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Patent 2314821 Summary

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(12) Patent Application: (11) CA 2314821
(54) English Title: METHODS FOR MAINTAINING OR RESTORING TISSUE-APPROPRIATE PHENOTYPE OF SOFT TISSUE CELLS
(54) French Title: METHODES PERMETTANT DE CONSERVER ET DE RESTAURER UN PHENOTYPE APPROPRIE AUX TISSUS DANS LES CELLULES DES TISSUS MOUS
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C07K 14/51 (2006.01)
  • A61K 38/18 (2006.01)
  • C07K 14/47 (2006.01)
  • C12N 15/86 (2006.01)
  • A61K 38/00 (2006.01)
  • A61K 48/00 (2006.01)
(72) Inventors :
  • SAMPATH, KUBER T. (United States of America)
  • COHEN, CHARLES M. (United States of America)
  • OEDA, EIICHI (United States of America)
  • MIYAZONO, KOHEI (Japan)
  • KAWABATA, MASAHIRO (Japan)
(73) Owners :
  • MARIEL THERAPEUTICS, INC. (Not Available)
(71) Applicants :
  • CREATIVE BIOMOLECULES, INC. (United States of America)
(74) Agent: MBM INTELLECTUAL PROPERTY LAW LLP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 1998-12-16
(87) Open to Public Inspection: 1999-06-24
Examination requested: 2003-11-07
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US1998/026788
(87) International Publication Number: WO1999/031136
(85) National Entry: 2000-06-14

(30) Application Priority Data:
Application No. Country/Territory Date
60/069,931 United States of America 1997-12-17
60/110,498 United States of America 1998-12-01

Abstracts

English Abstract




Methods for maintaining or restoring tissue-appropriate phenotype of diseased,
damaged, or aged mammalian soft tissue cells and methods for treating disorder
characterized by a decreased level of endogenous expression of a morphogen.
The methods of the invention serve to manipulate any one or several aspects of
morphogen-activated regulatory pathways of phenotype-specific protein
expression.


French Abstract

Cette invention concerne des méthodes permettant de conserver et de restaurer un phénotype approprié aux tissus dans des cellules pathologiques, lésées ou vieillies des tissus mous de mammifère ainsi que des méthodes permettant de traiter les troubles caractérisés par un niveau réduit d'expression endogène d'un morphogène. Les méthodes selon l'invention conviennent pour manipuler un ou plusieurs aspects des voies régulatrices, activées par un morphogène, de l'expression de la protéine spécifique du phénotype.

Claims

Note: Claims are shown in the official language in which they were submitted.



-34-

What is claimed is:

1. A method for restoring cellular phenotype in a cell affected by disease,
damage, or age,
the method comprising:
activating an intracellular pathway that induces expression of a phenotype-
specific
gene,
thereby to restore cellular phenotype.

2. The method of Claim 1, wherein said pathway is a pathway that is activated
by specific
binding of a morphogen to its transmembrane receptor.

3. The method of Claim 1, wherein said activating step comprises inducing
intracellular
formation of a Smad complex capable of inducing expression of a phenotype-
specific
gene.

4. The method of Claim 3, wherein said Smad complex comprises Smad1 and Smad4.

5. The method of Claim 3, wherein said inducing step comprises phosphorylation
of a Smad
molecule.

6. The method of Claim 1, wherein said activating step comprises exposing a
cell having
morphogen type-1 and morphogen type-II receptors to a small molecule capable
of being
an agonist of a morphogen type-I or morphogen type-II receptor.

7. The method of Claim 3, further comprising the step of inducing
translocation of said
Smad complex in to a cell nucleus.

8. The method of Claim 1, wherein the cell is a hepatocyte.

9. The method of Claim 1, wherein the cell is a renal cell.

10. The method of Claim 1, wherein said activating step comprises inducing the
expression
of a Smad protein.

11. The method of Claim 1, further comprising the step of transfecting the
cell with a DNA
encoding a Smad protein.

12. The method of Claim 11, wherein said transfecting step is performed by
using an
adenovirus-based vector.


-35-

13. The method of Claim 11, wherein said transfecting step is performed by
using a plasmid
including said DNA.

14. A method restoring cellular phenotype in a cell affected by disease,
damage, or age, the
method comprising:
inhibiting an intracellular pathway that induces expression of a gene that is
an
inhibitor of normal phenotype,
thereby to restore cellular phenotype.

15. The method of Claim 14, wherein said gene encodes TGF-.beta..

16. The method of Claim 14, wherein said inhibiting step comprises inducing
expression of
Smad6.

17. The method of Claim 14, wherein said inhibiting step comprises inducing
expression of
Smad7.

18. The method of Claim 1, wherein said activating step comprises
administering a
morphogen to a patient.

19. The method of Claim 18, wherein said morphogen is selected from the group
consisting
of OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-9,
BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, DPP, Vgl, Vgr-1, GDF-1, GDF-2,
GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, 60A, NODAL,
UNIVEN, SCREW, ADMP, and NEURAL.

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02314821 2000-06-14
WO 99/31136 PCT/US98I26788
METHODS FOR MAINTAINING OR RESTORING TISSUE-APPROPRIATE
PHENOTYPE OF SOFT TISSUE CELLS
This application claims the benefit of U.S. Provisional Application No.
60/069,931, filed
December 17, 1997, and U.S. Provisional Application No. 60/110,498, filed
December 1, 1998.
Field of the Invention
The present invention relates generally to methods for maintaining or
restoring tissue-
appropriate phenotype in soft tissue cells. More particularly, the invention
relates to methods for
maintaining or restoring tis sue-appropriate phenotype of diseased, damaged,
or aged soft tissue
by manipulating a regulatory pathway leading to phenotype-specific protein
expression.
Background of the Invention
Numerous factors are known to influence cellular growth, differentiation, and
maintenance. One of the most important groups of growth and differentiation
factors are
members of the TGF-(3 family, particularly the morphogens, including members
of the family of
bone morphogenic proteins, first identified by their ability to induce
endochondral bone
morphogenesis. However, they have now been recognized as one of the group of
general growth
and differentiation factors that are capable of sustaining growth and
differentiation in tissue
generally. In addition, morphogens have been implicated in cellular apoptosis.
As used herein, the terms "morphogen," "bone morphogen," "bone morphogenic
protein," "BMP," "morphogenic protein" and "morphogenetic protein" all embrace
the class of
proteins typified by human osteogenic protein 1 (hOP-1). Nucleotide and amino
acid sequences
for hOP-1 are shown in SEQ ID NO: 7. For ease of description, hOP-1 is a
representative
morphogen. It is appreciated that OP-1 is merely representative of the TGF-~i
subclass of true
tissue morphogens, and is not intended to limit the description. Preferred
morphogens are those
that share at least 60% amino acid sequence identity or preferably at least
70% amino acid
sequence homology with the C-terminal seven cysteine domain of hOP-1. Other
known and
useful morphogens include, but are not limited to, the mammalian osteogenic
protein-1 (OP-1,
also known as BMP-7, and the Drosophila homolog 60A), osteogenic protein-2 (OP-
2, also
known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or
CBMP-2A,


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and the Drosophila homolog DPP), BMP-3, BMP-4 (also known as BMP-2B or CBMP-
2B),
BMP-5, BMP-6 and its marine homolog Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, GDF3
(also known as Vgr2), GDF-8, GDF-9, GDF-10, GDF-1 l, GDF-12, BMP-13, BMP-14,
BMP-15,
GDF-5 (also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2), GDF-7
(also
known as CDMP-3), the Xenopus homolog Vgl and NODAL, UNIVIN, SCREW, ADMP, and
NEURAL, and morphogenically-active amino acid variants (such as conservative
substitution
variants) of any thereof. Typically, such morphogens share functional
features, such as the
ability to stimulate endochondral bone formation in an in vivo bone assay, or
the ability to
stimulate N-CAM or L1 isoform production in an NG108-15 neuronal cell culture.
See U.S.
4,968,590; Sampath et al., Proc. NatL Acad. Sci. USA 80: 6591-6595 (1983),
incorporatec: by
reference herein. Other functional assays for morphogen activity, useful in
identifying
morphogens are known in the art.
Morphogens include secretory peptides sharing common structural features.
Typically,
the mature form of the protein is processed from a precursor "pro-form." The
mature form is a
1 S dimer containing a carboxy terminal active domain having approximately 97-
106 amino acids,
containing a conserved pattern of cysteines. The active form is either a
disulfide-bonded
homodirner or a heterodimer. See, e.g., Massague, Annu. Rev. Cell Biol. 6:597
(1990); Sampath
et al., J. Biol. Chem. 265:13198 (1990). While the morphogens have significant
homologies and
similarities in structure, variants within the morphogenic protein genes may
have specific roles in
specific tissue involving, for example, stimulation of progenitor cell
multiplication, tissue
specific or tissue preferred phenotype maintenance, and/or stimulation or
modulation of the rate
of differentiation, growth or replication of tissue cells characterized by
high turnover.
The morphogenic activities of the TGF-(3 superfamily of proteins allow them to
initiate
and maintain the developmental cascade of tissue morphogenesis in an
appropriate,
morphogenically-permissive environment, stimulating stem cells to proliferate
and differentiate
in a tissue-specific manner, and inducing the progression of events that
culminate in new tissue
formation. Specifically, morphogens are capable of at least the following
biological functions in
a morphogenically permissive environment: stimulating proliferation of
progenitor cells;
stimulating the differentiation of progenitor cells; stimulating the
proliferation of differentiated


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cells; and supporting the growth and maintenance of differentiated cells,
including the
"redifferentiation" of transformed cells.
These morphogenic activities also allow the proteins to stimulate the
"redifferentiation"
of committed cells previously induced to alter their phenotype due to disease,
damage, or age.
Morphogens are useful in the replacement of diseased, damaged, or aged tissue,
particularly
when the damaged tissue interferes with normal tissue or organ function. For
example, elevated
morphogen expression induces repair of damaged lung tissue resulting from
emphysema;
damaged kidney cells; cirrhotic liver cells; damaged heart or blood vessel;
damaged stomach
tissue resulting from ulcers or their repair; damaged neural tissue (e.g.,
resulting from stroke) or
neuropathies such as Alzheimer's disease, Parkinson's disease, Huntington's
chorea, and
multiple sclerosis; damaged skeletal or orthopedic tissues; or damaged dentin
and periodontal
tissues as may result from disease or mechanical damage or injury.
Furthermore, morphogens
are useful in treating symptoms resulting from diseased, damaged, or aged soft
tissue cells, such
as pain, including neuropathy pain.
Morphogens act to induce an intracellular cascade that results in expression
of phenotype-
specific gene products. Such gene products include proteins necessary or
sufficient to maintain,
enhance, or restore phenotype, including structural proteins, enzymes, and the
like. Generally, a
morphogen acts as a ligand for specific Type I and/or Type II transmembrane
receptors, each
receptor typically being associated with a serine/threonine kinase. In a
common scenario, after
ligand binding, a Type II receptor phosphorylates an adjacent Type I receptor.
The activated
Type I receptor recognizes specific members of the Smad protein family,
phosphorylating them
at least at the carboxy-terminal serine residue. Eight different Smad proteins
have been
identified in mammals. These are classified into three subgroups, including
pathway-restricted
Smads (R-Smads), common-mediator Smads (co-Smads), and inhibitory Smads (anti-
Smads).
R-Smads are directly activated by Type I receptors, form complexes with co-
Smads, and
translocate into the nucleus. The Smad heteromers directly bind to DNA, and
also associate with
other DNA binding proteins, and thus regulate the transcription of target
genes. Smadl, 5, and 8
are activated by BMP receptors, and Smad2 and 3 are activated by TGF-/3 and
activin receptors.
Smad4 functions as a co-Smad. Smad6 and Smad7 are distantly related in terms
of structure
with other Smads, and serve as anti-Smads.


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The Smadl, Smad2, Smad3 and SmadS proteins consist of conserved amino- and
carboxy-terminal domains linked by a region that is more divergent among the
Smads. The
carboxy-terminal domain has an effector function. The amino-terminal domain
interacts
physically with the carboxy-terminal domain, inhibiting its effector activity,
and contributes to
DNA binding. Receptor-mediated phosphorylation of the serine residues at the
end of the
carboxy-terminal domain relieves the carboxy-terminal domain from the
inhibitory action of the
amino-terminal domain. Phosphorylated Smad molecules form a heteromeric
complex with at
least one other specific Smad family molecule. The resulting Smad complex then
translocates
into and accumulates in the cell nucleus. There, the heteromeric Smad
complexes regulate
transcriptional responses eitr_er alone or by specific interaction with a DNA-
binding protein, such
as forkhead activin signal transducer-1 (FAST1).
With particular reference to the OP-1 or BMP-2 activated pathway, as shown in
Figure 2,
morphogens are ligands for the Type I and Type II receptors. The Type II
receptor comprises a
constitutively-active kinase, which transphophorylates a Type I receptor upon
ligand binding.
Following phosphorylation of the Type I receptor by the Type II receptor, the
Type I receptor
specifically phosphorylates Smadl homodimers. The Type I receptor also
specifically
phosphorylates SmadS homodimers. The homodimers then separate to form, in
association with
a phosphorylated Smad4 molecule, a phosphorylated heteromeric complex
comprising at least a
Smadl and a Smad4. A phosphorylated Smadl/SmadS/Smad4 heterotrimer may
alternatively be
formed. The heteromeric complex then translocates into the nucleus, and
accumulates therein.
In the nucleus, the Smad complex binds operative DNA, either alone or in
association with a
specific DNA binding protein (the X-protein in Figure 2), to initiate DNA
transcription. The "X-
protein" acts as a DNA-binding protein, binding the Smad heteromeric complex
to the DNA.
The pathway leading to endogenous morphogen expression is similar to the one
described above,
with the Smad heteromeric complex inducing transcription of the morphogen-
encoding gene.
Other intracellular pathways are induced by morphogens, and may be affected in
the manner
described herein.
Diseased, damaged, or aged soft tissue cells are characterized in part by a
decrease in
endogenous expression of morphogenic protein, and OP-1 in particular. This
decrease in
endogenous expression of morphogenic protein causes the cells to
dedifferentiate, displaying


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tissue-inappropriate phenotype which, at the extreme, results in cell death.
For example, cells in
the substantia nigra of the brain progressively become dysfunctional in
patients with Alzheimer's
disease. Similarly, liver cells may lose their phenotype (i. e., become
cirrhotic) due to alcohol
abuse or other causes. Accordingly, there is a need in the art for methods to
stimulate diseased,
damaged, or aged cells to maintain or to restore tissue-appropriate phenotype.
Summary of the Invention
It has now been recognized that preservation and maintenance of cellular
phenotype is
accomplished by activation of pathways that normally are modulated by growth
and
differentiation factors. Moreover, inhibition of those pathways is now
recognized as an
additional means for preserving or inducing appropriate phenotype.
Normal phenotype is controlled not only by developmental cues, but also by
various
endogenous growth factors. However, disease, injury or aging may affect one or
more aspects of
cellular function, including the ability of growth and differentiation factors
to modulate gene
expression leading to normal cellular phenotype. For example, chronic
degenerative illness may
result not only in biochemical dysfunction, but in the inability of affected
tissue to replace lost
cells.
The present invention comprises activating and controlling phenotypic effects
through
action at various intracellular pathways. In so doing, morphogen-activated
pathways are used as
an example of the ways in which tissue growth and differentiation can be
modulated. However,
methods disclosed herein are useful in the restoration and/or maintenance of
phenotype through
action at any intracellular pathway that is normally modulated by any growth
and differentiation
factor.
Accordingly, the present invention provides methods for maintaining or
restoring tissue-
appropriate phenotype in a soft tissue cell. According to methods of the
invention, tissue-
appropriate phenotype is maintained or restored by increasing endogenous
expression of a
phenotype-specific protein. In a preferred embodiment, endogenous phenotype-
specific protein
expression is increased by manipulating an intracellular regulatory pathway
that modulates
expression of a gene encoding such a protein. Thus, methods of the invention
comprise
administering a composition that interacts with at least a portion of an
intracellular pathway by


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which growth and maintenance factors, such as morphogens, cause expression of
a phenotype-
specific protein (e.g., a protein associated with preservation, restoration,
or enhancement of
phenotype). Such methods also comprise exposing cells to a composition that
interacts with a
pathway by which endogenous growth and maintenance factor production is
stimulated, thereby
to stimulate an increase in growth and maintenance factor expression by the
cell. In a preferred
embodiment, methods of the invention comprise detecting the component or
components of
cellular biology that are lacking in a given cell or cell population, and then
targeting activation or
inhibition of a pathway, the result of which is compensation or restoration of
the lacking
component(s).
Methods of the invention involve exogenous stimulation of one or more
components of a
pathway that normally leads to the expression of a phenotype-specific gene
product. Expression
of a particular phenotype-specific gene may be the direct and immediate result
of the modulation
of a particular pathway or may be the result of any one or more biochemical
effects flowing from
such modulation. For example, modulation a particular pathway may result in an
increase of
endogenous expression of OP-1, which in turn may activate a different
regulatory pathway that
ultimately results in increased expression of a phenotype-specific gene
product. A prototypical
pathway is shown in Figure 2. As shown in the Figure, receptor binding
activates an intracellular
kinase, causing phosphorylation of intracellular messenger molecules called
Smads. The Smads
have been characterized, and are known in the art. See, e.g., Baker et al.,
Curr. Op. Genet.
Develop., 7: 467-473 (1997), incorporated by reference herein. Upon
phosphorylation, various
Smad subtypes form complexes which then translocate into the nucleus. Once in
the nucleus,
Smad complexes, either on their own or in association with a transcription
activator, modulate,
either directly or indirectly, expression of specific gene product that is
characteristic of the
stimulated cell. One such gene product is a morphogen itself. Thus, the
pathway may play a role
in positive feedback on morphogen expression, thus affecting another pathway
resulting in
phenotype-specific gene expression.
Methods of the invention may also be used to inhibit the effects of cellular
components
that diminish normal cellular phenotype. Whether any specific cellular
component enhances or
diminishes cellular phenotype depends upon the local environment, the
developmental state, and
the disease/injury status of the cell. For example, during wound healing
transforming growth


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-
factor-~i (TGF-(3) promotes formation of scar tissue via fibrosis. In wound
healing studies,
morphogens have been shown to counteract this effect. It has also been shown
that two
particular Smad proteins, Smad6 and Smad7, inhibit TGF-(3. Accordingly, in one
embodiment,
methods of the invention comprise activating Smad6/Smad7 to inhibit TGF-(3 at
the site of
S wound healing. Additional activation of morphogen-induced intracellular
pathways further
promotes healing, and the presentation of normal cells at the wound site. The
precise nature of
regulation according to methods of the invention depends upon the environment
in which the
tissue exists, the age of the tissue, and the disease or injury state of the
tissue. However,
activation of morphogen-induced pathways as described herein results in
restoration and
maintenance of normal phenotype. Precise control over biochemical functions is
achieved by
targeting specific pathways that have been adversely affected by age, disease,
and/or injury.
In another aspect, the invention provides methods for increasing the level of
endogenous
phenotype-specific protein comprising the step of introducing a small molecule
that regulates
some portion or portions of a cellular regulatory pathway, resulting in an
effective increase in
expression or activity of a phenotype-specific protein. This may result either
from stimulating an
increase in the endogenous expression of a phenotype-specific protein or a
decrease in the
expression or inhibitory activity of an inhibitor of normal (in the
appropriate developmental and
anatomical context) phenotype. For example, a small molecule may act at the
Type I or Type II
morphogen receptor; or at the serine/threonine kinase, or other kinase domains
of those
receptors. Another target of pathway activation is the Smad proteins,
including the monomeric,
dimeric (including heteromeric and homomeric complexes) or trimeric forms
(including
heteromeric and homomeric complexes). Alternately, activation of a
transcription factor (for
example, the X-protein shown in Figure 2) will lead to phenotype-specific
expression. A small
molecule may act to facilitate, mimic, or, if desired, prevent any one or
several of the following:
Type I and/or Type II receptor binding, phosphorylation of the Type I
receptor, phosphorylation
of the Smad molecules, Smad complex formation, Smad translocation into the
nucleus, nuclear
accumulation of the Smad complex, or transcription modulation of the Smad
complex.
Furthermore, a small molecule may act on Smads or Smad complexes to alter
tertiary structure,
thereby to facilitate or inhibit interaction of the Smad or Smad complex with
a receptor kinase
domain, other Smads, DNA binding proteins, or DNA itself.


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In a particularly-preferred embodiment, a small molecule is administered to a
patient,
wherein the small molecule facilitates formation of Smad complexes,
particularly complexes
comprising molecules of Smadl, Smad2, Smad3, Smad4, SmadS and/or Smad8 in
order to
facilitate phenotype-specific gene expression. Also in a preferred embodiment,
methods
comprise administering a composition that activates a serine/threonine kinase
domain associated
with a morphogen Type I or Type II receptor, thereby to activate the pathway
involved in
morphogen-induced gene expression. In another embodiment, methods of the
invention
comprise activating Smad4 association with Smadl, thereby to induce morphogen-
responsive
phenotypic gene expression. Methods of the invention may also facilitate Smad
interaction with
;.pecific nucleic acids, such as promoters of phenotype-specific gene
expression (i. e., expression
of genes for a phenotypic protein; a protein associated with preservation,
restoration, or
enhancement of phenotype, including a protein which is critical for production
of non-protein
phenotypic markers, such as characteristic lipids or carbohydrates; a protein
associated with
performance of a phenotypic function or morphology; or a morphogen). Such
interaction may
be, for example, in association with a transcription control factor that is
capable of binding to a
regulatory portion of a gene and, simultaneously, to one or more regulatory
proteins such as a
Smad complex (see Figure 2). As used herein, phenotype-specific gene
expression or
morphogen-induced gene expression refers to the expression of genes that are
under morphogen
control, or can be controlled by morphogens in a normal, healthy cell.
In another aspect of the invention, enhancement, preservation or restoration
of phenotype
may be achieved by providing a small molecule that acts as an agonist at the
morphogen Type I
or Type II receptor, thereby to stimulate activation of the pathway leading to
phenotype-specific
gene expression or to morphogen expression.
Methods of the invention also comprise the step of administering a composition
comprising a small molecule capable of decreasing inhibition of morphogen-
induced phenotype-
specific protein expression. Morphogen inhibition may be in the form of
endogenous inhibitory
compounds, such as leukemia inhibitory factor or cytokines, or may be in the
form of
exogenously applied inhibitors. Furthermore, Smad6 and/or Smad7 have
inhibitory activity on
the regulatory pathway of phenotype-specific protein expression. Accordingly,
methods of the
invention also comprise affecting Smad6 and/or Smad 7 activity.


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_g_
The present invention further provides methods for treating a soft tissue
disorder
characterized by decreased levels of morphogen expression. Disorders
characterized by
decreased levels of morphogen expression resulting in dedifferentiation of
soft tissue cells
include lung damage caused by emphysema; cirrhotic kidney or liver tissues;
damaged muscle
tissue; damaged heart or blood vessel tissues, as may result from
cardiomyopathies and/or
atherothrombotic or cardioembolic strokes; damaged stomach or intestinal
tissues resulting from
ulceric perforations or their repair; and damaged neural tissue (including
visual and auditory
sensory tissue) as may result from physical or chemical injury, such as
strokes, or neuropathies
such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, or
multiple sclerosis, or
neuropathic or other pain associated with any of the foregoing. Methods of the
invention may
comprise the step of administering a small molecule affecting a pathway
leading to expression of
phenotype-specific genes, or may comprise administering exogenous morphogenic
protein, or an
agonist thereof, including the monomer, dimer and/or soluble complex forms
(comprising one or
more morphogen pro domain noncovalently associated with a morphogen dimer), to
the tissue
locus having diseased, damaged, or aged soft tissue cells. The composition
comprising the
morphogenic protein may further comprise a matrix. Useful matrix materials
include collagen,
demineralized bone, hydroxyapatites, bioactive ceramics, calcium phosphate
ceramics or
mixtures comprising any one or more of the foregoing materials.
The present invention further provides methods for treating a soft tissue
disorder
characterized by decreased levels of morphogenic protein expression comprising
the step of
administering a composition comprising a morphogen and a small molecule
capable of releasing
inhibition of phenotype-specific protein expression.
In yet another aspect, the invention provides in vivo methods for increasing
the level of
endogenous expression of morphogenic protein or a phenotype-specific protein,
comprising the
step of administering naked DNA or mRNA encoding a morphogenic protein or a
phenotype-
specific protein directly to the locus of damaged, diseased or aged soft
tissue cells. See U.S.
Patent No. 5,580,859, the teachings of which are incorporated by reference
herein. In another
aspect, the invention provides ex vivo methods for increasing the level of
endogenous expression
of morphogenic protein or a phenotype-specific protein comprising introducing
DNA encoding a
morphogenic protein or a phenotype-specific protein into a soft tissue cell,
and placing the


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transformed soft tissue cell at the tissue locus having damaged, diseased or
aged soft tissue cells.
Introduction of the DNA encoding a morphogenic protein or a phenotype-specific
protein into a
soft tissue cell may be accomplished by a variety of means and methods,
including the use of
plasmid DNA; viral vectors, including retrovirus, adenovirus, adeno-associated
virus, herpes
simplex virus, SV40, polyoma virus, papillloma virus and picornavirus; DNA on
the interface of,
or encapsulated in liposomes or proteoliposomes; calcium phosphate, DEAF-
Dextran;
polybrene; polysine/DNA conjugates; or electroporation or microinjection.
Furthermore, the present invention provides methods for treating soft tissue
disorders by
affecting apoptosis by modulating a morphogen-activated regulatory pathway.
Apoptosis is a
distinctive form of cell death manifested by characteristic chromatin
condensation and DNA
fragmentation, resulting in the programmed death of cells as part of the
normal cell cycle.
However, apoptosis may also by induced by pathologic stimuli. In some
instances, gene
transcription and protein synthesis are required for the induction of
apoptosis, and the process is
regulated by a set of genes that are involved in normal cell growth and
differentiation.
Apoptosis is responsible for numerous physiologic and pathologic events. For
example,
apoptosis is responsible for the programmed destruction of cells during
embryogenesis
(including implantation, organogenesis, developmental involution) and
metamorphosis.
Apoptosis is also responsible for hormone-dependent involution in the adult,
such as endometrial
cell breakdown during the menstrual cycle, ovarian follicular atresia in the
menopause, and the
regression of the lactating breast after weaning. Apoptosis also functions in
cell deletion in
proliferating cell populations, such as intestinal crypt epithelia. In
addition, apoptosis is
responsible for cell death in tumors, most frequently during regression but
also in tumors with
active cell growth. Moreover, apoptosis is responsible for the death of immune
cells, both B and
T lymphocytes after cytokine depletion, as well as deletion of autoreactive T
cells in the
developing thymus. Furthermore, apoptosis is responsible for pathologic
atrophy of hormone-
dependent tissues, such as prostatic atrophy after castration and loss of
lymphocytes in the
thymus after glucocorticoid administration. Apoptosis is also responsible for
pathologic atrophy
in parenchyma) organs after duct obstruction cell death induced by cytotoxic T
cells, such as in
cellular immune rejection and graft-versus-host disease. In addition,
apoptosis is plays a role in
cell injury in certain viral diseases, as for.example in viral hepatitis, in
which apoptotic cell


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fragments in the liver are known as Councilman bodies. Apoptosis is also
responsible for cell
death produced by a variety of injurious stimuli, including mild thermal
injury, radiation,
cytotoxic anticancer drugs, and possible hypoxia, that are capable of
producing necrosis, but
when given in low doses, induce apoptosis.
The pathways by which apoptosis is induced vary, depending on the stimulus and
cell
type. One important feature of apoptosis is its dependence in many {but not
all) instances on
gene activation and new protein synthesis. A number of genes can be induced by
stimuli causing
apoptosis, such as heat-shock proteins and proto-oncogenes. Apoptosis-specific
genes that
stimulate or inhibit cell death have been described, Certain genes involved in
growth and in the
causation of cancer (oncogenes and suppressor genes) play a regulatory role in
the induction of
apoptosis. These include the bcl-2 oncogene which inhibits apoptosis induced
by hormones and
cytokines and thus extends cell survival; the c-myc oncogene, whose protein
produce can
stimulate either apoptosis of cell growth; and p53 which normally stimulates
apoptosis, but when
mutated or absent, favors cell survival. However, in many models of apoptosis,
new gene
expression is not required and indeed inhibition of gene expression causes
apoptosis.
Accordingly, the methods of the invention also provide for promoting or
inhibiting apoptosis in a
soft tissue cell by modulating a morphogen-activated regulatory pathway.
Methods of the invention are carried out in any tissue having diminished or
lost
phenotypic function as a result of disease, injury, or aging. Alternatively,
methods of the
invention are applicable in developing embryonic tissue.
The preferred methods and examples that will now be described are illustrative
only and
are not intended to be limiting. Other features and advantages of the
invention will be apparent
from the following detailed description and claims.
Brief Description of the Drawings
Figure 1 is a tabular presentation of the percent amino acid sequence identity
and percent
amino acid sequence homology ("similarity") that various members of the family
of
morphogenic proteins as defined herein share with hOP-1 in the C-terminal
seven cysteine
skeleton.


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Figure 2 is a schematic representation of a morphogen-activated regulatory
pathway for
expression of a phenotype-specific gene.
Figure 3 is a schematic representation of DIAPs and the truncated forms of
DIAP 1 with
amino acid positions indicated with numbers.
Figures 4 and S are tables summarizing interactions of various forms of DIAP 1
with
various DPP receptors.
Figure 6 is a schematic representation of the DIAP1 constructs used in
experiments
described herein.
Detailed Description
Methods of the invention rely, in part, on the role of morphogens and other
growth and
dii~erentiation factors in maintaining tissue-appropriate phenotype in soft
tissue cells. "Soft
tissue" as used herein includes all mammalian tissue except bone and
cartilage. Modulation of
morphogen-responsive pathways are exemplified herein. However, it is intended
that, because of
the underlying biology of growth and differentiation, other growth factor-
induced pathways are
modulated in the manner described herein. Decreased endogenous expression of
morphogen,
and especially of OP-1, is characteristic of diseased, damaged, or aged cells.
Methods of the
invention are useful to maintain or restore tissue-appropriate phenotype of
soft tissue cells which
have begun to dedifferentiate due to disease, damage, or age. Specifically,
methods of the
invention are useful to potentiate phenotype-specific protein expression by
manipulating
regulatory pathways that normally affect such protein expression.
Methods of the invention comprise activating regulatory pathways in order to
increase
expression of a phenotype-specific protein, such as a protein associated with
preservation,
restoration, or enhancement of phenotype; a protein associated with
performance of a phenotypic
function, or a protein characteristic of healthy cellular morphology. Cells
that express
endogenous morphogen (e.g., OP-1), and that are damaged due to injury,
disease, or age, lose
their ability to express certain tissue-specific phenotype markers. Inducing
such cells to express
morphogen, or morphogen-stimulated proteins restores cellular phenotype, as
evidenced by
expression of characteristic phenotype markers, performance of phenotypic
functions and display
of normal (healthy) phenotypic morphology.


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For example, hepatocytes are large, polyhedral cells that have a variable
cytoplasmic
appearance depending on the nutritive status of the body. The nuclei of
hepatocytes are large
with peripherally dispersed chromatin and prominent nucleoli. The nuclei,
however, vary greatly
in size. This reflects the fact that more than half the normal complement of
hepatocytes contain
twice the normal amount of chromosomal material, some contain four or even
eight times the
normal amount. Hepatocytes generally perform the phenotypic functions of
storing glycogen,
fat, and certain vitamins. Hepatocytes also transform nutritive substances in
the diet into one
another to deliver into the blood a needed nutrient. For example, hepatocytes
can transform
protein into carbohydrate, if needed. Furthermore, hepatocytes will transform
and/or conjugate
~ certain products in order to detoxify them. In addition, hepatocytes
regulate the concentration of
certain substances, such as sugar, in the blood. Hepatocytes also express such
phenotypic
proteins as albumins, fibrinogens and globulins. Morphogens restore healthy
phenotype to
hepatocytes that have lost one or more structural or biochemical function
characteristic of normal
function. Once identified, the diminished phenotypic function is restored, in
whole or in part, by
activation of pathways leading to phenotype-specific protein production.
Typically, such
pathways are under morphogen regulation.
Those practicing in the art will appreciate that phenotype-specific markers,
functions and
morphology for other cell types are well-known.
Small Molecule-Mediated Upregulation
The pathways that regulate gene expression are affected by a wide variety of
developmental and environmental stimuli, thus allowing each cell type to
express a unique and
characteristic subset of its genes, and to adjust the expression of particular
gene products as
needed. The importance of expression control is underscored by the fact that
targeted disruption
of key regulatory molecules in mice often result in drastic phenotypic
abnormalities, just as
inherited or acquired defects in the function of genetic regulatory mechanisms
contribute broadly
to human disease. Of interest in this regard is the usefulness of small
molecules capable of
controlling expression of phenotype-specific genes. Morphogen-activated
regulatory pathways
may be modulated by, for example, administering a small molecule capable of
stimulating the
expression of a phenotype-specific protein.


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A small molecule may be a morphogen analog that mimics activation of the
regulatory
pathway by a morphogen, such as OP-1. Small molecule morphogen analogs are
identified, for
example, as reported in co-pending patent application, U.S.S.N. 08/507,750,
incorporated by
reference herein. Any substance having such mimetic properties, regardless of
the chemical or
biochemical nature thereof, is useful as a morphogen analog as taught herein.
The present
morphogen analog may be a simple or complex substance produced by a living
system or
through chemical or biochemical synthetic techniques. It may be a substance
that occurs in
nature or a novel substance, e.g., prepared according to principles of
rational drug design. It may
be a substance that structurally resembles a solvent-exposed morphogen surface
epitope
implicated in receptor interactions, a substance that otherwise stimulates a
transmembrane
morphogen receptor, or a cell-membrane permeant substance that interacts with
any one or more
intracellular aspects of the signal transduction pathway of a morphogen
responsive cell. For
example, a naturally-sourced OP-1 or morphogen analog may comprise a
polypepdde,
polynucleotide, carbohydrate, lipid, amino acid, nucleic acid, sugar, fatty
acid, steroid, or a
derivative of any one of the aforementioned compounds. It may an intermediate
or end product
of metabolism of a eukaryotic or prokaryotic cell. Alternatively, the analog
may a biological
response modifier or a toxin.
Without being limited, one type of morphogen analog useful in the methods of
the
present invention can be prepared through application of the principles of
biosynthetic antibody
binding site (BABS) technology as set forth in U.S. Patent Nos. 5,132,405,
5,091,513 and
5,258,498, the teachings of which are incorporated by reference herein. BABS
analog constructs
are prepared from antibodies, preferably produced by hybridoma cells, that
bind specifically to a
morphogen transmembrane receptor. Alternatively, BABS analysis is based upon
anti-idiotypic
antibodies specifically reactive with the antigen binding site of an antibody
that blocks
morphogen biological activity. Vukicevic et al., Biochem. Biophys. Res. Comm.
198: 693-700
(1994), teaches the preparation of OP-1 specific monoclonal antibodies.
Skilled artisans will
appreciate that such antibodies can be used as immunogens in the routine
preparation of anti-
idiotypic antibodies from which BABS analogs of the present invention can be
prepared.
A structurally distinct class of morphogen analogs, again set forth herein for
illustration
and not for limitation, can be prepared through application of the principles
of directed molecular


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evolution as set forth in Tuerk et al., Science 249:505-S 10 ( 1990), Famulok
et al., Angew. Chem.
Intl. Ed. Engl. 31:979-988 (1992) and Bock et al., Nature 355:564-556 (1992),
the teachings of
each of which are incorporated by reference herein. The directed molecular
evolution process
involves isolation of a nucleic acid molecule, typically an RNA, that binds
with high affinity to a
selected ligand such as a protein. Such a nucleic acid molecule is referred to
in the art as an
"aptamer." The desired aptamer is initially present in a random pool of
nucleic acid molecules,
and is isolated by performing several rounds of ligand-affinity based
chromatography alternating
with PCR-based amplification of ligand-binding nucleic acids. Bock et al.,
(1992), above, have
demonstrated the preparations of aptamers, suitable for in vivo use in
mammals, that specifically
inhibit the blood clot promoting factor, thrombin.
Yet another structurally distinct class of morphogen analogs is prepared by
selecting
appropriate members of a random peptide library (Scott et al., (1990) Science
249:386-390), or a
combinatorially synthesized random library of organic or inorganic compounds.
Needels et al.,
Proc. Natl. Acad. Sci. USA, 90:10700-10704 (1993); Ohlmeyer et al., Proc.
Natl. Acad. Sci. USA
90:10922-10926 (1993). Skilled artisans appreciate that the foregoing and
other related
technologies, taken together with long-established principles of screening
biologically-produced
substances, offer a wide array of candidate compositions for screening for
morphogen analog
activity.
Thus, as used herein, a morphogen analog is a substance that mimics morphogen
activation of the regulatory pathway of phenotype-specific gene expression
inducing at least one
"morphogen-mediated biological effect" in a cell or tissue. The effect can be
any biological
effect resulting from exposure to or contact with a morphogen, including but
not limited to
maintenance or restoration of tissue-appropriate phenotype. Morphogen-mediated
biological
effects include cellular and molecular responses to morphogen exposure, as
described, for
example, in co-pending patent application U.S.S.N. 08/260;675 and U.S. Pat.
No. 5,656,593, the
disclosures of which are incorporated by reference herein. Thus, it is
appreciated that a
morphogen-mediated biological effect is any biological effect resulting from
exposure to or
contact of morphogen-responsive cells or tissue with a morphogen, whether in
vitro or in vivo.
A morphogen-mediated biological effect of particular interest herein includes
stimulation of the
expression of one or more phenotype-specific genes, including stimulation of
the binding of an


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intracellular substance to DNA expression regulation elements. Preferred
morphogen-mediated
biological effects include maintenance of a differentiated phenotype, or
induction of
redifferentiation, and/or stimulation of cellular proliferation and cellular
differentiation.
In a highly-preferred embodiment, the small molecule is a compound that
affects at least
one intracellular pathway that normally is under morphogen regulation. Such
small molecules
preferably have the ability to enter the cell and target one or more
intracellular pathway
components in order to stimulate or inhibit its activity. For example, a small
molecule that
promotes Smad complex formation between Smadl, Smad4, and SmadS will stimulate
pathways
leading to expression of genes encoding phenotype-specific proteins.
One way in which to identify a candidate small molecule is to assay for the
ability of the
candidate to modulate the effective systemic or local concentration of a
morphogen. This may be
done, for example, by incubating the candidate in a cell culture that produces
the morphogen, and
assaying the culture for a parameter indicative of a change in the production
level of the
morphogen according the methods of U.S. Pat. No. 5,741,641 and/or U.S. Pat.
No. 5,650,276,
the teachings of each of which are incorporated by reference herein.
Alternatively, candidate
compounds are screened for their ability to induce phenotype-specific protein
production in a cell
culture in which morphogen activity is not present. Examples of compositions
which may be
screened for their effect on the production of morphogens or other phenotype-
specific proteins
include but are not limited to chemicals, biological response modifiers (e.g.,
lymphokines,
cytokines, hormones, or vitamins), plant extracts, microbial broths and
extracts medium
conditioned by eukaryotic cells, body fluids, or tissue extracts. Useful
candidate compositions
then may be tested for in vivo efficacy in a suitable animal model. These
compositions then may
be used in vivo to upregulate morphogen-activated regulatory pathways of
phenotype-specific
protein expression.
A simple method of determining if a small molecule compositions has affected a
change
in the level of a phenotype-specific protein in cultured cells is provided in
U. S. Pat. No.
5,741,641, the disclosure of which is incorporated by reference herein. The
level of a target
phenotype-specific protein in a cell resulting from exposure to a small
molecule is measured.
Alternatively, a change in the activity or amount of an intracellular pathway
component is
measured in response to application of a candidate small molecule. Candidates
having the


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desired affect on protein production or pathway regulation are selected for
use in methods of the
invention. If, for example, a composition upregulates the production of OP-1
by a kidney cell
line, it would then be desirable to test systemic administration of this
compound in an animal
model to determine if it upregulates the production of OP-1 in vivo. The level
of morphogen in
the body may be a result of a wide range of physical conditions, e.g., tissue
degeneration such as
occurs in diseases including arthritis, emphysema, osteoporosis, kidney
diseases, lung diseases,
cardiomyopathy, and cirrhosis of the liver. The decrease in level of
morphogens in the body may
also occur as a result of the normal process of aging. The same strategy is
used for compositions
affecting intracellular pathway components. A composition selected by these
screening methods
is then used as a treatment or prophylactic.
An appropriate test cell is any cell comprising DNA defining a morphogen-
responsive
transcription activating element operatively associated with a reporter gene
encoding a detectable
phenotype-specific gene product. Such DNA can occur naturally in a test cell
or can be a
transfected DNA. The induced intracellular effect typically is characteristic
of morphogenic
biological activity, such as Smad activation, or activation of a cascade of
biochemical events,
such as described above, or involving, for example, cyclic nucleotides,
diacylglycerol, and/or and
other indicators of intracellular signal transduction such as activation or
suppression of gene
expression, including induction of mRNA resulting from gene transcription
and/or induction of
protein synthesis resulting from translation of mRNA transcripts indicative of
tissue
morphogenesis. Exemplary morphogen-responsive cells are preferably of
mammalian origin and
include, but are not limited to, osteogenic progenitor cells; calvaria-derived
cells; osteoblasts;
osteoclasts; osteosarcoma cells and cells of hepatic or neural origin. Any
such morphogen
responsive cell can be a suitable test cell for assessing whether a candidate
substance induced is a
morphogen analog.
A preferred identification method is carried out by exposing a test cell to at
least one
candidate substance, and detecting whether such exposure induces expression of
the detectable
phenotype-specific gene product that is in operative association with the
morphogen-responsive
transcription activating element. Expression of this gene product indicates
that the candidate
substance induces a morphogen-mediated biological effect. Skilled artisans
can, in light of
guidance provided herein, construct a test cell with a responsive element from
a morphogen-


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responsive cell and a reporter gene of choice, using recombinant vectors and
transfection
techniques well-known in the art. There are numerous well-known reporter genes
useful herein.
These include, for example, chloramphenicol acetyltransferase (CAT),
luciferase, human growth
hormone (hGH), beta-galactosidase, and assay systems and reagents which are
available through
commercial sources. As will be appreciated by skilled artisans, the listed
reporter genes
represent only a few of the possible reporter genes that can be used herein.
Examples of such
reporter genes can be found in Ausubel et al., Eds., Current Protocols in
Molecular Biology,
John Wiley & Sons, New York, (1989). Broadly, any gene that encodes a
detectable product,
e.g., any product having detectable enzymatic activity or against which a
specific antibody can
be raised, can be used as a reporter gene in the present identification
method.
A currently preferred reporter gene system is the firefly luciferase reporter
system. Gould
et al., Anal. Biochem., 7:404-408 ( 1988), incorporated herein by reference.
The luciferase assay
is fast and sensitive. In this assay system, a lysate of the test cell is
prepared and combined with
ATP and the substrate luciferin. The encoded enzyme luciferase catalyzes a
rapid, ATP-
dependent oxidation of the substrate to generate a light-emitting product. The
total light output
is measured and is proportional to the amount of luciferase present over a
wide range of enzyme
concentrations. CAT is another frequently used reporter gene system; a major
advantage of this
system is that it has been an extensively validated and is widely accepted as
a measure of
promoter activity. Gorman et al., Mol. Cell. Biol., 2:1044-1051 (1982),
incorporated by
reference herein. In this system, test cells are transfected with CAT
expression vectors and
incubated with the candidate substance within 2-3 days of the initial
transfection. Thereafter, cell
extracts are prepared. The extracts are incubated with acetyl CoA and
radioactive
chloramphenicol. Following the incubation, acetylated chloramphenicol is
separated from
nonacetylated form by thin layer chromatography. In this assay, the degree of
acetylation
reflects the CAT gene activity with the particular promoter.
Another suitable reporter gene system is based on immunologic detection of
hGH. This
system is also quick and easy to use. Selden et al., Mol. Cell, Biol., 6:3173-
3179 (1986),
incorporated by reference herein. The hGH system is advantageous in that the
expressed hGH
polypeptide is assayed in the media, rather than in a cell extract. Thus, this
system does not
require the destruction of the test cells. It will be appreciated that the
principle of this reporter


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gene system. is not limited to hGH but rather adapted for use with any
polypeptide for which an
antibody of acceptable specificity is available or can be prepared.
A small molecule composition may upregulate a morphogen-activated pathway by
acting
at any one or more point. For example, small molecule potentiation of the
pathway may be
S initiated at the receptor level. Depending on the pathway, the transmembrane
receptors may be
Type I and/or Type II, or may be comprise variations on either Type I or Type
II receptors. For
example, OP-1 is capable of activating regulatory pathways comprising at least
two variations of
both Type I and Type II receptors (ActR-1 and BMPR-1B, and ActRII and BMPR-II,
respectively). A small molecule may stimulate the pathway by acting as a
ligand and binding to
any of the receptors, thereby inducing phosphorylation of Type I receptors
and/or Smad
molecules. Similarly, a small molecule may activate the regulatory pathway at
the level of the
serine/threonine kinase domain of the receptors, thereby stimulating
phosphorylation of Type I
receptors and/or Smad molecules.
As a further alternative, a small molecule may activate the regulatory pathway
at the level
of Smad complex formation. A small molecule may stimulate the formation of
Smad family
homodimers, heterodimers, or other homomeric or heteromeric complexes.
Furthermore, a small
molecule may activate the pathway by interacting with a Smad molecule or Smad
complex,
thereby altering its tertiary structure.
Alternatively, or in addition, a small molecule may activate the regulatory
pathway by
facilitating translocation of a Smad molecule or Smad complex or accumulation
of the Smad
molecule or Smad complex within the nucleus of the cell. By acting as a DNA
binding protein
or a transcriptional activator, a small molecule may activate the regulatory
pathway by increasing
transcriptional activity caused by the Smad molecule or Smad complex.
Furthermore, a small molecule can act to stimulate the regulatory pathway by
interfering
with an inhibitor of the pathway. For example, Smad6 and Smad7, which are
structurally
different than Smadl, Smad2, Smad3 and SmadS, act as inhibitors of certain
types of desirable
phenotype-specific protein expression (e.g., by activating TGF-(3 to induce
scar tissue
formation). Smad6 forms a stable association with Type I receptors and
interferes with the
phosphorylation of other Smad proteins, including Smad2 and Smad l, and their
subsequent


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heteromerization with Smad4. Smad7 also forms a stable association with
activated Type I
receptors and blocks access and phosphorylation of certain Smad molecules,
thereby preventing
formation of certain Smad heteromeric complexes. Smad7 also inhibits nuclear
accumulation of
Smad heteromeric complexes. A small molecule may interfere with the inhibitory
activity of
these Smad proteins by, for example, tightly binding to either one or both
proteins and rendering
either protein incapable of stable association with Type I receptors, or by
competitively binding
and stimulating the morphogen-activated transmembrane receptors.
Alternatively, a small
molecule may activate the inhibitory effects of these Smads in order to
inhibit an undesirable
effect (e.g., TGF(3 activity).
Dedifferentiation of a diseased, damaged, or aged soft tissue cells may result
from a
disturbance in one or more components of a morphogen-activated regulatory
pathway. The most
appropriate therapy will become evident by screening the intracellular
processes in the diseased,
damaged, or aged cell. Upon elucidation of the precise nature of the
disturbance in the pathway,
a small molecule composition can be designed to rectify or bypass the
disturbance, thereby
allowing normal expression of phenotype-specific gene products to resume.
Examples
illustrating useful embodiments of the invention follow.


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Examples
Renal Phenotype Restoration Using Morphogens
Morphogens are expressed in the kidney during development. For example, BMP-3
has
been shown to be expressed in developing human kidney. Vukicevic et al., J.
Histochem.
S Cytochem. 42: 869-875 (1994). Also, OP-1 (BMP-7) has been shown
immunohistochemically to
be associated with basement membranes in the convoluted tubules of kidneys of
human embryos.
Vukicevic et al., Biochem. Biophys. Res. Commun. 198: 693-700 ( 1994). In
addition,
morphogens are expressed in the adult kidney, and high levels of marine OP-1
expression have
been observed in adult mouse kidneys. Ozkaynak et al., Biochem. Biophys. Res.
Commun. 179:
116-123 (1991). Morphogens aid in the preservation of renal phenotype, inter
alia, by causing
expression of phenotype-specific genes. Methods for increasing expression of
such genes by
activating a morphogen-induced pathway for phenotype-specific gene expression
is addressed
below.
A rat partial (S/6) nephrectomy or rat remnant kidney model (RRKM) model is
employed
essentially as described in Vukicevic et al., J. Bone Mineral Res. 2: 533
(1987). Male rats (2-3
months old, weighing about 150-200 g) are subjected to unilateral nephrectomy
(either left or
right kidney). After approximately one week, 2/3 of the remaining kidney is
surgically removed.
Immediately following surgery, plasma creatinine and BUN levels rise
dramatically due to the
loss of renal mass and function. Over the next several weeks of this "acute"
failure phase,
plasma creatinine and BUN levels of surviving animals decline somewhat toward
normal values
but remain elevated. Renal function then appears to remain relatively constant
or stable for a
period of variable duration. After this point, the animals enter a period of
chronic renal failure in
which there is an essentially linear decline in renal function ending in
death. As surgical
controls, additional rats are subjected to a "sham" operation in which the
kidneys are
decapsulated but no renal tissue is removed.
Both nephrectomized and sham-operated rats are maintained for approximately 5-
6
months after surgery. At that point, surviving nephrectomized animals have
entered chronic
renal failure.


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Rats are divided into 8 groups with 12 rats in each group. Two groups of
nephrectomized
rats are used as controls (Nx controls), with one of those groups receiving no
treatment at all,
while the other receives injections of only the vehicle buffer. In addition,
two groups of
sham-operated rats are used as controls (sham controls), with one group
receiving only the
vehicle buffer, while the other receives a small molecule activator of Smad
complex formation.
Four experimental groups of nephrectomized rats are employed, receiving the
small molecule in
solution by intraperitoneal injection. Treated and vehicle-only rats receive
three injections per
week for 4-8 weeks. Total injection volume is approximately 300 ~1. It is
expected that no
statistically-significant differences are observed between the two control
groups or between the
two sham control groups.
Compared to the sham group receiving only vehicle, the Nx control receiving
only
vehicle is expected to demonstrate significantly (p < 0.01 ) elevated serum
creatinine at the end of
the study, indicating a significant Loss of renal function. Although
nephrectomized rats treated
with Smad complex-inducing small molecules should not show significantly
reduced serum
creatinine when compared to the Nx control, nephrectomized rats treated with
the small molecule
should show significant reductions in creatinine values. Similar results
should be observed for
serum urea levels. All nephrectomized rats are expected to show significantly
higher serum urea
when compared to the sham-operated rats.
Histological observations are expected to indicate that, in contrast to the
vehicle treated
Nx control group, OP-1 treated nephrectomized rats exhibit relatively normal
glomerular
histology. Histomorphometric analysis is expected to indicate that small
molecule Nx rats show
reduced incidence of glomerular sclerosis and loop collapse, relatively
scattered sclerosis and
microaneurysms, and more viable glomeruli compared to Nx control rats. It is
expected that
such effects are due to increased production of one or mare phenotype-specific
gene products
due to small molecule activation of Smad complex formation in the morphogen-
induced pathway
described above.


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Upregulation of Morphogen-Activated Regulatory Pathway by Small Molecule
Interference of Inhibitory Activity of MAP Kinases
Smadl, a mediator of the morphogen-activated phenotype-specific gene
regulatory
pathway, is also a target of mitogenic growth-factor signaling through
epidermal growth factor
and hepatocyte growth factor receptor protein tyrosine kinases (RTKs).
Kretzschmar et al.,
Nature 389:618-621 (1997). Phosphorylation occurs at specific serines within
the region linking
the inhibitory and effector domains of Smadl and is catalyzed by the Erk
family of mitogen-
activated protein kinases (MAP kinases). In contrast to the morphogen-
stimulated
phosphorylation of Smadl, which affects carboxy-terminal serines and induces
nuclear
accumulation of Smadl, Erk-mediated phosphorylation specifically inhibits the
nuclear
accumulation of Smad 1. Smad 1 receives opposing regulatory inputs through
RTKs and
morphogen receptor serine/threonine kinases. Thus, the Erk family of MAP
kinases function to
inhibit phenotype-specific protein expression which would ordinarily result
from the stimulation
of morphogen-activated regulatory pathways.
To interfere with the RTK competitive inhibition of morphogen-activated
regulatory
pathways, a small molecule composition is prepared. The small molecule
composition
comprises a mutant growth factor protein molecule. The mutant growth factor
protein molecule
is capable of binding to epidermal growth factor receptors and/or hepatocyte
growth factor
receptor but incapable of activating the tyrosine kinase. By binding growth
factor receptors and
restricting tyrosine kinase activity, the linker domain of Smadl molecules
remains
unphosphorylated. The carboxy-terminal domain serines of these Smadl molecules
are
phosphorylated by the morphogen receptor serine/threonine kinases, thereby
permitting the
Smad 1 molecule to participate in the regulatory pathway, to translocate into
the cell nucleus,
where it induces transcription of phenotype-specific genes.


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The stimulating effect of the small molecule composition is further enhanced
by the
addition of a morphogen to activate the morphogen Type I and Type II
receptors. Thus, the
small molecule composition acts simultaneously to increase phosphorylation of
Smadl and
formation of Smad 1 heteromeric complexes and to release inhibition of nuclear
accumulation of
Smadl heteromeric complexes.
Stimulation of Morphogen-Regulated Transcriptional Activity by Delivery of
Naked
Smadl mRNA into Hepatocytes
Smadl and other Smad proteins stimulate transcriptional activator sequences in
association with phenotype-specific genes. This activity is located in the
carboxy-terminal
domain and is unmasked upon removal of the amino-terminal domain. The
transcriptional
activity of Smadl can be stimulated by morphogen-receptor-mediated signals.
Overexpression
of Smadl sensitizes cells to endogenous morphogen signals, or exogenous
morphogen
stimulation and increases transcriptional activity within the cell.
Using essentially the method of Liu et al., Nature, 381: 620-623 (1997), the
teachings of
which are incorporated by reference herein, mRNA encoding at least the carboxy-
terminal
domain of Smadl protein is prepared. Following the general methods of U.S.
5,580,859, and
Budker et al., Gene Therapy 3(7): 593-598 (1997); the teachings of both of
which are
incorporated herein by reference, naked mIRNA encoding full length Smadl
protein and a
reporter gene, such as CAT, is placed in a hypertonic solution. The solution
is injected
intraportally in a mammalian liver having transiently occluded hepatic veins.
Expression of the
naked mRNA is verified at least 48 hours later using the appropriate reporter
gene assay as
described above.
Methods of Treatment, Routes of Administration, and Compositions for Treatment
Methods of treating soft tissue having lost normal cellular phenotype comprise
the step of
administering a composition capable of stimulating one or more aspects of the
morphogen-
activated expression pathway described above. Administration may be by any
compatible route.
Thus, as appropriate, administration may be directly to a local environment of
a diseased,
damaged, or aged tissue. Other contemplated routes of administration include
oral or parenteral,
including intravenous and intraperitoneal routes of administration. In
addition, administration


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may be by periodic injections of a bolus of a composition, or may be made more
continuous by
intravenous or intraperitoneal administration from a reservoir which is
external (e.g., an
intravenous bag) or internal (e.g., a bioerodabie implant, or a colony of
implanted,
morphogen-producing cells).
Therapeutic compositions contemplated by the present invention may be provided
to an
individual by any suitable means, directly (e.g., locally, as by injection,
implantation or topical
administration to a tissue locus) or systemically (e.g., parenterally or
orally). Where the
composition is to be provided parenterally, such as by intravenous,
subcutaneous, intramolecular,
ophthalmic, intraperitoneal, intramuscular, buccal, rectal, vaginal,
intraorbital, intracerebral,
intracranial, intraspinal, intraventricular, intrathecal, intracisternal,
intracapsular, intranasal or by
aerosol administration, the composition may comprise part of an aqueous or
physiologically
compatible fluid suspension or solution. Thus, the carrier or vehicle is
physiologically
acceptable so that, in addition to delivery of the desired composition to the
patient, it does not
otherwise adversely affect the patient's electrolyte and/or volume balance.
The fluid medium for
the agent thus can comprise normal physiologic saline (e.g., 9.85% aqueous
NaCI, O.15M, pH
7-7.4).
Useful solutions for parenteral administration may be prepared by any of the
methods
well known in the pharmaceutical art, described, for example, in REMINGTON'S
PHARMACEUTICAL SCIENCES (Gennaro, A., ed.; Mack Publ., 1990). Formulations of
the
therapeutic agents of the invention may include, for example, polyalkylene
glycols such as
polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and
the like.
Formulations for direct administration, in particular, may include glycerol
and other
compositions of high viscosity to help maintain the agent at the desired
locus. Biocompatible,
preferably bioresorbable, polymers, including, for example, hyaluronic acid,
collagen, tricalcium
phosphate, polybutyrate, lactide, and glycolide polymers and lactide/glycolide
copolymers, may
be useful excipients to control the release of the agent in vivo. Other
potentially useful parenteral
delivery systems for these agents include ethylene-vinyl acetate copolymer
particles, osmotic
pumps, implantable infusion systems, and liposomes. Formulations for
inhalation administration
contain as excipients, for example, lactose, or may be aqueous solutions
containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily
solutions for


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administration in the form of nasal drops, or as a gel to be applied
intranasally. Formulations for
parenteral administration may also include glycocholate for buccal
administration,
methoxysalicylate for rectal administration, or citric acid for vaginal
administration.
Suppositories for rectal administration may also be prepared by mixing the
molecule capable of
releasing morphogen inhibition (alone or in combination with a morphogen) with
a non-irritating
excipient such as cocoa butter or other compositions which are solid at room
temperature and
liquid at body temperatures.
Formulations for topical administration to the skin surface may be prepared by
dispersing
the molecule capable of releasing morphogen inhibition (alone or in
combination with a
morphogen) with a dermatologically acceptable carrier such as a lotion, cream,
ointment or soap.
Particularly useful are carriers capable of forming a film or layer over the
skin to localize
application and inhibit removal. For topical administration to internal tissue
surfaces, the agent
may be dispersed in a liquid tissue adhesive or other substance known to
enhance adsorption to a
tissue surface. For example, hydroxypropylcellulose or fibrinogen/thrombin
solutions may be
used to advantage. Alternatively, tissue-coating solutions, such as pectin-
containing
formulations may be used.
Where the composition is intended for use as a therapeutic for disorders of
the CNS, an
additional problem must be addressed: overcoming the blood-brain barrier, the
brain capillary
wall structure that effectively screens out all but selected categories of
substances present in the
blood, preventing their passage into the brain. The blood-brain barrier can be
bypassed
effectively by direct infusion of the molecule capable of releasing morphogen
inhibition (alone
or in combination with a morphogen) into the brain, or by intranasal
administration or inhalation
of formulations suitable for uptake and retrograde transport by olfactory
neurons.
Modulation of Apoptosis
Modulation of a cellular pathway that controls expression of a phenotype-
specific gene
has been demonstrated in the Drosophila melanogaster model. A member of the
BMP
subfamily, DPP, plays an important role during Drosophida development to
establish the
patterning of the dorsal ectoderm, regulate gut morphogenesis, and regulate
the growth of
imaginal discs such as wings and eyes. The Drosophila inhibitor of apoptosis 1
(DIAP 1 ) protein


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in Drosophila is an interaction protein of a DPP Type I receptor, Thick veins
(Tkv). DIAP 1 is a
homolog of the baculovirus inhibitor of apoptosis (IAP) protein, also an
inhibitor of apoptosis.
While similar homologs have been reported in virus (OpIAP, VpIAP), mouse
(MIHA,
mc-IAP-1), and human (XIAP/nILP, MIHB/c-IAP1/hIAP2, MICH/c-IAP1/hIAPI, NAPI),
Drosophila was used as a model system herein for exemplification. Results
similar to those
shown below are expected in humans and other mammals.
IAPs share conserved regions, including two or three baculovirus IAP repeat
(BIR)
domains in their N-terminal region, and one RING finger domain in their C-
terminal region.
IAPs are able to prevent cell apoptosis induced by interleukin-1 (3 converting
enzyme (ICE), a
capase family protease. The DIAP1 protein associates with Tkv and others
(e.g., tumor necrosis
factor receptor associated factor (TRAF) 1 and 2) through the C-terminal RING
finger domain of
DIAP 1.
Apoptosis in Drosophila is under control of several genetic elements. Included
in these
are the doom gene, which induces apoptosis in insect cells. Doom is localized
in the nucleus, as
are its binding proteins. Also involved in apoptosis is Reaper, a 65 amino
acid polypeptide. The
DIAP2 protein prevents Reaper-induced cell death by binding the BIR domain of
DIAP2.
Reaper is localized in the cytoplasm, and accumulates in perinuclear locations
when the IAPs are
present.
Plasmid Construction
The bait plasmid, pEG-Tkv, encoding the fusion protein of the LexA DNA binding
domain and the cytoplasmic region of Tkv was constructed as follows. The
cytoplasmic region
of Tkv was amplified by polymerase chain reaction (PCR) from the full length
clone, Brk25D2
and inserted between the EcoRI and XhoI sites of pEG202. The prey plasmid of
Tkv, pJG-Tkv,
was constructed by inserting the EcoRI-XhoI fragment into pJG4-S. Tkv mutants
were
constructed by site-directed mutagenesis using the Chameleon mutagenesis kit
(Stratagene). Tkv
(OJM) lacks the juxtamembrane region (amino acids 205-254) of the wild type
Tkv. Tkv
(Q253D) and Tkv {K281R) have aspartic acid instead of glutamine 253, and
arginine instead of
lysine 281, respectively. pcDNA3-HA was made by inserting an annealed
oligonucleotide
between the XhoI and XbaI sites of pcDNA3 (Invitrogen). pcDNA3-FLAG was
described in


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Okadome et al., J. Biol. Chem, 271: 21687-21690 (1996). The whole coding
region of Tkv was
amplified by PCR. An EcoRI and an XhoI site was added before the starting
codon and in place
of the stop codon, followed by insertion between the EcoRI and XhoI sites of
pcDNA3-HA. The
internal EcoRI site of Tkv was removed by site-directed mutagenesis.
The yeast expression plasmids of DIAP 1 were made by subcloning the EcoRI-XhoI
fragment amplified by PCR into pEG202 and pJG4-5. The coding region of DIAP1,
without the
stop codon, was subcloned between the EcoRI and XhoI sites of pcDNA-FLAG,
yielding
FLAG-tagged DIAP 1. The DIAP2 plasmids were constructed in a similar manner to
DIAP 1.
Screening and Interaction Assay
To search for proteins that interact with Tkv, the interaction trap screen was
used,
essentially as described by Gyuris et al., Cell, 75: 791-803 (1993); Kawabata
et al., J. Biol.
Chem, 270: 29628-29631 (1995), both incorporated herein by reference. Briefly,
a Drosophila
imaginal disc cDNA library was screened with the cytoplasmic region of Tlcv as
a bait. The
yeast strain, EGY48, was transformed with the reporter, pSHl8-34, and pEG-
Tlcv. The cDNA
library was then introduced into EGY48. The transfolmants were grown on
appropriate selection
media, and positive clones were selected depending on (3-galactosidase
activity and leucine
prototrophy. Library plasmids were rescued from EGY48, amplified in bacteria,
and sequenced.
Interaction assays using the interaction trap were done as described before.
Kawabata et al., J.
Biol. Chem, 270: 29628-29631 ( 1995).
Cloning of the Full Length DIAPI
One of the positive clones contained a partial C-terminal region of DIAP 1
(Figure 3).
PCR was performed under standard conditions to amplify the missing N-terminal
region from a
Drosophila 4-8 hour embryo cDNA library in the pNB40 vector. The full coding
region of
DIAP1 was made by ligating the EcoRI-BanII fragment obtained from PCR and the
BanII-XhoI
fragment obtained from the interaction trap screen.
Protein Interaction in vivo
COS-7 cells were maintained in Dulbecco's modified Eagle's medium containing
10%
fetal bovine serum, 100 units/ml penicillin, and 4.5 g/liter glucose. Cells
were transiently


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transfected using DMRIE-C (GibcoBRL) with 10 ~g of plasmids. After two days,
cells were
labeled with 22.8 mCi/ml [35S]methionine and cysteine mixture {Amersham) for 5
hours, and
lysed in 150 mM NaCI, 20 mM Tris-HCI, pH 7.5, and 1% Triton X-100 containing
1.5% of
aprotinin. Cleared lysates were divide into two tubes and incubated with anti-
FLAG M2
(Eastman Kodak) or anti-HA 12CA5 (Boehringer Mannheim) monoclonal antibodies.
Immune
complexes were bound to protein G-Sepharose (Pharmacia) or protein A-Sepharose
(Pharmacia).
The precipitates were washed and subjected to SDS-polyacrylamide gel
electrophoresis
(SDS-PAGE) (8.5% or 10% gel) and analyzed by fluorography or with Fuji BAS
2000
Bio-Imaging Analyzer (Fuji Photo Film).
Results
The interaction trap screen was used to search for proteins that interact with
Tkv. A
Drosophila imaginal disc cDNA library was screened with the cytoplasmic region
of Tkv as a
bait. Of the one hundred and sixty thousand transformants screened, four
positive clones were
isolated. One clone encoded Drosophila FKBP12, a homolog of human FKBP12 which
is
known as a binding protein for mammalian Type I receptors, including T(3R-I.
Another clone
encoded a partial C-terminal region of DIAP 1 (PC 1 ), a homolog of
baculovirus IAP. See Figure
3. The remaining two positive clones were not analyzed.
PCR using a Drosophila 4-8 hour embryo cDNA library was performed to obtain
the
missing N-terminal region. The interaction of the full length DIAP 1 with Tkv
was examined
using the interaction trap. As summarized in Figure 4, DIAP1 strongly
interacted with the wild
type Tkv, although its interaction was slightly weaker than that of the
partial clone, PC1.
Mutants of Tkv with different signaling activities were also tested for the
interaction with
DIAP1. One Tkv mutant replacing glutamine 253 with aspartic acid (QD),
reported to have a
constitutively kinase activity, showed strong interaction with DIAP 1 as well
as with PC 1.
Another mutant replacing lysine 281 with arginine (KR), which is expected to
lack the kinase
activity, showed weak interaction with DIAP 1 and PC 1. Also tested was a
deletion mutant
lacking the juxtamembrane region {amino acids 205-254) at the Type II receptor
transphosphyorylation sites. The mutant, Tkv (~JM), did not interact with PC1
or DIAP1.
Contrary to Tkv, Saxophone (Sax), another DPP Type I receptor or Punt, a Type
II receptor for


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DPP, did not interact with PC 1 or DIAP 1. Thus, DIAP 1 specifically
interacted with Tkv in the
yeast system.
DIAP2, a second inhibitor of apoptosis in Drosophila, was also tested for
interactions
with Tkv. As depicted in Figure 3, DIAP2 has three BIR domains, while DIAP1
has two BIR
domains. DIAP2 did not show interaction with the wild type or mutants of Tkv,
Sax, or Punt in
the yeast system.
Various truncated forms (TF) of DIAP 1 were constructed to identify the
interacting
region of DIAP1 with Tkv. See Figure 3. The wild type Tkv only interacted with
TF4, but not
with the other truncated forms. See Figure 5. Since TF4 only has the RING
finger domain, the
interacting region is mapped to the RING finger domain in DIAP 1. Sax and Punt
did not interact
with TF4.
The interaction between DIAP 1 and Tkv in vivo were also examined. DIAP 1 was
epitope-tagged with FLAG at the C-terminus (DIAP1-FLAG). HA-tagged Tkv and/or
DIAP1-FLAG were transiently expressed in COS-7 cells. Labeled lysates were
immunoprecipitated with anti-HA or anti-FLAG monoclonal antibodies and then
subjected to
SDS-PAGE. Each antibody specifically recognized Tkv-HA and DIAP1-FLAG,
respectively.
Anti-FLAT antibody only coprecipitated Tkv when DIAP1 was expressed,
demonstrating that
DIAP 1 interacts with Tkv in vivo. Both constitutively active (QD) and kinase-
inactive (KR)
mutants interacted with DIAP 1 as efficiently as the wild type Tkv.
The interaction of DIAP2 with Tkv in vivo were also tested. Tkv-HA and/or
DIAP2-FLAG were transiently transfected in COS-7 cells. Although the
interaction of DIAP2
with Tkv was not detected in the yeast assay (Figure 4), DIAP2 coprecipitated
with the wild type
Tkv and also with the QD and KR mutants in vivo.
Expression plasmids of the BIR domain (BIR-FLAG) and C-terminal region of DIAP
1
(PC1-FLAG) were constructed (Figure 6) in order to determine the region of
DIAP1 required for
the interaction with Tkv in vivo. Tkv-HA was coexpressed with BIR-FLAG or PC1-
FLAG.
Tkv-HA was detected in a stable complex with PC1-FLAG but not with BIR-FLAG.
Consistent
with the results in the yeast assay, these results indicate that the
interaction region of DIAP 1 is
the C-terminus, which contains the RING finger domain.


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Thus, Tkv induces apoptosis by suppressing DIAP 1 function.
Transfection of Mesenchymal Cells with Smad Proteins and Activation of
Transfected Cells with BMPs
Biological effects of different Smad Proteins were examined in C2C 12
undifferentiated
mesenchymal cells using adenovirus-based vector system. Pathway-restricted
Smads (R-Smads)
activated by BMP receptors, such as Smadl and SmadS, induced the production of
alkaline
phosphatase in C2C 12 cells, whereas the R-Smads activated by TGF-~i/activin
pathway (Smad2
and Smad3) did not. Addition of BMP-6 dramatically enhanced the production of
alkaline
phosphatase induced by Smadl or 5, which may be due to the nuclear
translocation of R-Smads
induced by BMP-6. BMP Type I receptors such as ALK-3, ALK-6, and ALK-2, which
are
known to activate Smadl and 5, also induced the production of alkaline
phosphatase, in these
cells. Anti-Smads, i. e., Smad6 and Smad7, inhibited the production of
alkaline phosphatase
induced by Smads l and 5. R-Smads activated by BMP receptors were detected in
the cytoplasm
in the presence of Smad6 or Smad7. Thus, osteoblast-differentiation induced by
BMPs is mainly
mediated by R-Smads activated by BMPs, and the effect of R-Smads can be
interfered with by
anti- Smads.
The experimental procedures included plasmid construction, cell culture and
infection of
adenovirus, immunoblotting, assays for alkaline phosphatase and osteocalcin
production,
analysis for chondrogenesis, and subcellular localization of the Smad
proteins. All of these
procedures are well known in the art.
Results
D fferentiation Induction of C2C12 Cells into Osteoblasts by Smadl and SmadS--
C2C12
undifferentiated mesenchymal cells differentiate into osteoblast-like cells by
the treatment of
BMP-2, BMP-4, and OP-IBMP-7. The C2C12 cells were transfected with different
DNAs by
using the adenovirus-based vector, pAxCAwt. Transfection efficiency was very
high when
determined by staining of the cells by LacZ. Plasmids including cDNAs for
different Smads
were constructed and transfected into C2C12 cells. Expression of FLAG-epitope
tagged Smads
were analyzed by immunoblotting using anti-FLAG antibody, and production of
alkaline


CA 02314821 2000-06-14
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phosphatase was determined by staining of the cells after 6 days. Smadl, SmadS
and Smad4 are
highly expressed in the C2C 12 cells.
When the cells were stained for the production of alkaline phosphatase, the
cells transfected
with SmadS or Smadl were positively stained at high m.o.i. In contrast, Smad2
and Smad3,
S which are activated by TGF-(3 and activin receptors, did not induce the
synthesis of alkaline
phosphatase.
Smad4, the common-mediator Smad in mammals, did not induce the alkaline
phosphatase production when infected alone; however, co-infection of Smad4
into Smadi/5-
infected cells potentiated the effect of BMP-activated Smads. Smad4 weakly
induced the
production of alkaline phosphatase in the Smad3-infected C2C12 cells, thus,
Smad3 weakly, but
significantly, activates the transcription of alkaline phosphatase gene in the
presence of Smad4.
BMP-6 is structurally most similar to OP-1BMP-7. Two-hundred ng/ml of BMP-6
efficiently induced the differentiation of C2C 12 cells in osteoblasts. When
the C2C 12 cells were
infected with Smad 1 or SmadS, and treated with 200 ng/ml of BMP-6, production
of alkaline
phosphatase was dramatically enhanced. Smad2, however, did not facilitate the
production of
alkaline phosphatase.
D fferentiation Induction of C2C12 Cells by BMP Type I Receptors- Among the
seven
different Type I receptors in mammals (ALK-1 through 7), ALK-3 and ALK-6
function as
specific BMP Type I receptors. ALK-2 also binds OP-1BMP-7 and BMP6 and
functions as a
BMP Type I receptor. Constitutively active forms of HA-tagged ALK plasmids
were infected
into C2C12 cells and production of alkaline phosphatase was examined. ALK-
3(QD) and ALK-
6(QD), as well as ALK-2(QD) strongly induced the synthesis of alkaline
phosphatase. ALK-1
(QD), most similar structurally to ALK-2, also induced the production of
alkaline phosphatase.
In contrast, ALK-4(TD), ALK-5(TD) and ALK-7(TD) did not induce the alkaline
phosphatase
activity in the cells, indicating that TGF-~3 or activin receptors do not
efficiently induce the
osteoblast differentiation of C2C 12 cells..
Nuclear Translocation of Smads Induces Alkaline Phosphatase Synthesis in C2C12
Cells- Subcellular localization of Smad was determined by indirect
immunofluorescence staining
of the cells using the anti-FLAG antibody to SmadS. SmadS was observed mainly
in the


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cytoplasm. Treatment of the cells with SmadS and ALK-2(QD) with or without BMP-
6 strongly
induced the nuclear translocation of Smad4. ALK-3(QD) and ALK-6(QD) also
induced the
nuclear translocation of SmadS. Overexpression of SmadS does not result in the
nuclear
accumulation of Smalls, although small amounts of SmadS may spontaneously
translocate into
the nucleus. Potentiation of the effect of BMP-6 is thus induced by nuclear
translocation of
SmadS.
Anti-Smalls Block the Differentiation of C2C12 Cells into Osteoblasts Induced
by BMPs-
Smad6 and Smad7 inhibit the transcriptional activity of R-Smalls when assayed
using p3TP-lux
promoter and cyclin A promoter. It was also shown that Smad6 and Smad7 in
mammals as well
as Xenopus prevent the BMP activity in Xenopus embryo assays. However, the
effects of anti-
Smalls on differentiation of osteoblasts have not been examined. C2C 12 cells
were transfected
with Smad6 or Smad7 and treated with BMP-6. Expression of Smad6 and Smad7
correlated
with m.o.i. when determined by immunoblotting. Synthesis of alkaline
phosphatase was induced
by BMP-6, which was not affected by a control plasmid expressing LacZ. Both
Smad6 and
Smad7 inhibited the production of alkaline phosphatase induced by BMP-6.
When the ALK-3(QD), ALK-6(QD), or ALK-2(QD) were infected into C2C12 cells,
alkaline phosphatase was dramatically induced. Co-infection of Smad6 or Smad7
prevented the
differentiation of the cells into osteoblasts depending on the expression of
proteins.
Both Smadd and Smad7 were observed throughout the cells, although Smad6 was
observed more in the cytoplasm whereas Smad7 was detected more in the nucleus.
SmadS
translocated into the nucleus by the stimulation of ALK-3(QD) or ALK6(QD).
However, co-
infection of Smad6 or Smad7 blocked the nuclear translocation of SmadS, and
the protein was
observed only in the cytoplasm. When the cells were stained with anti-SmadS
antibody, Smad7
completely blocked nuclear translocation of SmadS, whereas in the Smad6-
infected cell, SmadS
was weakly stained in the nucleus, although alkaline phosphatase synthesis was
inhibited as in
the Smad7-infected cells. Thus, Smad6 and Smad7 prevent the nuclear
translocation of SmadS
and thereby inhibit the differentiation of C2C 12 cells into osteoblast-like
cells.
Additional aspects and embodiments of the invention are apparent to the
skilled artisan.


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-1-
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: SAMPATH, K. T.
COHEN, C.
EIICHI, O.
KOHEI, M.
KAViABATA, M.
(ii) TITLE OF INVENTION: METHODS FOR MAINTAINING OR RESTORING TISSUE-
APPROPRIATE PHENOTYPE
OF SOFT TISSUE CELLS
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CA 02314821 2000-06-14
' WO 99/31136 PCTNS98/26788
-2-
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
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(A) NAME/KEY: misc feature
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PROMOTER"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: l:
TCGATCCTAA AACACTTAAG GATATTTCTG TAAGGCTGTG AAAGAGAAAA CCAACTACTT 60
ACACGGATGG AGACCATGTT TATTTCTTTG GGAGAAAAGC CTAATTGGGA CGCTTCGAGA 120
TCCCTATAGG AAATTGCACC AGTAGTCAAC TGGATTTTTA AAAGGCAAAG CTTGAGGATT 180
TTTTTTTCCC TTTGAAATGA ATGTAGCAAA CTTATGTAAG CACGGAATAG GATTATTAGT 240
TAACAGTCTT TTCAATTATA TGGGAAAATG AAAACTAGGG GAGCGTCTAA GGCCACTTGC 300
TGACCTTTGT GCAGCTGTTA AGTAAAGAAA GTAAACCCTC CAGGGATACT GAACAGCCAA 360
CTGTCATAAG TCCAGGGTGT CTTGCACTTG CTGTGACAAG TTTAAAATAT TTAATATGAC 420
TATACCTGAA ATATTTAATG CTATCTTTTT CATGCACCAG CTTCTAAGAG CTTTCCCTAA 480
AATCCTGATA TGCAAAAGAA TATACCAATA TTTTCCCCCT TGCCCCTGGC GCTTGTCTCC 540
CAAGTTAGCA AACACTTAGCi TAAGCGATTT TTACAGAACT TTTTTCCCTA ATAACTGAAG 600
GACTAACATG ATGATTTAGA TCTATATTCT CCCCAAAAGG CGTCTCATAT TTTTGTATAT 660
TACCAAATAT TTTCAGTCAA ATAACACAAG AATGTATTTT AAAAATAAAA AGGGTGAATC 720
ATCATTCCAT CATGAACCAA CATTGGACTC AGAACTCCTA AAAGGAAAAC AGAAAAAAAA 780
AAAAAATCAT GCACAGCCGA AGCTATTART ATATAATGGA GACAAAGAGT TTATTTTTCA 840
ATGAGAATAA CAAGCiAAAAA AGCCTGATTT TGTACGCCTG CCCGTTAGGA CTTCCCACCA 900
TAATTAGTGC TTCTTGCCCC TGAGAGGAGG AGCTTCGGCT CAGGGGAACT TCATGCAATA 960
AGGGAAGAAA ACAGTATAAA TACTCCAGGG CAGCCGTGGG GAAGGCATTA TCCACTGCTC 1020

CA 02314821 2000-06-14
WO 99/31136 PCT/US98/26788
-3-
CTGGGCAGAG GAAGCCAGGA AAGCTGCCCC ACGCATCTCC CAGCACC 1067
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ixy FEATURE:
(A) NAME/KEY: miac feature
(B) LOCATION: 1..21
(D) OTHER INFORMATION: /product= "AP1 SEQUENCE A°
(xi) SEQUBNCE DESCRIPTION: SEQ ID N0:2:
CGCTTGATGA CTCAGCCGGA A 21
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomiC)
(ix) FEATURE:
(A) NAME/KEY: miac feature
(8) LOCATION: 1..10
(D) OTHER INFORMATION: /product= "AP1 SEQUENCE A MUTATION"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
TTCCTCATCA 10
(2) INFORMATION FOR SEQ ID N0:4:
(iy SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid

CA 02314821 2000-06-14
WO 99/31136 _4_ PCT/US98126788
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(1x) FEATURE:
(A) NAME/KEY: Peptide
IB) LOCATION: 1..25
(D) OTHER INFORMATION: /note= "Conserved domain of human
c-fos"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Lys Val Glu Gln Leu Ser Pro Glu Glu Glu Glu Lys Arg Arg Ile Arg
1 5 10 15
Arg Ile Arg Asn Lys Met Ala Ala Ala
20 25
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: miac feature
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /product= "AP-1 CONSENSUS SEQUENCE
Bn
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GTGACTCAGC GCGGA 15
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear

CA 02314821 2000-06-14
WO 99/31136 PCTNS98/26788
-S- -
(ix) FEATURE:
(A) NAME/KEY: miac feature
(B) LOCATION: 1..11
(D) OTHER INFORMATION: /product= "MEF-2 CONSENSUS"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CTAAAAATAA C 11
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1822 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSH: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(F) TISSUE TYPE: HIPPOCAMPUS
(ix) FEATURE:
(A) NAME/KEY: CDS
(8) LOCATION: 49..1341
(C) IDENTIFICATION METHOD: experimental
(D) OTHER INFORMATION: /function= "OSTHOCiBNIC PROTEIN"
/product= "OP1"
/evidence= EXPERIMENTAL
/standard name= "OP1"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57
Met H19 Val
1
CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TTC GTG GCG CTC TGG GCA 105
Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala

CA 02314821 2000-06-14
WO 99/31136 PCT/US98/26788
-6-
s l0 15
CCC CTG TTC CTG CTG CGC TCC GCC CTG GCC GAC TTC AGC CTG GAC AAC 153
Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Aap Aan
20 25 30 35
GAG GTG CAC TCG AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG 201
Glu Val Hia Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu Arg
40 45 50
COG GAG ATG CAG CGC GAG ATC CTC TCC ATT TTG GGC TTG CCC CAC CGC 249
Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg
55 60 65
CCG CGC CCG CAC CTC CAG GGC AAG CAC AAC TCG GCA CCC ATG TTC ATG 297
Pro Arg Pro His Leu Gln Gly Lys Hia Asn Ser Ala Pro Met Phe Met
70 75 80
CTG GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG GGC GGC GGG CCC GGC 345
Leu Asp Leu Tyr Aan Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly
85 90 95
GGC CAG GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC 393
Gly Gln Gly Phe Ser Tyr Pro Tyr Lya Ala Val Phe Ser Thr Gln Gly
100 105 110 115
CCC CCT CTG GCC AGC CTG CAA GAT AGC CAT TTC CTC ACC GAC GCC GAC 441
Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Aap Ala Asp
120 125 130
ATG GTC ATG AGC TTC GTC AAC CTC GTG GAA CAT GAC AAG GAA TTC TTC 489
Met Val Met Ser Phe Val Asn Leu Val Glu His Aap Lys Glu Phe Phe
i35 140 145
CAC CCA CGC TAC CAC CAT CGA GAG TTC CGG TTT GAT CTT TCC AAG ATC 537
His Pro Arg Tyr His His Arg Glu Phe Arg Phe Aap Leu Ser Lys Ile
150 155 160
CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TTC CGG ATC TAC AAG GAC 585
Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp
165 170 175
TAC ATC CGG GAA CGC TTC GAC AAT GAG ACG TTC CGG ATC AGC GTT TAT 633
Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile Ser Val Tyr
180 185 190 195
CAG GTG CTC CAG GAG CAC TTG GGC AGG GAA TCG GAT CTC TTC CTG CTC 681
Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu Phe Leu Leu
200 205 210

CA 02314821 2000-06-14
WO 99/31136 PCT/US98/26788
GAC AGC CGT ACC CTC TGG GCC TCG GAG GAG GGC TGG CTG GTG TTT GAC 729
Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp
215 220 225
ATC ACA GCC ACC AGC AAC CAC TGG GTG GTC AAT CCG CGG CAC AAC CTG 777
Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu
230 235 240
GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GGG CAG AGC ATC AAC CCC 825
Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile Asn Pro
245 250 255
AAG TTG GCG GGC CTG ATT GGG CGG CAC GGG CCC CAG AAC AAG CAG CCC 873
Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys Gln Pro
260 265 270 275
TTC ATG GTG GCT TTC TTC AAG GCC ACG GAG GTC CAC TTC CGC AGC ATC 921
Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser Ile
280 285 290
CGG TCC ACG GGG AGC AAA CAG CGC AGC CAG AAC CGC TCC AAG ACG CCC 969
Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro
295 300 305
AAG AAC CAG GAA GCC CTG CGG ATG GCC AAC GTG GCA GAG AAC AGC AGC 1017
Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser
310 315 320
AGC GAC CAG AGG CAG GCC TGT AAG AAG CAC GAG CTG TAT GTC AGC TTC 1065
Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe
325 330 335
CGA GAC CTG GGC TGG CAG GAC TGG ATC ATC GCG CCT GAA GCiC TAC GCC 1113
Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala
340 345 350 355
GCC TAC TAC TGT GAG GGG GAG TGT GCC TTC CCT CTG AAC TCC TAC ATG 1161
Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Aan Ser Tyr Met
360 365 370
AAC GCC ACC AAC CAC GCC ATC GTG CAG ACG CTG GTC CAC TTC ATC AAC 1209
Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val H1s Phe Ile Asn
375 380 385
CCG GAA ACG GTG CCC AAG CCC TGC TGT GCG CCC ACG CAG CTC AAT GCC 1257
Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala
390 395 400

CA 02314821 2000-06-14
WO 99/31136 PCT/US98/26788
_g_
ATC TCC GTC CTC TAC TTC GAT GAC AGC TCC AAC GTC ATC CTG AAG AAA 1305
Ile Ser Val Leu Tyr Phe Aap Asp Ser Ser Asn Val Ile Leu Lys Lys
405 410 415
TAC AGA AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCCTCC 1351
Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His
420 425 430
GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CC1TGGCCAG 1411
GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAAGG 1471
TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531
ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591
GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGCiGAA GTCTCAGCCA TGCACGGACT 1651
CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGAAGGG 1711
GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CCGGAAGTTC 1771
CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1822

Representative Drawing

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Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 1998-12-16
(87) PCT Publication Date 1999-06-24
(85) National Entry 2000-06-14
Examination Requested 2003-11-07
Dead Application 2015-07-10

Abandonment History

Abandonment Date Reason Reinstatement Date
2014-07-10 R30(2) - Failure to Respond

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $300.00 2000-06-14
Registration of a document - section 124 $100.00 2000-12-08
Maintenance Fee - Application - New Act 2 2000-12-18 $100.00 2000-12-08
Maintenance Fee - Application - New Act 3 2001-12-17 $100.00 2001-12-03
Maintenance Fee - Application - New Act 4 2002-12-16 $100.00 2002-12-05
Registration of a document - section 124 $100.00 2003-05-27
Registration of a document - section 124 $100.00 2003-05-27
Registration of a document - section 124 $100.00 2003-05-27
Registration of a document - section 124 $100.00 2003-05-27
Registration of a document - section 124 $100.00 2003-05-27
Request for Examination $400.00 2003-11-07
Maintenance Fee - Application - New Act 5 2003-12-16 $150.00 2003-12-08
Maintenance Fee - Application - New Act 6 2004-12-16 $200.00 2004-12-03
Maintenance Fee - Application - New Act 7 2005-12-16 $200.00 2005-12-12
Maintenance Fee - Application - New Act 8 2006-12-18 $200.00 2006-12-11
Maintenance Fee - Application - New Act 9 2007-12-17 $200.00 2007-12-13
Maintenance Fee - Application - New Act 10 2008-12-16 $250.00 2008-12-05
Registration of a document - section 124 $100.00 2009-03-13
Maintenance Fee - Application - New Act 11 2009-12-16 $250.00 2009-12-07
Maintenance Fee - Application - New Act 12 2010-12-16 $250.00 2010-12-07
Maintenance Fee - Application - New Act 13 2011-12-16 $250.00 2011-12-13
Maintenance Fee - Application - New Act 14 2012-12-17 $250.00 2012-12-05
Maintenance Fee - Application - New Act 15 2013-12-16 $450.00 2013-12-05
Registration of a document - section 124 $100.00 2014-09-15
Maintenance Fee - Application - New Act 16 2014-12-16 $450.00 2014-11-24
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
MARIEL THERAPEUTICS, INC.
Past Owners on Record
COHEN, CHARLES M.
CREATIVE BIOMOLECULES, INC.
CURIS, INC.
KAWABATA, MASAHIRO
MIYAZONO, KOHEI
OEDA, EIICHI
SAMPATH, KUBER T.
STRYKER CORPORATION
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Claims 2010-03-25 1 40
Description 2000-12-07 41 2,297
Description 2000-06-14 41 2,303
Abstract 2000-06-14 1 46
Claims 2000-06-14 2 73
Drawings 2000-06-14 5 98
Cover Page 2000-10-02 1 38
Claims 2004-03-09 5 148
Description 2007-04-02 41 2,232
Claims 2007-04-02 2 57
Claims 2011-11-22 1 38
Claims 2013-04-17 2 55
Correspondence 2000-09-15 1 3
Assignment 2000-06-14 3 111
PCT 2000-06-14 10 461
Prosecution-Amendment 2000-09-08 1 46
Assignment 2000-12-08 4 116
Correspondence 2000-12-07 9 238
Correspondence 2001-01-17 1 2
Correspondence 2001-09-18 1 49
Correspondence 2001-10-30 1 14
Correspondence 2002-09-18 1 39
Correspondence 2002-10-30 1 16
Assignment 2003-05-27 21 914
Prosecution-Amendment 2003-11-07 1 37
Prosecution-Amendment 2004-03-09 5 126
Prosecution-Amendment 2006-10-02 4 172
Prosecution-Amendment 2007-04-02 20 1,019
Fees 2007-12-13 1 45
Assignment 2009-03-13 16 789
Prosecution-Amendment 2009-10-06 4 154
Prosecution-Amendment 2010-03-25 7 328
Prosecution-Amendment 2011-05-25 3 133
Prosecution-Amendment 2011-11-22 6 241
Prosecution-Amendment 2012-10-18 6 310
Fees 2012-12-05 1 163
Prosecution-Amendment 2013-04-17 9 400
Assignment 2014-09-15 28 1,439
Prosecution-Amendment 2014-01-10 7 359

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