Note: Descriptions are shown in the official language in which they were submitted.
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TREATMENT AND PREVENTION OF VASCULAR DISEASE
BACKGROUND OF THE INVENTION
The vascular endothelium constitutes a major organ
that functions as a regulator of blood coagulation,
inflammation, and in the exchange of fluids and mediators
between the intravascular compartment and parenchyma
tissues. As such, the proper function of the endothelium is
critical to overall homeostasis. A dysfunction of the
endothelium resulting from an alteration in the expression
of important surface molecules, can result in coagulation
defects, local and systemic vascular inflammation, and
enhancement in the progression and rupture of
atherosclerotic plaque. These effects can further result in
conditions including myocardial infarction, deep venous
thrombosis, disseminated intravascular thrombosis, and
stroke. Certain cell surface proteins are altered in
response to a vascular injury or insult and can be used as
markers of a dysfunctional endothelium. Two such factors
are plasminogen activator inhibitor 1 (PAI-1) and
thrombomodulin (TM)
PAI-1 plays a critical role in the fibrinolytic
system by reducing the endogenous ability to remove fibrin
by inhibiting plasminogen activators such as tissue type
plasminogen activator (tPA). Studies have documented that
elevations of PAI-1 are associated with increased risk of
deep venous thrombosis. Further, elevations in PAI-1 are
found in patients suffering from myocardial infarction and
septicemia. Because impaired fibrinolytic capacity is
associated with increased cardiovascular risk, lowering PAI-
1 should result in lowered risk.
While PAI-1 can be produced in a variety of
tissues, substantial levels are secreted by the vascular
endothelial cell. Since PAI-1 can be increased in
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endothelial cells in response to certain stimuli, including
cytokines, it contributes to dysfunction of the endothelium
and the attendant problems.
TM plays a critical role in maintaining vessel
anticoagulant activity as a cofactor for thrombin-catalyzed
activation of protein C, the major endogenous antithrombotic
factor. Like PAI-1, the surface anticoagulant responses can
be impaired in states of endothelial dysfunction. In fact,
TM levels become suppressed in cytokine activated
endothelium.
Members of the transforming growth factor beta
(TGF~i) superfamily are involved in many physiological
activities including development, tissue repair, hormone
regulation, bone formation, and cell growth and
differentiation. Recent work has identified a family of
related gene products called "Smads" that function
downstream of the receptors of the TGF(3 family. Upon
activation, several of the Smads translocate directly to the
nucleus where they activate transcription. The Smads have
been implicated in the regulation of cell growth and
proliferation, development and differentiation of cell
types, and in endothelial cell response to shear stress.
Smadl is a bone morphogenetic protein (BMP) signal
transducer, Smad2 and Smad3 are TGF(3 and activin signal
transducers, and Smad4 (also known as DPC4) is a tumor
suppressor that functions as a mediator of TGF(3 signaling.
Smad6 (SEQ ID N0:2) and Smad7 {SEQ ID N0:4) were
initially discovered in endothelial cells to be implicated
in cardiovascular disease (US Pat. No. 5,834,248 and WO
96/24604, each entirely incorporated by reference herein).
Smad7 directly interferes with TGF(3 mediated activation of
Smad2 by preventing phosphorylation, interaction with Smad4,
and nuclear accumulation.
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Accordingly, there is a need to provide molecules,
such as polypeptides, nucleic acids, and/or organic
compounds that modulate expression, or at least one activity
of, proteins such as Smad6 and Smad7, which molecules can be
used to treat or affect cells, tissues, animals (e. g.,
humans) for various diseases or pathologies affected by such
proteins.
SiJN~ARY OF THE INVENTION
Since the local control of PAI-1 and TM at the
endothelial cell/plasma interface can play a major role in
many pathological processes, use of Smad6 and Smad7
polypeptides or agents that modulate their expression and/or
~5 activity in the endothelium can be used to treat conditions
such as, but not limited to, sepsis, injuries involving
major tissue damage and trauma, systemic inflammatory
response syndrome, sepsis syndrome, septic shock and
multiple organ dysfunction syndrome (including DIC), as well
as myocardial infarction, deep venous thrombosis,
disseminated intravascular thrombosis, atherosclerotic
plaque rupture, and associated sequela. Likewise, Smad6 and
Smad7 polypeptides or other molecules that modulate their
expression and/or activity can be used to prevent stroke.
Further, because of the critical role of fibrin in tumor
cell biology, Smad6 and Smad7 polypeptides or molecules that
modulate their expression and/or activity can be used as
anti-metastatic agents.
Moreover, the Smad6 polypeptide has been
determined to increase expression of TGF(3. Smad6-induced
TGF~i expression can be inhibited by inhibitors of protein
kinase C (PKC).
The current invention provides methods for treating or
preventing at least one aspect of at least one vascular
disease; the method comprising administering to a patient in
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need of such treatment, a compound that modulates at least
one TGFj3 regulated activity. Preferred TGF(3 regulatable
activities include but are not limited to an induction of
plasminogen activator inhibitor-1 expression, an increase in
plasminogen activator inhibitor-1 secretion, a suppression
of thrombomodulin activity, an increase in TGF(3 secretion,
and a decrease in TGF~i secretion.
Further, the current invention provides in one
aspect a method for treating or preventing a disease in
1o which TGF(3 is responsible for inducing cellular effects that
lead to at least one disease state; the method comprising
administering to a patient in need of such treatment, a
compound that modulates expression of, or the activity of
the protein product encoded by a nucleic acid molecule
selected from the group consisting of SEQ ID NO:1 (encoding
at least one Smad6 protein), SEQ ID N0:3 (encoding at least
one Smad7 protein), and a nucleic acid molecule that is
complementary to SEQ ID NO:1 or 3.
The current invention also provides methods for
identifying compounds that modulate anticoagulant and
fibrinolytic functions in the vasculature endothelium; the
method comprising contacting at least one cell or an
organism with a compound which modulates expression or
activity of PAI-I and TM.
As part of the current invention, there is also
provided methods for identifying compounds, such as
polypeptides or organic molecules, that modulate the
expression or activity of Smad6 or Smad7 (e.g., proteins or
polypeptides encoded by SEQ ID NO:1 and 2, respectively),
which in turn modulate expression or activity of
anticoagulant and fibrinolytic functions, namely PAI-I and
TM, in the vasculature endothelium.
The present invention also provides a method for
modulating TGF~i regulatable activities comprising
administering to a patient in need of such treatment, a
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protein product encoded by a nucleic acid molecule having at
least 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99 or 100% identity to a member selected
from the group consisting of at least 30 nucleotides of at
least one of SEQ ID NO:1, SEQ ID N0:3, and a nucleic acid
molecule that is complementary to SEQ ID N0:1 or SEQ ID
No:3. Preferred protein products for use in the current
invention include SEQ ID N0:2 and SEQ ID N0:4. A nucleic
acid molecule having at least 70% identity to the specified
sequences are preferred for use in the present invention;
identity of at least 95% is especially preferred. Preferred
TGF~3 regulatable activities include but are not limited to
an induction of plasminogen activator inhibitor-1
expression, an increase in plasminogen activator inhibitor-1
secretion, a suppression of thrombomodulin activity, an
increase in TGFj3 secretion, and a decrease in TGF(3
secretion.
The present invention also provides a method for
modulating TGF~i regulatable activities comprising
administering to a cell or cells, a protein product encoded
by a nucleic acid molecule having at least 70, 75, 80, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100% identity to a member selected from the group consisting
of at least 30 nucleotides of at least one of SEQ ID NO:1,
SEQ ID N0:3, and a nucleic acid molecule that is
complementary to SEQ ID N0:1 or SEQ ID N0:3. Preferred
protein products for use in the current invention include
SEQ ID N0:2 and SEQ ID N0:4. A nucleic acid molecule having
at least 70% identity to the specified sequences are
preferred for use in the present invention; identity of at
least 95% is especially preferred. Preferred TGF(3
regulatable activities include but are not limited to an
induction of plasminogen activator inhibitor-1 expression,
an increase in plasminogen activator inhibitor-1 secretion,
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a suppression of thrombomodulin activity, an increase in
TGF(3 secretion, and a decrease in TGF~ secretion.
In another embodiment the present invention
relates to a pharmaceutical formulation comprising as an
active ingredient a Smad6 or Smad7 polypeptide, associated
with one or more pharmaceutically acceptable carriers,
excipients, or diluents thereof. Preferred protein products
for use in the current invention include SEQ ID N0:2 and SEQ
ID N0:4.
The current invention also provides a method for
modulating TGF~i regulatable activities comprising
administering to at least one cell, an organism, or a
patient in need of such treatment, an antisense nucleic acid
molecule having a nucleotide sequence complementary to a
contiguous sequence of mRNA transcribed from a gene selected
from the group consisting of SEQ ID NO:1 and SEQ ID N0:3,
wherein said antisense nucleic acid molecule hybridizes to
said contiguous sequence such that translation of said mRNA
is inhibited. It is preferred if the contiguous sequence
includes at least fifteen nucleotides. Preferred nucleic
acid molecules for use in the current invention include SEQ
ID N0:5 and SEQ ID N0:7. Preferred TGF~i regulatable
activities include but are not limited to an induction of
plasminogen activator inhibitor-1 expression, an increase in
plasminogen activator inhibitor-1 secretion, a suppression
of thrombomodulin activity, an increase in TGF(3 secretion,
and a decrease in TGF(3 secretion.
The invention also provides methods for the
identification of compounds that modulate a TGF~i regulatable
activity comprising administering to at least one cell or an
organism a compound that modulates expression of, or the
activity of the protein product of, a nucleic acid molecule
selected from the group consisting of SEQ ID N0:1, SEQ ID
N0:3, and fragments thereof. Preferred TGF(3 regulatable
activities include but are not limited to an induction of
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plasminogen activator inhibitor-1 expression, an increase in
plasminogen activator inhibitor-1 secretion, a suppression
of thrombomodulin activity, an increase in TGF(3 secretion,
and a decrease in TGF(3 secretion.
The present invention also provides methods for
the identification of compounds that modulate a TGF(3
regulatable activity comprising administering to at least
one cell, or an organism a compound that results in an
induction of plasminogen activator inhibitor-1 expression,
an increase in plasminogen activator inhibitor-1 secretion,
a suppression of thrombomodulin activity, an increase in
TGF~i secretion, or a decrease in TGF(3 secretion.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
The terms ~complementary" or ~~complementarity~ as
used herein refer to the capacity of purine and pyrimidine
nucleotides to associate through hydrogen bonding to form
double stranded nucleic acid molecules. The following base
pairs are related by complementarity: guanine and cytosine;
adenine and thymine; and adenine and uracil. As used herein,
"complementary" means that the aforementioned relationship
applies to substantially all base pairs comprising two
single-stranded nucleic acid molecules over the entire
length of said molecules. "Partially complementary" refers
to the aforementioned relationship in which one of two
single-stranded nucleic acid molecules is shorter in length
than the other such that a portion of one of the molecules
remains single-stranded.
The term "conservative substitution" or
"conservative amino acid substitution" refers to a
replacement of one or more amino acid residues) in a parent
polypeptide as stipulated by Table 1.
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The term "fragment thereof" refers to a fragment,
piece, or sub-region of a nucleic acid or polypeptide
molecule whose sequence is disclosed herein, such that the
fragment comprises 5 or more amino acids, or 10 or more
nucleotides that are contiguous in the parent polypeptide or
nucleic acid molecule. "Fragment thereof" may or may not
retain biological activity. A fragment of a polypeptide
disclosed herein could be used as an antigen to raise a
specific antibody against the parent polypeptide molecule.
With reference to a nucleic acid molecule, "fragment
thereof" refers to 10 or more contiguous nucleotides,
derived from a parent nucleic acid. The term also
encompasses the complementary sequence. For example if the
fragment entails the sequence 5'-AGCTAG-3', then "fragment
thereof" would also include the complementary sequence, 3'-
TCGATC-5'.
The term "functional fragment" or "functionally
equivalent fragment", as used herein, refers to a region, or
fragment of a full length polypeptide, or sequence of amino
acids that, for example, comprises an active site, or any
other motif, relating to biological function. Functional
fragments are capable of providing a substantially similar
biological activity as a polypeptide disclosed herein, in
vivo or in vitro, viz. the capacity to modulate cell
proliferation. Functional fragments may be produced by
cloning technology, or as the natural products of
alternative splicing mechanisms.
The term "functionally related" is used herein to
describe polypeptides that are related to the Smad6 or Smad7
polypeptides used in the present invention, said
functionally related polypeptides constituting modifications
of said Smad6 or Smad7 polypeptides, in which conservative
amino acid changes are present as natural polymorphic
variants of the polypeptides disclosed herein. Conservative
amino acid substitutions and modifications may also be
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engineered using recombinant DNA techniques. Functionally
related polypeptides retain the biological activity of Smad6
or Smad7, such as the ability to inhibit cell proliferation,
and/or tumor growth in vivo or in vitro.
The symbol "N" in a nucleic acid sequence refers
to adenine ("A"), guanine ("G"), cytosine ("C"), thymine
(T"), or uracil ("U"); "Z" designates an unknown amino acid
residue.
The term "plasmid" refers to an extrachromosomal
1o genetic element. The plasmids disclosed herein are
commercially available, publicly available on an
unrestricted basis, or can be constructed from readily
available plasmids in accordance with published procedures.
A "primer" is a nucleic acid fragment which
functions as an initiating substrate for enzymatic or
synthetic elongation of, for example, a nucleic acid
molecule.
The term "promoter" refers to a nucleic acid
sequence that directs transcription, for example, of DNA to
RNA. An inducible promoter is one that is regulatable by
environmental signals, such as carbon source, heat, or metal
ions, for example. A constitutive promoter generally
operates at a constant level and is not regulatable.
The terms "protein" and "polypeptide" are used
interchangeably herein and are intended to mean a biopolymer
comprising a plurality of amino acid residues covalently
bound in peptide linkage.
The term "recombinant DNA cloning vector" as used
herein refers to any autonomously replicating agent,
3o including, but not limited to, plasmids and phages,
comprising a DNA molecule to which one or more additional
DNA segments can or have been incorporated.
The term "recombinant DNA expression vector" or
"expression vector" as used herein refers to any recombinant
DNA cloning vector, for example a plasmid or phage, in which
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a promoter and other regulatory elements are present thereby
enabling transcription of an inserted DNA, which may encode
a protein.
The term "substantially pure," used in reference
to a polypeptide, means substantial separation from other
cellular and non-cellular molecules, including other protein
molecules. A substantially pure preparation would be about
at least 85% pure; preferably about at least 95% pure. A
"substantially pure" protein can be prepared by a variety of
techniques, well known to the skilled artisan, including,
for example, the IMAC protein purification method.
The term "vector" as used herein refers to a
nucleic acid compound used for introducing exogenous or
endogenous DNA into host cells. A vector comprises a
nucleotide sequence which may encode one or more protein
molecules. Plasmids, cosmids, viruses, and bacteriophages,
in the natural state or which have undergone recombinant
engineering, are examples of commonly used vectors.
The various restriction enzymes disclosed and
described herein are commercially available and the manner
of use of said enzymes including reaction conditions,
cofactors, and other requirements for activity are well
known to one of ordinary skill in the art. Reaction
conditions for particular enzymes were carried out according
to the manufacturer's recommendation.
In accordance with an aspect of the present
invention, it has been discovered that a nucleic acid
molecule as set forth in SEQ ID NO:1 encodes a polypeptide
(SEQ ID N0:2) that mediates the intracellular signal induced
by the binding of TGF(3 to specific cell surface receptors.
The current invention arises from the discovery
that Smad6 and Smad7 mediate the expression of anticoagulant
and fibrinolytic functions in the vascular endothelium, as
well as modulation of TGF(3 secretion. Specifically, the
over-expression of Smad6 increases the secretion of PAI-1
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and suppresses the expression of TM (measured by the ability
of cells to support thrombin-mediated activation of protein
C) , similar to the direct affect of TGF~i itself .
Conversely, the inhibition of Smad6 by antisense decreases
TGF(3-induced PAI-1 levels/activity and increases TGFj3-
induced suppression of TM. Further, the effects of Smad6 on
these coagulation markers can be antagonized by Smad7.
These results with antisense and over-expression demonstrate
that Smad6 can mimic a TGF~i response in the absence of added
TGF(3, and is required for transducing the signal when TGF(3
is added. In addition, Smad6 acts as a feedback amplifier
of the pathway by up-regulating the secretion of TGF(31 and
TGF(33. Protein kinase C and inhibitors thereof also
modulate the activity of Smad6.
Other effects of inhibiting Smad6 expression and
activity include blocking suppression of thrombomodulin,
which in turn leads to an increase in activated protein C.
It has also been demonstrated by
immunolocalization and in situ hybridization studies of the
vasculature that Smad6 is predominately expressed in
atherosclerotic lesions, but not in the normal endothelium,
whereas Smad7 is predominately expressed in the normal
endothelium but not in atherosclerotic lesions. Strikingly,
Smad6 is also highly over-expressed in diseased heart, where
TGF~3 is known to play a critical role in congestive heart
failure .
Smad7 inhibits type I receptor kinase signaling by
acting as intracellular antagonist of the type I receptor
domain. Smad7 interacts directly with the activated type I
receptor, thereby blocking Smad2 phosphorylation, Smad2
association with Smad4, and nuclear accumulation of the
complex.
Therefore, agents that suppress the level and/or
function of Smad6, or increase the level and/or function of
Smad7 would be useful agents to increase anticoagulant or
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profibrinolytic functions in the vasculature for the
prevention and treatment of myocardial infarction and
peripheral vascular disease, as well as the prevention of
vascular dysfunction and thromboembolytic contributions to
stroke. Further, such agents would be useful in the
prevention and treatment of cardiac hypertrophy and
functional failure. Compounds that modulate expression of,
or the activity of the protein product of, a nucleic acid
molecule molecule selected from the group consisting of SEQ
ID NO:1, SEQ ID N0:3, and fragments or compounds having at
least 90% identity to SEQ ID N0:2, SEQ ID N0:4, or fragments
thereof can be used for the manufacture of a medicament for
the treatment and/or prevention of diseases, including but
not limited to, myocardial infarction, congestive heart
failure, dilated cardiomyopathy, deep venous thrombosis,
disseminated intravascular thrombosis, stroke, sepsis,
injuries involving major tissue damage and trauma, systemic
inflammatory response syndrome, sepsis syndrome, septic
shock, multiple organ dysfunction syndrome (including DIC),
atherosclerotic plaque rupture, and associated sequela
arising therefrom.
Because of the critical importance of PAI-1 and TM in
venous and microvascular functions, such agents would be
particularly useful in the treatment of hypercoagulable
states and disseminated intravascular coagulation associated
with systemic inflammatory responses, sepsis, burns,
obstetrical complications, and trauma.
' For therapeutic use in preventing or treating
vasculature disease an effective amount of SmadS or Smad7
polypeptide is administered to an organism in need thereof
in a dose between about 0.001 and 10,000 ~g/kg body weight.
In practicing the methods contemplated, Smad6 or Smad7 can
be administered in a single daily dose or in multiple doses
per day. The amount per administration will be determined by
the physician and depend on such factors as the nature and
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severity of the disease, and the age and general health of
the patient.
The present invention also provides a
pharmaceutical composition comprising as the active agent a
Smad6 or Smad7 polypeptide or fragment thereof, or a
pharmaceutically acceptable non-toxic salt thereof, and a
pharmaceutically acceptable solid or liquid carrier. For
example, compounds comprising Smad6 or Smad7 can be admixed
with conventional pharmaceutical carriers and excipients,
and used in the form of tablets, capsules, elixirs,
suspensions, syrups, wafers, and the like. The compositions
comprising Smad6 or Smad7 will contain from about 0.1% to
90% by weight of the active compound, and more generally
from about 10% to 30%. The compositions may contain common
carriers and excipients such as corn starch or gelatin,
lactose, sucrose, microcrystalline cellulose, kaolin,
mannitol, dicalcium phosphate, sodium chloride, and alginic
acid. The compounds can be formulated for oral or parenteral
administration.
For intravenous (IV) use, the Smad6 or Smad7
polypeptide is administered in commonly used intravenous
fluids) and administered by infusion. Such fluids, for
example, physiological saline, Ringer's solution or 5%
dextrose solution can be used.
For intramuscular preparations, a sterile formulation,
preferably a suitable soluble salt form of the Smad6 or
Smad7 polypeptide, for example SEQ ID NO: 2 or SEQ ID NO: 4,
such as the hydrochloride salt, can be dissolved and
administered in a pharmaceutical diluent such as pyrogen-
free water (distilled), physiological saline or 5% glucose
solution. A suitable insoluble form of the compound may be
prepared and administered as a suspension in an aqueous base
or a pharmaceutically acceptable oil base, e.g. an ester of
a long chain fatty acid such as ethyl oleate.
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Skilled artisans will recognize that the proteins
used in the present invention can be synthesized by a number
of different methods, such as chemical methods well known in
the art, including solid phase protein synthesis or
recombinant methods. Both methods are described in U.S.
Patent 4,617,189, entirely incorporated herein by reference.
The principles of solid phase chemical synthesis
of polyproteins are well known in the art and may be found
in general texts in the area. See, e.g., H. Dugas and C.
Penney, Bioorganic Chemistry (1981) Springer-Verlag, New
York, 54-92. For example, proteins may be synthesized by
solid-phase methodology utilizing an Applied Biosystems 430A
protein synthesizer (Applied Biosystems, Foster City, CA)
and synthesis cycles supplied by Applied Biosystems.
The proteins used in the present invention can
also be produced by recombinant DNA methods using a cloned
or other Smad6 or Smad7 nucleic acid template. Recombinant
methods are preferred if a high yield is desired. Expression
of a Smad6 or Smad7 gene can be carried out in a variety of
suitable host cells, well known to those skilled in the art.
For example, the Smad6 or Smad7 gene or fragments thereof
(e. g. SEQ ID NOS: 1 or 3) can be introduced into host cells
by any suitable means well known to those skilled in the
art. While chromosomal integration of the cloned genes is
possible, it is preferred that the gene or fragment thereof
be cloned into a suitable extra-chromosomally maintained
expression vector so that the coding region of the Smad6 or
Smad7 gene is operably-linked to a constitutive or inducible
promoter.
The basic steps in the recombinant production of a
Smad6 or Smad7 polypeptide are:
a) constructing a natural, synthetic or
semi-synthetic DNA encoding a Smad6 or Smad7
polypeptide;
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b) integrating said DNA into an expression
vector in a manner suitable for expressing
the Smad6 or Smad7 polypeptide, either alone
or as a fusion protein;
c) transforming or otherwise introducing
said vector into an appropriate eucaryotic or
prokaryotic host cell forming a recombinant
host cell,
d) culturing said recombinant host cell in
a manner to express the Smad6 or Smad7
polypeptide; and
e) recovering and substantially purifying
the Smad6 or Smad7 polypeptide by any
suitable means, well known to those skilled
in the art.
Procaryotes may be employed in the production of
recombinant Smad6 or Smad7 polypeptide. For example, the
Escherichia coli K12 strain 294 (ATCC No. 31846) is
particularly useful for the prokaryotic expression of
foreign proteins. Other strains of E. coli, bacilli such as
Bacillus subtilis, enterobacteriaceae such as Salmonella
typhimuriurrr or Serratia marcescens, various Pseudomonas
species and other bacteria, such as Streptomyces, may also
be employed as host cells in the cloning and expression of
the recombinant proteins of this invention.
Promoter sequences suitable for driving the
expression of genes in procaryotes include ~i-lactamase (e. g.
vector pGX2907 (ATCC 39344) which contains a replicon and X3-
lactamase gene), lactose systems (Chang et al., Nature
(London), 275:615 (1978); Goeddel et al., Nature (London),
281:544 (1979)), alkaline phosphatase, and the tryptophan
(trp) promoter system (vector pATHl (ATCC 37695) which is
designed to facilitate expression of an open reading frame
as a trpE fusion protein under the control of the trp
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promoter). Hybrid promoters such as the tac promoter
(isolatable from plasmid pDR540 (ATCC-37282)) are also
suitable. Still other bacterial promoters, whose nucleotide
sequences are generally known, may be ligated to DNA
encoding the protein of the instant invention, using linkers
or adapters to supply any required restriction sites.
Promoters for use in bacterial systems also will contain a
Shine-Dalgarno sequence operably-linked to the DNA encoding
the desired polyproteins. These examples are illustrative
1o rather than limiting.
The proteins used in this invention may be
synthesized either by direct expression or as a fusion
protein comprising the protein of interest as a
translational fusion with another protein or proteins. For
95 example, a glutathione-S-transferase (GST)-Smad6 or Smad7
fusion protein can be synthesized essentially as described
in Smith & Johnson, Gene, 67, 31, 1988, herein incorporated
by reference. Fusion partners can be removed by enzymatic
or chemical cleavage. It is often observed in the
2o production of certain proteins in recombinant systems that
expression as a fusion protein prolongs the lifespan,
increases the yield of the desired protein, or provides a
convenient means of purifying the protein. This is
particularly relevant when expressing mammalian proteins in
25 procaryotic hosts. A variety of peptidases (e. g.
enterokinase and thrombin) which cleave a polyprotein at
specific sites or digest the proteins from the amino or
carboxy termini (e. g. diaminopeptidase) of the protein chain
are known. Furthermore, particular chemicals (e. g. cyanogen
30 bromide) will cleave a polyprotein chain at specific sites.
The skilled artisan will appreciate the modifications
necessary to the amino acid sequence (and synthetic or semi-
synthetic coding sequence if recombinant means are employed)
to incorporate site-specific internal cleavage sites. See
35 e.g., P. Carter, "Site Specific Proteolysis of Fusion
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Proteins", Chapter 13, in Protein Purification: From
Molecular Mechanisms to Large Scale Processes, American
Chemical Society, Washington, D.C. (1990).
In addition to procaryotes, a variety of amphibian
expression systems such as frog oocytes, and mammalian cell
systems can be used in the production of recombinant Smad6
or Smad7 polypeptides. The choice of a particular host cell
depends to some extent on the particular expression vector
used. Exemplary mammalian host cells suitable for use in
the present invention include HepG-2 (ATCC HB 8065), CV-1
(ATCC CCL 70), LC-MK2 (ATCC CCL 7.1), 3T3 (ATCC CCL 92),
CHO-K1 (ATCC CCL 61), HeLa (ATCC CCL 2), RPMI8226 (ATCC CCL
155), H4IIEC3 (ATCC CCL 1600), C127I (ATCC CCL 1616), HS-
Sultan (ATCC CCL 1884), and BHK-21 (ATCC CCL 10), for
example.
A wide variety of vectors are suitable for
transforming mammalian host cells. For example, the pSV2-
type vectors comprise segments of the simian virus 40 (SV40)
genome required for transcription and polyadenylation. A
large number of plasmid pSV2-type vectors have been
constructed, such as pSV2-gpt, pSV2-neo, pSV2-dhfr, pSV2-
hyg, and pSV2- -globin, in which the SV40 promoter drives
transcription of an inserted gene. These vectors are widely
available from sources such as the American Type Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville,
Maryland, 20852, or the Northern Regional Research
Laboratory (NRRL), 1815 N. University Street, Peoria,
Illinois, 61604.
Promoters suitable for expression in mammalian
3o cells include the SV40 late promoter, promoters from
eukaryotic genes, such as, for example, the estrogen-
inducible chicken ovalbumin gene, the interferon genes, the
glucocorticoid-inducible tyrosine aminotransferase gene, the
thymidine kinase gene promoter, and the promoters of the
major early and late adenovirus genes.
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Plasmid pRSVcat (ATCC 37152) comprises portions of
a long terminal repeat of the Rous Sarcoma virus, a virus
known to infect chickens and other host cells. This long
terminal repeat contains a promoter which is suitable for
use in the vectors of this invention. H. German et al.,
Porch. Nat. Acad. SCI. (USA), 79, 6777 (1982). The plasmid
pMSVi (NRRL B-15929) comprises the long terminal repeats of
the Murine Sarcoma virus, a virus known to infect mouse and
other host cells. The mouse metallothionein promoter has
also been well characterized for use in eukaryotic host
cells and is suitable for use in the present invention.
This promoter is present in the plasmid pdBPV-MMTneo (ATCC
37224) which can serve as the starting material for the
construction of other plasmids.
Transfection of mammalian cells with vectors can
be performed by a plurality of well-known processes
including, but not limited to, protoplast fusion, calcium
phosphate co-precipitation, electroporation and the like.
See, e.g., Maniatis et al., supra.
2o Some viruses also make appropriate vectors.
Examples include the adenoviruses, the adeno-associated
viruses, vaccinia viruses, herpes viruses, baculoviruses,
and roux sarcoma viruses, as described in U.S. Patent
4,775,624, entirely incorporated herein by reference.
Eucaryotic microorganisms such as yeast and other
fungi are also suitable host cells. The yeast Saccharomyces
cerevisiae is the preferred eucaryotic microorganism. Other
yeasts such as Kluyveromyces Iactis and Pichia pastoris are
also suitable. For expression in Saccharomyces, the plasmid
YRp7 (ATCC-40053), for example, may be used. See, e.g., L.
Stinchcomb et al., Nature, 282, 39 (1979); J. Kingsman et
al., Gene, 7, 181 (1979); S. Tschemper et al., Gene, 10, 157
(1980). Plasmid YRp7 contains the TRPl gene which provides
a selectable marker for use in a trpl auxotrophic mutant.
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An expression vector carrying a cloned or isolated
or endogenous Smad6 or Smad7 genomic or coding sequence or
fragment thereof (e.g., SEQ ID NO: 1 or 3) is transformed or
transfected into a suitable host cell using standard
methods. Cells that contain the vector are propagated under
conditions suitable for expression of a recombinant Smad6 or
Smad7 polypeptide. For example, if the recombinant gene or
fragment thereof has been placed under the control of an
inducible promoter, suitable growth conditions would
incorporate the appropriate inducer. The recombinantly-
produced protein may be purified from cellular extracts of
transformed cells by any suitable means.
In a suitable process for protein purification, a
Smad6 or Smad7 gene or fragment thereof is modified at the
5~ end to incorporate several histidine codons. This
modification produces an "histidine tag" at the amino
terminus of the encoded protein, that enables a single- or
few-step protein purification method [i.e. "immobilized
metal ion affinity chromatography" (IMAC)], as described in
U.S. Patent 4,569,794, entirely incorporated by reference.
The IMAC method enables rapid isolation of substantially
pure recombinant Smad6 or Smad7 polypeptide starting from a
crude extract of cells that express a modified recombinant
protein, as described above.
The nucleic acids used in the present invention
(e. g. SEQ ID NOS: 1 and 3) and related nucleic acid
molecules may be produced by chemical synthetic methods.
The synthesis of nucleic acids is well known in the art.
See, e.g., E.L. Brown, R. Belagaje, M.J. Ryan, and H.G.
Khorana, Methods in Enzymology, 68:109-151 (1979). Nucleic
acids, including those disclosed herein, could be generated
using a conventional DNA synthesizing apparatus, such as the
Applied Biosystems Model 380A or 380B DNA synthesizers
(Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster
City, CA 94404) using phosphoramidite chemistry, thereafter
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ligating the fragments so as to reconstitute the entire
gene. Alternatively, phosphotriester chemistry may be
employed to synthesize the nucleic acids of this invention.
(See, e.g., M.J. Gait, ed., Oligonucleotide Synthesis, A
Practical Approach, (1984)).
In an alternative methodology, e.g., polymerase
chain reaction (PCR), the nucleic acid sequences disclosed
and described herein, can be produced from a plurality of
starting materials. For example, starting with an RNA or
1o cDNA preparation (e.g., a cDNA library) derived from a
tissue that expresses a Smad6 or Smad7 gene, suitable
oligonucleotide primers complementary to SEQ ID NO: 1, SEQ
ID NO: 3, or to a sub-region therein, for example, are
prepared as described in U.S. Patent No. 4,889,818, hereby
incorporated by reference. Other suitable protocols for the
PCR are disclosed in PCR Protocols: A Guide to Method and
Applications, Ed. Michael A. Innis et al., Academic Press,
Inc. (1990). Using PCR, any region of the Smad6 or Smad7
genes) can be targeted for amplification such that full or
2o partial length gene sequences may be produced.
The ribonucleic acids used in the present
invention may be prepared using polynucleotide synthetic
methods discussed supra, or they may be prepared
enzymatically, for example, using RNA polymerase to
transcribe a Smad6 or Smad7 DNA template.
The most preferred systems for preparing the
ribonucleic acids used in the present invention employ the
RNA polymerase from the bacteriophage T7 or the
bacteriophage SP6. These RNA polymerases are highly
specific, requiring the insertion of bacteriophage-specific
sequences at the 5~ end of the template to be transcribed.
See, Maniatis et al., supra.
This invention also requires nucleic acids, RNA or
DNA, that are complementary to Smad6 or Smad7 genes or
fragments thereof, for example, SEQ ID NOS: 1 or 3.
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Aspects of the present require recombinant DNA
cloning vectors and expression vectors comprising the
nucleic acids used in the present invention. The preferred
nucleic acid vectors are those which comprise DNA, for
example, SEQ ID NOS: 1 or 3 or a subregion therein.
The skilled artisan understands that choosing the
most appropriate cloning vector or expression vector depends
upon a number of factors including the availability of
restriction enzyme sites, the type of host cell into which
the vector is to be transfected or transformed, the purpose
of the transfection or transformation (e. g., stable
transformation as an extrachromosomal element, or
integration into the host chromosome), the presence or
absence of readily assayable or selectable markers (e. g.,
antibiotic resistance and metabolic markers of one type and
another), and the number of copies of the gene desired in
the host cell.
Vectors suitable to carry the nucleic acids used
in the present invention comprise RNA viruses, DNA viruses,
lytic bacteriophages, lysogenic bacteriophages, stable
bacteriophages, plasmids, viroids, and the like. The most
preferred vectors are plasmids.
When preparing an expression vector the skilled
artisan understands that there are many variables to be
considered, for example, whether to use a constitutive or
inducible promoter. The practitioner also understands that
the amount of nucleic acid or protein to be produced
dictates, in part, the selection of the expression system.
Regarding promoter sequences, inducible promoters are
preferred because they enable high level, regulatable
expression of an operably-linked gene. The skilled artisan
will recognize a number of suitable promoters that respond
to a variety of inducers, for example, carbon source, metal
ions, and heat. Other relevant considerations regarding an
expression vector include whether to include sequences for
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directing the localization of a recombinant protein. For
example, a sequence encoding a signal protein (e.g., SEQ ID
NO: 5) preceding the coding region of a gene is useful for
directing the extra-cellular export of a resulting
polyprotein.
The present invention may require constructing a
recombinant host cell capable of expressing the proteins
used in the invention, said method comprising transforming
or otherwise introducing into a host cell a recombinant DNA
vector that comprises an isolated DNA sequence that encodes
a protein of this invention. A suitable host cell is any
eucaryotic cell that can accomodate high level expression of
an exogenously introduced gene or protein, and that will
incorporate said protein into its membrane structure.
Vectors for expression are those which comprise a Smad6 or
Smad7 gene or fragment thereof, e.g. SEQ ID NO: 1, SEQ ID
NO: 3, or suitable subregion therein. Transformed host
cells may be cultured under conditions well known to skilled
artisans such that a protein used in the present invention
2o is expressed, thereby producing a recombinant Smad6 or Smad7
polypeptide in the recombinant host cell.
Embodiments of a Smad6 and Smad7 DNA sequence are
disclosed herein by SEQ ID NOS: 1 and 3, respectively.
Those skilled in the art will recognize that owing to the
degeneracy of the genetic code, numerous °silent"
substitutions of nucleotide base pairs could be introduced
into the sequences identified herein without altering the
identity of the encoded amino acids) or protein or protein
product.
Amino acids in a Smad6 or Smad7 polypeptide used
in the present invention that are essential for function can
be identified by methods known in the art, such as site-
directed mutagenesis or alanine-scanning mutagenesis
(Cunningham and Wells, Science 244:1081-1085 (1989)). The
latter procedure introduces single alanine mutations at
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every residue in the molecule. The resulting mutant
molecules are then tested for biological activity. Sites
that are critical for ligand-protein binding can also be
identified by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith
et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al.
Science 255:306-312 (1992)).
A Smad6 or Smad7 polypeptide can further comprise
a polypeptide encoded by 4-300 contiguous amino acids of SEQ
ID NO: 2 or SEQ ID NO: 4, or any range or value therein.
Also contemplated by the present invention is the
use of proteins that are functionally related to Smad6 or
Smad7. For example, proteins that are functionally related
to SEQ ID NO: 2 may be produced by conservative amino acid
substitutions, deletions, or insertions, at one or more
amino acid positions within Smad6 or Smad7, in accordance
with Table 1 presented herein.
Modifications of Smad6 or Smad7 polypeptides made
in accordance with Table 1 are generally expected to retain
the biological activity of the parent molecule based on art
recognized substitutability of the amino acids specified in
Table 1 (See e.g. M. Dayhoff, In Atlas of Protein Sequence
and Structure, Vol. 5, Supp. 3, pgs 345-352, 1978). Smad6
functionality is easily tested, for example, in an assay
such as that described in Example 1B herein.
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'ORIGINAL RESIDUE EXEMPLARY SUBSTITUTIONS
ALA SER, THR
ARG LYS
ASN HIS, SER
ASP GLU, ASN
CYS SER
GLN ASN, HIS
GLU ASP, GLU
GLY ALA, SER
HIS ASN, GLN
ILE LEU, VAL, THR
LEU ILE, VAL
LYS ARG, GLN, GLU, THR
MET LEU, ILE, VAL
PHE LEU, TYR
SER THR, ALA, ASN
THR SER, ALA
'TRP ARG, SER
TYR PHE
~VAL ILE, LEU, ALA
PRO
Fragments of the proteins used in the present
invention may be generated by any number of suitable
techniques, including chemical synthesis of any portion of
Smad6 or Smad7 (e. g. SEQ ID NO: 2 or SEQ ID N0:4),
proteolytic digestion of said proteins, or most preferably,
by recombinant DNA mutagenesis techniques, well known to the
skilled artisan. See. e.g. K. Struhl, "Reverse biochemistry:
Methods and applications for synthesizing yeast proteins in
vitro,~~ Meth. Enzymol. 194, 520-535. For example, in a
preferred method, a nested set of deletion mutations are
introduced into a gene encoding SmadS or Smad7 (e.g. SEQ ID
NOS: 1 or 3), or gene fragment thereof such that varying
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amounts of the protein coding region are deleted, either
from the amino terminal end, or from the carboxyl end of the
protein molecule. This method can also be used to create
internal fragments of the intact protein in which both
carboxyl and amino terminal ends are removed. Several
appropriate nucleases can be used to create such deletions,
for example Ba131, or in the case of a single stranded
nucleic acid molecule, mung bean nuclease. For simplicity,
it is preferred that the Smad6 or Smad7 gene be cloned into
a single-stranded cloning vector, such as bacteriophage M13,
or equivalent. If desired, the resulting deletion fragments
can be subcloned into any suitable vector for propagation
and expression in any suitable host cell.
Functional fragments of the Smad6 or Smad7
polypeptide of this invention may be produced as described
above, preferably using cloning techniques to engineer
smaller versions of the Smad6 or Smad7 gene, lacking
sequence from the 5' end, the 3' end, from both ends, or
from an internal site. Smaller fragments of the genes or
gene fragments of this invention can be used as a template
to produce the encoded proteins.
Those skilled in the art will recognize that the
Smad6 or Smad7 gene used in the present invention could be
obtained by a plurality of recombinant DNA techniques
including, for example, hybridization, polymerase chain
reaction (PCR) amplification, or de novo DNA synthesis.(See
e.g., T. Maniatis et al. Molecular Cloning: A Laboratory
Manual, 2d Ed. Chap. 18 (1989)).
Methods for constructing cDNA libraries in a
suitable vector such as a plasmid or phage for propagation
in procaryotic or eucaryotic cells are well known to those
skilled in the art. [See e.g. Maniatis et a1. Supra].
Suitable cloning vectors are well known and are widely
available.
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The Smad6 or Smad7 gene, or fragments thereof, can
be isolated from any tissue in which said gene is expressed.
In one method for gene isolation, mRNA is isolated from a
suitable tissue that expresses Smad6 or Smad7, and first
strand cDNA synthesis is carried out. Second round DNA
synthesis can be carried out for the production of the
second strand. If desired, the double-stranded cDNA can be
cloned into any suitable vector, for example, a plasmid,
thereby forming a cDNA library. Oligonucleotide primers
1o targeted to any suitable region of the sequences disclosed
herein can be used for PCR amplification of Smad6 or Smad7
genes. See e.g. PCR Protocols: A Guide to Method and
Application, Ed. M. Innis et al., Academic Press (1990).
The PCR amplification comprises template DNA, suitable
~5 enzymes, primers, and buffers, and is conveniently carried
out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk,
CT). A positive result is determined by detecting an
appropriately-sized DNA fragment following agarose gel
electrophoresis.
2o The present invention is also further described by
reference to the following examples, which are not intended
to limit the invention, but are to provide more specific,
non-limiting embodiments, which support the full scope of
the present invention as described herein.
EXAMPLE 1
SMAD GENE OVER-EXPRESSION
To elucidate their role on endothelial cell
anticoagulant and profibrinolytic activities, the effects of
Smad6 and Smad7 over-expression on two well-characterized
markers of endothelial cell function, thrombomodulin (TM)
and plasminogen activator inhibitor-1 (PAI-1), were
examined. Both markers are known to be responsive to TGF(3,
with TM being suppressed and PAI-1 being activated.
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The experiments were conducted in two independent
endothelial cell lines, SVHA-1 (an SV40 transformed human
aortic endothelial cell line) and ECV304 (a spontaneously
transformed human umbilical vein endothelial cell line)
(ATCC CRL-1998) with similar results. The cells were
maintained in DMEM/F-12 (3:1), a medium comprised of a 3:1
v/v mixture of Dulbecco~s Modified Eagle s Medium and Ham s
nutrient mixture F-12. The basal medium was supplemented
with 10 nM selenium, 50 E,~M 2-aminoethanol, 20 mM HEPES, 50
~g/ml gentamicin, and 5% fetal bovine serum (FBS).
A. PAI-1 Promoter activity
In order to determine the level of PAI-1 secretion
and promoter activity, the PAI-1 basal promoter driving the
expression of the CAT indicator gene (pOCAT2336) and a TGF(3
hyper-responsive PAI-1/TRE promoter construct (3TP-lux)
driving the expression of the luciferase indicator gene were
used. Smad6/Ha and Smad7/Ha vectors were constructed by
inserting the Smad6 coding sequence into the BamHI/Xhol
sites of HA Tag expression vector pN8e23/3xFlu2 and the
Smad7 coding sequence into the EcoRl/Xhol site of
pN8e23/SxFlu2, a standard expression vectors for epitope
tagging. ECV304 cells were seeded in 6-well plates to 80%
confluence. DNA was transfected at a concentration of 1 ~tg
per well for pOCAT2336 and 3TP-lux and 5 ~g per well for
Smad6 and Samd7 with lipofectin reagent (Gibco/Life
Technologies, Gaithersburg, MD). Expressed CAT protein from
the PAI-1 basal promoter construct was determined using a
CAT ELISA kit (Boehringer Mannheim; Indianapolis, IN).
Chemiluminescence resulting from expression of the
luciferase gene was determined as a measure of the effect on
the PAI-1/TRE promoter. The plates were read kinetically
and data expressed in terms of promoter activity relative to
control (Table 2).
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These experiments demonstrated a Smad6-dependent
induction of the PAI-1 promoter by both the highly TGF(3-
responsive transcriptional element based on the PAI-1
promoter (p3TP-Lux) as well as with the basal PAI-1 promoter
(pPAI-CAT). In particular, Smad6 over-expression resulted
in a higher induction with the more TGF~i-sensitive 3TP-lux
plasmid.
In addition to the ability of Smad6 over-
expression to activate artificial promoter constructs, the
1o actual level of PAI-1 secreted from the cells increased
almost 5-fold (Table 3).
B. Thrombomodulin activity
Determination of thrombomodulin anticoagulant
activity was done in confluent cultures of SVHA-1 cells.
The cultures were washed once with Hank s Basal Salt
Solution to remove serum proteins and incubated with serum-
free medium (DMEM/F-12 medium, 20 mrt-HEPES, pH 7.5, 50 mg/ml
gentamicin, 1 ~,g/ml human transferrin and 1 ~g/ml bovine
insulin) containing 400 nM recombinant human protein (made
according to techniques as set forth in U.S. Patent No.
4,981,952) and 10 nM human thrombin (Sigma; St. Louis, MO).
Cultures were incubated at 37°C, and at various times medium
was removed and added to an equal volume of a solution of 20
mM Tris-HC1, pH 7.5, 150 mM NaCl, 1 mg/ml BSA, and 10 U/ml
hirudin. The samples were incubated in the hirudin-
containing buffer for 5 minutes to inhibit thrombin
activity. In all experiments, samples of the protein
C/thrombin solution were incubated in wells without cells to
determine basal levels of thrombin-catalyzed activation of
protein C.
The amount of activated protein C generated was
determined by adding chromogenic substrate (52366)
(Chromogenix, Molndal, Sweden) to a final concentration of
0.75 mM, and measuring the change in absorbance units/minute
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at 405 nm in a kinetic micro-titer plate reader. Results
are expressed in terms of maximal response to TGF(3 (Table
4) .
The amount of activated protein C generated was
directly related to the level of surface TM. The over-
expression of Smad6 resulted in a suppression of TM activity
on the surface of the cells, to approximately 50% that
observed with TGF(3.
TABLE 2
Relative promoter Activity
plasmid 3TP-Lux
Control 1.0 t 0.5
+ Smad6 7.7 t 0.2
plasmid PAI-CAT
Control 1.0 t 0.2
+ Smad6 2.7 ~ 0.03
TABLE 3
Relative Level of PAI-1
Control 1.0 t 0.5
+ SmadS 4.7 t 0.1
TABLE 4
TM Activity
(% maximal TGF~i response)
Control (untreated) 0 ~ 21
TGF~ treated 100 f 25
SMAD-6 Transfected 52 t 21
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EXAMPLE 2
EFFECT OF INHIBITING SMAD WITH ANTISENSE DNA
The oligonucleotides used in antisense experiments
were synthesized using phosphorothioates and C-5 propyne
pyrimidines. Antisense oligodeoxynucleotides (oligos) were
designed to hybridize to the region of the Smad6 or the
Smad7 mRNA encompassing the initial ATG. The antisense
oligodeoxynucleotide for Smad6, was 5'-GGTTTGCCCATTCTGGACAT-
3' (SEQ ID N0:5). The sequence of the sense strand control
oligodeoxynucleotide for Smad6, was 5'-ATGTCCAGAATGGGCAAACC-
3' (SEQ ID N0:6). The antisense oligodeoxynucleotides for
Smad7, was 5'-GATCGTTTGGTCCTGAACAT-3' (SEQ ID N0:7). The
sense strand control oligodeoxynucleotide for Smad7, was
5'-ATGTTCAGGACCAAACGATC-3' (SEQ ID N0:8).
Cells were plated in 96-well plates at a density
of 2000 or 5000 cells/well and allowed to attach overnight
in DMEM/F-12 5% FBS. After washing monolayers with serum
free medium (SFM), 1 nmol of each oligo was introduced in
100 ~tl of SFM. Control wells containing SFM with vehicle
alone were included in addition to the sense strand oligo
controls. After an overnight incubation in the presence of
oligos, test wells were rinsed with SFM and re-charged with
oligo overnight as above. On the fourth day each
experimental condition was treated with or without TGF~i at a
concentration of 1 ng/ml (a concentration found to be
optimal for PAI-1 and TM response). PAI-1 levels were
assayed 16 hours later from the culture supernatant using a
commercially available PAI-1 ELISA kit (American Diagnostica
Inc., Greenwich, CT). Cell surface TM levels were assayed
in SVHA-1 cells indirectly by measuring the ability of the
cell surface TM to activate human protein C.
After the conditioned medium was removed for the
PAI-1 assay it was replaced with 100 ~.1 of SFM containing 25
~tg/m1 human protein C and 0.5 units/ml thrombin. After 1
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hour incubation at room temperature, 75 ~1 aliquots were
removed to 96-well plates, each test well containing 50 ~1
of 10 U/ml hirudin in activation buffer (20 mM tris pH 7.4,
150 mM NaCl), and incubated 5-10 minutes with agitation.
Activated human protein C was then assayed (as a measure of
TM activity) by adding 50 ~1 of the chromogenic substrate
S2366 and measuring the change in absorbance at 405 nm on a
5 minute kinetic run.
As shown in Table 5, antisense to Smad6 blocked
the TGF~i-dependent suppression of TM surface levels, as
measured by the ability of the cells to support the
thrombin-dependent activation of human protein.C. As shown
in Table 6, antisense to Smad6 suppressed the TGF(3-dependent
activation of PAI-1.
TABLE 5
TM activity (% of untreated)
Treatment control 10 uM sense 10 ~M antisense
Untreated 100 t 3 100 100
0.01 ng/ml TGF~i 91 t 7 82 99 t 3
0.1 ng/ml TGF(3 76 ~ 2 77 t 7 97 t 4
1.0 ng/ml TGF~i 59 t 4 62 t 6 92 t 2
TABLE 6
PAI-1 Induction
Sam le (% of TGF(3-treated)
TGF~3 (10 ng/ml) 100 t 6
TGF~i plus 10 ~,M sense 98 ~- 6.4
TGF~i plus 10 ~,M antisense 36 -~ 6.7
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EXAMPLE 3
SMAD GENE EXPRESSION IN HUMAN ENDOTHELIUM
The expression patterns for Smad6 and Smad 7 were
examined in multiple samples of normal and diseased human
arterial endothelium using immunohistochemical and in situ
hybridization techniques. Table 7 reports the percentage of
such samples that were positive for expression for the
indicated gene.
A. Immunohistochemistry
Human tissues collected from autopsy or surgery
were fixed overnight in Zn buffered formalin and then
transferred to 70% ethanol prior to processing through
paraffin. Five ~m sections were microtomed and the slides
baked overnight at 60°C. The slides were then
deparaffinized in xylene and rehydrated through graded
alcohols to water. Antigen retrieval was performed by
immersing the slides in tissue unmasking solution (Accurate
2o Chemical) for 10 min. at 90°C (in a water bath), cooling at
room temperature for 10 min., washing in water and then
proceeding with immunostaining. All subsequent staining
steps were performed on an automated stainer; incubations
were done at room temp., and Tris buffered saline (20mM
Tris, 150mM Na(1)) plus 0.05% Tween 20, pH 7.4 was used for
all washes and diluents. Thorough washing was performed
after each incubation. Slides were blocked with protein
blocking solution for 5 min.; after washing, 10 ~g/ml of the
particular Smad antibody (or irrelevant control antibody)
was added to the slides and incubated for 30 minutes. A
biotinylated secondary antibody plus streptavidin-
horseradish peroxidase was then utilized along With a
chromagenic peroxide substrate to detect the bound antibody
complexes. The slides were briefly counterstained with
hematoxylin, removed from the autostainer and dehydrated
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through graded alcohols to xylene. The slides were
coverslipped with a permanent mounting media and reviewed
using light microscopy to evaluate the intensity and
localization of the staining.
B. In situ hybridization
Zinc formalin fixed paraffin embedded tissues were
microtomed at 5 ~m and placed in an oven overnight at 60°C.
The hybridization protocol followed was the Super Sensitive
mRNA Detection System Kit from Biogenex. Briefly, slides
were deparaffinized in xylene and rehydrated through graded
alcohols to water (95%, 70% alcohols and water had RNAse
block (Biogenex) added). Tissues were then digested with
proteinase K solution (kit component) at room temperature
for 15 minutes. After rinsing in TBS (pH 9.5) with RNAse
block, the sections were dehydrated through graded alcohols
and dried at room temperature. The fluorescein labeled
riboprobes (both sense and anti-sense) were diluted in the
kit's hybridization solution and added to the tissue and
covered with a RNAse-free coverslip. Control slides had
only hybridization solution (no probe) added. Hybridization
was performed in a humidified thermal cycler for 10 minutes
at 95°C and then 2 hours at 37°C. After several Tris washes
to remove the coverslips and hybridization solution, the
slides were then placed on the autostainer and the
hybridized probes detected using an anti-fluorescein primary
antibody, a BCIP/NBT substrate, and a nuclear fast red
counterstain.
By both immunohistochemistry and in situ
hybridization, Smad6 gene expression was observed to be
significantly increased in 13 of 14 samples attained from
the endothelium of atherosclerotic lesions. Smad6 was not
found to be consistently expressed in normal, non-diseased
vessels. In contrast, Smad7 was not consistently found
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expressed in diseased vessels but was consistently expressed
in normal vessels.
These data associating Smad6 with vessel disease
and Smad7 with normal vessels, along with the functional
data, suggest that Smad6 contributes to vascular disease,
and further that suppression of Smad6 would be useful in the
treatment of this disorder. The data also suggest that
Smad7 protects against vascular disease.
TABLE 7
DIFFERENTIAL EXPRESSION OF SMAD6 AND SMAD7
Tissue Sample Numbers CD34a Smad6 Smad7
Normal Artery 35 100% 10% 90%
Atherosclerotic 35 100% 70-75% 5-10%
Plaque
CD34 is a marker of endothelial cells
EXAMPLE 4
SMAD GENE EXPRESSION IN HUMAN MYOCYTES
Smad gene expression was also determined in
myocytes from hearts of normal patients as compared to those
with congestive heart failure by means of
immunohistochemical and in situ hybridization (conducted as
in Example 3). These experiments demonstrated that Smad6 is
highly over-expressed in the myocytes of hearts of patients
with congestive heart failure.
Because of the well described role of TGF(3 in
promoting myocyte hypertrophy and dysfunction, agents that
suppress Smad6 and thus TGF(3 secretion, including kinase
inhibitors, have a positive benefit in the prevention and
treatment of heart failure. The ability of Smad7 to
3o antagonize the induction of TGF~i by Smad6 indicates that
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agents that increase the function or level of Smad7 are
useful in the treatment of heart failure.
EXAMPLE 5
EFFECT OF SMADS ON TGF(3 PRODUCTION
A. TFG(3/CAT Transfection
In order to determine Smad effects on TGF~i
promoter activity, a TGF(33 promoter construct driving CAT
expression (1 fig) was co-transfected into ECV304 cells with
Smad6 (5~g)/pcineo vector (5 ~,g), Smad7 (5~g)/pcineo vector
(5 fig), or both. Controls were co-transfected with 1 ~g
promoter and 10 ~g pcineo vector (Promega, Madison, WI).
Transfection was initiated by plating ECV304 cells
at 3 x 105 cells per well in a 6-well plate with DMEM/F12
media in 5% fetal bovine serum (FBS). The cells were
allowed to attach overnight at 37°C.
Twenty ~1 lipofectin were diluted with serum-free
medium to a total volume of 200 ~l per transfection and
placed at room temperature for 30 to 60 minutes. Eleven ~.g
DNA was diluted with 200 ~,1 of serum-free medium and mixed
with the lipofectin solution. The resulting reagent mixture
was incubated at room temperature for 15 minutes.
The medium was aspirated from the culture plates,
and the cultures were washed twice with PBS. Two ml of
medium were added per well, and the cells were incubated at
37°C 30 minutes prior to adding the lipofectin reagent
dropwise to the cultures. The cultures were then incubated
4-6 hours at 37°, the medium was aspirated, and fresh
DMEM/F12 5% FBS was added.
After culturing for 24 hours, the cells were
washed twice with phosphate buffered saline (PBS), and serum
free DMEM medium containing 100 ~g/ml Cohn~s fractionated
bovine serum albumin (BSA) and 2 ng/ml TGF(33 (R&D Systems)
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was added. The cells were incubated overnight at 37°C and
supernatants were collected and stored frozen. The cells
were washed twice with PBS, lysed, and expressed CAT
activity was measured kinetically as in Example 1. Lysates
were normalized in BCA assay measuring total protein
concentration.
B. Endogenous TGF(3
Endogenous levels of TGF~il and TGF~i3 secreted into
the supernatant of the above cultures were also evaluated by
ELISA.
96-well plates were coated with 0.5 ~g/ml TGF(3 RII
receptor (R&D systems) at 100 ~,1 per well in PBS. Plate
sealer was added and the plates were stored at 4°C
overnight. The plates were then washed three times with
0.1% Tween 20 in PBS, blocked with 300 ~,1 PBS containing 5%
Tween 20 and 5% sucrose for 1-3 hours at room temp.
Latent TGF(33 was activated by adding 0.1 ml 1N
HCL to 0.5 ml supernatant, and incubating 10 minutes at room
temperature. The mixture was neutralized with 0.1 ml 1.2 N
NaOH and 0.5 M Hepes. The blocking mixture was removed from
the prepared plates, and samples were added at 200 ~l per
well. Standard TGF(33, serially diluted from 2 ng/ml to
0.016 ng/ml in PBS 3% BSA, was added at 200 ~1 per well.
The plates were incubated for two hours and washed
as before. Rabbit anti-TGF(33 (Santa Cruz catalog# SC-082)
was prepared at 1 ~.g/ml in PBS 3% BSA, added to the plates
at 100 ~1 per well, incubated fox one hour at room temp.,
and washed as before. Goat anti-Rabbit IgG alkaline
3o phosphate conjugate at 1/250 dilution in PBS 3% goat serum
was added to the plates at 100 ~,1 per well, incubated one
hour, and washed as before. One PnPP tablet (Sigma) was
prepared in 3 ml H20, added at 100 ~.1 per well. The plates
were incubated for 20-30 minutes and read at OD 405 nm.
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Latent TGF~il was activated as described for TGF(33
and assayed according to directions in a commercial TGF(31
ELISA kit (R&D Systems).
Results show that in addition to the role of Smads
in the signaling of TGF(3, Smad6 induces TGF promoters and
the secretion of TGF~i. As shown in Table 8 (promoter) and
Table 9 (secretion), Smad6 induced both the promoter
activity and the secretion of TGF(33 by around three fold.
(Similar results were observed specifically for TGF~il as
well). In contrast, Smad7 had no significant affect on TGF~i
promoter activity or secretion, but could block the increase
in TGF~i secretion observed with Smad6.
Additionally, the ability of Smad6 to increase
endogenous TGF~3 secretion and promoter activity could be
blocked by the addition of a kinase inhibitor 24 hours prior
to the assay. Staurosporin inhibited Smad6-induced TGF(33
promoter activity, as measured by a decrease in CAT
expression, and the PKC specific inhibitor decreased the
endogenous secretion of Smad6-induced TGF~3 measured
directly (Table 10).
TABLE 8
TGF(3 PROMOTER ACTIVITY IN TRANSFECTED HUMAN ENDOTHELIAL
CELLS
Sample CAT Activity (OD)
Control 0.259 0.052
+Smad6 0.772 0.27
+SMAD7 0.353 t 0.12
+Smad6 +SMAD7 0.397 t 0.05
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TABLE 9
TGF(3 SECRETION FROM HUMAN ENDOTHELIAL CELLS
Sample Secreted TGF(33 (pg/nl )
Control 77 t 20
+Smad6 257 ~ 25
+SMAD7 79 t 7.5
+Smad6 +SMAD7 24 ~ g
TABLE 10
KINASE INHIBITION OF SMAD6-INDUCED ACTIVITY
Protein Kinase C Response
(pg/ml TGF(33 )
Control - 76.7 ~ 20
+ 88.7 t 15
+Smad6 - 257 t 25
+ 66.5 t 4.5
Staurosporin (10 nm) CAT Activity (OD)
Control - 0.179 t 0.036
+ 0.113 t 0.047 .
+Smad6 - 0.83 ~ 0.155
+ 0.18 ~ 0.105
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SEQUENCE LISTING
<110> Grinnell, Brian W
<120> Treatment and Prevention of Vascular Disease
<130> SMAD6 and 7
<140> X-11141
<141> 1999-03-26
<150> 60/079,681
<151> 1998-03-27
<160> 8
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gaaacggagg ctaccaactc cctcatcact gctccgggtg aattctcaga cgccagcatg 180
tctccggacg ccaccaagcc gagccactgg tgcagcgtgg cgtactggga gcaccggacg 240
cgcgtgggcc gcctctatgc ggtgtacgac caggccgtca gcatcttcta cgacctacct 300
cagggcagcg gcttctgcct gggccagctc aacetggagc agcgcagcga gtcggtgcgg 360
cgaacgcgca gcaagatcgg cttcggcatc ctgctcagca aggagcccga cggcgtgtgg 420
gcctacaacc gcggcgagca ccccatcttc gtcaactccc cgacgctgga cgcgcccggc 480
ggccgcgccc tggtcgtgcg caaggtgccc cccggctact ccatcaaggt gttcgacttc 540
gagcgctcgg gcctgcagca cgcgcccgag cccgacgccg ccgacggccc ctacgacccc 600
aacagcgtcc gcatcagctt cgccaagggc tgggggccct gctactcccg gcagttcatc 660
acctcctgcc cctgctggct ggagatcctc ctcaacaacc ccaga 705
<210> 2
<211> 235
<212> PRT
<213> Homo Sapiens
<400> 2
Met Ser Arg Met Gly Lys Pro Ile Glu Thr Gln Lys Ser Pro Pro Pro
1 5 10 15
Pro Tyr Ser Arg Leu Ser Pro Arg Asp Glu Tyr Lys Pro Leu Asp Leu
20 25 30
1
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Ser Asp Ser Thr Leu Ser Tyr Thr Glu Thr Glu Ala Thr Asn Ser Leu
35 40 45
Ile Thr Ala Pro Gly Glu Phe Ser Asp Ala Ser Met Ser Pro Asp Ala
50 55 60
Thr Lys Pro Ser His Trp Cys Ser Val Ala Tyr Trp Glu His Arg Thr
65 70 75 80
Arg Val Gly Arg Leu Tyr Ala Val Tyr Asp Gln Ala Val Ser Ile Phe
85 90 95
Tyr Asp Leu Pro Gln Gly Ser Gly Phe Cys Leu Gly Gln Leu Asn Leu
100 105 110
Glu Gln Arg Ser Glu Ser Val Arg Arg Thr Arg Ser Lys Ile Gly Phe
115 120 125
Gly Ile Leu Leu Ser Lys Glu Pro Asp Gly Val Trp Ala Tyr Asn Arg
13 0 13 5 14 0
Gly Glu His Pro Ile Phe Val Asn Ser Pro Thr Leu Asp Ala Pro Gly
145 150 155 160
Gly Arg Ala Leu Val Val Arg Lys Val Pro Pro Gly Tyr Ser Ile Lys
165 170 175
Val Phe Asp Phe Glu Arg Ser Gly Leu Gln His Ala Pro Glu Pro Asp
180 185 190
Ala Ala Asp Gly Pro Tyr Asp Pro Asn Ser Val Arg Ile Ser Phe Ala
195 200 205
Lys Gly Trp Gly Pro Cys Tyr Ser Arg Gln Phe Ile Thr Ser Cys Pro
210 215 220
Cys Trp Leu Glu Ile Leu Leu Asn Asn Pro Arg
225 230 235
<210>3
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2
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ggcgaggacg aggaggaggg cgcaggggga ggtggaggag gaggcgagct gcggggagaa 120
ggggcgacgg acagccgagc gcatggggcc ggtggcggcg gcccgggcag ggctggatgc 180
tgcctgggca aggcggtgcg aggtgccaaa ggtcaccacc atccccaccc gccagccgcg 240
ggcgccggcg cggccggggg cgccgaggcg gatctgaagg cgctcacgca ctcggtgctc 300
aagaaactga aggagcggca gctggagctg ctgctccagg ccgtggagtc ccgcggcggg 360
acgcgcaccg cgtgcctcct gctgcccggc cgcctggact gcaggctggg cccgggggcg 420
cccgccggcg cgcagcctgc gcagccgccc tcgtcctact cgctccccct cctgctgtgc 480
aaagtgttca ggtggccgga tctcaggcat tcctcggaag tcaagaggct gtgttgctgt 540
gaatcttacg ggaagatcaa ccccgagctg gtgtgctgca acccccatca ccttagccga 600
ctctgcgaac tagagtctcc cccccctcct tactccagat acccgatgga ttttctcaaa 660
ccaactgcag actgtccaga tgctgtgcct tcctccgctg aaacaggggg aacgaattat 720
ctggcccctg gggggctttc agattcccaa cttcttctgg agcctgggga tcggtcacac 780
tggtgcgtgg tggcatactg ggaggagaag acgagagtgg ggaggctcta ctgtgtccag 840
gagccctctc tggatatctt ctatgatcta cctcagggga atggcttttg cctcggacag 900
ctcaattcgg acaacaagag tcagctggtg cagaaggtgc ggagcaaaat cggctgcggc 960
atccagctga cgcgggaggt ggatggtgtg tgggtgtaca accgcagcag ttaccccatc 1020
ttcatcaagt ccgccacact ggacaacccg gactccagga cgctgttggt acacaaggtg 1080
ttccccggtt tctccatcaa ggctttcgac tacgagaagg cgtacagcct gcagcggccc 1140
aatgaccacg agtttatgca gcagccgtgg acgggcttta ccgtgcagat cagctttgtg 1200
aagggctggg gccagtgcta cacccgccag ttcatcagca gctgcccgtg ctggctagag 1260
gtcatcttca acagccgg 1278
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<212> PRT
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Met Phe Arg Thr Lys Arg Ser Ala Leu Val Arg Arg Leu Trp Arg Ser
1 5 10 15
Arg Ala Pro Gly Gly Glu Asp Glu Glu Glu Gly Ala Gly Gly Gly Gly
20 25 30
Gly Gly Gly Glu Leu Arg Gly Glu Gly Ala Thr Asp Ser Arg Ala His
35 40 45
Gly Ala Gly Gly Gly Gly Pro Gly Arg Ala Gly Cys Cys Leu Gly Lys
50 55 60
Ala Val Arg Gly Ala Lys Gly His His His Pro His Pro Pro Ala Ala
65 70 75 8U
Gly Ala Gly Ala Ala Gly Gly Ala Glu Ala Asp Leu Lys Ala Leu Thr
85 90 95
His Ser Val Leu Lys Lys Leu Lys Glu Arg Gln Leu Glu Leu Leu Leu
100 105 110
3
Ala Ala Asp Gly Pro Tyr Asp Pro Asn S
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Gln Ala Val Glu Ser Arg Gly Gly Thr Arg Thr Ala Cys Leu Leu Leu
115 120 125
Pro Gly Arg Leu Asp Cys Arg Leu Gly Pro Gly Ala Pro Ala Gly Ala
130 135 140
Gln Pro Ala Gln Pro Pro Ser Ser Tyr Ser Leu Pro Leu Leu Leu Cys
145 150 155 160
Lys Val Phe Arg Trp Pro Asp Leu Arg His Ser Ser Glu Val Lys Arg
165 170 175
Leu Cys Cys Cys Glu Ser Tyr Gly Lys Ile Asn Pro Glu Leu Val Cys
180 185 190
Cys Asn Pro His His Leu Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro
195 200 205
Pro Pro Tyr Ser Arg Tyr Pro Met Asp Phe Leu Lys Pro Thr Ala Asp
210 215 220
Cys Pro Asp Ala Val Pro Ser Ser Ala Glu Thr Gly Gly Thr Asn Tyr
225 230 235 240
Leu Ala Pro Gly Gly Leu Ser Asp Ser Gln Leu Leu Leu Glu Pro Gly
245 250 255
Asp Arg Ser His Tzp Cys Val Val Ala Tyr Trp Glu Glu Lys Thr Arg
260 265 270
Val Gly Arg Leu Tyr Cys Val Gln Glu Pro Ser Leu Asp Ile Phe Tyr
275 280 285
Asp Leu Pro Gln Gly Asn Gly Phe Cys Leu Gly Gln Leu Asn Ser Asp
290 295 300
Asn Lys Ser Gln Leu Val Gln Lys Val Arg Ser Lys Ile Gly Cys Gly
305 310 315 320
Ile Gln Leu Thr Arg Glu Val Asp Gly Val Trp Val Tyr Asn Arg Ser
325 330 335
Ser Tyr Pro Ile Phe Ile Lys Ser Ala Thr Leu Asp Asn Pro Asp Ser
340 345 350
Arg Thr Leu Leu Val His Lys Val Phe Pro Gly Phe Ser Ile Lys Ala
355 360 365
4
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Phe Asp Tyr Glu Lys Ala Tyr Ser Leu Gln Arg Pro Asn Asp His Glu
370 375 380
Phe Met Gln Gln Pro Trp Thr Gly Phe Thr Val Gln Ile Ser Phe Val
385 390 395 400
Lys Gly Trp Gly Gln Cys Tyr Thr Arg Gln Phe Ile Ser Ser Cys Pro
405 410 415
Cys Trp Leu Glu Val Ile Phe Asn Ser Arg
420 425
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ggtttgccca ttctggacat 2o
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<210>7
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gatcgtttgg tcctgaacat 20
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atgttcagga ccaaacgatc 20
