ile Thr 210 215 220
Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg Ile Met 225
230 235 240 Trp Ala Val His Asn Gly Thr Arg Pro Pro Leu Ile Lys Asn
Leu Pro 245 250 255 Lys Pro Ile Glu Ser Leu Met Thr Arg Cys Trp Ser
Lys Asp Pro Ser 260 265 270 Gln Arg Pro Ser Met Glu Glu Ile Val Lys
Ile Met Thr His Leu Met 275 280 285 Arg Tyr Phe Pro Gly Ala Asp Glu
Pro Leu Gln Tyr Pro Cys Gln Tyr 290 295 300 Ser Asp Glu Gly Gln Ser
Asn Ser Ala Thr Ser Thr Gly Ser Phe Met 305 310 315 320 Asp Ile Ala
Ser Thr Asn Thr Ser Asn Lys Ser Asp Thr Asn Met Glu 325 330 335 Gln
Val Pro Ala Thr Asn Asp Thr Ile Lys Arg Leu Glu Ser Lys Leu 340
345 350 Leu Lys Asn Gln Ala Lys Gln Gln Ser Glu Ser Gly Arg Leu Ser
Leu 355 360 365 Gly Ala Ser Arg Gly Ser Ser Val Glu Ser Leu Pro Pro
Thr Ser Glu 370 375 380 Gly Lys Arg Met Ser Ala Asp Met Ser Glu Ile
Glu Ala Arg Ile Ala 385 390 395 400 Ala Thr Thr Ala Tyr Ser Lys Pro
Lys Arg Gly His Arg Lys Thr Ala 405 410 415 Ser Phe Gly Asn Ile Leu
Asp Val Pro Glu Ile Val Ile Ser Gly Asn 420 425 430 Gly Gln Pro Arg
Arg Arg Ser Ile Gln Asp Leu Thr Val Thr Gly Thr 435 440 445 Glu Pro
Gly Gln Val Ser Ser Arg Ser Ser Ser Pro Ser Val Arg Met 450 455 460
Ile Thr Thr Ser Gly Pro Thr Ser Glu Lys Pro Thr Arg Ser His Pro 465
470 475 480 Trp Thr Pro Asp Asp Ser Thr Asp Thr Asn Gly Ser Asp Asn
Ser Ile 485 490 495 Pro Met Ala Tyr Leu Thr Leu Asp His Gln Leu Gln
Ala Arg Thr Ser 500 505 510 Cys Arg Thr Gly Pro Gly 515 14 1917 DNA
Homo Sapiens 14 gtgagctgca gagaagagga ggttggtgtg gagcacaggc
agcaccgagc ctgccccgtg 60 agctgagggc ctgcagtctg cggctggaat
caggatagac accaaggcag gacccccaga 120 gatgctgaag cctctttgga
aagcagcagt ggcccccaca tggccatgct ccatgccgcc 180 ccgccgcccg
tgggacagag aggctggcac gttgcaggtc ctgggagcgc tggctgtgct 240
gtggctgggc tccgtggctc ttatctgcct cctgtggcaa gtgccccgtc ctcccacctg
300 gggccaggtg cagcccaagg acgtgcccag gtcctgggag catggctcca
gcccagcttg 360 ggagcccctg gaagcagagg ccaggcagca gagggactcc
tgccagcttg tccttgtgga 420 aagcatcccc caggacctgc catctgcagc
cggcagcccc tctgcccagc ctctgggcca 480 ggcctggctg cagctgctgg
acactgccca ggagagcgtc cacgtggctt catactactg 540 gtccctcaca
gggcctgaca tcggggtcaa cgactcgtct tcccagctgg gagaggctct 600
tctgcagaag ctgcagcagc tgctgggcag gaacatttcc ctggctgtgg ccaccagcag
660 cccgacactg gccaggacat ccaccgacct gcaggttctg gctgcccgag
gtgcccatgt 720 acgacaggtg cccatggggc ggctcaccag gggtgttttg
cactccaaat tctgggttgt 780 ggatggacgg cacatataca tgggcagtgc
caacatggac tggcggtctc tgacgcaggt 840 gaaggagctt ggcgctgtca
tctataactg cagccacctg gcccaagacc tggagaagac 900 cttccagacc
tactgggtac tgggggtgcc caaggctgtc ctccccaaaa cctggcctca 960
gaacttctca tctcacttca accgtttcca gcccttccac ggcctctttg atggggtgcc
1020 caccactgcc tacttctcag cgtcgccacc agcactctgt ccccagggcc
gcacccggga 1080 cctggaggcg ctgctggcgg tgatggggag cgcccaggag
ttcatctatg cctccgtgat 1140 ggagtatttc cccaccacgc gcttcagcca
ccccccgagg tactggccgg tgctggacaa 1200 cgcgctgcgg gcggcagcct
tcggcaaggg cgtgcgcgtg cgcctgctgg tcggctgcgg 1260 actcaacacg
gaccccacca tgttccccta cctgcggtcc ctgcaggcgc tcagcaaccc 1320
cgcggccaac gtctctgtgg acgtgaaagt cttcatcgtg ccggtgggga accattccaa
1380 catcccattc agcagggtga accacagcaa gttcatggtc acggagaagg
cagcctacat 1440 aggcacctcc aactggtcgg aggattactt cagcagcacg
gcgggggtgg gcttggtggt 1500 cacccagagc cctggcgcgc agcccgcggg
ggccacggtg caggagcagc tgcggcagct 1560 ctttgagcgg gactggagtt
cgcgctacgc cgtcggcctg gacggacagg ctccgggcca 1620 ggactgcgtt
tggcagggct gaggggggcc tctttttctc tcggcgaccc cgccccgcac 1680
gcgccctccc ctctgacccc ggcctgggct tcagccgctt cctcccgcaa gcagcccggg
1740 tccgcactgc gccaggagcc gcctgcgacc gcccgggcgt cgcaaaccgc
ccgcctgctc 1800 tctgatttcc gagtccagcc ccccctgagc cccacctcct
ccagggagcc ctccaggaag 1860 ccccttccct gactcctggc ccacaggcca
ggcctaaaaa aaactcgtgg cttcaaa 1917 15 506 PRT Homo Sapiens 15 Met
Leu Lys Pro Leu Trp Lys Ala Ala Val Ala Pro Thr Trp Pro Cys 1 5 10
15 Ser Met Pro Pro Arg Arg Pro Trp Asp Arg Glu Ala Gly Thr Leu Gln
20 25 30 Val Leu Gly Ala Leu Ala Val Leu Trp Leu Gly Ser Val Ala
Leu Ile 35 40 45 Cys Leu Leu Trp Gln Val Pro Arg Pro Pro Thr Trp
Gly Gln Val Gln 50 55 60 Pro Lys Asp Val Pro Arg Ser Trp Glu His
Gly Ser Ser Pro Ala Trp 65 70 75 80 Glu Pro Leu Glu Ala Glu Ala Arg
Gln Gln Arg Asp Ser Cys Gln Leu 85 90 95 Val Leu Val Glu Ser Ile
Pro Gln Asp Leu Pro Ser Ala Ala Gly Ser 100 105 110 Pro Ser Ala Gln
Pro Leu Gly Gln Ala Trp Leu Gln Leu Leu Asp Thr 115 120 125 Ala Gln
Glu Ser Val His Val Ala Ser Tyr Tyr Trp Ser Leu Thr Gly 130 135 140
Pro Asp Ile Gly Val Asn Asp Ser Ser Ser Gln Leu Gly Glu Ala Leu 145
150 155 160 Leu Gln Lys Leu Gln Gln Leu Leu Gly Arg Asn Ile Ser Leu
Ala Val 165 170 175 Ala Thr Ser Ser Pro Thr Leu Ala Arg Thr Ser Thr
Asp Leu Gln Val 180 185 190 Leu Ala Ala Arg Gly Ala His Val Arg Gln
Val Pro Met Gly Arg Leu 195 200 205 Thr Arg Gly Val Leu His Ser Lys
Phe Trp Val Val Asp Gly Arg His 210 215 220 Ile Tyr Met Gly Ser Ala
Asn Met Asp Trp Arg Ser Leu Thr Gln Val 225 230 235 240 Lys Glu Leu
Gly Ala Val Ile Tyr Asn Cys Ser His Leu Ala Gln Asp 245 250 255 Leu
Glu Lys Thr Phe Gln Thr Tyr Trp Val Leu Gly Val Pro Lys Ala 260 265
270 Val Leu Pro Lys Thr Trp Pro Gln Asn Phe Ser Ser His Phe Asn Arg
275 280 285 Phe Gln Pro Phe His Gly Leu Phe Asp Gly Val Pro Thr Thr
Ala Tyr 290 295 300 Phe Ser Ala Ser Pro Pro Ala Leu Cys Pro Gln Gly
Arg Thr Arg Asp 305 310 315 320 Leu Glu Ala Leu Leu Ala Val Met Gly
Ser Ala Gln Glu Phe Ile Tyr 325 330 335 Ala Ser Val Met Glu Tyr Phe
Pro Thr Thr Arg Phe Ser His Pro Pro 340 345 350 Arg Tyr Trp Pro Val
Leu Asp Asn Ala Leu Arg Ala Ala Ala Phe Gly 355 360 365 Lys Gly Val
Arg Val Arg Leu Leu Val Gly Cys Gly Leu Asn Thr Asp 370 375 380 Pro
Thr Met Phe Pro Tyr Leu Arg Ser Leu Gln Ala Leu Ser Asn Pro 385 390
395 400 Ala Ala Asn Val Ser Val Asp Val Lys Val Phe Ile Val Pro Val
Gly 405 410 415 Asn His Ser Asn Ile Pro Phe Ser Arg Val Asn His Ser
Lys Phe Met 420 425 430 Val Thr Glu Lys Ala Ala Tyr Ile Gly Thr Ser
Asn Trp Ser Glu Asp 435 440 445 Tyr Phe Ser Ser Thr Ala Gly Val Gly
Leu Val Val Thr Gln Ser Pro 450 455 460 Gly Ala Gln Pro Ala Gly Ala
Thr Val Gln Glu Gln Leu Arg Gln Leu 465 470 475 480 Phe Glu Arg Asp
Trp Ser Ser Arg Tyr Ala Val Gly Leu Asp Gly Gln 485 490 495 Ala Pro
Gly Gln Asp Cys Val Trp Gln Gly 500 505 16 2048 DNA Homo Sapiens 16
ccgcaaagtg ctgggatgac aggtgtgagc caccgccccc ggcccctcgc ccgccttttg
60 aaggagcctt tcgtcctcaa gggagaggcc actccccccc cgcgagttcc
atgcccccta 120 gagggtcatc gttcccgacg gggaggtggc gccctccccc
gggccccggg ccccgaccgc 180 ccgtgctgcc tccttccggg ccatcatccg
cgatgacggc gccgccagca ggccaggcgg 240 actgggcggg gctccgagcg
gggactggga cccagaccga ctaggggact gggagcgggc 300 ggcgcggcca
tggcgggctg ctgcgccgcg ctggcggcct tcctgttcga gtacgacacg 360
ccgcgcatcg tgctcatccg cagccgcaaa gtggggctca tgaaccgcgc cgtgcaactg
420 ctcatcctgg cctacgtcat cgggtgggtg tttgtgtggg aaaagggcta
ccaggaaact 480 gactccgtgg tcagctccgt tacgaccaag gtcaagggcg
tggctgtgac caacacttct 540 aaacttggat tccggatctg ggatgtggcg
gattatgtga taccagctca ggaggaaaac 600 tccctcttcg tcatgaccaa
cgtgatcctc accatgaacc agacacaggg cctgtgcccc 660 gagattccag
atgcgaccac tgtgtgtaaa tcagatgcca gctgtactgc cggctctgcc 720
ggcacccaca gcaacggagt ctcaacaggc aggtgcgtag ctttcaacgg gtctgtcaag
780 acgtgtgarg tggcggcctg gtgcccggtg gaggatgaca cacacgtgcc
acaacctgct 840 tttttaaagg ctgcagaaaa cttcactctt ttggttaaga
acaacatctg gtatcccaaa 900 tttaatttca gcaagaggaa tatccttccc
aacatcacca ctacttacct caagtcgtgc 960 atttatgatg ctaaaacaga
tcccttctgc cccatattcc gtcttggcaa aatagtggag 1020 aacgcaggac
acagtttcca ggacatggcc gtggagggag gcatcatggg catccaggtc 1080
aactgggact gcaacctgga cagagccgcc tccctctgct tgcccaggta ctccttccgc
1140 cgcctcgata cacgggacgt tgagcacaac gtatctcctg gctacaattt
caggtttgcc 1200 aagtactaca gagacctggc tggcaacgag cagcgcacgc
tcatcaaggc ctatggcatc 1260 cgcttcgaca tcattgtgtt tgggaaggca
gggaaatttg acatcatccc cactatgatc 1320 aacatcggct ctggcctggc
actgctaggc atggcgaccg tgctgtgtga catcatagtc 1380 ctctactgca
tgaagaaaag actctactat cgggagaaga aatataaata tgtggaagat 1440
tacgagcagg gtcttgctag tgagctggac cagtgaggcc taccccacac ctgggctctc
1500 cacagcccca tcaaagaaca gagaggagga ggagggagaa atggccacca
catcacccca 1560 gagaaatttc tggaatctga ttgagtctcc actccacaag
cactcagggt tccccagcag 1620 ctcctgtgtg ttgtgtgcag gatctgtttg
cccactcggc ccaggaggtc agcagtctgt 1680 tcttggctgg gtcaactctg
cttttcccgc aacctggggt tgtcggggga gcgctggccc 1740 gacgcagtgg
cactgctgtg gctttcaggg ctggagctgg ctttgctcag aagcctcctg 1800
tctccagctc tctccaggac aggcccagtc ctctgaggca cggcggctct gttcaagcac
1860 tttatgcggc aggggaggcc gcctggctgc agtcactaga cttgtagcag
gcctgggctg 1920 caggcttccc cccgaccatt ccctgcagcc atgcggcaga
gctggcattt ctcctcagag 1980 aagcgctgtg ctaaggtgat cgaggaccag
acattaaagc gtgattttct taaaaaaaaa 2040 aaaaaaaa 2048 17 388 PRT Homo
Sapiens VARIANT (1)...(388) Xaa = Any Amino Acid 17 Met Ala Gly Cys
Cys Ala Ala Leu Ala Ala Phe Leu Phe Glu Tyr Asp 1 5 10 15 Thr Pro
Arg Ile Val Leu Ile Arg Ser Arg Lys Val Gly Leu Met Asn 20 25 30
Arg Ala Val Gln Leu Leu Ile Leu Ala Tyr Val Ile Gly Trp Val Phe 35
40 45 Val Trp Glu Lys Gly Tyr Gln Glu Thr Asp Ser Val Val Ser Ser
Val 50 55 60 Thr Thr Lys Val Lys Gly Val Ala Val Thr Asn Thr Ser
Lys Leu Gly 65 70 75 80 Phe Arg Ile Trp Asp Val Ala Asp Tyr Val Ile
Pro Ala Gln Glu Glu 85 90 95 Asn Ser Leu Phe Val Met Thr Asn Val
Ile Leu Thr Met Asn Gln Thr 100 105 110 Gln Gly Leu Cys Pro Glu Ile
Pro Asp Ala Thr Thr Val Cys Lys Ser 115 120 125 Asp Ala Ser Cys Thr
Ala Gly Ser Ala Gly Thr His Ser Asn Gly Val 130 135 140 Ser Thr Gly
Arg Cys Val Ala Phe Asn Gly Ser Val Lys Thr Cys Xaa 145 150 155 160
Val Ala Ala Trp Cys Pro Val Glu Asp Asp Thr His Val Pro Gln Pro 165
170 175 Ala Phe Leu Lys Ala Ala Glu Asn Phe Thr Leu Leu Val Lys Asn
Asn 180 185 190 Ile Trp Tyr Pro Lys Phe Asn Phe Ser Lys Arg Asn Ile
Leu Pro Asn 195 200 205 Ile Thr Thr Thr Tyr Leu Lys Ser Cys Ile Tyr
Asp Ala Lys Thr Asp 210 215 220 Pro Phe Cys Pro Ile Phe Arg Leu Gly
Lys Ile Val Glu Asn Ala Gly 225 230 235 240 His Ser Phe Gln Asp Met
Ala Val Glu Gly Gly Ile Met Gly Ile Gln 245 250 255 Val Asn Trp Asp
Cys Asn Leu Asp Arg Ala Ala Ser Leu Cys Leu Pro 260 265 270 Arg Tyr
Ser Phe Arg Arg Leu Asp Thr Arg Asp Val Glu His Asn Val 275 280 285
Ser Pro Gly Tyr Asn Phe Arg Phe Ala Lys Tyr Tyr Arg Asp Leu Ala 290
295 300 Gly Asn Glu Gln Arg Thr Leu Ile Lys Ala Tyr Gly Ile Arg Phe
Asp 305 310 315 320 Ile Ile Val Phe Gly Lys Ala Gly Lys Phe Asp Ile
Ile Pro Thr Met 325 330 335 Ile Asn Ile Gly Ser Gly Leu Ala Leu Leu
Gly Met Ala Thr Val Leu 340 345 350 Cys Asp Ile Ile Val Leu Tyr Cys
Met Lys Lys Arg Leu Tyr Tyr Arg 355 360 365 Glu Lys Lys Tyr Lys Tyr
Val Glu Asp Tyr Glu Gln Gly Leu Ala Ser 370 375 380 Glu Leu Asp Gln
385 18 1167 DNA Homo Sapiens 18 atggcgggct gctgcgccgc gctggcggcc
ttcctgttcg agtacgacac gccgcgcatc 60 gtgctcatcc gcagccgcaa
agtggggctc atgaaccgcg ccgtgcaact gctcatcctg 120 gcctacgtca
tcgggtgggt gtttgtgtgg gaaaagggct accaggaaac tgactccgtg 180
gtcagctccg ttacgaccaa ggtcaagggc gtggctgtga ccaacacttc taaacttgga
240 ttccggatct gggatgtggc ggattatgtg ataccagctc aggaggaaaa
ctccctcttc 300 gtcatgacca acgtgatcct caccatgaac cagacacagg
gcctgtgccc cgagattcca 360 gatgcgacca ctgtgtgtaa atcagatgcc
agctgtactg ccggctctgc cggcacccac 420 agcaacggag tctcaacagg
caggtgcgta gctttcaacg ggtctgtcaa gacgtgtgar 480 gtggcggcct
ggtgcccggt ggaggatgac acacacgtgc cacaacctgc ttttttaaag 540
gctgcagaaa acttcactct tttggttaag aacaacatct ggtatcccaa atttaatttc
600 agcaagagga atatccttcc caacatcacc actacttacc tcaagtcgtg
catttatgat 660 gctaaaacag atcccttctg ccccatattc cgtcttggca
aaatagtgga gaacgcagga 720 cacagtttcc aggacatggc cgtggaggga
ggcatcatgg gcatccaggt caactgggac 780 tgcaacctgg acagagccgc
ctccctctgc ttgcccaggt actccttccg ccgcctcgat 840 acacgggacg
ttgagcacaa cgtatctcct ggctacaatt tcaggtttgc caagtactac 900
agagacctgg ctggcaacga gcagcgcacg ctcatcaagg cctatggcat ccgcttcgac
960 atcattgtgt ttgggaaggc agggaaattt gacatcatcc ccactatgat
caacatcggc 1020 tctggcctgg cactgctagg catggcgacc gtgctgtgtg
acatcatagt cctctactgc 1080 atgaagaaaa gactctacta tcgggagaag
aaatataaat atgtggaaga ttacgagcag 1140 ggtcttgcta gtgagctgga ccagtga
1167 19 8 PRT Artificial Sequence Consensus Sequence VARIANT
(2)...(2) Xaa = Any amino acid VARIANT (4)...(7) Xaa = Any amino
acid 19 His Xaa Lys Xaa Xaa Xaa Xaa Asp 1 5 20 7 PRT Artificial
Sequence Heparin-binding segment VARIANT (2)...(2) Xaa = S or G
VARIANT (3)...(3) Xaa = any amino acid VARIANT (6)...(6) Xaa = any
amino acid 20 Trp Xaa Xaa Trp Ser Xaa Trp 1 5 21 6 PRT Artificial
Sequence Heparin-binding segment 21 Cys Ser Val Thr Cys Gly 1 5 22
7 PRT Artificial Sequence Heparin-binding segment 22 Trp Gly Pro
Trp Gly Pro Trp 1 5 23 6 PRT Artificial Sequence Heparin-binding
segment VARIANT (3)...(3) Xaa = R or K 23 Cys Ser Xaa Thr Cys Gly 1
5 24 4 PRT Artificial Sequence Cleavage Site 24 Arg Arg Arg Arg 1
25 4 PRT Artificial Sequence Cleavage Site 25 Arg Lys Lys Arg 1 26
4 PRT Artificial Sequence Consensus sequence VARIANT (2)...(2) Xaa
= any amino acid VARIANT (3)...(3) Xaa = K or R 26 Arg Xaa Xaa Arg
1 27 5 PRT Artificial Sequence Zinc-binding motif VARIANT (3)...(4)
Xaa = any amino acid 27 His Glu Xaa Xaa His 1 5 28 7 PRT Artificial
Sequence Catalytic signature 28 His Asp Val Asp His Pro Gly 1 5
* * * * *