Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
WA545 COMPOSITIONS
The present invention relates to a novel family of purified proteins
designated
WA545 and WA545-related proteins, DNA encoding them, and processes for
obtaining them. These proteins may be used to induce bone and/or cartilage or
other
connective tissue formation, and in wound healing and tissue repair. These
proteins
may also be used for augmenting the activity of bone morphogenetic proteins.
BACKGROUND OF THE INVENTION
The search for the molecule or molecules responsible for the bone, cartilage,
and other connective tissue-inductive activity present in bone and other
tissue extracts
has led to the discovery of a novel set of molecules called the Bone
Morphogenetic
Proteins (BMPs). The structures of several proteins, designated BMP-1 through
BMP-16, have previously been elucidated. The unique inductive activities of
these
proteins, along with their presence in bone, suggests that they are important
regulators
of bone repair processes, and may be involved in the normal maintenance of
bone
tissue. There is a need to identify whether additional proteins exist which
play a role
in these processes. The present invention relates to the identification of
such a
protein, which the inventors have designated WA545.
SUMMARY OF THE INVENTION
As used herein, the term "WA545-related protein" refers to the Xenopus
WA545 protein, having the amino acid sequence specified in SEQUENCE >D N0:2,
as well as homologues of this protein found in mammalian and other species;
and
other proteins which are closely related structurally and/or functionally to
WA545.
Examples of "WA545-related proteins" include murine, bovine and human WA545
protein, as well as homologues in other species, particularly human.
As used herein, the term "WA545 activity" refers to one or more of the
activities which are exhibited by the WA545 proteins of the present invention.
In
particular, "WA545 activity" includes the ability to induce, enhance and/or
inhibit the
formation, growth, proliferation, differentiation, maintenance of mesodermal
tissue,
including but not limited to neurons and/or related neural cells and tissues
such as
brain cells, Schwann cells, glial cells and astrocytes, as well as muscle
cells and
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tissues. "WA545 activity" also includes the ability to induce molecular
markers of
mesodermal tissue, such as Xbra, gsc, brachyury, Pintallavis, Xnot and muscle-
actin
as well as the ability to induce the formation of neurons and/or related
neural cells and
tissues such as brain cells, Schwann cells, glial cells and astrocytes. "WA545
activity"
may also include the ability to regulate the interaction ligands and their
protein
receptors. "WA545 activity" may further include the ability to regulate the
formation,
differentiation, proliferation and/or maintenance of other cells and/or
tissue, for
example connective tissue, organs and wound healing. In particular, "WA545
activity" may include the ability to enhance and/or inhibit angiogenesis, and
formation
and growth of capillaries, arteries and other blood vesssels, as well the
formation,
growth, proliferation, differentiation and/or maintenance of cardiac, spleen,
liver,
pancreas, stomach, kidney, lung and brain cells and tissue, osteoblasts and
bone,
chondrocytes and cartilage, tendon and epidermis. "WA545 activity" also
includes the
activities of WA545 protein in the described in the examples and specification
herein.
Xenopus WA545
The Xenopus WA545 DNA sequence (SEQ )D NO: 1 ) and amino acid
sequence (SEQ m NO: 2) are set forth in the Sequence Listings. WA545 proteins
are
capable of inducing the formation of cartilage, muscle, nerve, epidermis or
other
connective tissue, or combinations thereof. WA545 proteins may be further
characterized by the ability to demonstrate cartilage, bone, muscle, nerve,
epidermis
and/or other connective tissue formation activity in the assays described
below.
Xenopus WA545 protein may be produced by cuhuring a cell transformed with
a DNA sequence comprising nucleotide a DNA sequence encoding the mature WA545
polypeptide, comprising nucleotide #55, #775, or #811 to nucleotide #1113 or
#1116
as shown in SEQ >D NO: 1, and recovering and purifying from the culture medium
a protein characterized by the amino acid sequence comprising amino acids #-
240, # 1
or #13 to #113 or #114 as shown in SEQ ID N0:2 substantially free from other
proteinaceous materials with which it is co-produced. For production in
mammalian
cells, the DNA sequence further comprises a DNA sequence encoding a suitable
propeptide 5' to and linked in frame to the nucleotide sequence encoding the
mature
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WA545 polypeptide. The propeptide may be the native WA545 propeptide, or may
be a propeptide from another protein of the TGF-(3 superfamily.
Human WA545
It is expected that other species, particularly human, have DNA sequences
homologous to Xenopus WA545 protein. The invention, therefore, includes
methods
for obtaining the DNA sequences encoding human WA545, the DNA sequences
obtained by those methods, and the human protein encoded by those DNA
sequences.
This method entails utilizing the Xenopus WA545 nucleotide sequence or
portions
thereof to design probes to screen libraries for the human gene or coding
sequences
or functional fragments thereof using standard techniques. Thus, the present
invention
includes DNA sequences from other species, particularly, human, which are
homologous to Xenopus WA545 and can be obtained using the Xenopus WA545
sequence. A DNA sequence encoding the complete mature human WA545 protein
and the corresponding amino acid sequence can be obtained using the procedures
which are set forth herein. As described herein, these sequences are isolated
using a
portion of the Xenopus WA545 sequence as a probe. The human WA545 sequence
of may also be used in order to design probes to obtain the complete human
WA545
gene or coding sequences through standard techniques. The Xenopus WA545 and
human WA545 sequences, or portions thereof, may also be used as probes, or to
design probes, in order to obtain other related DNA sequences, such as
homologues
from other species. The WA545 proteins of the present invention, such as human
WA545, may be produced by culturing a cell transformed with the correlating
DNA
sequence, such as the WA545 DNA sequence, and recovering and purifying protein
from the culture medium. The purified expressed protein is substantially free
from
other proteinaceous materials with which it is co-produced, as well as from
other
contaminants. The recovered purified protein is contemplated to exhibit
cartilage,
bone, muscle, nerve, epidermis and/or connective tissue formation activity.
The
proteins of the invention may be further characterized by the ability to
demonstrate
cartilage, bone, muscle, nerve, epidermis and/or other connective tissue
formation
activity in the assays described below.
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Another aspect of the invention provides pharmaceutical compositions
containing a therapeutically effective amount of a WA545 protein, such as
Xenopus
or human WA545 protein, in a pharmaceutically acceptable vehicle or carrier.
These
compositions of the invention may be used in the formation of bone, cartilage,
muscle,
nerve, epidermis and/or other connective tissue, including tendon, ligament
and
meniscus, as well as combinations of the above, for example regeneration of
the
tendon-to-bone attachment apparatus. The compositions of the present
invention,
such as compositions of human WA545, may also be used for wound healing and
tissue repair. Compositions of the invention may further include at least one
other
therapeutically useful agent such as the BMP proteins BMP-l, BMP-2, BMP-3, BMP-
4, BMP-5, BMP-6 and BMP-7, disclosed for instance in United States Patents
5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8,
disclosed in PCT publication W091/18098; and BMP-9, disclosed in PCT
publication
W093/00432, BMP-10, disclosed in PCT application W094/26893; BMP-11,
disclosed in PCT application W094/26892, or BMP-12 or BMP-13, disclosed in PCT
application W095/16035, or BMP-15, disclosed in PCT application W096/36710 or
BMP-16, disclosed in co-pending patent application serial number 08/715/202,
filed
September 18, 1996.
Other compositions which may also be useful include Vgr-2, and any of the
growth and differentiation factors [GDFs], including those described in PCT
applications W094/15965; W094/15949; W095/01801; W095/01802;
W094/21681; W094/15966; and others. Also useful in the present invention may
be
BIP, disclosed in W094/01557; and MP52, disclosed in PCT application
W093/16099. The disclosures of all of the above applications are hereby
incorporated
by reference for the disclosure contained therein.
The compositions of the invention may comprise, in addition to a WA545
protein, other therapeutically useful agents including growth factors such as
epidermal
growth factor (EGF), fibroblast growth factor (FGF), transforming growth
factor
(TGF-a and TGF-~3), wnt proteins, hedgehog proteins such as sonic, Indian and
desert
hedgehog, activins, inhibins, and insulin-like growth factor (IGF). The
compositions
may also include an appropriate matrix, for instance, for supporting the
composition
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and providing a surface for bone, cartilage, muscle, nerve, epidermis andlor
other
connective tissue growth. The matrix may provide slow release of the
osteoinductive
protein and/or the appropriate environment for presentation thereof.
The WA545 compositions may be employed in methods for treating a number
of bone, cartilage, muscle, nerve, epidermis and/or other connective tissue
defects, as
well as periodontal disease and healing of various types of tissues and
wounds. The
tissue and wounds which may be treated include epidermis, nerve, including
spinal
chord, muscle, including cardiac, striated or smoothe muscle, and other
tissues and
wounds, and other organs such as liver, pancreas, spleen, brain, lung,
cardiac, and
kidney tissue. These methods, according to the invention, entail administering
to a
patient needing such bone, cartilage, muscle, nerve, epidermis and/or other
connective
tissue formation, wound healing or tissue repair, an effective amount of a
WA545
protein. The WA545 compositions may also be used to treat or prevent such
conditions as osteoarthritis, osteoporosis, and other abnormalities of bone,
cartilage,
muscle, nerve, epidermis or other connective tissue, organs such as liver,
pancreas,
spleen, lung, cardiac, and kidney and other tissues. These methods may also
entail the
administration of a protein of the invention in conjunction with at least one
other BMP
protein as described above. In addition, these methods may also include the
administration of a WA545 protein with other growth factors including EGF,
FGF,
TGF-a, TGF-~3, wnt, hedgehog, activin, inhibin and IGF.
Still a further aspect of the invention are DNA sequences coding for
expression
of a WA545 protein. Such sequences include the sequence of nucleotides in a 5'
to
3' direction illustrated in SEQ ID NO: l, as well as DNA sequences which, but
for the
degeneracy of the genetic code, are identical to the DNA sequence SEQ ID NO:
1, and
encode the protein of SEQ ID NO: 2. Further included in the present invention
are
DNA sequences which hybridize under stringent conditions with the DNA sequence
of SEQ ID NO: 1 and encode a protein having the ability to induce the
formation of
cartilage, bone, muscle, nerve, epidermis and/or other connective tissue, or
other
organs such as liver, pancreas, brain, spleen, lung, cardiac, and kidney
tissue.
Preferred DNA sequences include those which hybridize under stringent
conditions
[see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold Spring
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Harbor Laboratory ( 1982), pages 387 to 389]. It is generally preferred that
such DNA
sequences encode a polypeptide which is at least about 80% homologous, and
more
preferably at least about 90% homologous, to the mature human WA545 amino acid
sequence shown in SEQ )D N0:2. Finally, allelic or other variations of the
sequences
of SEQ ID NO: 1, whether such nucleotide changes result in changes in the
peptide
sequence or not, but where the peptide sequence still has WA545 activity, are
also
included in the present invention. The present invention also includes
functional
fragments of the DNA sequence of WA545 shown in SEQ ID NO: 1 which encode a
polypeptide which retains the activity of WA545 protein. The determination
whether
a particular variant or fragment of a WA545 protein of the present invention
will
maintain WA545 activity, is routinely performed using the assays described in
the
examples and specification herein.
The DNA sequences of the present invention are useful, for example, as probes
for the detection of mRNA encoding WA545 in a given cell population. The DNA
sequences may also be useful for preparing vectors for gene therapy
applications as
described below.
A further aspect of the invention includes vectors comprising a DNA sequence
as described above in operative association with an expression control
sequence
therefor. These vectors may be employed in a novel process for producing a
WA545
protein of the invention in which a cell line transformed with a DNA sequence
encoding a WA545 protein in operative association with an expression control
sequence therefor, is cultured in a suitable culture medium and a WA545
protein is
recovered and purified therefrom. This process may employ a number of known
cells
both prokaryotic and eukaryotic as host cells for expression of the
polypeptide. The
vectors may be used in gene therapy applications. In such use, the vectors may
be
transfected into the cells of a patient ex vivo, and the cells may be
reintroduced into
a patient. Alternatively, the vectors may be introduced into a patient in vivo
through
targeted transfection.
Still a further aspect of the invention are WA545 proteins or polypeptides.
Such polypeptides are characterized by having an amino acid sequence including
the
sequence illustrated in SEQ )D NO: 2, variants of the amino acid sequence of
SEQ >D
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NO: 2, including naturally occurring allelic variants, and other variants in
which the
protein retains the ability to induce the formation of cartilage, bone,
muscle, nerve,
epidermis and/or other connective tissue, or other organs such as liver,
pancreas, brain,
spleen, lung, cardiac, and kidney tissue, characteristic of WA545. Preferred
polypeptides include a polypeptide which is at least about 80% homologous, and
more
preferably at least about 90% homologous, to the mature Xenopus WA545 amino
acid
sequence shown in SEQ ID N0:2. Finally, allelic or other variations of the
sequences
of SEQ ID NO: 2, whether such amino acid changes are induced by mutagenesis,
chemical alteration, or by alteration of DNA sequence used to produce the
polypeptide, where the peptide sequence still has WA545 activity, are also
included
in the present invention. The present invention also includes functional
fragments of
the amino acid sequence of WA545 shown in SEQ )D NO: 2 which retains the
activity
of WA545 protein.
The purified proteins of the present inventions may be used to generate
antibodies, either monoclonal or polyclonal, to human WA545 and/or other WA545-
related proteins, using methods that are known in the art of antibody
production.
Thus, the present invention also includes antibodies to human WA545 and/or
other
WA545 proteins. The antibodies may be useful for purification of WA545 and/or
other WA545 proteins, or for inhibiting or preventing the effects of WA545
proteins.
The WA545 protein and related proteins may be useful for identifying and
isolating
a receptor protein which binds to WA545, and for inducing the growth and/or
differentiation of embryonic cells and/or stem cells. Thus, the present
invention also
includes WA545 receptors, methods of identifying receptors, and methods of
treating
cell populations, such as embryonic cells or stern cell populations, to
enhance or
enrich the growth and/or differentiation of the cells. The treated cell
populations may
be useful for implantation and for gene therapy applications.
Description of the Deposits
A clone encoding the full-length Xenopus WA545 protein was deposited with
the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville,
MD
20852, on May 9, 1997, and was accorded the ATCC designation 98428. This
deposit
fully satisfies the requirements of the Budapest Treaty.
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Description of the Sequences
SEQ >D NO: l is a nucleotide sequence encoding the entire mature Xenopus
WA545 polypeptide.
SEQ ID N0:2 is the amino acid sequence containing the mature Xenopus
WA545 polypeptide.
SEQ 1D N0:3 is an oligonucleotide probe to Xenopus WA545 signal sequence.
SEQ >D N0:4 is an oligonucleotide probe to Xenopus WA545 signal sequence.
SEQ ID NO:S is a consensus amino acid sequence derived from a highly
conserved region of BMP/TGF-(3/Vg-1 proteins
SEQ ID N0:6 is an oligonucleotide designed on the basis of the above
identified consensus amino acid sequence of SEQ ID NO:S
SEQ ID N0:7 is a consensus amino acid sequence derived from a highly
conserved region of BMP/TGF-(3/Vg-1 proteins.
SEQ ID N0:8 is an oligonucleotide designed on the basis of the above
identified consensus amino acid sequence of SEQ 1D N0:7.
SEQ )D N0:9 is an oligonucleotide probe to Xenopus WA545 mature peptide
sequence.
SEQ ID NO:10 is an oligonucleotide probe to Xenopus WA545 mature peptide
sequence.
Brief Description of the Fi ures
Figure 1. Expression Pattern of WA545. WA545 is expressed in the marginal
zone (mesoderm) and vegetal cells, and later becomes posteriorly restricted.
Whole mount in situ hybridization of Xenopus embryos with a digoxygenin-
labelled indecence RNA probe for WA545, showing localization of WA545 RNA.
Views from the vegetal pole or from the posterior (the vegetal pole eventually
becomes the posterior). (a) WA545 is first expressed during cleavage, by mid-
to-late
blastula {stage 9). (b) Expression increases by early gastrula (stage 10+) in
the entire
marginal zone (mesoderm) as well as in vegetal cells. {c,d) This high level
expression
is maintained during gastrulation. (e) The expression level starts to decline
during the
late stages of gastrulation. By this stage, expression is still present in
lateral and
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S ventral tissue but is excluded from the dorsal-most region which will form
the
notochord. (f) By early neurula, expression has declined and is still limited
to a region
just next to the closing blastopore.
Figure 2. WA545 is expressed during gastrulation.
Timing of WAS4S RNA expression, using a reverse transcriptase-PCR (RT-
PCR) based assay. RNA was isolated from the stages indicated and processed for
RT-
PCT. PCR reactions were done at 21 cycles for all samples using primers
specific to
WA54S or ornithine decarboxylase (ODC), which was used as a Loading control.
WAS4S expression could be detected at stage 9 (blastula) and remained at a
high level
1S until stage 13 (early neurula). St. 8, mid-blastula; st. 10.25, early
gastrula; st. 11.5,
mid-gastrula; st. 19, tailbud; st. 3S, hatching. The reactions for three
samples,
unfertilized egg, st. 8 and st. 9 were also done at 30 cycles to detect small
amounts of
RNA. No signal could be detected in both egg and stage 8 samples even after 30
cycles. This indicates that WAS4S does not have a maternal transcript and is
not
expressed until after mid-blastula transition. (stage 8.S).
Figure 3. WA545 induces a posterior secondary axis when misexpressed on
ventral side of embryos.
Embryos were injected in one ventral blastomere in the marginal zone at the
2S 4-cell stage with SO or 100 pg WAS4S in vitro transcribed RNA, or with
globin RNA
as a control. 80 pg lacZ RNA was included as a lineage tracer to determine
where the
WAS4S RNA became localized. Embryos were incubated until hatching (stage 3S)
before fixation and X-gal staining. Arrowheads point to a secondary axis that
is
induced on the injected side, as indicated by the blue staining, of the
embryos. A
secondary head was never observed. This indicates that WAS4S is capable of
inducing posterior, but not anterior regions. The secondary axis contains both
mesodermal and neural tissue.
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Figure 4. WA545 causes microcephaly when misexpressed on dorsal side of
embryos.
Embryos were injected in one dorsal blastomere in the marginal zone at the 4-
cell stage with 100 pg WA545 or globin RNA along with 80 pg lacZ RNA as
lineage
tracer. Embryos were incubated until tailbud stage (stage 22) before fixation
and X-
gal staining. Arrowheads indicate the chin primordium (cement gland), an
extreme
anterior structure. "Control" panel shows embryo injected with globin RNA.
"WA545" panel shows embryos injected with WA545 RNA. In WA545 injected
embryos, anterior structures are suppressed, as indicated by the smaller sizes
of the
cement glands. In later stages no eyes and no forebrain is present. This
indicates that
WA545 may convert anterior to more posterior tissue.
Figure 5. WA545 induces posterior mesodermal markers in an animal cap assay.
WA545 induces posterior mesoderm. Anterior mesoderm and neural gene
expression is not activated by this gene. Embryos were injected in one
blastomere in
the animal pole at 2-cell stage with 400 pg WA545 or globin RNA. Animal caps
were isolated as indicated and cultured until sibling embryos reached stage 14
(neurula) or 19 (tailbud) when various marker genes are maximally expressed.
Total
RNA was prepared from globin injected caps, WA545 injected caps and whole
embryos. RT-PCR was used to assay for the expression of marker genes. Lanes 1,
2
and 3 are samples harvested at stage 14 samples. Xbra is a general mesodermal
marker, gsc is a marker of anterior mesoderm, Pintallavis and Xnot are markers
of
posterior mesoderm. All genes were induced; however, gsc induction was very
weak
indicating that posterior mesoderm is predominantly induced (compare lanes 1
and 2).
Xvent-1 and odc are loading controls. Lanes 4, 5 and 6 are samples harvested
at stage
19. Muscle-specific actin is a lateral mesodermal marker and was strongly
induced.
HoxB9, a psoterior mesodermal marker was also induced. Krox20, a neural marker
whose expression is in the hindbrain and N-CAM, a general neural marker, were
not
induced.
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Figure 6. WA545 induces muscle in animal caps.
At the histological level, muscle formation can be observed after WA545
ectopic expression. Embryos were injected in one blastomere in the animal pole
at 2-
cell stage with 400 pg WA545 or globin RNA. Animal caps were isolated as
indicated
and cultured until sibling embryos reached hatching stage (stage 35) and
analysed
histologically. (a) Globin-injected caps are vesicular, with only epidermal
differentiation observed. (b) WA545-injected caps show extensive tissue
formation.
(c) At higher magnification, WA545-injected caps show muscle blocks and
possibly
blood formation.
Detailed Descriution of the Invention
WA545
The Xenopus WA545 nucleotide sequence (SEQ 1D NO: 1 ) and encoded
amino acid sequence (SEQ 1D NO: 2) are set forth in the Sequence Listing
herein.
The coding sequence of the mature Xenopus WA545 protein begins at nucleotide
#55
and continues through nucleotide #1116. Purified Xenopus WA545 proteins of the
present invention are produced by culturing a host cell transformed with a DNA
sequence comprising the DNA coding sequence of SEQ >D NO: 1 from nucleotide
#55
to #1116, or from nucleotide #775 to #1116, and recovering and purifying from
the
culture medium a protein which contains the amino acid sequence or a
substantially
homologous sequence as represented by amino acids #-240 to #114 or #1 to #114
of
SEQ )D NO: 2.
Other species, in particular human, are expected to have DNA sequences
homologous to Xenopus WA545. DNA sequences encoding human WA545 are
isolated by various techniques known to those skilled in the art. The
invention,
therefore, includes methods for obtaining DNA sequences encoding human WA545.
The methods utilize the Xenopus WA545 nucleotide sequences or portions in the
design of probes to screen libraries for the human gene or coding sequences or
fragments thereof using standard techniques.
Regions containing amino acid sequences which are highly conserved within
the WA545 family of proteins are identified and consensus amino acid sequences
of
these highly conserved regions are be constructed based on the similarity of
the
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corresponding regions of individual WA545 proteins. Oligonucleotide primers
designed on the basis of the amino acid sequence of such conserved sequences
allow
the specific amplification of the human WA545 encoding sequences. Two such
consensus amino acid sequences are set forth in the Sequence Listing. Once a
recombinant bacteriophage containing DNA encoding a portion of a human WA545
is obtained, the human coding sequence can be used as a probe to identify a
human
cell line or tissue which synthesizes WA545 mRNA. Alternatively, the Xenopus
coding sequence can be used as a probe to identify such human cell line or
tissue.
Alternatively, the Xerzopus WA545 coding sequence is used to design
oligonucleotide
primers which will specifically amplify a portion of the WA545 encoding
sequence
located in the region located between the primers utilized to perform the
specific
amplification reaction. Utilizing Xenopus and human WA545 sequences one can
specifically amplify corresponding human WA545 encoding sequences from mRNA,
cDNA or genomic DNA templates. Once a positive source has been identified by
one
of the above described methods, mRNA is selected by oligo (dT) cellulose
chromatography and cDNA is synthesized and cloned in Xgt 10 or other
bacteriophage
vectors known to those skilled in the art, for example, ZAP by established
techniques.
It is also possible to perform the oligonucleotide primer directed
amplification
reaction, described above, directly on a pre-established human cDNA or genomic
library which has been cloned into a bacteriophage vector. In such cases, a
library
which yields a specifically amplified DNA product encoding a portion of human
WA545 protein is screened directly, utilizing the fragment of amplified WA545
encoding DNA as a probe.
The human WA545 sequence of the present invention is obtained using the
whole or fragments of the Xenopus WA545 DNA sequence, or a partial human
WA545 sequence, as a probe. Thus, the human WA545 DNA sequence is expected
to comprise a DNA sequence highly homologous to nucleotides #55 or #775 to
#1116
of the Xenopus WA545 DNA sequence shown in SEQ ID NO: 1. The amino acid
sequence of the human WA545 protein is expected to comprise an amino acids
highly
homologous to the sequence of amino acids #-240 or #1 to #114 of SEQ >D NO: 2.
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It is expected that WA545 protein, as expressed by mammalian cells such as
CHO cells, exists as a heterogeneous population of active species of WA545
protein
with varying N-termini. It is expected that active species will comprise an
amino acid
sequence beginning with the cysteine residue at amino acid #13 of SEQ ID N0:2,
or
will comprise additional amino acid sequence further in the N-terminal
direction.
Thus, it is expected that DNA sequences encoding active WA545 proteins include
those comprising nucleotides #775 or #811 to #1113 or #1116 of SEQ )D NO: 1,
as
well as those including additional nucleotides at the 5'-terminal end.
Accordingly,
active species of WA545 are expected to include those comprising amino acids
#1 or
#13 to #113 or #114 of SEQ >D N0:2, as well as those including additional
amino
acids at the N-terminal end.
A host cell may be transformed with a coding sequence encoding a propeptide
suitable for the secretion of proteins by the host cell linked in proper
reading frame to
the coding sequence for the mature WA545 protein. For example, see United
States
Patent 5,168,050, in which a DNA encoding a precursor portion of a mammalian
protein other than BMP-2 is fused to the DNA encoding a mature BMP-2 protein.
See
also the specification of PCT application W095/16035, in which the propeptide
of
BMP-2 is fused to the DNA encoding a mature BMP-12 protein. Thus, the present
invention includes chimeric DNA molecules comprising a DNA sequence encoding
a propeptide or a regulatory sequence from a protein, such as a TGF-~3
protein, other
than WA545, linked in correct reading frame to a DNA sequence encoding a WA545
protein. The term "chimeric" is used to signify that the propeptide originates
from a
different polypeptide than the WA545 protein. A host cell which naturally
expresses
native WA545 may be transfected with a highly expressed or regulable
expression
sequence so as to recombine in order to increase or alter WA545 expression.
The N-terminus of one active species of WAS45 is expected to be
experimentally determined by expression in E. coli to be as follows:
[M]STHSSPPTP. Thus, it appears that the N-terminus of this species of WA545 is
at amino acid #1 of SEQ ID NO:1, and a DNA sequence encoding said species of
WA545 would comprise nucleotides #775 to #1116 of SEQ ff~ NO: 1. The apparent
molecular weight of WA545 monomer is expected to be experimentally determined
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by SDS-PAGE to be approximately 10-15 kd, more particularly, about 13 kd, on a
Novex 16% tricine gel. The WA545 protein is expected to exist as a clear,
colorless
solution in 0.1 % trifluoroacetic acid. The dimer is expected to have a
molecular
weight of approximately 20-30 kd.
It is expected that other WA545 proteins, as expressed by mammalian cells
such as CHO cells, also exist as a heterogeneous population of active species
of
WA545 protein with varying N-termini. For example, it is expected that active
species of WA545 will comprise an amino acid sequence beginning with the
cysteine
residue at amino acid #13 of SEQ ID N0:2, or will comprise additional amino
acid
sequence further in the N-terminal direction. Thus, it is expected that DNA
sequences
encoding active WA545 proteins include those which comprise a nucleotide
sequence
comprising nucleotides #55, #775, #811 to #1113 or #1116 of SEQ ID NO: 1.
Accordingly, active WA545 proteins include those comprising amino acids #-240,
#1,
or #13 to #113 or #114.
The WA545 proteins of the present invention, include polypeptides having a
molecular weight of about 10-15 kd in monomeric form, said polypeptide
comprising
the amino acid sequence of SEQ ID N0:2 and having the ability to induce the
formation of cartilage, bone, tendon, ligament, muscle, nerve, epidermis
and/or other
connective tissue in assays such as the Rosen-Modified Sampath-Reddi ectopic
implant assay, or in the other assays described below.
The WA545 proteins recovered from the culture medium are purified by
isolating them from other proteinaceous materials from which they are co-
produced
and from other contaminants present. WA545 proteins may be characterized by
the
ability to induce the formation of cartilage, bone, tendon, ligament, muscle,
nerve,
epidernus and/or other connective tissue, for example, in the assays described
below.
The WA545 proteins provided herein also include factors encoded by the
sequences similar to those of SEQ ID NO:1, but into which modifications or
deletions
are naturally provided (e.g. allelic variations in the nucleotide sequence
which may
result in amino acid changes in the polypeptide) or deliberately engineered.
For
example, synthetic polypeptides may wholly or partially duplicate continuous
sequences of the amino acid residues of SEQ »D N0:2. These sequences, by
virtue
14
CA 02295086 1999-12-21
WO 99/02678 PCT/US98/08334
of sharing primary, secondary, or tertiary structural and conformational
characteristics
with the polypeptide sequence of SEQ m NO: 2 may possess biological properties
in
common therewith. Thus, these modifications and deletions of the native WA545
may be employed as biologically active substitutes for naturally-occurring
WA545
polypeptides in therapeutic processes. It can be readily determined whether a
given
variant of WA545 maintains the biological activity of WA545 by subjecting both
WA545 and the variant of WA545 to the assays described in the examples and
specification herein.
Other specific mutations of the sequences of WA545 proteins described herein
involve modifications of glycosylation sites. These modifications may involve
O-
linked or N-linked glycosylation sites. For instance, the absence of
glycosylation or
only partial glycosylation results from amino acid substitution or deletion at
asparagine-linked glycosylation recognition sites. The asparagine-linked
glycosylation
recognition sites comprise tripeptide sequences which are specifically
recognized by
appropriate cellular glycosylation enzymes. These tripeptide sequences are
either
asparagine-X-threonine or asparagine-X-serine, where X is usually any amino
acid.
A variety of amino acid substitutions or deletions at one or both of the first
or third
amino acid positions of a glycosylation recognition site (and/or amino acid
deletion
at the second position) results in non-glycosylation at the modified
tripeptide
sequence. Additionally, bacterial expression of WA545 protein will also result
in
production of a non-glycosylated protein, even if the glycosylation sites are
left
unmodified.
The present invention also encompasses the novel DNA sequences, free of
association with DNA sequences encoding other proteinaceous materials, and
coding
for expression of WA545 proteins. These DNA sequences include those depicted
in
SEQ ID NO: 1 in a 5' to 3' direction and those sequences which hybridize
thereto
under stringent hybridization conditions [for example, O.1X SSC, 0.1% SDS at
65°C;
see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold Spring
Harbor
Laboratory ( 1982), pages 387 to 389] and encode a protein having cartilage,
bone,
tendon, ligament, muscle, nerve, epidermis and/or other connective tissue
inducing
activity. As used herein, the term "stringent hybridization conditions" also
refers to
CA 02295086 1999-12-21
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the use of initial low stringency hybridization conditions (such as 6X SSC,
0.5% SDS,
at about 60° C, overnight which is followed by higher stringency wash
condition (such
as 2X SSC, 0.1% SDS, at about 20°C) or even higher wash stringency
(such as O.1X
SSC, 0.1 % SDS, at about 65 °C, for less than an hour. The DNA
sequences of the
present invention also include those which comprise the DNA sequence of SEQ m
NO: 1 and those which hybridize thereto under stringent hybridization
conditions and
encode a protein having cartilage, bone, tendon, ligament, nerve, epidermis
and/or
other connective tissue inducing activity.
Similarly, DNA sequences which code for WA545 proteins coded for by the
sequences of SEQ >D NO: 1 or which encode the amino acid sequence of SEQ ID
NO:
2, but which differ in codon sequence due to the degeneracies of the genetic
code or
allelic variations (naturally-occurring base changes in the species population
which
may or may not result in an amino acid change) also encode the novel factors
described herein. Variations in the DNA sequences of SEQ m NO: 1 which are
caused by point mutations or by induced modifications (including insertion,
deletion,
and substitution) to enhance the activity, half life or production of the
polypeptides
encoded are also encompassed in the invention.
Another aspect of the present invention provides a novel method for producing
WA545 proteins. The method of the present invention involves culturing a
suitable
cell line, which has been transformed with a DNA sequence encoding a WA545
protein of the invention, under the control of known regulatory sequences. The
transformed host cells are cultured and the WA545 proteins recovered and
purified
from the culture medium. The purified proteins are substantially free from
other
proteins with which they are co-produced as well as from other contaminants.
Suitable cells or cell lines may be mammalian cells, such as Chinese hamster
ovary cells (CHO). The selection of suitable mammalian host cells and methods
for
transformation, culture, amplification, screening, product production and
purification
are known in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625
(1981),
or alternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759 (1985) or
Howley et
al, U.S. Patent 4,419,446. Another suitable mammalian cell line, which is
described
16
CA 02295086 1999-12-21
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in the accompanying examples, is the monkey COS-1 cell line. The mammalian
cell
CV-1 may also be suitable.
Bacterial cells may also be suitable hosts. For example, the various strains
of
E. coli (e.g., HB 101, MC 1061 ) are well-known as host cells in the field of
biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and
the like
may also be employed in this method. For expression of the protein in
bacterial cells,
DNA encoding the propeptide of WA545 is generally not necessary.
Many strains of yeast cells known to those skilled in the art may also be
available as host cells for expression of the polypeptides of the present
invention.
Additionally, where desired, insect cells may be utilized as host cells in the
method
of the present invention. See, e.g. Miller et al, Genetic En ineering, 8:277-
298
(Plenum Press 1986) and references cited therein.
Another aspect of the present invention provides vectors for use in the method
of expression of these novel WA545 polypeptides. Preferably the vectors
contain the
full novel DNA sequences described above which encode the novel factors of the
invention. Additionally, the vectors contain appropriate expression control
sequences
permitting expression of the WA545 protein sequences. Alternatively, vectors
incorporating modified sequences as described above are also embodiments of
the
present invention. Additionally, the sequence of SEQ m NO:1 or other sequences
encoding WA545 proteins could be manipulated to express a mature WA545 protein
by deleting WA545 propeptide sequences and replacing them with sequences
encoding the complete propeptides of other BMP proteins or members of the TGF-
~3
superfamily. Thus, the present invention includes chimeric DNA molecules
encoding
a propeptide from a member of the TGF-~i superfamily linked in correct reading
frame
to a DNA sequence encoding a WA545 polypeptide.
The vectors may be employed in the method of transforming cell lines and
contain selected regulatory sequences in operative association with the DNA
coding
sequences of the invention which are capable of directing tissue specific or
inducible
replication and expression thereof in selected host cells. Regulatory
sequences for
such vectors are known to those skilled in the art and may be selected
depending upon
the host cells. Thus, such specialized vectors constitute part of the present
invention.
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A protein of the present invention, which induces cartilage, bone, tendon,
ligament, muscle, nerve, epidermis and/or other connective tissue formation in
circumstances where such tissue is not normally formed, has application in the
healing
of bone fractures and cartilage or other connective tissue defects in humans
and other
animals. Such a preparation employing a WA545 protein may have prophylactic
use
in closed as well as open fracture reduction and also in the improved fixation
of
artificial joints. De novo bone formation induced by an osteogenic agent
contributes
to the repair of congenital, trauma induced, or oncologic resection induced
craniofacial
defects, and also is useful in cosmetic plastic surgery. A WA545 protein may
be used
in the treatment of periodontal disease, and in other tooth repair processes.
Such
agents may provide an environment to attract bone-forming cells, stimulate
growth of
bone-forming cells or induce differentiation of progenitors of bone-forming
cells, and
may also support the regeneration of the periodontal ligament and attachment
apparatus, which connects bone and teeth. WA545 polypeptides of the invention
may
also be useful in the treatment of osteoporosis. A variety of osteogenic,
cartilage-inducing and bone inducing factors have been described. See, e.g.,
European
patent applications 148,155 and 169,016 for discussions thereof.
The proteins of the invention may also be used in wound healing and related
tissue repair. The types of wounds include, but are not limited to burns,
incisions and
ulcers. (See, e.g. PCT Publication W084/Ol 106 for discussion of wound healing
and
related tissue repair). It is further contemplated that proteins of the
invention may
increase neuronal and glial cell survival and therefore be useful in
transplantation and
treatment of conditions exhibiting a decrease in neuronal survival and repair.
The
proteins of the invention may further be useful for the treatment of
conditions related
to other types of tissue, such as nerve, including spinal chord, epidermis,
muscle,
including cardiac, striated and smoothe muscle, and other organs such as
liver,
pancreas, brain, spleen, lung, cardiac, and kidney tissue. The proteins of the
present
invention may also have value as a dietary or nutrient supplement. For this
use, the
proteins may be used in intact form, or may be predigested to provide a more
readily
absorbed supplement.
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The proteins of the invention may also have other useful properties
characteristic of the TGF-~3 superfamily of proteins. Such properties include
angiogenic, chemotactic and/or chemoattractant properties, and effects on
cells
including induction of collagen synthesis, fibrosis, differentiation
responses, cell
proliferative responses and responses involving cell adhesion, migration and
extracellular matrices. These properties make the proteins of the invention
potential
agents for wound healing, reduction of fibrosis and reduction of scar tissue
formation.
When dimerized as a homodimer or as a heterodimer with other BMPs, with
other members of the TGF-~3 superfamily of proteins, or with inhibin-a
proteins or
inhibin-~3 proteins, the WA545 heterodimer is expected to demonstrate effects
on the
production of follicle stimulating hormone (FSH), as described further herein.
It is
recognized that FSH stimulates the development of ova in mammalian ovaries
(Ross
et al., in Textbook of Endocrinology, ed. Williams, p. 355 ( 1981 ) and that
excessive
stimulation of the ovaries with FSH will lead to multiple ovulations. FSH is
also
important in testicular function. Thus, WA545 may be useful as a contraceptive
based
on the ability of inhibins to decrease fertility in female mammals and
decrease
spermatogenesis in male mammals. Administration of sufficient amounts of other
inhibins can induce infertility in mammals. WA545 may also be useful as a
fertility
inducing therapeutic, based upon the ability of activin molecules in
stimulating FSH
release from cells of the anterior pituitary. See, for example, United States
Patent
4,798,885. WA545 may also be useful for advancement of the onset of fertility
in
sexually immature mammals, so as to increase the lifetime reproductive
performance
of domestic animals such as cows, sheep and pigs. It is further contemplated
that
WA545 may be useful in modulating hematopoiesis by inducing the
differentiation
of erythroid cells [see, e.g., Broxmeyer et al, Proc. Natl. Acad. Sci. USA,
85:9052-
9056 ( 1988) or Eto et al, Biochem. Biophys. Res. Comm., 142:1095-1103 (
1987)], for
suppressing the development of gonadal tumors [see, e.g., Matzuk et al.,
Nature,
360:313-319 ( 1992)] or for augmenting the activity of bone morphogenetic
proteins
[see, e.g., Ogawa et al., J. Biol. Chem., 267:14233-14237 (1992)].
WA545 proteins may be further characterized by their ability to modulate the
release of follicle stimulating hormone (FSH) in established in vitro
bioassays using
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CA 02295086 1999-12-21
WO 99/02678 PCT/US98/08334
rat anterior pituitary cells as described [see, e.g., Vale et al,
Endocrinology, 91:562-
572 (1972); Ling et al., Nature, 321:779-782 (1986) or Vale et al., Nature,
321:776-
779 (1986)]. It is contemplated that the WA545 protein of the invention, when
composed as a heterodimer with inhibin ~i chains will exhibit stimulatory
effects on
the release of follicle stimulating hormone (FSH) from anterior pituitary
cells as
described [Ling et al., Nature, 321:779-782 (1986) or Vale et al., Nature,
321:776-
779 ( 1986)]. Additionally, it is contemplated that the WA545 protein of the
invention,
when composed as a heterodimer with the inhibin a chain, will inhibit the
release of
follicle stimulating hormone (FSH) from anterior pituitary cells as described
[see, e.g.,
Vale et al, Endocrinology, 91:562-572 ( 1972). Therefore, depending on the
particular
composition, it is expected that the WA545 protein of the invention may have
contrasting and opposite effects on the release of follicle stimulating
hormone (FSH)
from the anterior pituitary.
Activin A (the homodimeric composition of inhibin ~iA) has been shown to
have erythropoietic-stimulating activity [see e.g. Eto et al., Biochem.
Biophys. Res.
Comm., 142:1095-1103 (1987) and Murata et al., Proc. Natl. Acad. Sci. U.S.A.,
85:2434-2438 (1988) and Yu et al., Nature, 330:765-767 (1987)]. It is
contemplated
that the WA545 protein of the invention has a similar erythropoietic-
stimulating
activity. This activity of the WA545 protein may be further characterized by
the
ability of the WA545 protein to demonstrate erythropoietin activity in the
biological
assay performed using the human K-562 cell line as described by [Lozzio et
al., Blood,
45:321-334 (1975) and U.S. Pat. No. 5,071,834].
A further aspect of the invention is a therapeutic method and composition for
repairing fractures and other conditions related to cartilage, bone, tendon,
ligament,
muscle, nerve, epidermis and/or other connective tissue defects or periodontal
dis-
eases. The invention further comprises therapeutic methods and compositions
for
wound healing and tissue repair. Such compositions comprise a therapeutically
effective amount of at least one of the WA545 proteins of the invention in
admixture
with a pharmaceutically acceptable vehicle, carrier or matrix. It is further
contemplated that compositions of the invention may increase neuronal survival
and
therefore be useful in transplantation and treatment of conditions exhibiting
a decrease
CA 02295086 1999-12-21
WO 99/02678 PCT/US98/08334
in neuronal survival. Compositions of the invention may further include at
least one
other therapeutically useful agent, such as members of the TGF-~i superfamily
of
proteins, which includes the BMP proteins BMP-1, BMP-2, BMP-3, BMP-4, BMP-5,
BMP-6 and BMP-7, disclosed for instance in United States Patents 5,108,922;
5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in
PCT
publication W091/18098; BMP-9, disclosed in PCT publication W093/00432; BMP-
10, disclosed in PCT application W094/26893; BMP-11, disclosed in PCT
application W094/26892, BMP-12 or BMP-13, disclosed in PCT application WO
95/16035, BMP-15, disclosed in PCT application W096/36710, and BMP-16,
disclosed in co-pending patent application serial number 08/715/202, filed
September
18, 1996. Other compositions which may also be useful include Vgr-2, and any
of the
GDFs, including those described in PCT applications W094/15965; W094/15949;
W095/01801; W095/01802; W094/21681; W094/15966; and others. Also useful
in the present invention may be BIP, disclosed in W094/01557; and MP52,
disclosed
in PCT application W093/16099. The disclosures of the above applications are
hereby incorporated by reference herein.
It is expected that WA545 proteins may exist in nature as homodimers or
heterodimers. To promote the formation of dimers of WA545 with increased
stability,
one can genetically engineer the DNA sequence of SEQUENCE ID NO:1 to provide
one or more additional cysteine residues to increase potential dimer
formation. The
resulting DNA sequence would be capable of producing a "cysteine added
variant" of
WA545 protein. Alternatively, one can produce "cysteine added variants" of
WA545
proteins by altering the sequence of the protein at the amino acid level, for
example,
by altering the amino acid sequences of one or more amino acid residues to
Cys.
Production of "cysteine added variants" of proteins is described in United
States
Patent 5,166,322, the disclosure of which is hereby incorporated by reference.
It is expected that the proteins of the invention may act in concert with or
perhaps synergistically with other related proteins and growth factors.
Further
therapeutic methods and compositions of the invention therefore comprise a
therapeutic amount of at least one WA545 protein of the invention with a
therapeutic
amount of at least one other member of the TGF-~3 superfamily of proteins,
such as the
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WO 99/02678 PCT/US98/08334
BMP proteins disclosed in the applications described above. Such combinations
may
comprise separate molecules of the BMP proteins or heteromolecules comprised
of
different BMP moieties. For example, a method and composition of the invention
may comprise a disulfide linked dimer comprising a WA545 protein subunit and a
subunit from one of the "BMP" proteins described above. Thus, the present
invention
includes a purified WA545 polypeptide which is a heterodimer wherein one
subunit
comprises an amino acid sequence of SEQ ID N0:2, and one subunit comprises an
amino acid sequence for a bone morphogenetic protein selected from the group
consisting of BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9,
BMP-10, BMP-1 l, BMP-12 or BMP-13, disclosed in PCT application WO 95/16035,
or BMP-15, disclosed in PCT application W096/36710 or BMP-16, disclosed in co-
pending patent application serial number 08/715/202, filed September 18, 1996.
A
further embodiment may comprise a heterodimer of WA545 moieties, for example,
of Xenopus WA545 and the human homologue of Xenopus WA545 protein. Further,
WA545 may be combined with other agents beneficial to the treatment of the
bone,
cartilage, tendon, ligament, muscle, nerve, epidermis and/or other conn:,ctive
tissue
defect, wound, or tissue in question. These agents include various growth
factors such
as epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet
derived
growth factor (PDGF), transforming growth factors (TGF-a and TGF-~3), wnt
proteins,
hedgehog proteins, such as sonic, Indian and desert hedgehog, activins,
inhibins, and
k-fibroblast growth factor (kFGF), parathyroid hormone (PTH), leukemia
inhibitory
factor (LIF/HILDA/DIA), insulin-like growth factors (IGF-I and IGF-II).
Portions of
these agents may also be used in compositions of the present invention.
The preparation and formulation of such physiologically acceptable protein
compositions, having due regard to pH, isotonicity, stability and the like, is
within the
skill of the art. The therapeutic compositions are also presently valuable for
veterinary
applications due to the lack of species specificity in BMP proteins.
Particularly
domestic animals and thoroughbred horses in addition to humans are desired
patients
for such treatment with the WA545 proteins of the present invention.
The therapeutic method includes administering the composition topically,
systemically, or locally as an implant or device. When administered, the
therapeutic
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S composition for use in this invention is, of course, in a pyrogen-free,
physiologically
acceptable form. Further, the composition may desirably be encapsulated or
injected
in a viscous form for delivery to the site of bone, cartilage, tendon,
ligament, muscle,
nerve, epidermis or other connective tissue or other tissue damage. Topical
administration may be suitable for wound healing and tissue repair.
Therapeutically
useful agents other than the WAS4S proteins which may also optionally be
included
in the composition as described above, may alternatively or additionally, be
administered simultaneously or sequentially with the BMP composition in the
methods of the invention.
Preferably for bone, cartilage, tendon, ligament, muscle, nerve, epidermis
1S and/or other connective tissue formation, the composition includes a matrix
capable
of delivering WAS4S or other BMP proteins to the site of bone, cartilage,
tendon,
ligament, muscle, nerve, epidermis and/or other connective tissue damage,
providing
a structure for the developing bone, cartilage, tendon, ligament, muscle,
nerve,
epidermis and/or other connective tissue and optimally capable of being
resorbed into
the body. The matrix may provide slow release of WAS4S and/or other inductive
protein, as well as proper presentation and appropriate environment for
cellular
infiltration. Such matrices may be formed of materials presently in use for
other
implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability,
2S mechanical properties, cosmetic appearance and interface properties. The
particular
application of the WAS4S compositions will define the appropriate formulation.
Potential matrices for the compositions may be biodegradable and chemically
defined
calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and
polyanhydrides. Other potential materials are biodegradable and biologically
well
defined, such as bone or dermal collagen. Further matrices are comprised of
pure
proteins or extracellular matrix components. Other potential matrices are
nonbiodegradable and chemically defined, such as sintered hydroxyapatite,
bioglass,
aluminates, or other ceramics. Matrices may be comprised of combinations of
any of
the above mentioned types of material, such as polylactic acid and
hydroxyapatite or
3S collagen and tricalcium phosphate. The bioceramics may be altered in
composition,
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WO 99/02678 PCT/US98/08334
such as in calcium-aluminate-phosphate and processing to alter pore size,
particle size,
particle shape, and biodegradability.
The dosage regimen will be determined by the attending physician considering
various factors which modify the action of the WA545 protein, e.g., amount of
tissue
weight desired to be formed, the site of tissue damage, the condition of the
damaged
tissue, the size of a wound, type of damaged tissue, the patient's age, sex,
and diet, the
severity of any infection, time of administration and other clinical factors.
The dosage
may vary with the type of matrix used in the reconstitution and the types of
BMP
proteins in the composition. The addition of other known growth factors, such
as IGF
I (insulin like growth factor I), to the final composition, may also affect
the dosage.
Progress can be monitored by periodic assessment of tissue growth and/or
repair. The progress can be monitored, for example, x-rays, histomorphometric
determinations and tetracycline labeling.
The following examples illustrate practice of the present invention in
recovering and characterizing Xenopus WA545 protein and employing the DNA it
to
recover human WA545 proteins, obtaining the human proteins and expressing the
proteins via recombinant techniques. The examples are not limiting, and the
invention
includes many variations as described herein, or will be apparent to those
skilled in
the art upon consideration of the detailed description of the invention and
preferred
embodiments thereof. The techniques and tools for such variations are known to
those skilled in the art, and such variations constitute part of the present
invention.
EXAMPLES
Example 1. Isolation of Xenonus cDNA
The Xenopus WA545 full-length cDNA was isolated from a dT-primed cDNA
library constructed in the plasmid vector CS2+. cDNA was made from Xenopus
embryos (stage 11.5-12). The probe sequences used to isolate the clone were
derived
from an sEST, an EST used to allow secretion of invertase in the signal
sequence trap,
which is described in United States Patents 5,536,637, the disclosure of which
is
hereby incorpated herein. The sequences of the probes for WA545 were as
follows:
5' - GAAAGTGATAGCCACAACTCTGCCATG - 3' (SEQ m NO 3) and
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WO 99/02678 PCT/US98/08334
5' - GATTTAGGTACAGGAGCTGGAGCAATG - 3' (SEQ >D NO 4).
Both probes were antisense sequence to the sEST. The DNA probes were
radioactively labelled with 32P and used to screen the Xenopus dT-primed cDNA
library, under high stringency hybridization/washing conditions, to identify
clones
containing sequences of the WA545 gene.
Approximately 61,000 library transformants were plated at a density of
approximately 4350 transformants per plate on selective plates to screen for
WA545.
Nitrocellulose replicas of the transformed colonies were hybridized to the ~zP-
labelled
DNA probe in standard hybridization buffer (6X SSC, 0.5% SDS, SX Denhardt's,
IOmM EDTA pHB, 100mg/ml Bakers Yeast ribonucleic acid) under high stringency
conditions (65°C for 2 hours). After 2 hours hybridization, the filters
were removed
from the hybridization solution and washed under high stringency conditions
(2X
SSC, 0.5% SDS 21 °C for 5 minutes; followed by 2X SSC, 0.1 % SDS 21
°C for 15
minutes; followed by a 2nd 2X SSC, 0.1 % SDS 21 °C for 15 minutes;
followed by 2X
SSC, 0.1 % SDS 65°C for 10 minutes). The filters were wrapped in Saran
wrap and
exposed to X-ray film for overnight to 3 days at room temperature. The
autoradiographs were developed and positively hybridizing transformants of
various
signal intensities were identified. These positive clones were picked, grown
for 5
hours in selective medium and plated at low density (approximately 100
colonies per
plate). Nitrocellulose replicas of the colonies were hybridized to the ~ZP-
labelled
probe in standard hybridization buffer (6X SSC, 0.5% SDS, SX Denhardt's, IOmM
EDTA pHB, 100mg/ml Bakers Yeast ribonucleic acid) under high stringency
conditions (65°C for 2 hours). After 2 hours hybridization, the filters
were removed
from the hybridization solution and washed under high stringency conditions
(e.g., 2X
SSC, 0.5% SDS 21°C for 5 minutes; followed by 2X SSC, 0.1% SDS
21°C for 15
minutes; followed by a second wash at 2X SSC, 0.1% SDS 21°C for 15
minutes;
followed by 2X SSC, 0.1% SDS 65°C for 10 minutes). The filters were
wrapped in
Saran wrap and exposed to X-ray film for overnight to 3 days at room
temperature.
The autoradiographs were developed and positively hybridizing transformants
were
identified. Bacterial stocks of purified hybridization positive clones were
made and
CA 02295086 1999-12-21
WO 99/02678 PCT/US98/08334
plasmid DNA was isolated. The sequence of the cDNA insert was determined. The
cDNA insert contained the sequences of the DNA probe used in the
hybridization.
Example 2. Isolation of WA545 Homolotzues
Using the DNA sequence reported herein for the newly isolated WA545
protein, DNA sequences encoding WA545 homologues and related proteins, such as
the murine and human WA545 protein, may be isolated by various techniques
known
to those skilled in the art. As described below, oligonucleotide primers may
be
designed on the basis of amino acid sequences present in other BMP proteins,
Vg-1
related proteins and other proteins of the TGF-(3 superfamily. Regions
containing
amino acid sequences which are highly conserved within the BMP family of
proteins
and within other members of the TGF-~i superfamily of proteins can be
identified and
consensus amino acid sequences of these highly conserved regions can be
constructed
based on the similari ty of the corresponding regions of individual BMP/TGF-
~3/Vg-1
proteins. It is contemplated that the WA545 protein of the invention and other
BMP/TGF-~3/Vg-1 related proteins may contain amino acid sequences similar to
the
consensus amino acid sequences described above and that the location of those
sequences within a WA545 protein or other novel related proteins would
correspond
to the relative locations in the proteins from which they were derived. It is
further
contemplated that this positional information derived from the structure of
other
BMP/1'GF-(3/Vg-1 proteins and the oligonucleotide sequences which have been
derived from consensus amino acid sequences could be utilized to specifically
amplify
DNA sequences encoding the corresponding amino acids of a WA545 protein or
other
BMP/TGF-~i/Vg-1 related proteins.
An example of such a consensus amino acid sequence is indicated below.
Consensus amino acid sequence:
Trp Xaa Xaa Trp Ile Xaa Ala (SEQ ID NO: 5) wherein the first Xaa is Glu,
Asn or Asp; the second Xaa is Asp, Glu or Asn; and the third Xaa is Val or
Ile.
Where X/Y indicates that either amino acid residue may appear at that
position.
The following oligonucleotide is designed on the basis of the above identified
consensus amino acid sequence ( 1 ):
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#1: GCGGATCCTGGVANGABTGGATHRTNGC (SEQ ID N0:6)
This oligonucleotide sequence is synthesized on an automated DNA
synthesizer. The standard nucleotide symbols in the above identified
oligonucleotide
primer are as follows: A, adenosine; C, cytosine; G, guanine; T, thymine; N,
adenosine or cytosine or guanine or thymine; R, adenosine or cytosine; Y,
cytosine or
thymine; H, adenosine or cytosine or thymine; V, adenosine or cytosine or
guanine;
D, adenosine or guanine or thymine.
The first eight nucleotides of oligonucleotide #1 (underlined) contain the
recognition sequence for the restriction endonuclease BamHI in order to
facilitate the
manipulation of a specifically amplified DNA sequence encoding the WA545 or
WA545-related proteins and are thus not derived from the consensus amino acid
sequence ( 1 ) presented above.
A second consensus amino acid sequence is derived from another highly
conserved region of BMP/TGF-~3/Vg-1 proteins as described below:
Asn His Ala Ile Xaa Gln Thr (SEQ ID N0:7) wherein Xaa is equal to Val or
Ixu.
The following oligonucleotide is designed on the basis of the above identified
consensus amino acid sequence (2):
#2: GCTCTAGAGTYTGNAYNATNGCRTGRTT (SEQ ID N0:8)
This oligonucleotide sequence is synthesized on an automated DNA
synthesizer. The same nucleotide symbols are used as described above.
The first eight nucleotides of oligonucleotide #2 (underlined) contain the
recognition sequence for the restriction endonuclease XbaI in order to
facilitate the
manipulation of a specifically amplified DNA sequence encoding the WA545 or
WA545-related proteins and are thus not derived from the consensus amino acid
sequence (2) presented above.
It is contemplated that the WA545 or WA545-related proteins of the invention
and other BMP/TGF-~i/Vg-1 related proteins may contain amino acid sequences
similar to the consensus amino acid sequences described above and that the
location
of those sequences within a WA545 or WA545-related protein or other novel
related
proteins would correspond to the relative locations in the proteins from which
they
27
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were derived. It is further contemplated that this positional information
derived from
the structure of other BMP/TGF-~3/Vg-1 proteins and the oligonucleotide
sequences
# 1 and #2 which have been derived from consensus amino acid sequences ( 1 )
and (2),
respectively, can be utilized to specifically amplify DNA sequences encoding
the
corresponding amino acids of a WA545 or WA545-related protein or other
BMP/TGF-~3/Vg-1 related proteins.
Based on the knowledge of the gene structures of BMP/TGF-(3/Vg-1 proteins,
it is further contemplated that Xenopus, human or murine genomic DNA can be
used
as a template to perform specific amplification reactions which would result
in the
identification of WA545 encoding sequences and WA545 proteins. Such specific
amplification reactions of a human or murine genomic DNA template can be
initiated
with the use of oligonucleotide primers as described earlier. Oligonucleotides
such
as those set forth in SEQ ID N0:6 and SEQ ~ N0:8 are utilized as primers to
allow
the specific amplification of a specific nucleotide sequence from Xenopus,
human,
murine or other genomic DNA. The amplification reaction is performed as
follows:
Genomic DNA is sheared by repeated passage through a 25 gauge needle,
denatured at 100°C for 5 minutes and then chilled on ice before adding
to a reaction
mixture containing 200 pM each deoxynucleotide triphosphates (dATP, dGTP, dCTP
and dTTP), 10 mM Tris-HCl pH 8.3, 50 mM KCI, 1.5 mM MgClz, 0.001 % gelatin,
1.25 units Taq DNA polymerase, 50 pM of each oligonucleotide, such as those
set
forth in SEQ ID N0:6 and SEQ m N0:8, to the consensus sequence, in a total
reaction volume of 50 pl. This reaction mixture is subjected to thermal
cycling in the
following manner: 1 minute at 94°C, 1 minute at 37°C, 2 minutes
at 72°C for thirty
cycles; followed by a 7 minute incubation at 72°C.
The DNA which is specifically amplified by this reaction is ethanol
precipitated, digested with the restriction endonucleases BamHI and XbaI and
subjected to agarose gel electrophoresis. A region of the gel, corresponding
to the
predicted size of the human or murine WA545 or WA545-related encoding DNA
fragment, is excised and the specifically amplified DNA fragments contained
therein
are electroeluted and subcloned into a suitable vector, for example, the
plasmid vector
pGEM-3 between the XbaI and BamHI sites of the polylinker. DNA sequence
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analysis of the resulting human or murine WA545 or WA545-related subclones is
conducted to determine whether the specifically amplified DNA sequence product
contained therein encode a portion of the human or murine WA545 or WA545-
related
protein of the invention.
Oligonucleotide probes, preferably 30-50 nucleotides in length, can be
designed on the basis of the human or murine WA545 or WA545-related
specifically
amplified DNA sequence described above. The oligonucleotide probes are
radioactively labeled with 32P and employed to screen a murine or human
genomic
library constructed in the vector ,FIX II (Stratagene catalog #946309) or a
comparable
substitute. 500,000 recombinants of the human genomic library are plated at a
density
I5 of approximately 10,000 recombinants per plate on 50 plates. Duplicate
nitrocellulose
replicas of the recombinant bacteriophage plaques are made one set of
nitrocellulose
filters is hybridized to one oligonucleotide probe and a duplicate set of
nitrocellulose
filters is hybridized to a second oligonucleotide probe, both in a
hybridization buffer
consisting of 5X SSC, I% SDS, 10% dextran sulfate, 2X Denhardt's, 100 pg/ml
herring salmon sperm DNA) at 60°C overnight. The following day the r
adioactively
labelled oligonucleotide containing hybridization solution is removed an the
filters are
washed with 5X SSC, 0.1 % SDS at 60°C. Recombinants which hybridize to
both
oligonucleotide probes are identified and plaque purified. The plaque purified
recombinant bacteriophage clones which hybridize to the Xenopus WA545
oligonucleotide probes and are analyzed to confirm that they encode a WA545
protein
of the invention using sequence analysis and the assays described herein.
Bacteriophage plate stocks are made and bacteriophage DNA is isolated from the
murine or human genomic clone. The complete insert of the murine or human
genomic recombinant is excised with restriction endonucleases, subcloned into
a
plasmid vector (pBluescript) and DNA sequence analysis is performed.
Based on the knowledge of other BMP proteins and other proteins within the
TGF-(3 family, it is predicted that the Xenopus WA545 precursor polypeptide
would
be cleaved at the multibasic sequence Arg-Ala-Lys-Arg in agreement with a
proposed
consensus proteolytic processing sequence of Arg-X-X-Arg. Cleavage of the
Xenopus
WA545 precursor polypeptide is expected to generate a 114 amino acid mature
29
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peptide beginning with the amino acid Ser at position #1 of SEQ 1D N0:2. The
processing of Xenopus and mammalian WA545 and WA545-related proteins into the
mature form is expected to involve dimerization and removal of the N-terminal
region
in a manner analogous to the processing of the related protein TGF-~3 [Gentry
et al.,
Molec & Cell. Biol., 8:4162 (1988); Derynck et al. Nature, 316:701 (1985)].
It is contemplated therefore that the mature active species of mammalian
WA545 proteins will comprise a homodimer of two polypeptide subunits, each
subunit comprising an amino acid sequence which correlates to a portion of the
sequence of SEQ ID 2, such as amino acids #1 through #114. Further active
species
are contemplated comprising at least amino acids # 13 to #I 14 of SEQ ID N0:2,
1 S thereby including the first conserved cysteine residue. As with other
members of the
TGF-~3BMP family of proteins, the carboxy-terminal portion of the murine WA545
protein exhibits greater sequence conservation than the more amino-terminal
portion.
The percent amino acid identity of the Xenopus WA545 protein in the cysteine-
rich
C-terminal domain (amino acids #24-#125) to the corresponding region of human
BMP proteins and other proteins within the TGF-a family is as follows: BMP-2,
60%;
BMP-3, 43%; BMP-4, 57%; BMP-5, 57%; BMP-6, 59%; BMP-7, 57%; BMP-8, 55%;
BMP-9, 49%; BMP-10, 51%; BMP-11, 41%; BMP-12, 51%; Vgl, 82%; GDF-1, 63%;
TGF-(31, 39°l0; TGF-[32, 40%; TGF-~i3, 43%; inhibin 13(B), 42%; inhibin
13(A), 43%.
The Xenopu.s WA545 DNA sequence (SEQ >D NO:1 ), or a portion thereof,
such as the portion of the Xenopus WA545 sequence corresponding to the mature
peptide encoding region, can be used as probe to identify corresponding
homologues
or related proteins, such as the human or murine WA545 or WA545-related
proteins.
Nucleotides #775 through #1116 of SEQ 1D NO:1 can be specifically amplified
with
oligonucleotide primers designed on the basis of the Xenopus WA545 sequence
(SEQ
ID NO:1). The following oligonucleotide primer is designed on the basis of
nucleotide #775 through #794 of the DNA sequence set forth in SEQ )D NO: l and
synthesized on an automated DNA synthesizer:
AGTACTCATTCATCACCTCC (SEQ >D N0:9)
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S The following oligonucleotide primer is designed on the basis of the reverse
compliment of nucleotide #1116 through #1097 of the DNA sequence set forth in
SEQ
ID NO:1 and synthesized on an automated DNA synthesizer.
CTTGCAACCACACTCATCCA (SEQ ID NO:10)
The amplification reaction is performed as follows: 10 ng of a bacterial
plasmid DNA containing the Xenopus WA545 full-length cDNA is added to a
reaction
mixture containing 200 pM each deoxynucleotide triphosphates (dATP, dGTP, dCTP
and dTTP) 10 mM Tris-HCl pH 8.3, 50 mM KCI, 1.5 mM MgCl2, 0.001 % gelatin,
1.25 units Taq DNA polymerase, and 100 pM of each oligonucleotide primer. The
reaction mixture is then subjected to thermal cycling in the following manner:
1
minute at 94°C, 1 minute at 55°C, 1 minute at 72°C for
thirty cycles. This
amplification reaction would be expected to generate a DNA fragment of
approximately 344 base pairs which encodes the entire mature peptide of the
Xenopus
WA545 protein of the invention. The resulting 344 by DNA product is visualized
following electrophoresis of the reaction products through a 2% agarose gel.
The
region of the gel containing the 344 base pair Xenopus WA545 DNA fragment is
excised and the specifically amplified DNA fragments contained therein are
extracted
(by electroelution or by other methods known to those skilled in the art). The
gel-
extracted 344 base pair DNA amplification product was radioactively labelled
with
32P and employed to screen a human genomic library constructed in the vector
eDASH
II (Stratagene catalog #945203). The same probe can be used to screen a murine
genomic library constructed in the vector eFIX II (Stratagene catalog
#946309).
Murine or Human WA545 or WA545-related genes
One million recombinants of the human or murine genomic library are plated
at a density of approximately 20,000 recombinants per plate on 50 plates.
Duplicate
nitrocellulose replicas of the recombinant bacteriophage plaques are
hybridized, under
reduced stringency conditions, to the 32P-labelled specifically amplified 344
by probe
in standard hybridization buffer (SHB = SX SSC, 0.1 % SDS, SX Denhardt's, 100
pg/ml salmon sperm DNA) at 60°C overnight. The following day the
radioactively
labelled oligonucleotide containing hybridization solution is removed an the
filters are
washed, under reduced stringency conditions, with 2X SSC, 0.1 % SDS at
60°C.
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Multiple positively hybridizing recombinants can be identified and plaque
purified.
These plaque purified recombinant bacteriophage are used to prepare
bacteriophage
plate stocks from which recombinant bacteriophage DNA can be isolated and
purified.
The resulting recombinant bacteriophage DNA, isolated from either marine or
human
genomic clones which were originally identified by hybridization to the
Xenopus
WA545 probe are analyzed for the presence of either human or marine WA545 or
WA545-related sequences by DNA sequence characterization.
The Xenopus WA545 DNA sequence (SEQ ID NO:1), or a portion thereof, can
be used as a probe to identify a human cell line or tissue which synthesizes
WA545
mRNA. Briefly described, RNA is extracted from a selected cell or tissue
source and
either electrophoresed on a formaldehyde agarose gel and transferred to
nitrocellulose,
or reacted with formaldehyde and spotted on nitrocellulose directly. The
nitrocellulose is then hybridized to a probe derived from the coding sequence
of
Xenopus WA545 DNA.
Alternatively, the Xenopus WA545 sequence is used to design oligonucleotide
primers which will specifically amplify a portion of the human or marine WA545
or
WA545-related encoding sequence located in the region between the human or
marine
primers utilized to perform the specific amplification reaction. It is
contemplated that
these Xenopus WA545 derived primers would allow one to specifically amplify
corresponding human WA545 or other mammalian WA545 encoding sequences from
mRNA, cDNA or genomic DNA templates. Once a positive source has been
identified by one of the above described methods, mRNA is selected by oligo
(dT)
cellulose chromatography and cDNA is synthesized and cloned in ~,gtl0 or other
~,
bacteriophage vectors known to those skilled in the art, for example, RZAP by
established techniques (Toole et al., supra). It is also possible to perform
the
oligonucleotide primer directed amplification reaction, described above,
directly on
a pre-established human cDNA or genomic library which has been cloned into a
~,
bacteriophage vector. In such cases, a library which yields a specifically
amplified
DNA product encoding a portion of the human WA545 protein could be screened
directly, utilizing the fragment of amplified human WA545 protein encoding DNA
as
a probe.
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Oligonucleotide primers designed on the basis of the DNA sequence of the
Xenopus WA545 genomic clone are predicted to allow the specific amplification
of
human WA545 encoding DNA sequences from pre-established human cDNA libraries
which are commercially available (i.e., Stratagene, La Jolla, CA or Clonetech
Laboratories, Inc., Palo Alto, CA). The oligonucleotide primers are designed
on the
basis of the DNA sequence set forth in SEQ a7 NO:1 and synthesized on an
automated DNA synthesizer.
Approximately 1 x 108 pfu (plaque forming units) of ~.bacteriophage libraries
containing human cDNA inserts corresponding to the primers are denatured at
95°C
for five minutes prior to addition to a reaction mixture containing 200 pM
each
deoxynucleotide triphosphates (dATP, dGTP, dCTP and dTTP) 10 mM Tris-HCl pH
8.3, 50 mM KCI, 1.5 mM MgCl2, 0.001 % gelatin, 1.25 units Taq DNA polymerise,
100 pM oligonucleotide primers. The reaction mixture is then subjected to
thermal
cycling in the following manner: 1 minute at 94°C, 1 minute at
50°C, 1 minute at 72°C
for thirty-nine cycles followed by 10 minutes at 72°C.
The resulting DNA product which is specifically amplified by this reaction is
visualized following electrophoresis of the reaction products through a 2%
agarose
gel. Once a positive cDNA source has been identified in this manner, the
corresponding cDNA library from which a WA545 specific sequence was amplified
could be screened directly with the other WA545 specific probes in order to
identify
and isolate cDNA clones encoding the full-length WA545 protein of the
invention.
Additional methods known to those skilled in the art may be used to isolate
other
full-length eDNAs encoding human WA545 proteins, or full length cDNA clones
encoding WA545 proteins of the invention from species other than humans,
particularly other mammalian species.
Alternatively, oligonucleotides are utilized as primers to allow the specific
amplification of human or murine WA545 specific nucleotide sequences from
Xenopus WA545 encoding plasmids. The amplification reaction is performed as
follows: Approximately 25 ng of Xenopus WA545 DNA is added to a reaction
mixture containing 200 uM each deoxynucleotide triphosphates (dATP, dGTP, dCTP
and dTTP) 10 mM Tris-HCl pH 8.3, 50 mM KCI, 1.5 mM MgClz, 0.001% gelatin,
33
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1.25 units Taq DNA polymerise, 100 pM each of oligonucleotide primers to the
Xenopus WA545 DNA sense and complementary orientations. The reaction mixture
is then subjected to thermal cycling in the following manner: 1 minute at
94°C, 1
minute at 53°C, 1 minute at 72°C for thirty cycles.
The DNA which is specifically amplified by this reaction would be expected
to generate a WA545 encoding product. The resulting DNA product is visualized
following electrophoresis of the reaction products through a 2% agarose gel.
The
region of the gei containing the WA545 DNA fragment is excised and the
specifically
amplified DNA fragments contained therein are extracted (by electroelution or
by
other methods known to those skilled in the art). The gel-extracted DNA
amplification product is radioactively labelled with 32P and employed to
screen a
human genomic library constructed in the vector ~. DASH II (Stratagene catalog
#945203).
Additional methods known to those skilled in the art can also be used to
isolate
human and other species homologues of WA545 and related proteins using the DNA
and amino acid sequences of the present invention.
Example 3. W-20 Bioassays
A. Description of W-20 cells
Use of the W-20 bone marrow stromal cells as an indicator cell line is based
upon the conversion of these cells to osteoblast-like cells after treatment
with a BMP
protein [Thies et al, Journal of Bone and Mineral Research, 5:305 ( 1990); and
Thies
et al, Endocrinolo~y, 130:1318 ( 1992)]. Specifically, W-20 cells are a clonal
bone
marrow stromal cell line derived from adult mice by researchers in the
laboratory of
Dr. D. Nathan, Children's Hospital, Boston, MA. Treatment of W-20 cells with
certain BMP proteins results in ( 1 ) increased alkaline phosphatase
production, (2)
induction of PTH stimulated cAMP, and (3) induction of osteocalcin synthesis
by the
cells. While (1) and (2) represent characteristics associated with the
osteoblast
phenotype, the ability to synthesize osteocalcin is a phenotypic property only
displayed
by mature osteoblasts. Furthermore, to date we have observed conversion of W-
20
stromal cells to osteoblast-like cells only upon treatment with BMPs. In this
manner,
34
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the in vitro activities displayed by BMP treated W-20 cells correlate with the
in vivo
bone forming activity known for BMPs.
Below two in vitro assays useful in comparison of BMP activities of novel
osteoinductive molecules, such as WA545, are described.
B. W-20 Alkaline Phosphatase Assay Protocol
W-20 cells are plated into 96 well tissue culture plates at a density of
10,000
cells per well in 200 pl of media (DME with 10% heat inactivated fetal calf
serum, 2
mM glutamine and 100 Units/ml penicillin + 100 pg/ml streptomycin. The cells
are
allowed to attach overnight in a 95% air, 5% COZ incubator at 37°C.
The 200 pl of media is removed from each well with a multichannel pipettor
and replaced with an equal volume of test sample delivered in DME with 10%
heat
inactivated fetal calf serum, 2 mM glutamine and 1 % penicillin-streptomycin.
Test
substances are assayed in triplicate.
The test samples and standards are allowed a 24 hour incubation period with
the W-20 indicator cells. After the 24 hours, plates are removed from the
37°C
incubator and the test media are removed from the cells.
The W-20 cell layers are washed 3 times with 200 pl per well of
calcium/magnesium free phosphate buffered saline and these washes are
discarded.
50 pl of glass distilled water is added to each well and the assay plates are
then
placed on a dry ice/ethanol bath for quick freezing. Once frozen, the assay
plates are
removed from the dry ice/ethanol bath and thawed at 37°C. This step is
repeated 2
more times for a total of 3 freeze-thaw procedures. Once complete, the
membrane
bound alkaline phosphatase is available for measurement.
50 NI of assay mix (50 mM glycine, 0.05% Triton X-100, 4 mM MgCl2, 5 mM
p-nitrophenol phosphate, pH = 10.3) is added to each assay well and the assay
plates
are then incubated for 30 minutes at 37 °C in a shaking waterbath at 60
oscillations per
minute.
At the end of the 30 minute incubation, the reaction is stopped by adding 100
pl of 0.2 N NaOH to each well and placing the assay plates on ice.
The spectrophotometric absorbance for each well is read at a wavelength of
405 nanometers. These values are then compared to known standards to give an
CA 02295086 1999-12-21
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estimate of the alkaline phosphatase activity in each sample. For example,
using
known amounts of p-nitrophenol phosphate, absorbance values are generated.
This
is shown in Table I.
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Table I
Absorbance Values for Known Standards
of P-Nitrophenol Phosphate
P-nitrophenol phosphate umoles Mean absorbance (405 nm)
0.000 0
0.006 0.261 +/- .024
0.012 0.521 +/- .031
0.018 0.797 +/- .063
0.024 1.074 +/- .061
0.030 1.305 +/- .083
Absorbance values for known amounts of BMPs can be determined and
converted to pmoles of p-nitrophenol phosphate cleaved per unit time as shown
in
Table II.
Table II
Alkaline Phosphatase Values for W-20 Cells
Treating with BMP-2
BMP-2 concentration Absorbance Reading umoles substrate
n /m~ 405 nmeters per hour
0 0.645 0.024
1.56 0.696 0.026
3.12 0.765 0.029
6.25 0.923 0.036
12.50 1.121 0.044
25.0 1.457 0.058
50.0 1.662 0.067
100.0 1.977 0.080
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These values are then used to compare the activities of known amounts of
WA545 to BMP-2.
C. Osteocalcin RIA Protocol
W-20 cells are plated at 106 cells per well in 24 well multiwell tissue
culture
dishes in 2 mls of DME containing 10% heat inactivated fetal calf serum, 2 mM
glutamine. The cells are allowed to attach overnight in an atmosphere of 95%
air 5%
"' COZ at 37 °C.
The next day the medium is changed to DME containing 10% fetal calf serum,
2 mM glutamine and the test substance in a total volume of 2 ml. Each test
substance
is administered to triplicate wells. The test substances are incubated with
the W-20
cells for a total of 96 hours with replacement at 48 hours by the same test
medias.
At the end of 96 hours, 50 ~l of the test media is removed from each well and
assayed for osteocalcin production using a radioimmunoassay for mouse
osteocalcin.
The details of the assay are described in the kit manufactured by Biomedical
Technologies Inc., 378 Page Street, Stoughton, MA 02072. Reagents for the
assay
are found as product numbers BT-431 (mouse osteocalcin standard), BT-432 (Goat
anti-mouse Osteocalcin), BT-431 R (iodinated mouse osteocalcin), BT-415
(normal
goat serum) and BT-414 (donkey anti goat IgG). The RIA for osteocalcin
synthesized
by W-20 cells in response to BMP treatment is carried out as described in the
protocol
provided by the manufacturer.
The values obtained for the test samples are compared to values for known
standards of mouse osteocalcin and to the amount of osteocalcin produced by W-
20
cells in response to challenge with known amounts of BMP-2. The values for BMP-
2
induced osteocalcin synthesis by W-20 cells is shown in Table III.
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Table III
Osteocalcin Synthesis by W-20 Cells
BMP-2 Concentration n~/mlOsteocalcin Synthesis n,/g
well
0 0.8
2 0.9
4 0.8
8 2.2
16 2.7
31 3.2
62 5.1
125 6.5
250 8.2
500 9.4
1000 10.0
Example 4. Rosen Modified Sampath-Reddi Assay
A modified version of the rat bone formation assay described in Sampath and
Reddi, Proc. Natl. Acad. Sci. USA, 80:6591-6595 (1983) is used to evaluate
hone
and/or cartilage and/or other connective tissue activity of BMP proteins. This
modified assay is herein called the Rosen-modified Sampath-Reddi assay. The
ethanol precipitation step of the Sampath-Reddi procedure is replaced by
dialyzing (if
the composition is a solution) or diaflltering (if the composition is a
suspension) the
fraction to be assayed against water. The solution or suspension is then
equilibrated
to 0.1 % TFA. The resulting solution is added to 20 mg of rat matrix. A mock
rat
matrix sample not treated with the protein serves as a control. This material
is frozen
and lyophilized and the resulting powder enclosed in #5 gelatin capsules. The
capsules are implanted subcutaneously in the abdominal thoracic area of 21-49
day old
male Long Evans rats. The implants are removed after 7-14 days. Half of each
implant is used for alkaline phosphatase analysis [see, Reddi et al, Proc.
Natl. Acad.
Sci., 69:1601 (1972)].
The other half of each implant is fixed and processed for histological
analysis.
1 pm glycolmethacrylate sections are stained with Von Kossa and acid fuschin
to
score the amount of induced bone and cartilage and other connective tissue
formation
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present in each implant. The terms +1 through +5 represent the area of each
histological section of an implant occupied by new bone and/or cartilage cells
and
matrix. A score of +5 indicates that greater than 50% of the implant is new
bone
and/or cartilage produced as a direct result of protein in the implant. A
score of +4,
+3, +2, and +1 would indicate that greater than 40%, 30%, 20% and 10%
respectively
of the implant contains new cartilage and/or bone.
Alternatively, the implants are inspected for the appearance of tissue
resembling embryonic tendon, which is easily recognized by the presence of
dense
bundles of fibroblasts oriented in the same plane and packed tightly together.
[Tendon/ligament-like tissue is described, for example, in Ham and Cormack,
Histolo~y (JB Lippincott Co. ( 1979), pp. 367-369, the disclosure of which is
hereby
incorporated by reference]. These findings may be reproduced in additional
assays in
which tendon/ligament-like tissues are observed in the WA545 protein
containing
implants.
The WA545 proteins of this invention may be assessed for activity on this
assay.
Example 5. Expression of WA545
In order to produce murine, human or other mammalian WA545 proteins, the
DNA encoding it is transferred into an appropriate expression vector and
introduced
into mammalian cells or other preferred eukaryotic or prokaryotic hosts by
conventional genetic engineering techniques. The preferred expression system
for
biologically active recombinant human WA545 is contemplated to be stably
transformed mammalian cells.
One skilled in the art can construct mammalian expression vectors by
employing the sequence of SEQ >D NO: 1, or other DNA sequences encoding WA545
proteins or other modified sequences and known vectors, such as pCD [Okayama
et
al., Mol. Cell Biol., 2:161-170 (1982)], pJL3, pJL4 [Gough et al., EMBO J.,
4:645-
653 (1985)] and pMT2 CXM.
The mammalian expression vector pMT2 CXM is a derivative of p91023(b)
(along et al., Science 228:810-815, 1985) differing from the latter in that it
contains
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the ampicillin resistance gene in place of the tetracycline resistance gene
and further
contains a XhoI site for insertion of cDNA clones. The functional elements of
pMT2
CXM have been described (Kaufman, R.J., 1985, Proc. Natl. Acad. Sci. USA
82:689-
693) and include the adenovirus VA genes, the SV40 origin of replication
including
the 72 by enhancer, the adenovirus major late promoter including a 5' splice
site and
the majority of the adenovirus tripartite leader sequence present on
adenovirus late
mRNAs, a 3' splice acceptor site, a DHFR insert, the SV40 early
polyadenylation site
(SV40), and pBR322 sequences needed for propagation in E. coli.
Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF, which
has been deposited with the American Type Culture Collection (ATCC),
Rockville,
MD (USA) under accession number ATCC 67122. EcoRI digestion excises the
cDNA insert present in pMT2-VWF, yielding pMT2 in linear form which can be
ligated and used to transform E. coli HB 101 or DH-5 to ampicillin resistance.
Plasmid pMT2 DNA can be prepared by conventional methods. pMT2 CXM is then
constructed using loopout/in mutagenesis [Morinaga, et al., Biotechnolo~y 84:
636
(1984). This removes bases 1075 to 1145 relative to the Hind III site near the
SV40
origin of replication and enhancer sequences of pMT2. In addition, it inserts
the
following sequence:
5' PO-CATGGGCAGCTCGAG-3'
at nucleotide 1145. This sequence contains the recognition site for the
restriction
endonuclease Xho I. A derivative of pMT2CXM, termed pMT23, contains
recognition sites for the restriction endonucleases PstI, Eco RI, SaII and
XhoI.
Plasmid pMT2 CXM and pMT23 DNA may be prepared by conventional methods.
pEMC2~i 1 derived from pMT21 may also be suitable in practice of the
invention. pMT21 is derived from pMT2 which is derived from pMT2-VWF. As
described above EcoRI digestion excises the cDNA insert present in pMT-VWF,
yielding pMT2 in linear form which can be ligated and used to transform E.
Coli HB
101 or DH-5 to ampicillin resistance. Plasmid pMT2 DNA can be prepared by
conventional methods.
pMT21 is derived from pMT2 through the following two modifications. First,
76 by of the 5' untranslated region of the DHFR cDNA including a stretch of 19
G
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residues from G/C tailing for cDNA cloning is deleted. In this process, a XhoI
site is
inserted to obtain the following sequence immediately upstream from DHFR: 5' -
CTGCAGGCGAGCCTGAATTCCTCGAGCCATCATG-3'
PstI Eco RI XhoI
Second, a unique CIaI site is introduced by digestion with EcoRV and XbaI,
treatment
with Klenow fragment of DNA polymerase I, and ligation to a CIaI linker
(CATCGATG). This deletes a 250 by segment from the adenovirus associated RNA
(VAI) region but does not interfere with VAI RNA gene expression or function.
pMT21 is digested with EcoRI and Xhol, and used to derive the vector pEMC2B 1.
A portion of the EMCV leader is obtained from pMT2-ECAT1 [S.K. Jung, et
al, J. Virol 63:1651-1660 (1989)] by digestion with Eco RI and PstI, resulting
in a
2752 by fragment. This fragment is digested with TaqI yielding an Eco RI-TaqI
fragment of 508 by which is purified by electrophoresis on low melting agarose
gel.
A 68 by adapter and its complementary strand are synthesized with a 5' TaqI
protruding end and a 3' XhoI protruding end which has the following sequence:
5'-CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT
TaqI
GAAAAACACGATTG_C-3'
X6oI
This sequence matches the EMC virus leader sequence from nucleotide 763 to
827.
It also changes the ATG at position 10 within the EMC virus leader to an ATT
and is
followed by a XhoI site. A three way ligation of the pMT21 Eco RI-XhoI
fragment,
the EMC virus EcoRI-TaqI fragment, and the 68 by oligonucleotide adapter TaqI-
XhoI adapter resulting in the vector pEMC2(31.
This vector contains the SV40 origin of replication and enhancer, the
adenovirus major late promoter, a cDNA copy of the majority of the adenovirus
tripartite leader sequence, a small hybrid intervening sequence, an SV40
polyadenylation signal and the adenovirus VA I gene, DHFR and (3-lactamase
markers
and an EMC sequence, in appropriate relationships to direct the high level
expression
of the desired cDNA in mammalian cells.
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The construction of vectors may involve modification of the WA545 DNA
sequences. For instance, WA545 cDNA can be modified by removing the non-coding
nucleotides on the 5' and 3' ends of the coding region. The deleted non-coding
nucleotides may or may not be replaced by other sequences known to be
beneficial for
expression. These vectors are transformed into appropriate host cells for
expression
of WA545 proteins. Additionally, the sequence of SEQ 1D NO:1 or other
sequences
encoding WA545 proteins can be manipulated to express a mature WA545 protein
by
deleting WA545 encoding propeptide sequences and replacing them with sequences
encoding the complete propeptides of other BMP proteins.
One skilled in the art can manipulate the sequences of SEQ ID NO: 1 by
eliminating or replacing the mammalian regulatory sequences flanking the
coding
sequence with bacterial sequences to create bacterial vectors for
intracellular or
extracellular expression by bacterial cells. For example, the coding sequences
could
be further manipulated (e.g. ligated to other known linkers or modified by
deleting
non-coding sequences therefrom or altering nucleotides therein by other known
techniques). The modified WA545 coding sequence could then be inserted into a
known bacterial vector using procedures such as described in T. Taniguchi et
al., Proc.
Natl Acad. Sci. USA, 77:5230-5233 ( 1980). This exemplary bacterial vector
could
then be transformed into bacterial host cells and a WA545 protein expressed
thereby.
For a strategy for producing extracellular expression of WA545 proteins in
bacterial
cells, see, e.g. European patent application EPA 177,343.
Similar manipulations can be performed for the construction of an insect
vector [See, e.g. procedures described in published European patent
application
155,476] for expression in insect cells. A yeast vector could also be
constructed
employing yeast regulatory sequences for intracellular or extracellular
expression of
the factors of the present invention by yeast cells. [See, e.g., procedures
described in
published PCT application W086/00639 and European patent application EPA
123,289].
A method for producing high levels of a WA545 protein of the invention in
mammalian cells may involve the construction of cells containing multiple
copies of
the heterologous WA545 gene. The heterologous gene is linked to an ampiifiable
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marker, e.g. the dihydrofolate reductase (DHFR) gene for which cells
containing
increased gene copies can be selected for propagation in increasing
concentrations of
methotrexate (MTX) according to the procedures of Kaufman and Sharp, J. Mol.
Biol., 159:601-629 ( 1982). This approach can be employed with a number of
different
cell types.
For example, a plasmid containing a DNA sequence for a WA545 protein of
the invention in operative association with other plasmid sequences enabling
expression thereof and the DHFR expression plasmid pAdA26SV(A)3 [Kaufman and
Sharp, Mol. Cell. Biol., 2:1304 (1982)] can be co-introduced into DHFR-
deficient
CHO cells, DUKX-BII, by various methods including calcium phosphate
coprecipitation and transfection, electroporation or protoplast fusion. DHFR
expressing transformants are selected for growth in alpha media with dialyzed
fetal
calf serum, and subsequently selected for amplification by growth in
increasing
concentrations of MTX (e.g. sequential steps in 0.02, 0.2, 1.0 and 5uM MTX) as
described in Kaufman et al., Mol Cell Biol., 5:1750 ( 1983). Transformants are
cloned,
and biologically active WA545 expression is monitored by the Rosen-modified
Sampath-Reddi rat bone formation assay described above in Example 4. WA545
protein expression should increase with increasing levels of MTX resistance.
WA545
polypeptides are characterized using standard techniques known in the art such
as
pulse labeling with [35S] methionine or cysteine and polyacrylamide gel
electrophoresis. Similar procedures can be followed to produce other related
WA545
proteins.
Example 6 Bioloeical Activity of Expressed WA545
To measure the biological activity of the expressed WA545 proteins obtained
in Example 5 above, the proteins are recovered from the cell culture and
purified by
isolating the WA545 proteins from other proteinaceous materials with which
they are
co-produced as well as from other contaminants. The purified protein may be
assayed
in accordance with the rat bone formation assay described in Example 4.
Purification is carried out using standard techniques known to those skilled
in
the art.
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Protein analysis is conducted using standard techniques such as SDS-PAGE
acrylamide [Laemmli, Nature 227:680 ( 1970)] stained with silver [Oakley, et
al. Anal.
Biochem. 105:361 ( 1980)] and by immunoblot [Towbin, et al. Proc. Natl. Acad.
Sci.
USA 76:4350 (1979)]
Example 7. Northern Analysis of WA545
Using Northern analysis, WA545 proteins can be tested for their effects on
various cell lines. Suitable cell lines include cell lines derived from E 13
mouse limb
buds. After 10 days of treatment with WA545 protein, the cell phenotype is
examined
histologically for indications of tissue differentiation. In addition,
Northern analysis
of mRNA from WA545 protein treated cells can be performed for various markers
including one or more of the following markers for bone, cartilage and/or
tendon/ligament, as described in Table IV:
Table IV
Marker Bone Cartilage Tendon/Ligament
Osteocalcin 1 - -
Alkaline Phosphatase + - -
Proteoglycan Core Protein +/-' + +2
Collagen Type I + + +
Collagen Type II +/-' + +2
Decorin + + +
Elastin +/-3 ? +
1- Marker seen early, marker not seen as mature bone tissue forms
2- Marker depends upon site of tendon; strongest at bone interface
3- Marker seen at low levels
Example 8. Embryonic Stem Cell Assay
In order to assay the further effects of the WA545 proteins of the present
invention, it is possible to assay the growth and differentiation effects in
vitro on a
number of available embryonic stem cell lines. One such cell line is ES-
E14TG2,
which is available from the American Type Culture Collection in Rockville, Md.
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In order to conduct the assay, cells may be propagated in the presence of 100
units of LIF to keep them in an undifferentiated state. Assays are setup by
first
removing the LIF and aggregating the cells in suspension, in what is known as
embryoid bodies. After 3 days the embryoid bodies are plated on gelatin coated
plates
( 12 well plates for PCR analysis, 24 well plates for immunocytochemistry) and
treated
with the proteins to be assayed. Cells are supplied with nutrients and treated
with the
protein factor every 2-3 days. Cells may be adapted so that assays may be
conducted
in media supplemented with 15% Fetal Bovine Serum (FBS) or with CDM defined
media containing much lower amounts of FBS.
At the end of the treatment period (ranging from 7-21 days) RNA is harvested
from the cells and analyzed by quantitative multiplex PCR for the following
markers:
Brachyury, a mesodermal marker, AP-2, an ectodermal marker, and HNF-3 a an
endodermal marker. Through immunocytochemistry, it is also possible to detect
the
differentiation of neuronal cells (glia and neurons), muscle cells
(cardiomyocytes,
skeletal and smooth muscle), and various other phenotype markers such as
proteoglycan core protein (cartilage), and cytokeratins (epidermis). Since
these cells
have a tendency to differentiate autonomously when LIF is removed, the results
are
always quantitated by comparison to an untreated control.
Example 9. In Situ Hybridization of WA545 with Embryos of Xenonus laevis
Albino embryos were collected at various stages for fixation, permeabilized
with proteinase K and pre-hybridized. They were then hybridized overnight with
digoxygenin-labeled riboprobes. Embryos were washed, treated with RNase A and
Tl to remove background and blocked with Boehringer Mannheim Blocking Reagent.
Embryos were incubated with alkaline phosphatase-conjugated anti-digoxygenin
antibody for four hours at room temperature, washed extensively before
chromogenic
reaction with alkaline phosphatase substrate. Embryos were then re-fixed and
de-
stained to remove background for photography.
Results: Expression profile.
Results from in situ hybridization (Figure 1 ) and developmental RT-PCR
(Figure 2) show that WA545 has no maternal transcript and is first expressed
at late
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blastula in the entire marginal zone and in some of the vegetal cells. The
expression
level increases at the onset of gastrulation. This high level expression is
maintained
during gastrulation and starts to decline during late stages of gastrulation.
By late
gastrula, WA545 expression is still present in lateral and ventral mesoderm
but is
excluded from the dorsal-most region which will form the notochord. The early
expression of this gene in the entire marginal zone and later posterior
restriction
correlates well with the conclusion that WA545 is involved in induction of
mesoderm
with posterior characteristics and modification of neural tissue formation.
Example 10. Whole embryo assay of WA545
Frog embryos were microinjected with SOpg or 100pg in vitro synthesized,
capped RNA at 2-cell or 4-cell stages. Beta-galactosidase RNA was included as
lineage tracer to determine the extent of diffusion of the injected RNA. The
product
of the b-gal RNA was visualized histochemically, by X-gal staining. The target
area
of these micro-injections were dorsal marginal zone or ventral marginal zone
of one
of the 2 or 4 cells. Embryos were left to develop until desired stages before
harvested
for fixation, X-gal staining and photography.
Results: WA545 lain-of-function phenotype in whole embryos.
Ventral microinjection of early embryos results in the formation of a
secondary
axis at later stages (Figure 3). This secondary axis does not contain a head,
implying
that WA545 is an inducer of posterior mesoderm. Dorsal micro-injection of
early
embryos results in a loss of anterior structures (Figure 4) including cement
gland
(chin), hatching gland, eyes, and forebrain. This observation indicates that
WA545
may convert anterior to more posterior tissue.
Example 11. Animal cap assay
Embryos were micro-injected with 50 to 400pg capped RNA in animal pole
of one cell at 2-cell stage. Globin RNA was used as a control. Embryos were
left to
develop until stage 8. Cells at the animal pole of embryos were microdissected
(animal caps) and cultured until sibling intact, uninfected embryos reach
stages 14 or
19. 15 animal caps microinjected with the same RNA (experimental or globin)
were
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pooled for total RNA preparation. 5 intact embryos were used for preparation
of
whole embryo control RNA. These RNA samples were reverse transcribed using
random hexamer as primers for cDNA. These DNA samples were subjected to PCR
using gene-specific primer pairs at the presence of 3zP-dCTP to assay for the
presence
of corresponding mRNAs in the original RNA samples. The primer pairs used and
cycle number for each pair were optimized previously. The products of these
PCR
reactions were subsequently resolved on polyacrylamide gels.
Results: Animal cap gain-of function assay.
Examination of a panel of molecular markers indicates that most mesodermal
markers are induced in animal cap assays (Figure 5). These markers include
brachyury, Pintallavis, Xnot and muscle-actin. Histologically, large blocks of
muscle
are formed. Of the neural markers examined, Krox20 is not induced while HoxB9
is.
Krox20 is normally expressed in rhombomeres 3 and 5 and HoxB9 is expressed in
the
posterior spinal cord. These results indicate that WA545 induces posterior
mesoderm
while anterior mesoderm (goosecoid) and neural (NCAM) genes are not activated.
This again lends support to the idea that WA545 is an inducer of mesoderm. of
posterior characteristics, and may modify neural fates from brain to spinal
cord.
Example 12. Summary of Results for WA545
WA545 is expressed from late blastula throughout the mesoderm and
endoderm. It is later expressed in posterior mesoderm. It is able to
efficiently induce
posterior and lateral mesoderm, including muscle. Thus, WA545 may be involved
in
formation of posterior regions and may be useful for ectopic activation of
muscle and
spinal cord development.
* * * * *
The foregoing descriptions detail presently preferred embodiments of the
present invention. Numerous modifications and variations in practice thereof
are
expected to occur to those skilled in the art upon consideration of these
descriptions.
Those modifications and variations are part of the present invention, and
believed to
be encompassed within the claims appended hereto.
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The disclosure of all of the publications and patent applications which are
cited
in this specification are hereby incorporated by reference for the disclosure
contained
therein.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: RACIE, LISA A.
LaVALLIE, EDWARD R.
SIVE, HAZEL
SUN, BENJAMIN
(ii) TITLE OF INVENTION: WA545 COMPOSITIONS
(iii) NUMBER OF SEQUENCES: 10
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: Massachusetts
(E) COUNTRY: USA
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
{B) COMPUTER: IBM PC compatible
{C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: L1S TBD
(B) FILING DATE: 10-JUL-1997
(C) CLASSIFICATION:
{viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: LAZAR, STEVEN R.
(B) REGISTRATION NUMBER: 32,618
(C) REFERENCE/DOCKET NUMBER: GI 5292
{ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8260
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1554 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
( ix ) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: 55..1119
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 55..774
(ix) FEATURE:
SO
T
CA 02295086 1999-12-21
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(A) NAME/KEY: mat~peptide
(B) LOCATION: 775..1119
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:1:
GAATTCCCAT CATAGCAACAAACA GTAGAGAAGT AACATG
57
AGCAACAAAC C
AGTACC
Met
-240
GCA GAGTTGTGG CTATCACTT TCTTGCATG TTCTCC TTGCTT CTACTG 105
Ala GluLeuTrp LeuSerLeu SerCysMet PheSer LeuLeu LeuLeu
-235 -230 -225
ACA AATTCATCT CCACTTACC TTCCAGGAA AGAATG CTCCTT AAAGCC 153
Thr AsnSerSer ProLeuThr PheGlnGlu ArgMet LeuLeu LysAla
-220 -215 -210
TTG GGGCTGAAC ACCAGACCA AACCCCATT GCTCCA GCTCCT GTACCT 201
Leu GlyLeuAsn ThrArgPro AsnProIle AlaPro AlaPro ValPro
-205 -200 -195
AAA TCTTTAAGA GACATTTTT GAGAAGGGG ATAAAC CAGGAC AATCCC 249
Lys SerLeuArg AspIlePhe GluLysGly IleAsn GlnAsp AsnPro
-190 -185 -180
TGC ATGATGGAA GGTTTCGGA GTACCTGGA AATATT GTCCGC TCATAT 297
Cys MetMetGlu GlyPheGly ValProGly AsnIle ValArg SerTyr
-175 -170 -165 -160
CGA GATCAAGGA ACCATAGCA GCCATAGAG GAGCCA CAAGGA 'rCTCTG 345
Arg AspGlnGly ThrIleAla AlaIleGlu GluPro GlnGly SerLeu
-155 -150 -145
TGC TTAAAGAAA TTTCTCTTT TTTGACCTA TCAGCA GTGGAG AACAAG 393
Cys LeuLysLys PheLeuPhe PheAspLeu SerAla ValGlu AsnLys
-140 -135 -130
GAG CAATTGACC CTAGGCCAA CTGGAAATT AAGTTC AAGCAC AACACA 441
Glu GlnLeuThr LeuGlyGln LeuGluIle LysPhe LysHis AsnThr
-125 -120 -115
TAT TATGGACAA CAGTTCCAT CTCCGCCTC TACCGC ACCCTT CAGCTA 489
Tyr TyrGlyGln GlnPheHis LeuArgLeu TyrArg ThrLeu GlnLeu
-110 -105 -100
TCT CTAAAAGGG ATGAGAGAC AGCAAGATG AACAGG AAGCTC CTGGTG 537
Ser LeuLysGly MetArgAsp SerLysMet AsnArg LysLeu LeuVal
-95 -90 -85 -80
ACT CAGTCTTTC CGTCTCCTT CACAAGTCC CTCTAT TTCAAC TTGACC 585
Thr GlnSerPhe ArgLeuLeu HisLysSer LeuTyr PheAsn LeuThr
-75 -70 -65
AAG GTGGCAGAG GACTGGAAA AACCCTGAG AAGAAT ATGGGT CTGATA 633
Lys ValAlaGlu AspTrpLys AsnProGlu LysAsn MetGly LeuIle
-60 -55 -50
CTG GAAATATAT GCAAGCAGT GAACTTGCA GGAGGC AATCGA TCATTT 681
Leu GluIleTyr AlaSerSer GluLeuAla GlyGly AsnArg SerPhe
-45 -40 -35
GTA GTATGTGAA CCAATACAG TCTTTCATT TACACT TCTCTG CTCACT 729
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Val Val Cys Glu Pro Ile Gln Ser Phe Ile Tyr Thr Ser Leu Leu Thr
-30 -25 -20
GTG TCC CTA GAC CCA TCC AAT TGC AAA ACT CAA CGA GCC AAG AGG AGT 777
Val Ser Leu Asp Pro Ser Asn Cys Lys Thr Gln Arg Ala Lys Arg Ser
-15 -10 -5 1
ACT CAT TCA TCA CCT CCA ACC CCA AGC AAT ATC TGC AAG AAA AGG AGA 825
Thr His Ser Ser Pro Pro Thr Pro Ser Asn Ile Cys Lys Lys Arg Arg
10 15
>.~. TTG TAC ATT GAC TTC AAG GAT GTT GGA TGG CAG AAC TGG GTC ATT GCA 873
Leu Tyr Ile Asp Phe Lys Asp Val Gly Trp Gln Asn Trp Val Ile Ala
20 25 30
CCCCGT GGTTACATG GCA TACTGCCAT GGAGAGTGC CCCTATCCA 921
AAC
ProArg GlyTyrMet AlaAsn TyrCysHis GlyGluCys ProTyrPro
35 40 45
CTGACG GAAATGCTA AGGGGC ACAAATCAT GCTGTTTTA CAGACTCTG 969
LeuThr GluMetLeu ArgGly ThrAsnHis AlaValLeu GlnThrLeu
50 55 60 65
GTGCAT TCTGTAGAA CCAGAA AACACCCCA TTGCCTTGC TGTGCCCCC 1017
ValHis SerValGlu ProGlu AsnThrPro LeuProCys CysAlaPro
70 75 80
ACTAAG CTGTCTCCT ATCTCC ATGCTATAT TATGACAAC AATGACAAT 1065
ThrLys LeuSerPro IleSer MetLeuTyr TyrAspAsn AsnAspAsn
85 90 95
GTGGTA CTGAGGCAC TATGAA GATATGGTA GTGGATGAG TGTGGTTGC 1113
ValVal LeuArgHis TyrGlu AspMetVal ValAspGlu CysGlyCys
100 105 110
AAGTGA GTTTGCTTTG TAAACTTATC 1169
GAGATTGTTC
TCATTCCCTT
ATCTAAGCCT
Lys
115
CTCTAAAGGGACTGCTGCCAACCTAGTTATGAAGCCTCGCGCCTCGTGCGACAGTGACTT 1229
TAACCATCTTACATAACATTAATTGATAAGACTATATTTATTTTGGGGTGTACTTGCCCT 1289
TTAGGTGGTTTGGCAAATGCCATGCGTGGCTCTTAACAGAGCTGCTGGATGAAACACATT 1349
TTTAAAAAAG TATATTGTTG TCAATAAATG TTTTTATCTT TATATATTGG GCATAGAGCT 1409
AGGTTGGTGC CTGAAAATTG CCTAGCACTT GCAAGTACAG CTGATTGTTG GAAATAAATG 1469
TGATTTAACC CP~AAAAAAAA P,~~~AAAAAAA F~t?1AAAAAAA AAAAAAAAAA AAAAAAAAAA 1529
C TCGAG 1554
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 355 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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{xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ala Glu Leu Trp Leu Ser Leu Ser Cys Met Phe Ser Leu Leu Leu
-240 -235 -230 -225
Leu Thr Asn Ser Ser Pro Leu Thr Phe Gln Glu Arg Met Leu Leu Lys
-220 -215 -210
Ala Leu Gly Leu Asn Thr Arg Pro Asn Pro Ile Ala Pro Ala Pro Val
-205 -200 -195
~~ Pro Lys Ser Leu Arg Asp Ile Phe Glu Lys Gly Ile Asn Gln Asp Asn
-190 -185 -180
Pro Cys Met Met Glu Gly Phe Gly Val Pro Gly Asn Ile Val Arg Ser
-175 -170 -165
Tyr Arg Asp Gln Gly Thr Ile Ala Ala Ile Glu Glu Pro Gln Gly Ser
-160 -155 -150 -145
Leu Cys Leu Lys Lys Phe Leu Phe Phe Asp Leu Ser Ala Val Glu Asn
-140 -135 -130
Lys Glu Gln Leu Thr Leu Gly Gln Leu Glu Ile Lys Phe Lys His Asn
-125 -120 -115
Thr Tyr Tyr Gly Gln Gln Phe His Leu Arg Leu Tyr Arg Thr Leu Gln
-110 -105 -100
Leu Ser Leu Lys Gly Met Arg Asp Ser Lys Met Asn Arg Lys Leu Lea
-95 -90 -85
Val Thr Gln Ser Phe Arg Leu Leu His Lys Ser Leu Tyr Phe Asn Leu
-80 -75 -70 -65
Thr Lys Val Ala Glu Asp Trp Lys Asn Pro Glu Lys Asn Met Gly Leu
-60 -55 -50
Ile Leu Glu Ile Tyr Ala Ser Ser Glu Leu Ala Gly Gly Asn Arg Ser
-45 -40 -35
Phe Val Val Cys Glu Pro Ile Gln Ser Phe Ile Tyr Thr Ser Leu Leu
-30 -25 -20
Thr Val Ser Leu Asp Pro Ser Asn Cys Lys Thr Gln Arg Ala Lys Arg
-15 -10 -5
Ser Thr His Ser Ser Pro Pro Thr Pro Ser Asn Ile Cys Lys Lys Arg
1 5 10 15
Arg Leu Tyr Ile Asp Phe Lys Asp Val Gly Trp Gln Asn Trp Val Ile
20 25 30
Ala Pro Arg Gly Tyr Met Ala Asn Tyr Cys His Gly Glu Cys Pro Tyr
35 40 45
Pro Leu Thr Glu Met Leu Arg Gly Thr Asn His Ala Val Leu Gln Thr
50 55 60
Leu Val His Ser Val Glu Pro Glu Asn Thr Pro Leu Pro Cys Cys Ala
65 70 75 80
Pro Thr Lys Leu Ser Pro Ile Ser Met Leu Tyr Tyr Asp Asn Asn Asp
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85 90 95
Asn Val Val Leu Arg His Tyr Glu Asp Met Val Val Asp Glu Cys Gly
100 105 110
Cys Lys
115
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GAAAGTGATA GCCACAACTC TGCCATG 27
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GATTTAGGTA CAGGAGCTGG AGCAATG 27
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:5:
Trp Xaa Xaa Trp Ile Xaa Ala
1 5
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
GCGGATCCTG GVANGABTGG ATHRTNGC 2g
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Asn His Ala Ile Xaa Gln Thr
1 5
(2) INFORMATION FOR SEQ ID NO:$:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
GCTCTAGAGT YTGNAYNATN GCRTGRTT 28
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
CA 02295086 1999-12-21
WO 99/02678 PCT/US98/08334
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
AGTACTCATT CATCACCTCC 20
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
CTTGCAACCA CACTCATCCA 20
56
CA 02295086 1999-12-21
WO 99/02678 PCT/US98/08334
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule l3bis)
A. The indications made below relate
to the microorganism referred to
in the description
on page 8 , lines 5-23
B. IDENTIFICATION OF DEPOSIT Further
deposits are identified on an additional
sheet
y:ame of depository institution
American Type Culture Collection
Address of depository institution
(including postal code and country)
10801 University Boulevard
Manassas, Virginia 20110-2209
United States of America
Date of deposit Accession Number
09 May 1997 98428, 98429, 98430, 98431, and
98432
C. ADDITIONAL INDICATIONS (lurvebtankif
not applicable) This information
is continued on an additional sheet
D. DESIGNATED STATES FOR WHICH INDICATIONS
ARE MADE (if tlw indications are
not jor all designated States)
I E. SEPARATE FURNISHING OF INDICATIONS
(leave blank if not applicable)
The indications listed below will
be submitted to the International
Bureau later (specifythegeneral
natureojtheindicationre.g., "Accession
Number of Deposit")
- For receiving Office use only For International Bureau use only ---
This sheet was received with the international application ~ This sheet was
received by the international Bureau on:
Authorized officer ~ ~ Authorized officer
/Nc,~~n~
Fotvt PCT/RO/134 (July 1992)
56/1
