U.S. patent application number 10/458839 was filed with the patent office on 2003-11-20 for methods for using 22045, a human cyclic nucleotide phosphodiesterase.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Hunter, John Joseph, Kapeller-Libermann, Rosana, Williamson, Mark.
Application Number | 20030215898 10/458839 |
Document ID | / |
Family ID | 23665445 |
Filed Date | 2003-11-20 |
United States Patent
Application |
20030215898 |
Kind Code |
A1 |
Kapeller-Libermann, Rosana ;
et al. |
November 20, 2003 |
Methods for using 22045, a human cyclic nucleotide
phosphodiesterase
Abstract
The present invention relates to methods for using a human
cyclic nucleotide phosphodiesterase belonging to the superfamily of
mammalian phosphodiesterases. The invention also relates to methods
for using polynucleotides encoding the phosphodiesterase. The
invention relates to methods using the phosphodiesterase
polypeptides and polynucleotides as a target for diagnosis and
treatment in phosphodiesterase-mediated or -related disorders. The
invention further relates to drug-screening methods using the
phosphodiesterase polypeptides and polynucleotides to identify
agonists and antagonists for diagnosis and treatment. The invention
further encompasses agonists and antagonists based on the
phosphodiesterase polypeptides and polynucleotides. The invention
further relates to agonists and antagonists identifiefd by drug
screening methods with the phosphodiesterase polypeptides and
polynucleotides as a target.
Inventors: |
Kapeller-Libermann, Rosana;
(Chestnut Hill, MA) ; Hunter, John Joseph;
(Somerville, MA) ; Williamson, Mark; (Saugus,
MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
75 Sidney Street
Cambridge
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
23665445 |
Appl. No.: |
10/458839 |
Filed: |
June 11, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10458839 |
Jun 11, 2003 |
|
|
|
09420190 |
Oct 18, 1999 |
|
|
|
Current U.S.
Class: |
435/19 ;
514/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 9/10 20180101; C12N 9/16 20130101; A61P 5/00 20180101; A61P
9/04 20180101; A61K 38/00 20130101 |
Class at
Publication: |
435/19 ;
514/1 |
International
Class: |
A61K 031/00; C12Q
001/44 |
Claims
That which is claimed:
1. A method of identifying an inhibitor of a human cyclic
phosphodiesterase protein comprising: a) combining an agent to be
tested with a host cell expressing human cyclic phosphodiesterase
protein having an amino acid sequence as set forth in SEQ ID NO:1
under conditions suitable for detecting a phosphodiesterase
activity; and b) assessing the ability of the agent to inhibit the
phosphodiesterase activity, whereby inhibition of the
phosphodiesterase activity by the agent is indicative that the
agent is an inhibitor; wherein the host cell is selected from the
group consisting of breast carcinoma cells and colon carcinoma
cells.
2. The method of claim 1, wherein the phosphodiesterase activity is
a signaling activity or a cellular response.
3. The method of claim 1, wherein detecting of a phosphodiesterase
activity comprises measuring substrate consumption or measuring the
production of end product or intermediates.
4. The method of claim 1, wherein the agent is selected from the
group consisting of a small molecule, a peptide, an antibody and an
antibody fragment.
5. An inhibitor of a human cyclic phosphodiesterase protein
identified according to the method of claim 1, wherein the
inhibitor is an antagonist.
6. The method of claim 1, wherein the human cyclic
phosphodiesterase protein is encoded by the nucleotide sequence set
forth in SEQ ID NO:2.
7. A method of identifying an inhibitor of a human cyclic
phosphodiesterase protein comprising: a) combining an agent to be
tested with a host cell expressing a fusion protein, wherein one or
more domains or subregions of the fusion protein comprise(s) the
amino acid sequence set forth in SEQ ID NO:1, under conditions
suitable for detecting a phosphodiesterase activity; and b)
assessing the ability of the agent to inhibit the phosphodiesterase
activity, whereby inhibition of the phosphodiesterase activity by
the agent is indicative that the agent is an inhibitor; wherein the
host cell is selected from the group consisting of breast carcinoma
cells and colon carcinoma cells.
8. The method of claim 7, wherein the phosphodiesterase activity is
a signaling activity or a cellular response.
9. The method of claim 7, wherein detecting of a phosphodiesterase
activity comprises measuring substrate consumption or measuring the
production of end product or intermediates.
10. The method of claim 7, wherein the agent is selected from the
group consisting of a small molecule, a peptide, an antibody and an
antibody fragment.
11. An inhibitor of a human cyclic phosphodiesterase protein
identified according to the method of claim 7, wherein the
inhibitor is an antagonist.
12. The method of claim 7, wherein the one or more domains or
subregions of the fusion protein comprising the amino acid sequence
set forth in SEQ ID NO:1, is/are encoded by the nucleotide sequence
set forth in SEQ ID NO:2.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional application of U.S.
patent application Ser. No. 09/420,190, filed Oct. 18, 1999, which
is hereby incorporated herein in its entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods for using a human
cyclic nucleotide phosphodiesterase. The invention also relates to
methods for using polynucleotides encoding the phosphodiesterase.
The invention further relates to methods using the
phosphodiesterase polypeptides and polynucleotides as a target for
diagnosis and treatment in phosphodiesterase-mediated or -related
disorders. The invention further relates to drug-screening methods
using the phosphodiesterase polypeptides and polynucleotides to
identify agonists and antagonists for diagnosis and treatment. The
invention further encompasses agonists and antagonists based on the
phosphodiesterase polypeptides and polynucleotides. The invention
further relates to agonists and antagonists identified by drug
screening methods with the phosphodiesterase polypeptides and
polynucleotides as a target.
BACKGROUND OF THE INVENTION
[0003] 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).
[0004] 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. A
conserved motif of the sequence HDXXHXX has been identified in the
catalytic domain of all cyclic nucleotide phosphodiesterases
isolated to date. 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.
[0005] 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.
[0006] 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,
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, incorporated herein,
for that teaching. A Type 8 family is reported in U.S. Pat. No.
5,798,246.
[0007] Many functions of the immune and inflammatory response
system are inhibited by agents that increase intracellular levels
of cAMP (Verghese (1995) Mol. Pharmacol. 47:1164-1171). 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.
[0008] 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).
[0009] 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, incorporated herein for that
teaching.
[0010] Cyclic nucleotide phosphodiesterases have also been reported
to affect cellular proliferation in 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.
[0011] 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 tissues and disorders in which cyclic
phosphodiesterases are differentially expressed. The present
invention advances the state of the art by providing tissues and
disorders in which expression of a human cyclic nucleotide
phosphodiesterase is relevant. Accordingly, the invention provides
methods directed to expression of the phosphodiesterase.
SUMMARY OF THE INVENTION
[0012] It is an object of the invention to identify tissues and
disorders in which expression of the cyclic nucleotide
phosphodiesterase is relevant.
[0013] It is a further object of the invention to provide methods
wherein the cyclic nucleotide phosphodiesterase polypeptides are
useful as reagents or targets in phosphodiesterase assays
applicable to treatment and diagnosis of disorders mediated by or
related to the cyclic nucleotide phosphodiesterase.
[0014] It is a further object of the invention to provide methods
wherein polynucleotides corresponding to the phosphodiesterase
polypeptide are useful as targets or reagents in phosphodiesterase
assays applicable to treatment and diagnosis of disorders mediated
by or related to the phosphodiesterase.
[0015] A specific object of the invention is to identify compounds
that act as agonists and antagonists and modulate the expression of
the phosphodiesterase in specific tissues and disorders.
[0016] A further specific object of the invention is to provide
compounds that modulate expression of the phosphodiesterase for
treatment and diagnosis of phosphodiesterase-mediated or related
disorders.
[0017] The invention is thus based on the expression of a human
cyclic nucleotide phosphodiesterase in specific tissues and
disorders.
[0018] The invention provides methods of screening for compounds
that modulate expression or activity of the phosphodiesterase
polypeptides or nucleic acid (RNA or DNA) in the specific tissues
or disorders.
[0019] The invention also provides a process for modulating
phosphodiesterase polypeptide or nucleic acid expression or
activity, especially using the screened compounds.
[0020] Modulation may be used to treat conditions related to
aberrant activity or expression of the phosphodiesterase
polypeptides or nucleic acids.
[0021] The invention also provides assays for determining the
activity of or the presence or absence of the phosphodiesterase
polypeptides or nucleic acid molecules in specific biological
samples, including for disease diagnosis.
[0022] The invention also provides assays for determining the
presence of a mutation in the polypeptides or nucleic acid
molecules, including for disease diagnosis.
[0023] The invention utilizes isolated phosphodiesterase
polypeptides, including a polypeptide having the amino acid
sequence shown in SEQ ID NO 1.
[0024] The invention also utilizes an isolated phosphodiesterase
nucleic acid molecule having the sequence shown in SEQ ID NO 2.
[0025] The invention also utilizes variant polypeptides having an
amino acid sequence that is substantially homologous to the amino
acid sequence shown in SEQ ID NO 1.
[0026] The invention also utilizes variant nucleic acid sequences
that are substantially homologous to the nucleotide sequence shown
in SEQ ID NO 2.
[0027] The invention also utilizes fragments of the polypeptide
shown in SEQ ID NO 1 and nucleotide sequence shown in SEQ ID NO 2,
as well as substantially homologous fragments of the polypeptide or
nucleic acid.
[0028] The invention further utilizes 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.
[0029] The invention also utilizes vectors and host cells that
express the phosphodiesterase and provides methods for expressing
the phosphodiesterase nucleic acid molecules and polypeptides in
specific cell types and disorders, and particularly recombinant
vectors and host cells.
[0030] The invention also utilizes methods of making the vectors
and host cells and provides methods for using them to assay
expression and cellular effects of expression of the
phosphodiesterase nucleic acid molecules and polypeptides in
specific cell types and disorders.
[0031] The invention also utilizes antibodies or antigen-binding
fragments thereof that selectively bind the phosphodiesterase
polypeptides and fragments.
DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows the phosphodiesterase nucleotide sequence (SEQ
ID NO 2) and the deduced amino acid sequence (SEQ ID NO 1). BLAST
analysis showed the top BLAST scores to AB020593, AF127479,
AF127480 human dual specificity cAMP/cGMP, and mouse PDE10A,
AF110507. AF110507, AF127480, AF127479, AB020593=GenBank accession.
See, for example, Sonderling, et al., Proc. Nati'l. Acad. Sci.
U.S.A. 96:7071-7076 (1999).
[0033] FIG. 2 shows a hydrophobicity plot of the
phosphodiesterase.
[0034] FIG. 3 shows an analysis of the phosphodiesterase amino acid
sequence: .alpha..beta.turn and coil regions; hydrophilicity;
amphipathic regions; flexible regions; antigenic index; and surface
probability plot.
[0035] FIG. 4 shows an analysis of the phosphodiesterase open
reading frame for amino acids corresponding to specific functional
sites. Glycosylation sites are shown in the figure with the actual
modified residue being the first amino acid. Protein kinase C
phosphorylation sites are shown in the figure with the actual
modified residue being the first amino acid. Casein kinase II
phophorylation sites are shown in the figure with the actual
modified residue being the first amino acid. Tyrosine kinase
phosphorylation sites are shown in the figure with the actual
modified residue being the last amino acid. N-myristoylation sites
are shown in the figure. In addition, amino acids corresponding to
the phosphodiesterase signature, HDXXHXX, are found in the sequence
HDLDHRG at amino acids 553-559.
[0036] FIG. 5 shows expression of the 22045 phosphodiesterase in
various normal human tissues.
[0037] FIG. 6 shows expression of the 22045 phosphodiesterase in
various cardiovascular tissues. Int. Mamm.: internal mammary
artery; CHF: congestive heart failure; ISCH: ischemic heart; myop:
myopathic heart.
[0038] FIG. 7 shows expression of the phosphodiesterase in breast,
lung, and colon carcinoma as well as colon metastases to the
liver.
DETAILED DESCRIPTION OF THE INVENTION
[0039] 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.
[0040] 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.
[0041] The cAMP turnover pathway is a signaling 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.
[0042] The cGMP turnover pathway is also a signaling pathway. As
used herein, "cyclic GMP turnover and metabolism" refers to the
molecules involved in the turnover and metabolism of cGMP as well
as to the activities of these molecules. Cyclic GMP is a second
messenger produced in response to ligand-induced stimulation of
certain receptors. In the cGMP signaling pathway, binding of a
ligand can lead to the activation of the enzyme guanyl cyclase,
which catalyzes the synthesis of cGMP. Synthesized cGMP can in turn
activate a cGMP-dependent protein kinase.
[0043] The invention is directed to methods, uses and reagents
applicable to methods and uses that are applied to cells, tissues
and disorders of these cells and tissues wherein phosphodiesterase
expression is relevant. The phosphodiesterase is expressed in a
variety of tissues as shown in FIGS. 5 and 6. Accordingly, the
methods and uses of the invention as disclosed in greater detail
below apply to these tissues, disorders involving these tissues,
and particularly to the disorders with which gene expression is
associated, as shown in these figures and as disclosed herein.
Accordingly, the methods, uses and reagents disclosed in greater
detail below especially apply to breast, lung, colon carcinoma, and
colonic metastases to the liver, especially apply also to
expression in thyroid, heart, kidney, fetal kidney, fetal heart and
testes, especially apply to diseased heart and associated vessels,
specifically to endothelial cells and vascular smooth muscle cells,
applying particularly to congestive heart failure and ischemia. In
situ hybridization shows 22045 expression is moderate to low in
normal and tumor lung samples, specifically in the large vessels,
macrophages, and tumor cells. It also shows low positive expression
in normal and diseased heart, specifically in the endothelial and
vascular smooth muscle cells. Accordingly, the uses, reagents and
methods disclosed in detail herein below apply especially to these
tissues, cell types, and disorders.
[0044] Methods of Using the Polypeptide
[0045] The invention provides methods using the cyclic nucleotide
phosphodiesterase, variants, or fragments, including but not
limited to use in the cells, tissues, and disorders as disclosed
herein.
[0046] The invention provides biological assays related to cyclic
nucleotide phosphodiesterases. Such assays involve any of the known
functions or activities or properties useful for diagnosis and
treatment of cyclic phosphodiesterase-related conditions. These
include, but are not limited to, hydrolysis of cAMP and cGMP,
ability to be bound by specific antibody, cGMP or cAMP binding, and
protein kinase A interaction as well as the various other
properties and functions disclosed herein and disclosed in the
references cited herein.
[0047] The invention provides 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, cell-based assays
involve recombinant host cells expressing the phosphodiesterase.
Accordingly, cells that are useful in this regard include, but are
not limited to, those disclosed herein as expressing or
differentially expressing the phosphdiesterase, such as those shown
in FIGS. 5-7. These include, but are not limited to, cells or
tissues derived from heart, kidney, testis, aortic endothelial
cells, aortic smooth muscle cells, cells derived from the internal
mammary artery, lung small cell carcinoma, breast carcinoma, colon
carcinoma, and colon metastases in the liver. Such cells can
naturally express the gene or can be recombinant, containing one or
more copies of exogenously-introduced phosphodiesterase sequences
or genetically modified to modulate expression of the endogenous
phosphodiesterase sequence.
[0048] This aspect of the invention particularly relates to cells
derived from subjects with disorders involving the tissues in which
the phosopodiesterase is expressed or derived from tissues subject
to disorders including, but not limited to, those disclosed herein.
These disorders may naturally occur, as in populations of human
subjects, or may occur in model systems such as in vitro systems or
in vivo, such as in non-human transgenic organisms, particularly in
non-human transgenic animals.
[0049] Such assays can involve the identification of agents that
interact with the phosphodiesterase protein. This interaction can
be detected by functional assays, such as the ability to be
affected by an effector molecule, such as phosphorylation by an
effector or phosphorylating a substrate. Such interaction can also
be measured by ultimate biological effects, such as increasing or
decreasing the levels of cAMP or cGMP or having biological effects
on immunity/inflammation or cell proliferation, i.e., any of the
effects of modulating the intracelluar levels of the second
messengers cAMP and cGMP.
[0050] 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., a GMP or cAMP) to bind to the polypeptide.
[0051] In yet another aspect of the invention, the invention
provides methods to identify proteins that interact with the
phosphodiesterase in the tissues and disorders disclosed. 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.
[0052] The invention provides methods to identify compounds that
modulate phosphodiesterase activity. Such compounds, for example,
can increase or decrease affinity or rate of binding to cGMP or
cAMP, compete with cGMP or cAMP for binding to the
phosphodiesterase, or displace cGMP or 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. The subject can be a human subject, for example, a subject
in a clinical trial or undergoing treatment or diagnosis, or a
non-human transgenic subject, such as a transgenic animal model for
disease.
[0053] The invention provides methods 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 or
cGMP 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 or cGMP turnover, and biological endpoints of
the pathway.
[0054] 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.
[0055] 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).
[0056] 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).
[0057] 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).
[0058] One candidate compound is a soluble full-length
phosphodiesterase or fragment that competes for cGMP or cAMP
binding. Other candidate compounds include mutant
phosphodiesterases or appropriate fragments containing mutations
that affect phosphodiesterase function and thus compete for cGMP or
cAMP. Accordingly, a fragment that competes for cAMP or cGMP, for
example with a higher affinity, or a fragment that binds cAMP or
cGMP but does not hydrolyze it, is encompassed by the
invention.
[0059] 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.
[0060] 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.
[0061] In the case of the phosphodiesterase, specific end points
can include cAMP and cGMP hydrolysis and a decrease in protein
kinase A activation.
[0062] Assays for phosphodiesterase function include, but are not
limited to, those that are well known in the art and available to
the person of ordinary skill in the art, for example, those found
in Soderling et al., (Proc. Nat'l Acad. Sci. U.S.A. 96:7071-7076
(1999), for example, page 7072, which also discloses cGMP-binding
assays. Assays for phosphodiesterase function are also disclosed in
U.S. Pat. Nos. 5,798,246; 5,581,784; 5,702,936, all of which are
incorporated by reference for these assays. Assays are also
disclosed in Houslay et al. (1997), TIBS 22:217-224, Bloom et al.
(1996), Proc. Natl. Acad. Sci, USA 93:14188-14192, Zhu et al.
(1997) J. Biol. Chem. 272:16152-16157, and Beavo (1995),
Physiological Reviews 75:725-748, also incorporated by reference
for these assays.
[0063] 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 phosphodiesterase isoforms of the same family 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.
[0064] The invention provides competition binding assays 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.
[0065] 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 or cGMP. A compound that competes with cAMP or cGMP will
reduce the amount of hydrolysis or binding of the cAMP or cGMP to
the phosphodiesterase. Accordingly, compounds can be discovered
that directly interact with the phosphodiesterase and compete with
cAMP or cGMP. Such assays can involve any other component that
interacts with the phosphodiesterase.
[0066] 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.
[0067] 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.
[0068] Modulators of phosphodiesterase level or activity identified
according to these assays can be used to test the effects of
modulation of expression of the enzyme on the outcome of clinically
relevant disorders. This can be accomplished in vitro, in vivo,
such as in human clinical trials, and in test models derived from
other organisms, such as non-human transgenic subjects. Modulation
in such subjects includes, but is not limited to, modulation of the
cells, tissues, and disorders particularly disclosed herein.
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 those disclosed
herein, especially in FIGS. 5-7, as well as those disorders
disclosed in the references cited herein above. In one embodiment,
the cells that are treated are derived from heart, kidney, testes,
and thyroid, and as such, modulation is particularly relevant to
disorders involving these tissues. In another embodiment,
modulation is in aortic endothelial cells, aortic smooth muscle
cells and cells of the internal mammary artery. Accordingly,
disorders in which modulation is particularly relevant can include
these tissues. Since the gene is also over-expressed in breast,
lung, and colon carcinoma, modulation is also particularly relevant
in these tissues. 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.
[0069] Disorders involving the spleen include, but are not limited
to, splenomegaly, including nonspecific acute splenitis, congestive
spenomegaly, and spenic infarcts; neoplasms, congenital anomalies,
and rupture. Disorders associated with splenomegaly include
infections, such as nonspecific splenitis, infectious
mononucleosis, tuberculosis, typhoid fever, brucellosis,
cytomegalovirus, syphilis, malaria, histoplasmosis, toxoplasmosis,
kala-azar, trypanosomiasis, schistosomiasis, leishmaniasis, and
echinococcosis; congestive states related to partial hypertension,
such as cirrhosis of the liver, portal or splenic vein thrombosis,
and cardiac failure; lymphohematogenous disorders, such as Hodgkin
disease, non-Hodgkin lymphomas/leukemia, multiple myeloma,
myeloproliferative disorders, hemolytic anemias, and
thrombocytopenic purpura; immunologic-inflammatory conditions, such
as rheumatoid arthritis and systemic lupus erythematosus; storage
diseases such as Gaucher disease, Niemann-Pick disease, and
mucopolysaccharidoses; and other conditions, such as amyloidosis,
primary neoplasms and cysts, and secondary neoplasms.
[0070] 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.
[0071] Disorders involving the colon include, but are not limited
to, congenital anomalies, such as atresia and stenosis, Meckel
diverticulum, congenital aganglionic megacolon-Hirschsprung
disease; enterocolitis, such as diarrhea and dysentery, infectious
enterocolitis, including viral gastroenteritis, bacterial
enterocolitis, necrotizing enterocolitis, antibiotic-associated
colitis (pseudomembranous colitis), and collagenous and lymphocytic
colitis, miscellaneous intestinal inflammatory disorders, including
parasites and protozoa, acquired immunodeficiency syndrome,
transplantation, drug-induced intestinal injury, radiation
enterocolitis, neutropenic colitis (typhlitis), and diversion
colitis; idiopathic inflammatory bowel disease, such as Crohn
disease and ulcerative colitis; tumors of the colon, such as
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[0072] Disorders involving the liver include, but are not limited
to, hepatic injury; jaundice and cholestasis, such as bilirubin and
bile formation; hepatic failure and cirrhosis, such as cirrhosis,
portal hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, such as viral hepatitis,
including hepatitis A-E infection and infection by other hepatitis
viruses, clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and
toxin-induced liver disease, such as alcoholic liver disease;
inborn-errors of metabolism and pediatric liver disease, such as
hemochromatosis, Wilson disease, .alpha..sub.1-antitrypsin
deficiency, and neonatal hepatitis; intrahepatic biliary tract
disease, such as secondary biliary cirrhosis, primary biliary
cirrhosis, primary sclerosing cholangitis, and anomalies of the
biliary tree; circulatory disorders, such as impaired blood flow
into the liver, including hepatic artery compromise and portal vein
obstruction and thrombosis, impaired blood flow through the liver,
including passive congestion and centrilobular necrosis and
peliosis hepatis, hepatic vein outflow obstruction, including
hepatic vein thrombosis (Budd-Chiari syndrome) and veno-occlusive
disease; hepatic disease associated with pregnancy, such as
preeclampsia and eclampsia, acute fatty liver of pregnancy, and
intrehepatic cholestasis of pregnancy; hepatic complications of
organ or bone marrow transplantation, such as drug toxicity after
bone marrow transplantation, graft-versus-host disease and liver
rejection, and nonimmunologic damage to liver allografts; tumors
and tumorous conditions, such as nodular hyperplasias, adenomas,
and malignant tumors, including primary carcinoma of the liver and
metastatic tumors.
[0073] Disorders involving the uterus and endometrium include, but
are not limited to, endometrial histology in the menstrual cycle;
functional endometrial disorders, such as anovulatory cycle,
inadequate luteal phase, oral contraceptives and induced
endometrial changes, and menopausal and postmenopausal changes;
inflammations, such as chronic endometritis; adenomyosis;
endometriosis; endometrial polyps; endometrial hyperplasia;
malignant tumors, such as carcinoma of the endometrium; mixed
Mullerian and mesenchymal tumors, such as malignant mixed Mullerian
tumors; tumors of the myometrium, including leiomyomas,
leiomyosarcomas, and endometrial stromal tumors.
[0074] Disorders involving the brain include, but are not limited
to, disorders involving neurons, and disorders involving glia, such
as astrocytes, oligodendrocytes, ependymal cells, and microglia;
cerebral edema, raised intracranial pressure and herniation, and
hydrocephalus; malformations and developmental diseases, such as
neural tube defects, forebrain anomalies, posterior fossa
anomalies, and syringomyelia and hydromyelia; perinatal brain
injury; cerebrovascular diseases, such as those related to hypoxia,
ischemia, and infarction, including hypotension, hypoperfusion, and
low-flow states--global cerebral ischemia and focal cerebral
ischemia--infarction from obstruction of local blood supply,
intracranial hemorrhage, including intracerebral (intraparenchymal)
hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms,
and vascular malformations, hypertensive cerebrovascular disease,
including lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicalla-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer disease and Pick
disease, degenerative diseases of basal ganglia and brain stem,
including Parkinsonism, idiopathic Parkinson disease (paralysis
agitans), progressive supranuclear palsy, corticobasal degenration,
multiple system atrophy, including striatonigral degenration,
Shy-Drager syndrome, and olivopontocerebellar atrophy, and
Huntington disease; spinocerebellar degenerations, including
spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease.
[0075] Disorders involving T-cells include, but are not limited to,
cell-mediated hypersensitivity, such as delayed type
hypersensitivity and T-cell-mediated cytotoxicity, and transplant
rejection; autoimmune diseases, such as systemic lupus
erythematosus, Sjogren syndrome, systemic sclerosis, inflammatory
myopathies, mixed connective tissue disease, and polyarteritis
nodosa and other vasculitides; immunologic deficiency syndromes,
including but not limited to, primary immunodeficiencies, such as
thymic hypoplasia, severe combined immunodeficiency diseases, and
AIDS; leukopenia; reactive (inflammatory) proliferations of white
cells, including but not limited to, leukocytosis, acute
nonspecific lymphadenitis, and chronic nonspecific lymphadenitis;
neoplastic proliferations of white cells, including but not limited
to lymphoid neoplasms, such as precursor T-cell neoplasms, such as
acute lymphoblastic leukemia/lymphoma, peripheral T-cell and
natural killer cell neoplasms that include peripheral T-cell
lymphoma, unspecified, adult T-cell leukemia/lymphoma, mycosis
fungoides and Sezary syndrome, and Hodgkin disease.
[0076] Diseases of the skin, include but are not limited to,
disorders of pigmentation and melanocytes, including but not
limited to, vitiligo, freckle, melasma, lentigo, nevocellular
nevus, dysplastic nevi, and malignant melanoma; benign epithelial
tumors, including but not limited to, seborrheic keratoses,
acanthosis nigricans, fibroepithelial polyp, epithelial cyst,
keratoacanthoma, and adnexal (appendage) tumors; premalignant and
malignant epidermal tumors, including but not limited to, actinic
keratosis, squamous cell carcinoma, basal cell carcinoma, and
merkel cell carcinoma; tumors of the dermis, including but not
limited to, benign fibrous histiocytoma, dermatofibrosarcoma
protuberans, xanthomas, and dermal vascular tumors; tumors of
cellular immigrants to the skin, including but not limited to,
histiocytosis X, mycosis fungoides (cutaneous T-cell lymphoma), and
mastocytosis; disorders of epidermal maturation, including but not
limited to, ichthyosis; acute inflammatory dermatoses, including
but not limited to, urticaria, acute eczematous dermatitis, and
erythema multiforme; chronic inflammatory dermatoses, including but
not limited to, psoriasis, lichen planus, and lupus erythematosus;
blistering (bullous) diseases, including but not limited to,
pemphigus, bullous pemphigoid, dermatitis herpetiformis, and
noninflammatory blistering diseases: epidermolysis bullosa and
porphyria; disorders of epidermal appendages, including but not
limited to, acne vulgaris; panniculitis, including but not limited
to, erythema nodosum and erythema induratum; and infection and
infestation, such as verrucae, molluscum contagiosum, impetigo,
superficial fungal infections, and arthropod bites, stings, and
infestations.
[0077] In normal bone marrow, the myelocytic series
(polymorphoneuclear cells) make up approximately 60% of the
cellular elements, and the erythrocytic series, 20-30%.
Lymphocytes, monocytes, reticular cells, plasma cells and
megakaryocytes together constitute 10-20%. Lymphocytes make up
5-15% of normal adult marrow. In the bone marrow, cell types are
add mixed so that precursors of red blood cells (erythroblasts),
macrophages (monoblasts), platelets (megakaryocytes),
polymorphoneuclear leucocytes (myeloblasts), and lymphocytes
(lymphoblasts) can be visible in one microscopic field. In
addition, stem cells exist for the different cell lineages, as well
as a precursor stem cell for the committed progenitor cells of the
different lineages. The various types of cells and stages of each
would be known to the person of ordinary skill in the art and are
found, for example, on page 42 (FIGS. 2-8) of Immunology,
Imunopathology and Immunity, Fifth Edition, Sell et al. Simon and
Schuster (1996), incorporated by reference for its teaching of cell
types found in the bone marrow. According, the invention is
directed to disorders arising from these cells. These disorders
include but are not limited to the following: diseases involving
hematopoetic stem cells; committed lymphoid progenitor cells;
lymphoid cells including B and T-cells; committed myeloid
progenitors, including monocytes, granulocytes, and megakaryocytes;
and committed erythroid progenitors. These include but are not
limited to the leukemias, including B-lymphoid leukemias,
T-lymphoid leukemias, undifferentiated leukemias; erythroleukemia,
megakaryoblastic leukemia, monocytic; [leukemias are encompassed
with and without differentiation]; chronic and acute lymphoblastic
leukemia, chronic and acute lymphocytic leukemia, chronic and acute
myelogenous leukemia, lymphoma, myelo dysplastic syndrome, chronic
and acute myeloid leukemia, myelomonocytic leukemia; chronic and
acute myeloblastic leukemia, chronic and acute myelogenous
leukemia, chronic and acute promyelocytic leukemia, chronic and
acute myelocytic leukemia, hematologic malignancies of
monocyte-macrophage lineage, such as juvenile chronic myelogenous
leukemia; secondary AML, antecedent hematological disorder;
refractory anemia; aplastic anemia; reactive cutaneous
angioendotheliomatosis; fibrosing disorders involving altered
expression in dendritic cells, disorders including systemic
sclerosis, E-M syndrome, epidemic toxic oil syndrome, eosinophilic
fasciitis localized forms of scleroderma, keloid, and fibrosing
colonopathy; angiomatoid malignant fibrous histiocytoma; carcinoma,
including primary head and neck squamous cell carcinoma; sarcoma,
including kaposi's sarcoma; fibroadanoma and phyllodes tumors,
including mammary fibroadenoma; stromal tumors; phyllodes tumors,
including histiocytoma; erythroblastosis; neurofibromatosis;
diseases of the vascular endothelium; demyelinating, particularly
in old lesions; gliosis, vasogenic edema, vascular disease,
Alzheimer's and Parkinson's disease; T-cell lymphomas; B-cell
lymphomas.
[0078] 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.
[0079] 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.
[0080] Disorders involving red cells include, but are not limited
to, anemias, such as hemolytic anemias, including hereditary
spherocytosis, hemolytic disease due to erythrocyte enzyme defects:
glucose-6-phosphate dehydrogenase deficiency, sickle cell disease,
thalassemia syndromes, paroxysmal nocturnal hemoglobinuria,
immunohemolytic anemia, and hemolytic anemia resulting from trauma
to red cells; and anemias of diminished erythropoiesis, including
megaloblastic anemias, such as anemias of vitamin B12 deficiency:
pernicious anemia, and anemia of folate deficiency, iron deficiency
anemia, anemia of chronic disease, aplastic anemia, pure red cell
aplasia, and other forms of marrow failure.
[0081] 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.
[0082] Disorders involving B-cells include, but are not limited to
precursor B-cell neoplasms, such as lymphoblastic
leukemia/lymphoma. Peripheral B-cell neoplasms include, but are not
limited to, chronic lymphocytic leukemia/small lymphocytic
lymphoma, follicular lymphoma, diffuse large B-cell lymphoma,
Burkitt lymphoma, plasma cell neoplasms, multiple myeloma, and
related entities, lymphoplasmacytic lymphoma (Waldenstr{overscore
(o)}m macroglobulinemia), mantle cell lymphoma, marginal zone
lymphoma (MALToma), and hairy cell leukemia.
[0083] 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
(hypernephroma, adenocarcinoma of kidney), which includes
urothelial carcinomas of renal pelvis.
[0084] Disorders of the breast include, but are not limited to,
disorders of development; inflammations, including but not limited
to, acute mastitis, periductal mastitis, periductal mastitis
(recurrent subareolar abscess, squamous metaplasia of lactiferous
ducts), mammary duct ectasia, fat necrosis, granulomatous mastitis,
and pathologies associated with silicone breast implants;
fibrocystic changes; proliferative breast disease including, but
not limited to, epithelial hyperplasia, sclerosing adenosis, and
small duct papillomas; tumors including, but not limited to,
stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas,
and epithelial tumors such as large duct papilloma; carcinoma of
the breast including in situ (noninvasive) carcinoma that includes
ductal carcinoma in situ (including Paget's disease) and lobular
carcinoma in situ, and invasive (infiltrating) carcinoma including,
but not limited to, invasive ductal carcinoma, no special type,
invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)
carcinoma, tubular carcinoma, and invasive papillary carcinoma, and
miscellaneous malignant neoplasms.
[0085] Disorders in the male breast include, but are not limited
to, gynecomastia and carcinoma.
[0086] 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.
[0087] Disorders involving the prostate include, but are not
limited to, inflammations, benign enlargement, for example, nodular
hyperplasia (benign prostatic hypertrophy or hyperplasia), and
tumors such as carcinoma.
[0088] Disorders involving the thyroid include, but are not limited
to, hyperthyroidism; hypothyroidism including, but not limited to,
cretinism and myxedema; thyroiditis including, but not limited to,
hashimoto thyroiditis, subacute (granulomatous) thyroiditis, and
subacute lymphocytic (painless) thyroiditis; Graves disease;
diffuse and multinodular goiter including, but not limited to,
diffuse nontoxic (simple) goiter and multinodular goiter; neoplasms
of the thyroid including, but not limited to, adenomas, other
benign tumors, and carcinomas, which include, but are not limited
to, papillary carcinoma, follicular carcinoma, medullary carcinoma,
and anaplastic carcinoma; and cogenital anomalies.
[0089] Disorders involving the skeletal muscle include tumors such
as rhabdomyosarcoma.
[0090] Disorders involving the pancreas include those of the
exocrine pancreas such as congenital anomalies, including but not
limited to, ectopic pancreas; pancreatitis, including but not
limited to, acute pancreatitis; cysts, including but not limited
to, pseudocysts; tumors, including but not limited to, cystic
tumors and carcinoma of the pancreas; and disorders of the
endocrine pancreas such as, diabetes mellitus; islet cell tumors,
including but not limited to, insulinomas, gastrinomas, and other
rare islet cell tumors.
[0091] Disorders involving the small intestine include the
malabsorption syndromes such as, celiac sprue, tropical sprue
(postinfectious sprue), whipple disease, disaccharidase (lactase)
deficiency, abetalipoproteinemia, and tumors of the small intestine
including adenomas and adenocarcinoma.
[0092] Disorders related to reduced platelet number,
thrombocytopenia, include idiopathic thrombocytopenic purpura,
including acute idiopathic thrombocytopenic purpura, drug-induced
thrombocytopenia, HIV-associated thrombocytopenia, and thrombotic
microangiopathies: thrombotic thrombocytopenic purpura and
hemolytic-uremic syndrome.
[0093] Disorders involving precursor T-cell neoplasms include
precursor T lymphoblastic leukemia/lymphoma. Disorders involving
peripheral T-cell and natural killer cell neoplasms include T-cell
chronic lymphocytic leukemia, large granular lymphocytic leukemia,
mycosis fungoides and Sezary syndrome, peripheral T-cell lymphoma,
unspecified, angioimmunoblastic T-cell lymphoma, angiocentric
lymphoma (NK/T-cell lymphoma.sup.4a), intestinal T-cell lymphoma,
adult T-cell leukemia/lymphoma, and anaplastic large cell
lymphoma.
[0094] Disorders involving the ovary include, for example,
polycystic ovarian disease, Stein-leventhal syndrome, Pseudomyxoma
peritonei and stromal hyperthecosis; ovarian tumors such as, tumors
of coelomic epithelium, serous tumors, mucinous tumors,
endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma,
brenner tumor, surface epithelial tumors; germ cell tumors such as
mature (benign) teratomas, monodermal teratomas, immature malignant
teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma;
sex cord-stomal tumors such as, granulosa-theca cell tumors,
thecoma-fibromas, androblastomas, hill cell tumors, and
gonadoblastoma; and metastatic tumors such as Krukenberg
tumors.
[0095] 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, osteoporois, paget
disease, rickets, osteomalacia, high-turnover osteodystrophy,
low-turnover of 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 saracoma, primitive neuroectodermal
tumor, giant cell tumor, and metastatic tumors.
[0096] The invention thus provides methods for treating a 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 downregulates) 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.
[0097] 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 cGMP or protein kinase A. These
phosphodiesterases or fragments can have a higher affinity for the
target so as to provide effective competition.
[0098] 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).
[0099] The invention also provides methods 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 heart, thyroid, kidney, testes, aortic endothelial
cells, aortic smooth muscle cells, internal mammary artery, breast,
lung, and colon carcinoma, especially colonic metastases to the
liver. In addition, as indicated in FIG. 6, low positive expression
occurs in diseased heart tissue from patients with congestive heart
failure and ischemia. In view of these results, in one embodiment
of the invention, these disorders are treated by modulating the
level or activity of the phosphodieserase gene in diseased hearts.
Since expression has been shown (by in situ hybridization)
specifically in endothelial cells and vascular smooth muscle cells,
treatment is especially directed to these cells. Likewise, in one
embodiment, diagnosis is directed to cells and tissues involved in
these disorders. As mentioned above, treatment and diagnosis can be
in human subjects in which the disease normally occurs and in model
systems, both in vitro and in vivo, such as in transgenic
animals.
[0100] Accordingly, methods are directed to detecting the presence,
or levels of, the phosphodiesterase in a cell, tissue, or organism.
The methods involve contacting a biological sample with a compound
capable of interacting with the phosphodiesterase such that the
interaction can be detected.
[0101] 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.
[0102] The invention also provides methods 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 or cGMP 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.
[0103] 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.
[0104] The invention also provides methods of pharmacogenomic
analysis including, but not limited to, in the cells, tissues and
disorders disclosed herein in which expression of the
phosphodiesterase either occurs or shows differential expression.
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 can be used as a target to ascertain a
genetic predisposition that can affect treatment modality. Thus, in
a cGMP- or 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.
[0105] The invention also provides 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.
[0106] Polypeptides
[0107] The methods and uses herein disclosed can be based on
polypeptide reagents and targets. The invention is thus based on
the use of a 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 fetal testis 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 homologous
to GenBank human fetal lung sequence AB020593.
[0108] The invention thus relates to expression of a
phosphodiesterase having the deduced amino acid sequence shown in
FIG. 1 (SEQ ID NO 1).
[0109] "Phosphodiesterase polypeptide" or "phosphodiesterase
protein" refers to the polypeptide in SEQ ID NO 1. 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.
[0110] Tissues and/or cells in which the phosphodiesterase is found
include, but are not limited to those shown in FIGS. 5-7, and
particularly in thyroid, heart, kidney, testes, aortic endothelial
cells, aortic smooth muscle cells, and internal mammary artery. In
addition, the phosphodiesterase is expressed in diseased tissues,
including but limited to, heart tissue derived from patients with
congestive heart failure and ischemia, and in breast, colon, and
lung carcinoma. Expression has been confirmed by Northern blot
analysis. In situ hybridization results show moderate to low
expression in normal and tumor lung samples, specifically in the
large vessels, macrophages, and tumor cells. Furthermore, low
positive expression in normal and diseased heart has been shown
specifically in the endothelial cells and vascular smooth muscle
cells.
[0111] The present invention thus utilizes an isolated or purified
phosphodiesterase polypeptide and variants and fragments
thereof.
[0112] 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."
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] In one embodiment, the phosphodiesterase polypeptide
comprises the amino acid sequence shown in SEQ ID NO 1. 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.
[0118] Variants also encompass proteins derived from other genetic
loci in an organism, but having substantial homology to the
phosphodiesterase of SEQ ID NO 1. 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.
[0119] 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 2 under stringent conditions as more fully described
below.
[0120] 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% or more of the
length of the reference sequence. 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.
[0121] 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).
1TABLE 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
[0122] 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).
[0123] 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. See www.ncbi.nlm.nih.gov. 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).
[0124] 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
(available at www.gcg.com), 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) (available at
www.gcg.com), 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.
[0125] 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.
[0126] 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.
[0127] 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 the cyclic nucleotide (e.g., AMP, GMP)-dependent
signal transduction pathways.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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 or cGMP. A further useful variation at the same
site can result in altered affinity for cAMP or cGMP. 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.
[0132] 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 or cGMP hydrolysis in vitro or cGMP- or cAMP-dependent
in vitro activity, such as proliferative activity. Sites that are
critical for cGMP or 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).
[0133] Substantial homology can be to the entire nucleic acid or
amino acid sequence or to fragments of these sequences.
[0134] The invention thus also includes polypeptide fragments of
the phosphodiesterase. 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 phosphodiesterase as
described herein.
[0135] 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 cGMP
or cAMP, as well as fragments that can be used as an immunogen to
generate phosphodiesterase antibodies.
[0136] 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, protein kinase C phosphorylation, casein kinase II
phosphorylation, tyrosine kinase phosphorylation, and
N-myristoylation. Further possible fragments include the catalytic
site or domain including HDXXX, an allosteric binding site, sites
important for cellular and subcellular targeting, sites functional
for interacting with components of other cGMP or cAMP-dependent
signal transduction pathways, and aminoterminal and carboxyterminal
regulatory sites.
[0137] Such domains or motifs can be identified by means of routine
computerized homology searching procedures.
[0138] 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.
[0139] These regions can be identified by well-known methods
involving computerized homology analysis.
[0140] 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.
[0141] 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. Regions having a high
antigenicity index are shown in FIG. 3. However,
intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular peptide
regions.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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 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 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.
[0146] 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
utilizes 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.
[0147] 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.
[0148] 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
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 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.
[0149] Additionally, chimeric phosphodiesterase proteins can be
produced in which one or more functional sites is derived from a
different isoform, or from another phosphodiesterase family. 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 cyclic AMP- and
cyclic GMP-dependent signal transduction pathways, protein kinase A
phosphorylation sites, glycosylation sites, and other functional
sites disclosed herein.
[0150] The isolated phosphodiesterases can be purified from cells
that naturally express it, such as from those shown in FIGS. 5-7
and/or specifically disclosed herein above, among others,
especially purified from cells that have been altered to express it
(recombinant), or synthesized using known protein synthesis
methods.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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).
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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.
[0160] Methods of Using Antibodies
[0161] Methods for using antibodies as disclosed herein are
particularly applicable to the cells, tissues and disorders shown
in FIGS. 5-7 and as otherwise discussed herein above.
[0162] The invention provides methods using 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.
[0163] The invention provides methods of using antibodies to
isolate a phosphodiesterase by standard techniques, such as
affinity chromatography or immunoprecipitation. The antibodies can
facilitate the purification of the phosphodiesterase from cells
naturally expressing it and cells recombinantly producing it.
[0164] The antibodies can be used 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.
[0165] 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.
[0166] The antibodies can be used to assess abnormal tissue
distribution or abnormal expression during development.
[0167] Antibody detection of circulating fragments of the full
length phosphodiesterase can be used to identify phosphodiesterase
turnover.
[0168] 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.
[0169] The antibodies can also be used to assess normal and
aberrant subcellular localization in cells in the various tissues
in an organism. Antibodies can be developed against the whole
phosphodiesterase or portions of the phosphodiesterase.
[0170] 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.
[0171] 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.
[0172] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic
phosphodiesterase can be used to identify individuals that require
modified treatment modalities.
[0173] Antibodies can also be used in diagnostic procedures 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.
[0174] The antibodies are also useful for tissue typing. Thus,
where the phosphodiesterase is expressed in a specific tissue,
antibodies that are specific for this phosphodiesterase can be used
to identify the tissue type.
[0175] The antibodies are also useful for inhibiting
phosphodiesterase function, for example, blocking binding of cGMP
or cAMP, protein kinase A, or the catalytic site.
[0176] These uses can also be applied in a therapeutic context in
which treatment involves inhibiting phosphodiesterase function.
Antibodies can be prepared against specific fragments containing
sites required for function or against intact
phosphodiesterase.
[0177] 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.
[0178] 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.
[0179] Antibodies
[0180] The methods for using antibodies described above are based
on the generation of antibodies that specifically bind to the
phosphodiesterase or its variants or fragments.
[0181] 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. Regions having a high antigenicity index are
shown in FIG. 3.
[0182] 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 or cGMP 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.
[0183] 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.
[0184] Antibodies can be polyclonal or monoclonal. An intact
antibody, or a fragment thereof (e.g. Fab or F(ab').sub.2) can be
used.
[0185] 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.
[0186] An appropriate immunogenic preparation can be derived from
native, recombinantly expressed, or chemically synthesized
peptides.
[0187] Methods for Using the Polynucleotide
[0188] The methods and uses described herein below for the
phosphodiesterase polynucleotide are particularly applicable to the
cells, tissues, and disorders shown in FIGS. 5-7, and specifically
discussed herein above.
[0189] The nucleic acid fragments useful to practice the invention
provide probes or primers in assays, such as those described
herein. "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 2 and
the complements thereof. More typically, the probe further
comprises a label, e.g., radioisotope, fluorescent compound,
enzyme, or enzyme co-factor.
[0190] 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.
[0191] The phosphodiesterase polynucleotides can be utilized as
probes and primers in biological assays.
[0192] 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.
[0193] The invention utilizes the phosphodiesterase polynucleotides
as a hybridization probe for cDNA and genomic DNA to isolate a
full-length cDNA and genomic clones encoding variant polypeptides
and to isolate cDNA and genomic clones that correspond to variants
producing the same polypeptides shown in SEQ ID NO 1 or the other
variants described herein. This method is useful for isolating
variant genes and cDNA that are expressed in the cells, tissues,
and disorders disclosed herein.
[0194] 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.
[0195] The nucleic acid probe can be, for example, the full-length
cDNA of SEQ ID NO 2, 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.
[0196] Fragments of the polynucleotides can also be used 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.
[0197] Fragments can also be used to synthesize antisense molecules
of desired length and sequence.
[0198] Antisense nucleic acids, useful in treatment and diagnosis,
can be designed using the nucleotide sequences of SEQ ID NO 2, 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-carboxymethylaminomet- hyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopente- nyladenine,
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).
[0199] Additionally, the nucleic acid molecules useful to practice
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.
[0200] The nucleic acid molecules and fragments useful to practice
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).
[0201] The phosphodiesterase polynucleotides can also be used as
primers for PCR to amplify any given region of a phosphodiesterase
polynucleotide.
[0202] The phosphodiesterase polynucleotides can also be used to
construct 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.
[0203] The phosphodiesterase polynucleotides can also be used to
express antigenic portions of the phosphodiesterase protein.
[0204] The phosphodiesterase polynucleotides can also be used 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 sequence to chromosomes is important in
correlating these sequences with genes associated with disease,
especially where translocations and/or amplification has
occurred.
[0205] 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.
[0206] 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).
[0207] 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.
[0208] The phosphodiesterase polynucleotide probes can also be used
to determine patterns of the presence of the gene encoding the
phosphodiesterase with respect to tissue distribution, for example,
whether gene duplication has occurred and whether the duplication
occurs in all or only a subset of cells in a tissue. The genes can
be naturally occurring or can have been introduced into a cell,
tissue, or organism exogenously.
[0209] The phosphodiesterase polynucleotides can also be used to
design ribozymes corresponding to all, or a part, of the mRNA
produced from genes encoding the polynucleotides described herein,
the ribozymes being useful to treat or diagnose a disorder or
otherwise modulate expression of the nucleic acid.
[0210] The phosphodiesterase polynucleotides can also be used to
make vectors that express part, or all, of the phosphodiesterase
polypeptides.
[0211] The phosphodiesterase polynucleotides can also be used to
construct host cells expressing a part, or all, of the
phosphodiesterase polynucleotides and polypeptides.
[0212] The phosphodiesterase polynucleotides can also be used to
construct transgenic animals expressing all, or a part, of the
phosphodiesterase polynucleotides and polypeptides.
[0213] The phosphodiesterase polynucleotides can also be used as
hybridization probes to determine 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.
DNA or RNA level can be determined. Probes 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 gene.
[0214] Alternatively, the probe can be used in an in situ
hybridization context to assess the position of extra copies of the
phosphodiesterase gene, 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.
[0215] 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, such as in the
cells and tissues shown in FIGS. 5-7 and otherwise specifically
discussed herein. Thus in one embodiment, disorders include
diseases of the heart, such as congestive heart failure and
ischemia, as well as carcinoma of the lung, breast and colon.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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.
[0221] 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 gene to a subject) in patients or in
transgenic animals.
[0222] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
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 excessive or deficient phosphodiesterase nucleic
acid expression.
[0223] The assays can be performed in cell-based and cell-free
systems, such as systems using the tissues described herein, in
which the gene is expressed or in model systems for the disorders
to which the invention pertains. Cell-based assays include cells
naturally expressing the phosphodiesterase nucleic acid or
recombinant cells genetically engineered to express specific
nucleic acid sequences.
[0224] Alternatively, candidate compounds can be assayed in vivo in
patients or in transgenic animals.
[0225] 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
cAMP or cGMP 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.
[0226] 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.
[0227] 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.
[0228] The gene is particularly relevant for the treatment of
disorders involving the tissues shown in FIGS. 5-7, particularly
thyroid, heart, kidney, testes, aortic endothelial cells, aortic
smooth muscle cells, internal mammary artery, as well as tissues
and cells involved in congestive heart failure and ischemia, and in
lung, colon and breast carcinoma.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] The phosphodiesterase polynucleotides can be used 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.
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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.
[0237] Alternatively, mutations in a phosphodiesterase gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[0238] 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.
[0239] Perfectly matched sequences can be distinguished from
mismatched sequences by nuclease cleavage digestion assays or by
differences in melting temperature.
[0240] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method.
[0241] 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).
[0242] 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.
[0243] 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.
[0244] The phosphodiesterase polynucleotides can also be used 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 or cGMP could result in
an excessive or decreased drug effect with standard concentrations
of cAMP or cGMP. 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.
[0245] 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.
[0246] 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.
[0247] 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.
[0248] 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).
[0249] 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.
[0250] 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.
[0251] 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 2.
[0252] 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.
[0253] 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.
[0254] 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.
[0255] Polynucleotides
[0256] The methods and uses described herein can be based on the
phosphodiesterase polynucleotide as a reagent or as a target.
[0257] The invention thus provides methods and uses for the
nucleotide sequence in SEQ ID NO 2.
[0258] The specifically disclosed cDNA comprises the coding region
and 5' and 3' untranslated sequences in SEQ ID NO 2.
[0259] The invention provides isolated polynucleotides encoding the
phosphodiesterase. The term "phosphodiesterase polynucleotide" or
"phosphodiesterase nucleic acid" refers to the sequences shown in
SEQ ID NO 2 or SEQ ID NO 4 or in the deposited cDNAs. The term
"phosphodiesterase polynucleotide" or "phosphodiesterase nucleic
acid" further includes variants and fragments of the
phosphodiesterase polynucleotides.
[0260] 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.
[0261] 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.
[0262] 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.
[0263] 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.
[0264] 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.
[0265] 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.
[0266] 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.
[0267] 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).
[0268] In one embodiment, the phosphodiesterase nucleic acid
comprises only the coding region.
[0269] The invention further provides variant phosphodiesterase
polynucleotides, and fragments thereof, that differ from the
nucleotide sequence shown in SEQ ID NO 2 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 2.
[0270] 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.
[0271] Typically, variants have a substantial identity with a
nucleic acid molecule of SEQ ID NO 2 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.
[0272] 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 2 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
2 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, or all cyclic nucleotide
phosphodiesterases.
[0273] 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 1 or SEQ ID NO 3
corresponds to a naturally-occurring nucleic acid molecule. As used
herein, a "naturally-occuring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein).
[0274] 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.
[0275] 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 2 or the complement of SEQ ID NO 2. In one embodiment,
the nucleic acid consists of a portion of the nucleotide sequence
of SEQ ID NO 2 and the complement of SEQ ID NO 2. 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.
[0276] Furthermore, the invention provides polynucleotides that
comprise a fragment of the full-length phosphodiesterase
polynucleotide. 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.
[0277] 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.
[0278] 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.
[0279] Where the location of the domains or sites have been
predicted by computer analysis, one of ordinary skill would
appreciate that the amino acid residues constituting these domains
can vary depending on the criteria used to define the domains.
[0280] However, it is understood that a phosphodiesterase fragment
includes any nucleic acid sequence that does not include the entire
gene.
[0281] The invention also provides phosphodiesterase nucleic acid
fragments that encode epitope bearing regions of the
phosphodiesterase proteins described herein.
[0282] Methods Using Vectors and Host Cells
[0283] The methods using vectors and host cells are particularly
relevant where vectors are expressed in the cells, tissues, and
disorders shown in FIGS. 5-7, and otherwise discussed herein, or
where the host cells are those that naturally express the gene, as
shown in these figures and which may be the native or a recombinant
cell expressing the gene.
[0284] 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.
[0285] 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, as well as cells producing significant amounts of the
polypeptide, for example, the high-expressors shown in FIG. 6, in
other words, fetal heart cell and testes.
[0286] 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 or cGMP binding,
gene expression at the level of transcription or translation,
protein kinase A interaction, and components of the signal
transduction pathway.
[0287] 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.
[0288] 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.
[0289] 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.
[0290] In another embodiment, the cells provide a phosphodiesterase
that is abnormally inactive. This phosphodiesterase can compete
with endogenous phosphodiesterase in the individual.
[0291] In another embodiment, cells expressing phosphodiesterases
that cannot be activated are introduced into an individual in order
to compete with endogenous phosphodiesterase 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.
[0292] Homologously recombinant host cells can also be produced
that allow the in situ alteration of endogenous phosphodiesterase
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 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. Alternatively, increased expression of
phosphodiesterase 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
phosphodiesterase 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 phosphodiesterase
proteins. Such mutations could be introduced, for example, into the
specific functional regions such as the cyclic nucleotide-binding
site.
[0293] 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.
[0294] 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.
[0295] Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[0296] In one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which phosphodiesterase polynucleotide
sequences have been introduced.
[0297] 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.
[0298] 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.
[0299] 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.
[0300] 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.
[0301] 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.
[0302] 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.
[0303] 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.
[0304] Vectors/Host Cells
[0305] The methods using the vectors and host cells discussed above
are based on the vectors and host cells including, but not limited
to, those described below.
[0306] The invention also provides methods using 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.
[0307] 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.
[0308] 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).
[0309] 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.
[0310] It is understood, however, that in some embodiments,
transcription and/or translation of the phosphodiesterase
polynucleotides can occur in a cell-free system.
[0311] 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 .lambda., 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.
[0312] 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.
[0313] 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.).
[0314] 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, poxyiruses, 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.
[0315] 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.
[0316] 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.
[0317] 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.
[0318] 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).
[0319] 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).
[0320] 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.).
[0321] 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).
[0322] 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).
[0323] 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.
[0324] 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).
[0325] 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.
[0326] 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.).
[0327] 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.
[0328] 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.
[0329] 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.
[0330] 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.
[0331] 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.
[0332] 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.
[0333] 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.
[0334] Pharmaceutical Compositions
[0335] The invention encompasses use of the polypeptides, nucleic
acids, and other agents in pharmaceutical compositions to
administer to the cells in which expression of the
phosophodiesterase is relevant and in disorders as disclosed
herein. Uses are both diagnostic and therapeutic. The
phosphodiesterase nucleic acid molecules, protein, 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.
It is understood however, that administration can also be to cells
in vitro as well as to in vivo model systems such as non-human
transgenic animals.
[0336] 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."
[0337] 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.
[0338] 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.
[0339] 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.
[0340] 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.
[0341] 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.
[0342] 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.
[0343] 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.
[0344] 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.
[0345] 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.
[0346] 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.
[0347] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0348] 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.
[0349] 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.
[0350] 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.
[0351] 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.
[0352] 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.
Sequence CWU 1
1
2 1 779 PRT Homo sapiens 1 Met Arg Ile Glu Glu Arg Lys Ser Gln His
Leu Thr Gly Leu Thr Asp 1 5 10 15 Glu Lys Val Lys Ala Tyr Leu Ser
Leu His Pro Gln Val Leu Asp Glu 20 25 30 Phe Val Ser Glu Ser Val
Ser Ala Glu Thr Val Glu Lys Trp Leu Lys 35 40 45 Arg Lys Asn Asn
Lys Ser Glu Asp Glu Ser Ala Pro Lys Glu Val Ser 50 55 60 Arg Tyr
Gln Asp Thr Asn Met Gln Gly Val Val Tyr Glu Leu Asn Ser 65 70 75 80
Tyr Ile Glu Gln Arg Leu Asp Thr Gly Gly Asp Asn Gln Leu Leu Leu 85
90 95 Tyr Glu Leu Ser Ser Ile Ile Lys Ile Ala Thr Lys Ala Asp Gly
Phe 100 105 110 Ala Leu Tyr Phe Leu Gly Glu Cys Asn Asn Ser Leu Cys
Ile Phe Thr 115 120 125 Pro Pro Gly Ile Lys Glu Gly Lys Pro Arg Leu
Ile Pro Ala Gly Pro 130 135 140 Ile Thr Gln Gly Thr Thr Val Ser Ala
Tyr Val Ala Lys Ser Arg Lys 145 150 155 160 Thr Leu Leu Val Glu Asp
Ile Leu Gly Asp Glu Arg Phe Pro Arg Gly 165 170 175 Thr Gly Leu Glu
Ser Gly Thr Arg Ile Gln Ser Val Leu Cys Leu Pro 180 185 190 Ile Val
Thr Ala Ile Gly Asp Leu Ile Gly Ile Leu Glu Leu Tyr Arg 195 200 205
His Trp Gly Lys Glu Ala Phe Cys Leu Ser His Gln Glu Val Ala Thr 210
215 220 Ala Asn Leu Ala Trp Ala Ser Val Ala Ile His Gln Val Gln Val
Cys 225 230 235 240 Arg Gly Leu Ala Lys Gln Thr Glu Leu Asn Asp Phe
Leu Leu Asp Val 245 250 255 Ser Lys Thr Tyr Phe Asp Asn Ile Val Ala
Ile Asp Ser Leu Leu Glu 260 265 270 His Ile Met Ile Tyr Ala Lys Asn
Leu Val Asn Ala Asp Arg Cys Ala 275 280 285 Leu Phe Gln Val Asp His
Lys Asn Lys Glu Leu Tyr Ser Asp Leu Phe 290 295 300 Asp Ile Gly Glu
Glu Lys Glu Gly Lys Pro Val Phe Lys Lys Thr Lys 305 310 315 320 Glu
Ile Arg Phe Ser Ile Glu Lys Gly Ile Ala Gly Gln Val Ala Arg 325 330
335 Thr Gly Glu Val Leu Asn Ile Pro Asp Ala Tyr Ala Asp Pro Arg Phe
340 345 350 Asn Arg Glu Val Asp Leu Tyr Thr Gly Tyr Thr Thr Arg Asn
Ile Leu 355 360 365 Cys Met Pro Ile Val Ser Arg Gly Ser Val Ile Gly
Val Val Gln Met 370 375 380 Val Asn Lys Ile Ser Gly Ser Ala Phe Ser
Lys Thr Asp Glu Asn Asn 385 390 395 400 Phe Lys Met Phe Ala Val Phe
Cys Ala Leu Ala Leu His Cys Ala Asn 405 410 415 Met Tyr His Arg Ile
Arg His Ser Glu Cys Ile Tyr Arg Val Thr Met 420 425 430 Glu Lys Leu
Ser Tyr His Ser Ile Cys Thr Ser Glu Glu Trp Gln Gly 435 440 445 Leu
Met Gln Phe Thr Leu Pro Val Arg Leu Cys Lys Glu Ile Glu Leu 450 455
460 Phe His Phe Asp Ile Gly Pro Phe Glu Asn Met Trp Pro Gly Ile Phe
465 470 475 480 Val Tyr Met Val His Arg Ser Cys Gly Thr Ser Cys Phe
Glu Leu Glu 485 490 495 Lys Leu Cys Arg Phe Ile Met Ser Val Lys Lys
Asn Tyr Arg Arg Val 500 505 510 Pro Tyr His Asn Trp Lys His Ala Val
Thr Val Ala His Cys Met Tyr 515 520 525 Ala Ile Leu Gln Asn Asn His
Thr Leu Phe Thr Asp Leu Glu Arg Lys 530 535 540 Gly Leu Leu Ile Ala
Cys Leu Cys His Asp Leu Asp His Arg Gly Phe 545 550 555 560 Ser Asn
Ser Tyr Leu Gln Lys Phe Asp His Pro Leu Ala Ala Leu Tyr 565 570 575
Ser Thr Ser Thr Met Glu Gln His His Phe Ser Gln Thr Val Ser Ile 580
585 590 Leu Gln Leu Glu Gly His Asn Ile Phe Ser Thr Leu Ser Ser Ser
Glu 595 600 605 Tyr Glu Gln Val Leu Glu Ile Ile Arg Lys Ala Ile Ile
Ala Thr Asp 610 615 620 Leu Ala Leu Tyr Phe Gly Asn Arg Lys Gln Leu
Glu Glu Met Tyr Gln 625 630 635 640 Thr Gly Ser Leu Asn Leu Asn Asn
Gln Ser His Arg Asp Arg Val Ile 645 650 655 Gly Leu Met Met Thr Ala
Cys Asp Leu Cys Ser Val Thr Lys Leu Trp 660 665 670 Pro Val Thr Lys
Leu Thr Ala Asn Asp Ile Tyr Ala Glu Phe Trp Ala 675 680 685 Glu Gly
Asp Glu Met Lys Lys Leu Gly Ile Gln Pro Ile Pro Met Met 690 695 700
Asp Arg Asp Lys Lys Asp Glu Val Pro Gln Gly Gln Leu Gly Phe Tyr 705
710 715 720 Asn Ala Val Ala Ile Pro Cys Tyr Thr Thr Leu Thr Gln Ile
Leu Pro 725 730 735 Pro Thr Glu Pro Leu Leu Lys Ala Cys Arg Asp Asn
Leu Ser Gln Trp 740 745 750 Glu Lys Val Ile Arg Gly Glu Glu Thr Ala
Thr Trp Ile Ser Ser Pro 755 760 765 Ser Val Ala Gln Lys Ala Ala Ala
Ser Glu Asp 770 775 2 4381 DNA Homo sapiens CDS (67)...(2403) 2
catccacaga gatgttacag ttgaagagat gggggtagag aagactttga aggaaaagaa
60 tgtaga atg agg ata gaa gag agg aaa tcc caa cat tta aca ggt ttg
108 Met Arg Ile Glu Glu Arg Lys Ser Gln His Leu Thr Gly Leu 1 5 10
aca gat gaa aaa gtg aag gca tat ctt tct ctt cac ccc cag gta tta 156
Thr Asp Glu Lys Val Lys Ala Tyr Leu Ser Leu His Pro Gln Val Leu 15
20 25 30 gat gaa ttt gta tct gaa agt gtt agt gca gag aca gta gag
aaa tgg 204 Asp Glu Phe Val Ser Glu Ser Val Ser Ala Glu Thr Val Glu
Lys Trp 35 40 45 ctg aag agg aag aac aac aaa tca gaa gat gaa tcg
gct cct aag gaa 252 Leu Lys Arg Lys Asn Asn Lys Ser Glu Asp Glu Ser
Ala Pro Lys Glu 50 55 60 gtc agc agg tac caa gat acg aat atg cag
gga gtt gta tat gaa cta 300 Val Ser Arg Tyr Gln Asp Thr Asn Met Gln
Gly Val Val Tyr Glu Leu 65 70 75 aac agc tat ata gaa caa cgg ttg
gac aca gga gga gac aac cag cta 348 Asn Ser Tyr Ile Glu Gln Arg Leu
Asp Thr Gly Gly Asp Asn Gln Leu 80 85 90 ctc ctc tat gaa ctg agc
agc atc att aaa ata gcc aca aaa gcc gat 396 Leu Leu Tyr Glu Leu Ser
Ser Ile Ile Lys Ile Ala Thr Lys Ala Asp 95 100 105 110 gga ttt gca
ctg tat ttc ctt gga gag tgc aat aat agc ctg tgt ata 444 Gly Phe Ala
Leu Tyr Phe Leu Gly Glu Cys Asn Asn Ser Leu Cys Ile 115 120 125 ttc
acg cca cct ggg ata aag gaa gga aaa ccc cgc ctc atc cct gct 492 Phe
Thr Pro Pro Gly Ile Lys Glu Gly Lys Pro Arg Leu Ile Pro Ala 130 135
140 ggg ccc atc act cag ggc acc acc gtc tct gct tat gtg gcc aag tcc
540 Gly Pro Ile Thr Gln Gly Thr Thr Val Ser Ala Tyr Val Ala Lys Ser
145 150 155 agg aaa aca ctg cta gta gaa gac atc ctt gga gat gaa cga
ttt cca 588 Arg Lys Thr Leu Leu Val Glu Asp Ile Leu Gly Asp Glu Arg
Phe Pro 160 165 170 aga ggt act gga ctg gaa tca ggg act cgt atc cag
tct gtt ctt tgc 636 Arg Gly Thr Gly Leu Glu Ser Gly Thr Arg Ile Gln
Ser Val Leu Cys 175 180 185 190 tta cca att gtc act gca att ggt gac
ttg att ggt att ctc gag ctg 684 Leu Pro Ile Val Thr Ala Ile Gly Asp
Leu Ile Gly Ile Leu Glu Leu 195 200 205 tat cgg cac tgg ggc aaa gaa
gcc ttc tgt ctt agt cac cag gag gtt 732 Tyr Arg His Trp Gly Lys Glu
Ala Phe Cys Leu Ser His Gln Glu Val 210 215 220 gca aca gca aat ctt
gcc tgg gct tca gta gca ata cat cag gtg cag 780 Ala Thr Ala Asn Leu
Ala Trp Ala Ser Val Ala Ile His Gln Val Gln 225 230 235 gta tgc aga
ggc ctt gcc aaa cag aca gaa ttg aat gac ttc cta ctc 828 Val Cys Arg
Gly Leu Ala Lys Gln Thr Glu Leu Asn Asp Phe Leu Leu 240 245 250 gac
gta tca aaa aca tat ttt gat aac ata gtt gca ata gat tct cta 876 Asp
Val Ser Lys Thr Tyr Phe Asp Asn Ile Val Ala Ile Asp Ser Leu 255 260
265 270 ctt gaa cac ata atg ata tat gca aaa aac ctg gtg aat gcc gat
cgt 924 Leu Glu His Ile Met Ile Tyr Ala Lys Asn Leu Val Asn Ala Asp
Arg 275 280 285 tgt gca ctt ttc cag gtg gac cat aag aac aag gag tta
tat tca gac 972 Cys Ala Leu Phe Gln Val Asp His Lys Asn Lys Glu Leu
Tyr Ser Asp 290 295 300 ctt ttt gat att gga gag gaa aag gaa gga aaa
cct gtc ttc aag aag 1020 Leu Phe Asp Ile Gly Glu Glu Lys Glu Gly
Lys Pro Val Phe Lys Lys 305 310 315 acc aaa gag ata aga ttt tca att
gag aaa gga att gct ggc caa gta 1068 Thr Lys Glu Ile Arg Phe Ser
Ile Glu Lys Gly Ile Ala Gly Gln Val 320 325 330 gca aga aca ggg gaa
gtc ctg aac att cca gat gcc tat gca gac cca 1116 Ala Arg Thr Gly
Glu Val Leu Asn Ile Pro Asp Ala Tyr Ala Asp Pro 335 340 345 350 cgc
ttt aac aga gaa gta gac ttg tac aca ggc tac acc acg cgg aac 1164
Arg Phe Asn Arg Glu Val Asp Leu Tyr Thr Gly Tyr Thr Thr Arg Asn 355
360 365 atc ctg tgc atg ccc atc gtc agc cga ggc agc gtg ata ggt gtg
gtg 1212 Ile Leu Cys Met Pro Ile Val Ser Arg Gly Ser Val Ile Gly
Val Val 370 375 380 cag atg gtc aac aaa atc agt ggc agt gcc ttc tct
aaa aca gat gaa 1260 Gln Met Val Asn Lys Ile Ser Gly Ser Ala Phe
Ser Lys Thr Asp Glu 385 390 395 aac aac ttc aaa atg ttt gcc gtc ttt
tgt gct tta gcc tta cac tgt 1308 Asn Asn Phe Lys Met Phe Ala Val
Phe Cys Ala Leu Ala Leu His Cys 400 405 410 gct aat atg tat cat aga
att cgc cac tca gag tgc att tac cgg gta 1356 Ala Asn Met Tyr His
Arg Ile Arg His Ser Glu Cys Ile Tyr Arg Val 415 420 425 430 acg atg
gaa aag ctg tcc tac cat agc att tgt act tca gaa gag tgg 1404 Thr
Met Glu Lys Leu Ser Tyr His Ser Ile Cys Thr Ser Glu Glu Trp 435 440
445 caa ggt ctc atg caa ttc acc ctt ccc gtg cgt ctc tgc aaa gaa att
1452 Gln Gly Leu Met Gln Phe Thr Leu Pro Val Arg Leu Cys Lys Glu
Ile 450 455 460 gaa tta ttc cac ttt gac att ggt cct ttt gaa aac atg
tgg cct gga 1500 Glu Leu Phe His Phe Asp Ile Gly Pro Phe Glu Asn
Met Trp Pro Gly 465 470 475 att ttt gtc tac atg gtt cat cgg tcc tgt
ggg aca tcc tgc ttt gag 1548 Ile Phe Val Tyr Met Val His Arg Ser
Cys Gly Thr Ser Cys Phe Glu 480 485 490 ctt gaa aag ttg tgt cgt ttt
att atg tct gtg aag aag aac tat cgg 1596 Leu Glu Lys Leu Cys Arg
Phe Ile Met Ser Val Lys Lys Asn Tyr Arg 495 500 505 510 cgg gtt cct
tat cac aac tgg aag cat gcg gtc act gta gca cac tgc 1644 Arg Val
Pro Tyr His Asn Trp Lys His Ala Val Thr Val Ala His Cys 515 520 525
atg tat gcc ata ctt cag aac aat cac acg ctt ttc aca gac ctt gag
1692 Met Tyr Ala Ile Leu Gln Asn Asn His Thr Leu Phe Thr Asp Leu
Glu 530 535 540 cgc aaa gga ctg ctg att gcg tgt ctg tgt cat gac ctg
gac cac agg 1740 Arg Lys Gly Leu Leu Ile Ala Cys Leu Cys His Asp
Leu Asp His Arg 545 550 555 ggc ttc agt aac agc tac ctg cag aag ttc
gac cac cct ctg gcc gct 1788 Gly Phe Ser Asn Ser Tyr Leu Gln Lys
Phe Asp His Pro Leu Ala Ala 560 565 570 ctc tac tcc act tcc acc atg
gag cag cac cac ttc tcc cag act gtg 1836 Leu Tyr Ser Thr Ser Thr
Met Glu Gln His His Phe Ser Gln Thr Val 575 580 585 590 tcc atc ctc
cag ttg gaa ggg cac aat atc ttc tcc act ctg agc tcc 1884 Ser Ile
Leu Gln Leu Glu Gly His Asn Ile Phe Ser Thr Leu Ser Ser 595 600 605
agt gaa tat gag cag gtg ctt gag atc atc cgc aaa gcc atc att gcc
1932 Ser Glu Tyr Glu Gln Val Leu Glu Ile Ile Arg Lys Ala Ile Ile
Ala 610 615 620 aca gac ctt gct tta tac ttt gga aac agg aag cag ttg
gaa gag atg 1980 Thr Asp Leu Ala Leu Tyr Phe Gly Asn Arg Lys Gln
Leu Glu Glu Met 625 630 635 tac cag acc gga tca cta aac ctt aat aat
caa tca cat aga gac cgt 2028 Tyr Gln Thr Gly Ser Leu Asn Leu Asn
Asn Gln Ser His Arg Asp Arg 640 645 650 gta att ggt ttg atg atg act
gcc tgt gac ctt tgt tct gtg aca aaa 2076 Val Ile Gly Leu Met Met
Thr Ala Cys Asp Leu Cys Ser Val Thr Lys 655 660 665 670 ctg tgg ccc
gtt aca aaa ttg acg gca aat gat ata tat gca gaa ttc 2124 Leu Trp
Pro Val Thr Lys Leu Thr Ala Asn Asp Ile Tyr Ala Glu Phe 675 680 685
tgg gct gag ggt gat gaa atg aag aaa ttg gga ata cag cct att cct
2172 Trp Ala Glu Gly Asp Glu Met Lys Lys Leu Gly Ile Gln Pro Ile
Pro 690 695 700 atg atg gac aga gac aag aag gat gaa gtc ccc caa ggc
cag ctt ggg 2220 Met Met Asp Arg Asp Lys Lys Asp Glu Val Pro Gln
Gly Gln Leu Gly 705 710 715 ttc tac aat gcc gtg gcc att ccc tgc tat
aca acc ctt acc cag atc 2268 Phe Tyr Asn Ala Val Ala Ile Pro Cys
Tyr Thr Thr Leu Thr Gln Ile 720 725 730 ctc cct ccc acg gag cct ctt
ctg aaa gca tgc agg gat aat ctc agt 2316 Leu Pro Pro Thr Glu Pro
Leu Leu Lys Ala Cys Arg Asp Asn Leu Ser 735 740 745 750 cag tgg gag
aag gtg att cga ggg gag gag act gca acc tgg att tca 2364 Gln Trp
Glu Lys Val Ile Arg Gly Glu Glu Thr Ala Thr Trp Ile Ser 755 760 765
tcc cca tcc gtg gct cag aag gca gct gca tct gaa gat tgagcactgg 2413
Ser Pro Ser Val Ala Gln Lys Ala Ala Ala Ser Glu Asp 770 775
tcaccctgac acgctgtccc acctacagat cctcatcttg cttctttgac attcttttcc
2473 tttttttggg gggggtgggg ggaacctgca cctggtaact ggggtgcaaa
cctcttcaag 2533 aaggtaacat caaataaata agtcaagcag aggacttcct
gccaatctct tctgtgaggc 2593 atcatagaca ctgagcaacc aggaccaccc
ccacgttcag aaatcagctg gccaagtgac 2653 tccatttgac ttgcaaacca
gccttttcta ataggctaat attgctgagg ccttaaagga 2713 aatggacaaa
aattatccag aaggggtact tttccattgt atctttctaa taagggttta 2773
aaatggtact attatggtat tgtacttggg ctttaacatc aatgttgctt tgatgttgtt
2833 ggatataaat aggaattttt acacattact attgtgaatg gtgaatgttc
atgtatgacc 2893 tacttgtaat taacttgagt tgtagtccac agcctcagga
caaatgtcgt tgaggttaca 2953 gagtaagaaa tgatggcaaa acgtcaaact
cttatttcag agcttcatga atttagttag 3013 actaaacata attctttaag
ttcaacctaa agggctgaga tcaataaatt taacactaga 3073 cgaagtagac
ttcctgtctt tttgagaaga gatgaggtat atgttacaat aaatctcaga 3133
acttcaagta gcagttcaaa agatgtcagt ttttaaaatt gtttttgttg ttgtcttggc
3193 agttttactg aaccctttgc ataaagaaca aaataaaagc tcggcattgt
aattttttta 3253 atggacaagt cttatggata cgaagggtac atttttcata
atgattcctt tatattttca 3313 ctttgtgtca ttgcagaatt ttagactctc
attcacaatg aaaagtttat tttaaacatt 3373 gtttaattaa aataccatac
agttctcttt taaacatcaa accataaaaa gtgtattttg 3433 taattttact
ctgacctgcc gcagtcacct ctcacttatc tcttccacgt actgcacggt 3493
cgtatttcat gagctttctg tccatagcac agaaacagag cagaaagtag tacaatcatg
3553 ttggaccttc tttctgttct ctttactctt ctcacagatc agatcactcc
atagaagcct 3613 gtgggtttcg atggtttctt ctatacacct ttttggttga
ccagtattac tatacaatgt 3673 aagtgtttta aaaaatacga aagtaatact
ctgcacccct tcctacaaag atgataaagc 3733 agtcacttct ggcgcatttt
aataatttaa agatttttag tgcaatggca cggtaacctc 3793 caaacctgaa
ttagacagag actcactcag gaagtgacag gcccatcata tcaaataact 3853
tattcacttt tcatgtggca ggaaactgga atatcgcttt taataaaatg gaaaaatatg
3913 cttctacata tttaccacca taggcgtttt gttcatatga gcctggtttg
tgcaaaatta 3973 aatcagaggc ttctacaaca tggtttattt atgttgtagc
aaagttggct ctacataaac 4033 attgttctta ttttaaaatt aacactatgt
gttcagtttt cttgtgggct tctgaaagtt 4093 gccatcttcc ctccgtggag
ctccatttgc tattttcatt atacactatg aggtaaaatg 4153 taataacaaa
agagagagaa gtaccactgt ggctagatat atacacacac atatatatat 4213
ggatggatgt aatatatgta gaacacacac atagatgtat ataggataca cactcatgta
4273 tgtaaacgta tacatatgtg tatatatgat acatacacat acacacacac
gagagacaga 4333 aggaaagaga ggaagagaga agcaaacatg taggaaaaaa
tataaatc 4381
* * * * *
References