U.S. patent application number 09/730772 was filed with the patent office on 2001-08-02 for dna molecules encoding cartilage-derived morphogenetic proteins.
Invention is credited to Chang, Steven Chao-Huan, Luyten, Frank P., Moos, Malcolm JR..
Application Number | 20010011131 09/730772 |
Document ID | / |
Family ID | 25271192 |
Filed Date | 2001-08-02 |
United States Patent
Application |
20010011131 |
Kind Code |
A1 |
Luyten, Frank P. ; et
al. |
August 2, 2001 |
DNA molecules encoding cartilage-derived morphogenetic proteins
Abstract
Nucleotide and amino acid sequences of cartilage-derived
morphogenetic proteins (CDMP-1 and CDMP-2) from human and bovine
cartilage extracts. These proteins exhibit chondrogenic activity
and can be used to repair cartilage defects in a mammal.
Inventors: |
Luyten, Frank P.;
(Rockville, MD) ; Moos, Malcolm JR.; (Bethesda,
MD) ; Chang, Steven Chao-Huan; (Chicago, IL) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
620 NEWPORT CENTER DRIVE
SIXTEENTH FLOOR
NEWPORT BEACH
CA
92660
US
|
Family ID: |
25271192 |
Appl. No.: |
09/730772 |
Filed: |
December 5, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09730772 |
Dec 5, 2000 |
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08836081 |
Jul 28, 1997 |
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08836081 |
Jul 28, 1997 |
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PCT/US94/12814 |
Nov 7, 1994 |
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Current U.S.
Class: |
536/23.5 ;
424/426 |
Current CPC
Class: |
C07K 14/51 20130101;
Y10S 930/12 20130101; A61K 38/1875 20130101; C07K 14/78 20130101;
A61K 35/32 20130101 |
Class at
Publication: |
536/23.5 ;
514/12; 424/426 |
International
Class: |
A61K 038/00; C07H
021/04; A61F 002/00 |
Claims
We claim:
1. A purified cartilage extract that stimulates local cartilage
formation when combined with a matrix and implanted into a mammal,
said extract being produced by a method comprising: (a) obtaining
cartilage tissue; (b) homogenizing said cartilage tissue in the
presence of chaotropic agents under conditions that permit
separation of proteins from proteoglycans; (c) separating said
proteins from said proteoglycans; and (d) obtaining said
proteins.
2. The extract of claim 1, wherein said extract is obtained by a
method in which step (c) comprises use of a sepharose column.
3. The extract of claim 1, wherein said extract is obtained by a
method which additionally comprises the steps of separating said
proteins on a molecular sieve and obtaining those proteins having a
molecular weight between 30 kDa and 60 kDa.
4. The extract of claim 1, wherein said extract is an extract of
articular cartilage.
5. The extract of claim 1, wherein said extract is an extract of
epiphyseal cartilage.
6. A method of preparing a partially purified articular cartilage
extract having chondrogenic activity, comprising the steps: (a)
obtaining cartilage tissue; (b) homogenizing said cartilage tissue
in the presence of chaotropic agents under conditions that permit
separation of proteins from proteoglycans; (c) separating said
proteins from said proteoglycans; and (d) obtaining said
proteins.
7. The method of claim 6, wherein step (c) comprises use of a
sepharose column.
8. The method of claim 7, wherein step (c) comprises isolating the
proteins that bind heparin Sepharose in the presence of 0.15 M NaCl
but not in the presence of 1 M NaCl.
9. The method of claim 6, additionally comprising the steps of
separating said proteins on a molecular sieve and obtaining those
proteins having a molecular weight between 30 kDa and 60 kDa.
10. An isolated DNA molecule encoding a protein having chondrogenic
activity in vivo but substantially no osteogenic activity in vivo,
said molecule having a nucleotide sequence that can hybridize to a
polynucleotide having a nucleotide sequence selected from the group
consisting of SEQ ID NO:11 and SEQ ID NO:12 at 55.degree. C. with
0.4.times.SSC and 0.1% SDS.
11. The isolated DNA molecule of claim 10, wherein said molecule
has a nucleotide sequence selected from the group consisting of SEQ
ID NO:13 and SEQ ID NO:14.
12. A recombinant protein having chondrogenic activity in vivo but
substantially no osteogenic activity in vivo, wherein said protein
has an amino acid sequence selected from the group consisting of
SEQ ID NO:13 and SEQ ID NO:14.
13. A method of stimulating cartilage formation in a mammal,
comprising the steps: a) supplying cartilage-derived morphogenetic
proteins having in vivo chondrogenic activity; b) mixing said
partially purified proteins with a matrix to produce a product that
facilitates administration of said partially purified proteins; and
c) implanting the product of step (b) into the body of mammal to
stimulate cartilage formation at the site of implantation.
14. The method of claim 13, wherein said partially purified
cartilage-derived morphogenetic proteins are obtained from
articular cartilage or epiphyseal cartilage.
15. The method of claim 13, wherein the matrix of step (b)
comprises a cellular material.
16. The method of claim 13, wherein mixing step (b) additionally
comprises mixing of viable chondroblast or chondrocytes.
17. The method of claim 5, wherein the implanting step comprises
implanting subcutaneously.
18. The method of claim 5, wherein the implanting step compises
implanting subcutaneously.
19. The method of claim 5, wherein the implanting step comprises
implanting intramuscularly.
20. A composition that can be administered to a mammal for the
purpose of stimulating chondrogenic activity at the site of
administration without substantially stimulating osteogenic
activity, said composition comprising at least one
cartilage-derived morphogenetic protein and a matrix.
21. The composition of claim 20, wherein said cartilage-derived
morphogenetic protein is derived from an extract of cartilage
tissue.
22. The composition of claim 20, wherein said cartilage tissue is
selected from the group consisting of articular cartilage and
epiphyseal cartilage.
23. The composition of claim 20, wherein said cartilage-derived
morphogenetic protein is a recombinant protein.
24. The composition of claim 20, wherein said recombinant protein
has a sequence selected from the group consisting of SEQ ID NO:13
and SEQ ID NO:14.
25. The composition of claim 20, wherein said matrix is selected
from the group consisting of fibrin glue, freeze-dried cartilage,
collagens and guanidinium-insoluble collagenous residue of
demineralized bone.
26. The composition of claim 20, wherein said matrix is a
non-resorbable matrix selected from the group comprising
tetracalcium phosphate and hydroxyapatite.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the field of
cartilage and bone development. More specifically, the invention
relates to cartilage-derived morphogenetic proteins that stimulate
development and repair of cartilage in vivo.
BACKGROUND OF THE INVENTION
[0002] Bone morphogenetic proteins (BMPs) are members of the
TGF-.beta. superfamily that can induce endochondral bone formation
in adult animals. This superfamily includes a large group of
structurally related signaling proteins that are secreted as dimers
and then cleaved to result in biologically active carboxy terminal
domains of the proteins. These bioactive proteins are characterized
by 7 highly conserved cysteine residues. Interestingly, these
proteins have different roles at various stages of embryogenesis
and in adult animals. Recombinant BMPs are now available and have
been shown to induce endochondral bone formation when assayed in
vivo.
[0003] Indeed, the initial discovery of the BMPs was facilitated by
such in vivo assays for cartilage and bone development. These
assays were based on the observation that bone development could be
initiated by subcutaneous or intramuscular implantation of
compositions comprising an extract of demineralized bone and
residual bone powder. The novel proteins identified in the extracts
were termed "bone morphogenetic proteins." These proteins were
subsequently classified as members of the TGF-.beta. superfamily by
virtue of amino acid sequence relatedness. Screening of genomic and
cDNA libraries led to the isolation of polynucleotides encoding
BMP-2, -3, -4, -5, -6 and -7.
[0004] One deficiency of the bone induction assay regards its
inability to distinguish the physiological roles of different BMP
family members. The cartilage and bone inducing activity of the
BMPs is remarkable because the normal stages of endochondral bone
formation that occur during ontogeny are recapitulated in the adult
animal. These stages include mesenchymal condensation, cartilage
and bone and bone marrow formation and eventual mineralization to
produce mature bone.
[0005] Several observations suggest that BMPs have wide-ranging
extraskeletal roles in development. First, localization studies in
both human and mouse tissues have demonstrated high levels of mRNA
expression and protein synthesis for various BMPs in kidney (BMPs
-3, -4, -7), lung (BMPs -3, -4, -5, -6), small intestine (BMPs -3,
-4, -7), heart (BMPs -2, -4, -6, -7), limb bud (BMPs -2, -4, -5,
-7) and teeth (BMPs -3, -4, -7). Second, several members of the
family, including BMP-4 and -7, are key molecules in
epithelial-mesenchymal interactions, for instance during
odontogenesis. Third, BMP-2 and BMP-4 are involved in the signaling
pathway that controls patterning in the developing chick limb and
BMP-4 is a ventralizing factor in early Xenopus development.
Fourth, Drosophila homologs of the BMPs, the decapentaplegic (dpp)
and 60 A gene products, have the capacity to induce bone in mammals
whereas human BMP-4 confers normal embryonic dorso-ventral
patterning in Drosophila transformants defective in dpp expression.
Thus, the BMPs are now appreciated as pleiotropic cytokines.
[0006] Interestingly, none of the known BMPs are strongly expressed
in the chondroblasts and chondrocytes of the cartilage core of
developing long bones. The hypertrophic chondrocytes, where both
Vgr-1 (BMP-6, (Lyons et al., Development 109:833 (1990)) and OP-1
(BMP-7)(Vukicevic et al., Biochem. Biophys. Res. Commun. 198:693
(1994)) have been found are exceptions in this regard.
SUMMARY OF THE INVENTION
[0007] One aspect of the present invention is a purified cartilage
extract that stimulates local cartilage formation when combined
with a matrix and implanted into a mammal. This extract can
conveniently be produced by a method which includes the steps of:
obtaining cartilage tissue; homogenizing the cartilage tissue in
the presence of chaotropic agents under conditions that permit
separation of proteins from proteoglycans; separating the proteins
from the proteoglycans and then obtaining the proteins. The step
for separating the proteins from the proteoglycans can be carried
out using a sepharose column. The extract can also be obtained by
additionally including the steps of separating the proteins on a
molecular sieve and then collecting the proteins having molecular
weights in the 30 kDa to 60 kDa size range. Articular cartilage or
epiphyseal cartilage can be used in the preparation of this
purified extract.
[0008] A second aspect of the present invention is a method of
preparing a partially purified articular cartilage extract having
chondrogenic activity. This method includes the steps of first
obtaining cartilage tissue; homogenizing the cartilage tissue in
the presence of chaotropic agents under conditions that permit
separation of proteins from proteoglycans; separating the proteins
from the proteoglycans and finally obtaining the proteins. The
separation of proteins and proteoglycans can be accomplished using
a sepharose column. In particular, the step for separating proteins
from proteoglycans can include isolating the proteins that bind
heparin Sepharose in the presence of 0.15 M NaCl but not in the
presence of 1 M NaCl. An additional step in the purification
procedure can include separating the proteins on a molecular sieve
and then obtaining the proteins having molecular weights between 30
kDa and 60 kDa.
[0009] A third aspect of the present invention is an isolated DNA
molecule that encodes a protein having chondrogenic activity in
vivo but substantially no osteogenic activity in vivo. More
particularly, this aspect of the invention regards a molecule
having a nucleotide sequence that can hybridize to a polynucleotide
which has the nucleotide sequence SEQ ID NO:11 or SEQ ID NO:12 at
55.degree. C. with 0.4.times.SSC and 0.1% SDS. The proteins encoded
by such DNA molecules can have the amino acid sequences of SEQ ID
NO:13 or SEQ ID NO:14.
[0010] A forth aspect of the present invention is a recombinant
protein having chondrogenic activity in vivo but substantially no
osteogenic activity in vivo. This protein can have the amino acid
sequence of SEQ ID NO:13 or SEQ ID NO:14.
[0011] A fith aspect of the present invention is a method of
stimulating cartilage formation in a mammal. This method includes
the steps: supplying cartilage-derived morphogenetic proteins
having in vivo chondrogenic activity; mixing the partially purified
proteins with a matrix to produce a product that facilitates
administration of thet partially purified proteins and implanting
this mixture into the body of mammal to stimulate cartilage
formation at the site of implantation. The partially purified
cartilage-derived morphogenetic proteins can be obtained from
either articular cartilage or epiphyseal cartilage. The matrix can
also include non-cellular material. Viable chondroblast or
chondrocytes can also be included in the mixture prior to
implantation. The mixture can be implanted either subcutaneously or
intramuscularly.
[0012] A sixth aspect of the present invention is a composition
that can be administered to a mammal for the purpose of stimulating
chondrogenic activity at the site of administration without
substantially stimulating osteogenic activity. This composition
comprises at least one cartilage-derived morphogenetic protein and
a matrix. The cartilage-derived morphogenetic protein can be
derived from an extract of either articular cartilage or epiphyseal
cartilage. In another embodiment, the cartilage-derived
morphogenetic protein is a recombinant protein. This recombinant
protein can have the amino acid sequence of either SEQ ID NO:13 or
SEQ ID NO:14. The matrix used to create the composition can be
either fibrin glue, freeze-dried cartilage, collagens or the
guanidinium-insoluble collagenous residue of demineralized bone.
Alternatively the matrix can be a non-resorbable matrix such as
tetracalcium phosphate or hydroxyapatite.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 presents the nucleotide and predicted amino acid
sequence encoded by the full length human CDMP-1 cDNA.
[0014] FIG. 2 presents the nucleotide and predicted amino acid
sequence encoded by the bovine CDMP-2.
[0015] FIG. 3 presents the genetic maps of chromosome 2 showing the
localization of CDMP-1. The map on the right is based on the data
from two separate crosses.
[0016] FIG. 4 shows an alignment of segments from predicted CDMP
amino acid sequences in standard one letter code.
DETAILED DESCRIPTION OF THE INVENTION
[0017] We discovered that partially purified extracts of newborn
calf articular cartilage contained an activity that induced
cartilage formation when implanted subcutaneously in rats. This
biological activity was reminiscent of that which characterized the
BMPs. Degenerate oligonucleotide primer sets derived from the
highly conserved carboxy-terminal region of the BMP family were
employed in reverse transcription-polymerase chain reactions
(RT-PCR) using poly(A).sup.+ RNA from articular cartilage as a
template. These procedures allowed us to determine which BMPs were
expressed in chondrocytes.
[0018] Two novel members of the TGF-.beta. superfamily were
identified and designated Cartilage-Derived Morphogenetic Protein-1
(CDMP-1), and -2 (CDMP-2). The C-terminal TGF-.beta. domains of
these proteins were 82% identical, thus defining a novel subfamily
most closely related to BMP-5, BMP-6 and osteogenic protein-1.
Northern analyses showed that postnatally both genes were
predominantly expressed in cartilaginous tissues. In situ
hybridization and immunostaining of sections from human embryos
showed that CDMP-1 was predominantly found at the stage of
precartilaginous mesenchymal condensation and throughout the
cartilaginous cores of the developing long bones. CDMP-2 expression
was restricted to the hypertrophic chondrocytes of ossifying long
bone centers. Neither gene was detectable in the axial skeleton
during human embryonic development. The cartilage-specific
localization pattern of these novel TGF-.beta. superfamily members,
which contrasts with the more ubiquitous presence of other BMP
family members, suggested a role for these proteins in chondrocyte
differentiation and growth of long bones.
[0019] The discovery of a novel subfamily of cartilage derived
morphogenetic proteins suggested the existence of morphogens that
primarily functioned in the induction and maintenance (i.e.,
balancing cartilage and bone formation at articular surfaces) of
cartilaginous and bony tissues. This subfamily may also include key
molecules that govern bone marrow differentiation.
[0020] The cartilage-derived morphogenetic proteins contained in
the cartilage extract of the present invention, and the recombinant
CDMP-1 and CDMP-2 proteins described herein are contemplated for
use in the therapeutic induction and maintenance of cartilage. For
example, local injection of CDMPs as soluble agents is contemplated
for the treatment of subglottic stenosis, tracheomalacia,
chondromalacia patellae and osteoarthritic disease. Other
contemplated utilities include healing of joint surface lesions
(e.g. temporomandibular joint lesions or lesions induced
posttraumatically or by osteochondritis) using biological delivery
systems such as fibrin glue, freeze-dried cartilage grafts, and
collagens mixed with CDMPs and locally applied to fill the lesion.
Such mixtures can also be enriched with viable cartilage progenitor
cells, chondroblasts or chondrocytes. We also contemplate repair or
reconstruction of cartilaginous tissues using resorbable or
non-resorbable matrices (tetracalcium phosphate, hydroxyapatite) or
biodegradable polymers (PLG, polylactic acid/polyglycolic acid)
coated or mixed with CDMPs. Such compositions may be used in
maxilofacial and orthopedic reconstructive surgery. Finally, the
CDMPs disclosed herein have utility as growth factors for cells of
the chondrocyte lineage in vitro. Cells expanded ex vivo can be
implanted into an individual at a site where chondrogenesis is
desired.
[0021] We also anticipate the polynucleotides disclosed herein will
also have utility as diagnostic reagents for detecting genetic
abnormalities associated genes encoding CDMPs. Diagnostic testing
could be performed prenatally using material obtained during
amniocentesis. Any of several genetic screening procedures could be
adapted for use with probes enabled by the present invention. These
procedures include restriction fragment length polymorphism (RFLP),
ligase chain reaction (LCR) or polymerase chain reaction (PCR).
[0022] We began our investigations by considering whether there
were differences between the chondrogenic/osteogenic
differentiation factors that characterized calcifying (epiphyseal,
scapular cartilage) and non-calcifying (articular, nasal septum)
cartilage tissues. It had been previously established that tail
tendon, achilles tendon, cartilage and skin matrices were devoid of
chondrogenic/osteogenic activity (originally described as
"transforming potency") as measured in an in vivo subcutaneous
implantation model in rats (Reddi A. H., 1976, "Collagen and Cell
differentiation" in Biochemistry of Collagen, eds. Ramachandran G.
N. and Reddi, A. H., pp449-478, Plenum Press, New York and
London.).
[0023] We confirmed the absence of chondrogenic or osteogenic
activity in crude 4 M guanidine HCl (GdnHCl) extracts of cartilage
matrices, but unexpectedly discovered in vivo chondrogenic activity
in the 0.15 M NaCl eluate of the cartilage extracts after ion
exchange chromatography. The development of a unique extraction
procedure (1.2 M GdnHCl and 0.5% CHAPs) followed by a heparin
Sepharose affinity chromatography step confirmed the presence of in
vivo chondrogenic activity in cartilaginous tissues. This was
especially true in bovine articular and epiphyseal cartilage. When
the bioactive heparin Sepharose eluates (1M NaCl eluate) were
further purified using previously established procedures, molecular
sieve chromatography and Con A affinity chromatography steps
followed by SDS polyacrylamide gel electrophoresis and gel elution,
chondrogenic activity was established. Implantation of 0.5 to 1
.mu.g gel eluted material resulted in in vivo chondrogenesis.
Surprisingly, and in contrast to the bone matrix purified activity,
none of the peptide sequences that were found in tryptic digests of
the highly purified cartilage extracts corresponded to any of the
known BMPs. However, the biological activity present in the
extracts was reminiscent of BMP-like activity by virtue of its loss
of activity upon reduction and alkylation, its affinity for heparin
Sepharose and Con A.
[0024] Although other materials and methods similar or equivalent
to those described herein can be used in the practice or testing of
the present invention, the preferred methods and materials are now
described. General references for methods that can be used to
perform the various nucleic acid manipulations and procedures
described herein can be found in Molecular Cloning: A Laboratory
Manual (Sambrook et al. eds. Cold Spring Harbor Lab Publ. 1989) and
Current Protocols in Molecular Biology (Ausubel et al. eds., Greene
Publishing Associates and Wiley-lnterscience 1987). The disclosures
contained in these references are hereby incorporated by reference.
A description of the experiments and results that led to the
creation of the present invention follows.
[0025] We initially discovered that an extract of cartilage
possessed a unique chondrogenic activity. In particular, we
discovered that newborn articular cartilage contained chondrogenic
activities when assayed in the in vivo subcutaneous implantation
model. Using a procedure adapted from that used for the isolation
of BMPs from demineralized bone matrix, we partially purified this
activity and thereby provided evidence for the presence of BMP-like
molecules in cartilage.
[0026] Example 1 describes the biochemical methods used to
characterize a chondrogenic activity present in bovine
cartilage.
EXAMPLE 1
Characterization of Cartilage Derived Chondrogenic Activity
[0027] Articular (metatarsophalangeal joints), scapular and nasal
cartilage (300 grams wet weight per tissue) were prepared from
newborn calves. Epiphyseal cartilage was dissected from fetal
bovine femurs (7-8 months). The tissues were finely minced and
homogenized with a Polytron (top speed, 2.times.30 seconds) in 20
volumes of 1.2 M GdnHCl, 0.5% CHAPS, 50 mM Tris-HCl pH 7.2,
containing protease inhibitors and extracted overnight at 4.degree.
C. as described by Luyten et al., in J. Biol. Chem. 264:13377
(1989), which is hereby incorporated by reference. The disclosure
of this article is hereby incorporated by reference. Sorgente et
al., (Biochem Biophys. Acta. 282:441 (1972)) disclosed these
procedures extract >90% of the lower molecular weight matrix
while leaving most of the high molecular weight proteoglycans
behind. The extracts were concentrated and exchanged with 6 M urea
by diafiltration using an Ultrasette.TM. (Filtron Technology Inc.,
MA) and loaded on a 0.5 L heparin Sepharose (Pharmacia/LKB, NJ)
column. Thereafter, the column was washed with 5 bed volumes of 6 M
urea, Tris HCl pH 7.4 with 0.15 M NaCl, and then eluted with 2 vol
1 M NaCl in the same buffer. Chondrogenic activity was assayed by
reconstituting a portion of the eluate with 25 mg of
guanidine-insoluble collagenous residue of demineralized rat bone
matrix according to procedures described by Luyten et al., in J.
Biol. Chem 264:13377 (1989). Implants were recovered after 10 days
and alkaline phosphatase activity was measured as a biochemical
indicator of cartilage and/or bone formation. The specific activity
was expressed as units of alkaline phosphatase/mg of protein used
for reconstitution in the bioassay. Implants were also examined
histologically for evidence of cartilage formation using standard
procedures known to those of ordinary skill in the art.
[0028] Additional purification steps were also performed. The 1 M
NaCl eluate of articular cartilage, which contained biological
activity, was concentrated by diafiltration and loaded onto a
Sephacryl S-200 HR gel filtration column (XK 50/100, Pharmacia/LKB,
NJ). After molecular sieve chromatography, the bioactive fractions
were pooled and exchanged with 50 mM Hepes, pH 7.4, containing 0.15
M NaCl, 10 mM MgSO.sub.4, 1 mM CaCl.sub.2 and 0.1% (w/v) CHAPS
using Macrosep.TM. concentrators (Filtron Technology Inc.,
Northborough, Mass.). The equilibrated sample was mixed with 1 ml
Con A Sepharose (Pharmacia-LKB) previously washed with 20 volumes
of the same buffer according to the procedure described by Paralkar
et al., in Biochem. Biophys. Res. Comm. 131:37 (1989). After
overnight incubation on an orbital shaker at 4.degree. C., the
slurry was packed into a disposable 0.7 cm ID Bio-Rad column and
washed with 20 volumes of the Hepes buffer to remove unbound
proteins. Bound proteins were eluted with 20 volumes of the same
buffer containing 500 mM methyl-D-mannopyronaside. The eluate was
concentrated to 200 .mu.l using Macrosep.TM. concentrators.
Macromolecules were then precipitated overnight with 9 volumes of
absolute ethanol at 4.degree. C. The precipitate was redissolved in
1 ml 6 M urea, Tris HCl pH 7.4. The bioactive bound protein was
then mixed with 2 X Laemmli sample buffer (without reducing agents)
and electrophoresed on a 12% preparative SDS/polyacrylamide gel.
Gel elution of the separated protein fractions and testing for
biological activity was performed as described by Luyten et al., in
J. Biol. Chem. 264:13377 (1989). We also observed that, after
reduction with dithiothreitol and alkylation with iodoacetamide,
substantially all of the cartilage-forming activity contained in
the protein sample was lost.
[0029] Results indicated that each of the crude extracts of the
different cartilaginous tissues (articular, nasal, scapular or
epiphyseal) were inactive when tested directly in the in vivo
cartilage and bone inducing assay. This finding confirmed
previously described results published by Reddi in "Collagen and
Cell differentiation" in Biochemistry of Collagen (eds.
Ramachandran G. N. and Reddi, A. H., pp449-478, Plenum Press, New
York and London (1976)). However, after heparin affinity
chromatography (Sampath et al., Proc. Natl. Acad. Sci. U.S.A.
84:7109 (1987)), chondrogenic activity was recovered in the 1 M
NaCl eluate from articular cartilage extracts. An additional
molecular sieve chromatography step (S200) was required to recover
chondrogenic activity from epiphyseal cartilage extracts. Similar
results were obtained upon ion exchange chromatography using DEAE
Sephadex (0.15 M NaCl eluate). Significantly, no activity was
detected in the extracts of the other cartilaginous tissues.
[0030] The highest specific activity was obtained for material
derived from articular cartilage (1 U alkaline phosphatase/mg
protein). This material was used for characterization of the
bioactivity. Further purification of the active fraction by
molecular sieve chromatography on Sephacryl S-200HR (specific
activity 112 U/mg), and affinity chromatography on Concanavalin A
(specific activity 480 U/mg), established the presence of cartilage
and bone inducing activity characteristic of the members of the BMP
family. Gel elution experiments with the Con A bound bioactive
fraction demonstrated that the activity resided between roughly 34
and 38 kDa (specific activity of the gel eluted fractions was 2143
U/mg). We have also demonstrated that size separation by molecular
sieve chromatography can be used to purify biological activity in
the 30-60 kDa size range. In addition, loss of activity that was
observed following reduction and alkylation suggested that the
bioactivity was induced either by a known or a new member(s) of the
BMP family.
[0031] Given the demonstration that cartilage contained a BMP-like
activity, we proceeded to isolate polynucleotides encoding the
responsible proteins. Specifically, degenerate primers
corresponding to conserved regions of known BMPs were designed.
These primers were then employed to amplify polynucleotides using
reverse transcribed mRNA from articular cartilage as a template.
These procedures ultimately led to the identification of two novel
cDNAs, which we called CDMP-1 and CDMP-2.
[0032] Example 2 describes the methods used to amplify
polynucleotides corresponding to mRNAs that were expressed in
cartilage tissue and that exhibited at least weak sequence
similarity to conserved regions of the BMP mRNAs.
EXAMPLE 2
PCR Amplification of cDNAs Encoding Cartilage-Derived Morphogenetic
Proteins
[0033] Total RNA from bovine articular chondrocytes
(metatarsophalangeal joints) was extracted using a modified acid
guanidine-phenol-chloroform method described by Chomczynski et al.,
in Anal. Biochem. 162:156 (1987) and by Luyten et al., in Exp.
Cell. Res. 210:224 (1994). Poly(A).sup.+ RNA was isolated using
magnetic beads (PolyATract.TM., Promega, Madison, Wis.). Four
degenerate oligonucleotide primers corresponding to highly
conversed motifs in the C-terminal region of the BMPs were used;
S1: 5'-GGITGG(C/A)AIGA(C/T)TGGAT(A/C/T)(A/G)TIGC(A/C/G/T)CC-3' (SEQ
ID NO:1) corresponding to amino acids [GW(Q/N)DWI(I/V)AP] (SEQ ID
NO:2); S2:
5'-GGITGG(A/T)(G/C)(I)GA(G/A)TGGAT(T/C/A)ATI(A/T)G(A/C/G/T)CC-3'
(SEQ ID NO:3) corresponding to amino acids [GWSEWIISP] (SEQ ID
NO:4); AS1:
5'-A(A/G)A/G)GT(C/T)TG(A/C/G/T)AC(A/G)AT(A/G)GC(A/G)TG(A/G)T -3'
(SEQ ID NO:5) corresponding to amino acids [NHAIVQTL] (SEQ ID
NO:6);
AS2:5'-CAI(C/G)C(A/G)CAI(G/C)(A/C/T)I(C/T)(C/G/T)IACIA(C/T)CAT-3'
(SEQ ID NO:7) corresponding to amino acids
[M(V/I)V(E/R)(G/S/A)C(G/A)C] (SEQ ID NO:8). Nucleotides in
parenthesis denote sites of degeneracy and I denotes inosine. First
strand cDNA synthesis was performed using 1 .mu.g Poly(A).sup.+ or
5 .mu.g total RNA with oligo dT, random hexanucleotide primers, or
the antisense degenerate primers, AS1 and AS2. Successful PCR
amplifications were performed with the degenerate sense primers, S1
or S2 in combination with the AS 1 antisense primer were performed
using conditions described by Wharton et al., in Proc. Natl. Acad.
Sci. U.S.A. 88:9214 (1991). The reaction products were
electrophoresed on 1.2% agarose gels, and DNA fragments of
appropriate sizes were excised and purified using the Magic PCR
Prep DNA purification system (Promega, Madison, Wis.).
Reamplification was performed with the same primers and each PCR
product was subcloned into the PCR II vector using the TA
Cloning.TM. System (In Vitrogen Corporation, San Diego, Calif.).
Results of RT-PCR using poly(A).sup.+ RNA isolated from newborn
bovine articular cartilage as template and sets of degenerate
oligonucleotide primers (S1/AS1 and S1/AS2) yielded amplification
products of 120 bp and 280 bp.
[0034] Subcloned inserts were sequenced according to the dideoxy
DNA sequencing method of Sanger et al., (Proc. Natl. Acad. Sci.
U.S.A. 74:5463 (1977)). Both DNA strands were sequenced using
Sequenase Version 2.0 DNA polymerase according to manufacturer's
instructions (USB, Cleveland, Ohio) with at least two-fold
redundancy. Confirmatory data in ambiguous regions were obtained by
automated thermal cycle sequencing with an Applied Biosystems Model
370A sequencer and by using 7-deaza-GTP (USB, Cleveland, Ohio). The
sequencing data were obtained from restriction fragments subcloned
into pBluescript (Stratagene, La Jolla, Calif.) using either M13
forward and reverse primers or synthetic oligonucleotide
primers.
[0035] The results from a computer-assisted search of the nucleic
acid sequence databases indicated the cloned inserts encoded BMP-2,
-6, BMP-7 (OP-1), and several other BMP-like sequences.
Identification of these latter gene fragments led us to isolate
larger cDNAs that included the entire protein coding region of the
transcript. The availability of such clones facilitated both a more
precise analysis of the encoded BMP-like protein and permitted
studies aimed at localizing the expression of these genes. Thus,
cloned inserts having novel BMP-like sequences were isolated,
radiolabeled and used to screen both human and bovine articular
cartilage cDNA libraries.
[0036] Example 3 describes the methods used to isolate human and
bovine cDNAs that corresponded to a segment of one of the BMP-like
gene segments that were amplified from cartilage mRNA
templates.
EXAMPLE 3
Library Screening
[0037] A 120 bp PCR fragment encoding part of the C-terminal domain
of novel BMP like genes (dashed line, FIG. 1) was used to screen
two cDNA libraries. One library, from adolescent human articular
cartilage poly(A).sup.+ RNA (kindly provided by Dr. Bjorn Olsen,
Harvard, Boston, Mass.), was primed with oligo dT and constructed
in the .lambda.gtl 1 vector. The other was a bovine oligo dT and
random primed articular cartilage cDNA library constructed in the
UNIZAP.RTM.XR vector (Stratagene, La Jolla, Calif.). Approximately
1.times.10.sup.6 plaques from each library were screened by
standard procedures. Hybridizations were performed for 20 hours at
42.degree. C. in 6.times.SSC, 1.times.Denhardt's solution, 0.01%
tRNA, 0.05% sodium pyrophosphate and the membranes (DuPont 137 mm
nylon membranes, New England Nuclear, MA) were washed to final
stringency of 6.times.SSC, 0.1% SDS at 55.degree. C. for 20
minutes.
[0038] Thus, cloned inserts having novel BMP-like sequences were
isolated, radiolabeled and used to screen both human and bovine
articular cartilage cDNA libraries. Six clones were isolated from
the human cDNA library. The sizes of the EcoRI inserts (2.1 kb) and
their restriction maps were found to be identical for all six
clones. One clone was used for nucleotide sequencing. An open
reading frame encoding a BMP related protein, designated CDMP-1,
was identified. It appeared that the human cDNA clone lacked the
coding region for the first methionine and signal peptide. The 5'
end of the human CDMP-1 was subsequently obtained from a human
genomic clone isolated from a library constructed in the EMBL-3
vector (Clontech, Palo Alto, Calif.). The 5' end of human CDMP-1
contained a consensus translation initiation sequence disclosed by
Kozak (J. Biol. Chem. 266:19867 (1991)) immediately followed by a
putative transmembrane signal sequence described by Von Heijne
(Nucl. Acids Res. 14:4683 (1986)). The nucleotide sequence and the
translation of the open reading frame of CDMP-1 are presented in
FIG. 1. As shown in the figure, the CDMP-1 protein was predicted to
have 500 amino acids, to consist of a pro-region of 376 amino
acids, a typical cleavage site (Arg-Xaa-Xaa-Arg/Ala) (SEQ ID NO:9),
and a C-terminal domain of 120 amino acids containing the seven
highly conserved cysteines characteristic of the TGF-.beta. gene
family. A single N-linked glycosylation site is located in the
pro-region (marked by an asterisk in the figure). A putative signal
peptide is underlined in bold. A termination codon (TGA) is shown
in the 5' untranslated region. The bold dashed underline indicates
the fragment obtained by RT-PCR that was subsequently used to
screen cDNA libraries. The 13 amino acid peptide used to raise
polyclonal antibodies in rabbits is underlined. A vertical
arrowhead marks the boundary between the sequence obtained from
genomic DNA and cDNA.
[0039] Two clones with inserts of 2.8 kb were isolated from a
bovine articular cartilage cDNA library. Both clones were sequenced
and the open reading frame was found to encode another novel
TGF-.beta. related protein, designated CDMP-2. The CDMP-2 cDNA and
predicted protein sequences are presented in FIG. 2. As shown in
the figure, the open reading frame contained a putative proteolytic
processing site (boxed), preceding a 120 amino acid mature
C-terminal region containing seven highly conserved cysteines. The
5' end with the first methionine and signal peptide were missing.
The product obtained by RT-PCR (bold dashed underline) was used to
screen a bovine cDNA articular cartilage library. The ApaI sites
used to release a cDNA fragment for hybridization experiments are
underlined. At the 5' end, the pro-region lacked the first
methionine and signal peptide. The mature C-terminal domain of 120
amino acids showed 82% identity with CDMP-1.
[0040] Alignment of the carboxy terminal domains of CDMP-1 and -2
with other members of the BMP family revealed an amino acid
identity of about 50% with BMP-5, BMP-6 and OP-1 (BMP-7). These
results suggested that CDMP-1 and CDMP-2 are members of a new
subfamily.
[0041] The amino acid sequence similarity between the human CDMP-1
and bovine CDMP-2 proteins prompted us to further investigate
conservation of the CDMPs across different species. In particular,
we employed a PCR amplification protocol to isolate CDMP cDNA
sequences from a variety of species. Based on alignments of the
predicted proteins encoded by these cDNAs, we identified a highly
conserved amino acid sequence spanning 31 residues. Only 5 amino
acid positions within this sequence showed variability. All
remaining positions were identical for all isolates. As disclosed
in the following Example, even the 5 variable positions showed a
high degree of conservation. This structural conservation likely
represents a functional domain that is characteristic of the CDMP
family of proteins. Those of ordinary skill in the art will
appreciate that such extraordinary amino acid sequence conservation
is indicative of a functional domain. We therefore believe the
consensus amino acid sequence presented in the following Example is
critical to the biological activity of the CDMPs.
[0042] Example 4 describes the procedures used to identify an amino
acid consensus sequence that characterizes the CDMPs from several
different species.
EXAMPLE 4
Identification of a Highly Conserved Consensus Sequence in CDMP
Proteins
[0043] RNA isolated from chicken sternal cartilage, bovine
articular cartilage and human articular cartilage was employed as
the template in RT-PCR protocols using the primers S1 and AS1 and
procedures described under Example 2. Genomic DNA isolated from
Xenopus and zebrafish was also used as the template for
amplification of related gene sequences in a PCR protocol that
employed the same primer sets. Amplified DNA fragments were
subcloned according to standard procedures. The inserts from
various isolates were sequenced by standard dideoxy chain
termination protocols. Aligned segments of the predicted proteins
encoded by the cloned cDNAs are presented in FIG. 4.
[0044] Results of the protein alignments clearly indicated that
CDMP family members from several species shared a common amino acid
sequence motif in the region of the proteins encoded by the
amplified cDNA segments. Of the 31 amino acid positions presented
in FIG. 4, all but 5 were occupied by identical amino acid residues
for all of the isolates. The variable amino acids were located at
positions 3, 7, 11, 16 and 18. Position 3 was occupied either by I,
M or V. Position 7 was occupied by either D or E, both of which
have acidic side groups. Position 11 was occupied by either Y, F or
H. Position 16 was occupied by L or V, and position 18 was occupied
by D or E. The consensus deduced from this alignment was:
W-I-(I/M/V)-A-P-L-(D/E)-Y-E-A-(Y/F/H)-H-C-E-G-(L/V)-C-(D/E-
)-F-P-L-R-S-H-L-E-P-T-N-H-A (SEQ ID NO:15). This consensus sequence
is slightly broader than the one shown in FIG. 4, as it encompasses
all the variations observed in the sequenced polynucleotides. The
consensus sequence in the figure indicates predominating amino
acids.
[0045] We believe that biologically active CDMPs will possess this
highly conserved amino acid sequence motif. Proteins having
different amino acids in the variable positions in the consensus
will likely represent novel family members having distinct
functions. We also believe that polynucleotide hybridization probes
or PCR primers designed based on this conserved protein motif can
be used to isolate cDNAs encoding CDMP family members or related
proteins.
[0046] Southern analyses were also carried out to investigate
possible sequence conservation across species and to localize the
CDMP-1 gene to a particular chromosome.
[0047] Example 5 describes the Southern blotting protocols used to
detect DNA sequences corresponding to the CDMP-1 cDNA.
EXAMPLE 5
Genetic Mapping of CDMP-1
[0048] Southern hybridization was performed using the evolutionary
relatedness blot (Bios Laboratories, New Haven, Conn.) under
conditions recommended by the manufacturer. The panel of
EcoRI-digested genomic DNAs included human (homo sapiens), mouse
(Mus musculus), chicken (Gallus domesticus), frog (Xenopus laevis),
lobster (Homarus americanus), mussel (Mytilus edulis), fish
(Tautoga onitis), fruit fly (Drosophila melanogaster), nematode
(Caenorhabditis elegans), yeast (Saccharomyces cerevisiae) and
bacteria (E. coli). The 2.1 kb CDMP-1 EcoRI fragment originally
obtained from the cDNA library was used as a probe, and the blot
was washed to a final stringency of 0.4.times.SSC, 0.1% SDS, at
55.degree. C.
[0049] Results from these Southern analyses using the original 2.1
kb human CDMP-1 cDNA probe (starting from amino acid position 40),
showed 5.9 and 2.6 kb bands in humans and strong hybridization in
both mouse and chicken. Fainter bands were seen in fish, frog and
lobster after 5 days autoradiographic exposure. No hybridization
was detected to Drosophila DNA.
[0050] The 2.1 kb ApaI fragment of CDMP-1 was used as a
hybridization probe on Southern blots to type mouse genomic DNAs
from two genetic crosses: (NFS/N or C58/J.times.M. m.
musculus).times.M. m. musculus (see Joseph et al., Mol. Immunol.
30:733 (1990)) and (NFS/N.times.M. spretus).times.M. spretus or
C58/J (see Adamson et al., Virology 183:778 (1991)). DNAs from
these crosses have been typed for over 650 markers including the
Chr 2 markers Snap (synaptosomal associated protein 25), Psp
(parotid secretory protein), Emv15 (ecotropic murine leukemia virus
15), Src (src oncogene), and Cd40 (cluster designation 40). Probes
for these markers and restriction fragment length polymorphisms
used to type these crosses have been described by Joseph et al., in
Mol. Immunol. 30:733 (1990) and by Grimaldi et al., in J. Immunol.
149:3921 (1992). Src was typed using a mouse Src probe obtained
from E. Rassart (U. Quebec, Montreal) following XbaI digestion in
the musculus cross and BamHI digestion in the spretus cross.
[0051] Results from Southern blotting with the 2.1 kb cDNA
described above identified EcoRI fragments of 7.1 and 2.0 kb in M.
m. spretus and M. m. musculus and 6.8 and 3.2 kb in NFS/N and
C58/J.
[0052] Inheritance of the polymorphic fragments in the progeny of
the two crosses used for mapping was compared with inheritance of
over 650 markers previously mapped to all 19 autosomes and the X
chromosome. The gene encoding CDMP-1 was found to be linked to
markers on Chr 2 just proximal to Src. The closest linkage was
observed with Psp and Emv15. No recombination was observed between
Cdmp1 and Psp in the 100 mice typed for both markers indicating
that these genes are within 3.0 cM at the 95% confidence level.
Similarly, the absence of recombination between Gdf5 (Storm et al.,
Nature 368:639 (1994)) and Cdmp1 in 125 mice suggested these genes
colocalized within 2.4 cM. This map location suggested close
proximity to the brachypodism locus (bp). A genetic map that
presents the localization of CDMP-1 on chromosome 2 is shown in
FIG. 3. Recombination fractions are given to the right of each map
of the diagram for each adjacent locus pair or cluster. Numbers in
parenthesis represent the percent recombination and standard error
calculated as described by Green in Genetics and Probability in
Animal Breeding Experiments, Oxford University Press, New York
(1981). The map on the left is an abbreviated version of the Chr 2
Committee Map disclosed by Siracusa et al., in Mammal Genome 4:S31
(1993), and shows the map location of bp relative to the other
markers typed in the crosses used here.
[0053] The brachypodism (bp mice) disorder is characterized by a
distinct shortening of the limbs without other tissue
abnormalities. The defect has previously been attributed to lack of
production of a chondrogenic signal by mesenchymal cells at the
time of chondrogenesis (Owens et al., Dev. Biol. 91:376 (1982)).
During the course of our investigation, an independent study by
Storm et al. (Nature 368:639 (1994)) described the isolation of the
mouse CDMP-1 homolog, called Gdf-5, and established its linkage to
the bracypodism (bp) mutation. The types of mutations observed in
bp mice were found to be effective null-mutations for the gene
encoding Gdf-5/CDMP-1. The pattern of expression of CDMP-1
throughout the cartilaginous core observed during human embryonic
long bone development, coupled with the bp mutation in mice,
indicated that its primary physiological role was most likely at
the stage of early chondrogenesis and chondrocyte differentiation
in the developing limb.
[0054] The foregoing results indicated the CDMP-1 and CDMP-2 cDNAs
were novel, exhibited moderate sequence conservation across species
as judged by evolutionary hybridization studies and that the CDMP-1
gene localized to mouse chromosome 2. We proceeded to examine the
pattern of CDMP expression at the mRNA level.
[0055] Example 6 demonstrates the methods used to determine the
pattern of CDMP mRNA expression.
EXAMPLE 6
CDMPs are Predominantly Expressed in Cartilage During Postnatal
Life
[0056] Equal amounts of poly(A).sup.+ RNA (2 .mu.g) from bovine
criocoid and articular cartilage were electrophoresed on 1.2%
agarose-formaldehyde gels and then transferred to Nytran membranes
(Schleicher and Schuell, Kenne, N H) according to standard
laboratory procedures. Multiple Tissue Northern blots were obtained
from Clontech (Palo Alto, Calif.). The membranes were prehybridized
for 3 hours at 42.degree. C. in hybridization buffer (5.times.SSPE,
5.times.Denhardt's solution, 50% formamide, 1% SDS and 100 .mu.g/ml
freshly denatured salmon sperm DNA). Hybridizations with
[.sup.32P]dCTP labeled probes, having specific activities of at
least 1.times.10.sup.9 CPM/.mu.g, were performed overnight under
the same conditions as the prehybridization. Probes included the
cDNA probe for human glyceraldehyde-3-phosphate dehydrogenase (1.1
kb, G3PDH (Clontech, Palo Alto, Calif.), an ApaI fragment (bp
470-1155) of CDMP-1, and an ApaI fragment (bp 194-677) of CDMP-2.
The CDMP-1 and CDMP-2 probes were chosen to avoid the highly
conserved carboxy-terminal domain, thereby minimizing the potential
for cross hybridization with other members of the gene family.
Following hybridization, the filters were washed to a final
stringency of 55.degree. C., 0.4.times.SSC, 0.1% SDS. The mRNA
expression levels were quantified using a Phosphorimager (Molecular
Dynamics, Sunnyvale, Calif.).
[0057] Results from Northern analyses of a number of postnatal
tissues indicated that CDMP-1 could predominantly be detected in
newborn articular and cricoid cartilage. In both cases a single
mRNA transcript of approximately 3 kb was observed. The CDMP-1 mRNA
was not detected in pancreas, kidney, skeletal muscle, liver, lung,
placenta, brain or heart. In contrast, BMP-3 and BMP-7 transcripts
were detected in subsequent hybridizations of the same blots in
mRNA samples from lung, kidney, brain and small intestine. This
finding was consistent with previous results disclosed by Vukicevic
et al., (J. Histochem. Cytochem. 42:869 (1994)). CDMP-2 mRNA was
detected in postnatal bovine articular and cricoid cartilage as a
4.6 kb mRNA band. After prolonged exposure, weak hybridization
signals were detected at 4.6 kb and 4.0 kb in mRNA from colon and
small intestine, skeletal muscle and placenta.
[0058] Two other procedures were used to localize and visualize
expression of the CDMP-1 and CDMP-2 gene products. These approaches
relied on detection of mRNA and protein in tissue sections prepared
for analysis by microscopy.
[0059] Example 7 describes the methods used to demonstrate the
preferential expression of CDMPs during human embryogenesis.
EXAMPLE 7
CDMPs are Preferentially Expressed in the Cartilaginous Cores of
Long Bone During Human Embryogenesis
[0060] In Situ Hybridization
[0061] Tissues from human embryos were obtained after pregnancy
termination at from 5 to 14 weeks of gestation. Embryo age was
estimated in weeks (W) on the basis of crown-rump length (CRL) and
pregnancy records of the conceptual age. They were fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.2), embedded in
paraffin, sectioned serially at 5-7 .mu.m, and mounted on silanated
slides. The tissues used in the present study were obtained from
legally sanctioned procedures performed at the University of Zagreb
Medical School. The procedure for obtaining the human autopsy
material was approved and controlled by the Internal Review Board
of the Ethical Committee at the School of Medicine, University of
Zagreb and Office of Human Subjects Research (OHSR) at the National
Institutes of Health, Bethesda, Md. In situ hybridization was done
as described by Vukicevic et al., (J. Histochem. Cytochem. 42:869
(1994)) and by Pelton et al. (Development 106:759 (1989)). Briefly,
sections were incubated overnight at 50.degree. C. in a humidified
chamber in 50% formamide, 10% dextran sulfate, 4.times.SSC, 10 mM
dithiothreitol, 1.times.Denhardt's solution, 500 .mu.g/ml of
freshly denatured salmon sperm DNA and yeast tRNA with 0.2-0.4
ng/ml .sup.35S labeled riboprobe (1.times.10.sup.9 CPM/.mu.g). ApaI
fragments of CDMP-1 and of CDMP-2 (described above) from the pro
region, subcloned in both sense and anti-sense direction into
pBluescript II (SK).sup.+ vector (Strategene, Calif.), were used as
transcription templates. Riboprobes were then prepared using T7 RNA
polymerase (Sure Site Kit, Novagen, Madison, Wis.) according to the
manufacturer's instructions and used with and without prior
alkaline hydrolysis. After hybridization, the sections were washed
as described by Lyons et al., in Development 109:833 (1990), to a
final stringency of 0.1.times.SSC, 65.degree. C. for 2.times.15
minutes. After dehydration through a graded ethanol series
containing 0.3 M ammonium acetate, slides were covered with NTB-2
emulsion (Kodak) and exposed between 1 and 3 weeks. After
development, the slides were stained with 0.1% toluidine blue,
dehydrated, cleared with xylene and mounted with Permount.
[0062] Immunostaining
[0063] A polyclonal antibody to the peptide QGKRPSKNLKARC (SEQ ID
NO:10) (amino acids 388-400; prepared by Peptide Technologies,
Gaithersburg, Md.), which belongs to the mature secreted protein of
CDMP-1, was raised in rabbits. Before immunization, the peptide was
conjugated to Imject.RTM. Malemide Activated Keyhole Limpet
Hemocyanin (Pierce, Rockfor, Ill.). Searches performed using the
BLAST (Altschul et al., J. Mol. Biol. 215:403 (1990)) network
service available through the National Center for Biotechnology
Information indicated that the peptide does not show sequence
identity with any known protein or BMP. The embryonic tissue
sections were stained as recommended by the manufacturer using
immunogold as a detection system (Auroprobe L M; Janssen, Belgium)
and counterstained with 0.1% toluidine blue. The primary antibody
(crude antiserum) was used at a concentration of 15 .mu.g/ml in PBS
with 0.5% bovine serum albumin (BSA) for 1 hour. In the controls,
the primary antibody was replaced with BSA, normal rabbit serum, or
secondary antibody alone.
[0064] Results indicated that, at 6 weeks of gestation, CDMP-1
transcripts were detected in precartilage condensations within the
developing limbs. At 7.5-8.5 weeks of gestation, CDMP-1 mRNA
expression was found in the cartilaginous cores of long bones,
including the articular surfaces. In areas of active cartilage
degradation and bone matrix formation, CDMP-1 expression was also
detected in hypertrophic chondrocytes. Remarkably, no expression
was detected in the axial skeleton and only low mRNA levels were
observed in other tissues, such as distal convoluted tubules of the
developing kidney, brain and placenta. Immunohistochemical staining
indicated that CDMP-1 protein colocalized with the mRNA. However,
in addition to the sites of transcription, the protein was also
found in the surrounding cartilaginous matrix and in
osteoblast-like cells from the primary ossification centers of long
bones.
[0065] Between 9 and 10 weeks of gestation, CDMP-2 expression was
predominantly localized in the more mature and hypertrophic
chondrocytes in regions of invasion by blood vessels through the
periosteal bony collar of the developing long bone. Again, as for
CDMP-1, no hybridization was detected in the vertebral bodies in
the corresponding sections and stages of human embryonic
development. Low expression levels were detected in the
periosteum.
[0066] The expression pattern of CDMP-2 suggests it is involved in
the terminal differentiation of chondrocytes (hypertrophic and
mineralizing) and at the earliest stages of endochondral bone
formation, including angiogenesis and osteoblast differentiation.
In addition, the relatively high levels of expression (detectable
in total RNA blots) in postnatal cartilage suggest possible roles
in the maintenance and stabilization of the cartilage phenotype
after birth.
[0067] We have also designed experiments aimed at determining
whether all of the chondrogenic activity contained in cartilage
extracts can be attributed to the proteins encoded by the CDMP-1
and CDMP-2 cDNAs. Our approach involves the production and use of
neutralizing antibodies using synthetic peptides or recombinant
CDMP-1 and CDMP-2 proteins as immunogens. Antibodies raised against
these peptides or proteins will be tested for their ability to
deplete cartilage extracts of chondrogenic activity. If antibodies
specific for the recombinant proteins fail to deplete the extracts
of cartilage-forming activity, then residual activity will be due
to factors within the extract that are separate from proteins
encoded by the CDMP-1 and CDMP-2 proteins. Alternatively, if
antibodies raised against the peptides or recombinant proteins can
remove cartilage-inducing activity from the extracts, this will
confirm that CDMP-1 and/or CDMP-2 must be responsible for the
active agents contained in the extracts.
[0068] Example 8 describes the methods that will be used to raise
antibodies against synthetic peptides and recombinant CDMP-1 and
CDMP-2 proteins. Antibodies produced in this fashion will be tested
for their ability to deplete extracts containing CDMP activity.
EXAMPLE 8
Production and Use of Anti-CDMP Antibodies
[0069] Specific monoclonal and polyclonal antibodies will be raised
against peptides designed from the mature protein of the CDMPs.
Preferentially, the region between the protein cleavage site and
the first cysteine of the CDMP-1 and CDMP-2 proteins will be used
to design the peptides. In addition, the cDNAs encoding the mature
region of the CDMPs will be subcloned in the bacterial pET
expression vector, and expressed as monomers in the bacterial
expression system. The protein expressed in this system will be
used to raise additional antibodies, and to determine the
immunoreactivity of the various antisera in Western blots. The
bacterially expressed monomers will be refolded into biologically
active dimers using standard protocols. This approach may afford
another source of recombinant protein.
[0070] The antisera obtained in this fashion will be used to
further establish the synthesis of the CDMPs by chondrocytes in
vivo and in vitro, and to link the cloned CDMPs to the chondrogenic
activity found in cartilage extracts. Conditioned media obtained
from chondrocyte cultures and partially purified chondrogenic
cartilage extracts after heparin sepharose affinity chromatography,
molecular sieve chromatography and Con A chromatography, will be
analyzed for the presence of CDMPs by Western blot analysis. Due to
the possible heterogeneity of the highly purified chondrogenic
cartilage extracts, the antibodies will be used to reduce or
deplete the chondrogenic/osteogenic activity in purified fractions
in a standard immunoprecipitation experiment.
[0071] An important aspect of our invention regards the production
and use of recombinant proteins that possess the biological
activities of the CDMPs. The following Example describes methods
and results that illustrate the production of recombinant CDMP-1
and CDMP-2 in transfected 293 cells, COS-1 cells, and CHO-1 cells.
We discovered that 293 cells express BMP-7 that could conceivably
contaminate preparations of recombinant CDMPs. To avoid possible
ambiguities in the interpretation of our results, recombinant
CDMP-1 produced in COS-1 cells was used to demonstrate cartilage
forming activity. Although the production of recombinant CDMPs in
this fashion was rather inefficient, the key finding illustrated by
our results was that recombinant protein had the desired
cartilage-forming activity. Unexpectedly, and in contrast to the
related BMPs, recombinant CDMP-1 induced cartilage formation
without noticeable bone formation.
[0072] Example 9 describes the procedures used to produce
recombinant CDMP proteins. The results presented in the Example
confirm that the recombinant cartilage-derived proteins stimulated
cartilage formation.
EXAMPLE 9
Production of Recombinant CDMPs and Assessment of Bioactivity
[0073] Full length CDMP-1 was subcloned into the mammalian
expression vector pcDNA3 (Invitrogen Corporation, San Diego,
Calif.) containing the cytomegalovirus early gene promotor and
other elements required for expression in mammalian cells. COS 1
cells were cultured in Opti-MEM I (Gibco/BRL, Gaithersburg, Md.) in
the presence of 5% fetal bovine serum and antibiotics. The cells
were grown to approximately 70-80% confluency in 150 mm dishes and
transfections of the respective plasmids (20 .mu.g plamid) were
carried out by the calcium phosphate method using the transfection
MBS mammalian transfection kit (Stratagene, La Jolla, Calif.). The
cells were incubated with the calcium phosphate-DNA mixture for 3
hours at 35.degree. C. Supernatants were removed and the plates
were washed 3 times with PBS. 15 ml of Opti-MEM I (serum reduced
medium) was added in the absence of serum, and the dishes were
incubated overnight. Transfection efficiencies were tested by
transfection of a control plasmid, pCMV.beta.-gal and cell extracts
were assayed for .beta.-galactosidase activity. Conditioned media
were collected at 24 hour intervals for a period of 96 hours. The
pooled media was centrifuged to remove cell debris and then
concentrated using Mascrosep 10 concentrators (Filtron Technology
Inc., Northborough, Mass.). Further purification of recombinantly
expressed protein was performed as described in preceding Examples.
In one exemplary procedure, the conditioned media was adjusted to 4
M urea, 25 mM Tris HCl (pH 7.0) and applied to a heparin Sepharose
column. The column was washed with the same buffer containing 0.1 M
NaCl, and eluted with 1 M NaCl. The heparin Sepharose unbound and
eluted fractions were assayed for biological activity as described
by Luyten et al., in J. Biol. Chem. 264:13377 (1989).
[0074] Biological activity of the recombinantly expressed protein
was investigated using in vitro and in vivo chondrogenic/osteogenic
assays. For the in-vivo assay, fractions containing the CDMPs were
precipitated with ethanol, or dried onto a carrier such as bone
matrix residue (mainly collagen type I particles) and cartilage
matrix residue (cartilage tissue after extraction with chaotropic
agents, and powderized to particles with a size of 75-400 .mu.m).
The dried pellet (about 25 mg) was implanted subcutaneously in
rats. Implants were harvested after 11 and 21 days, and analyzed
for chondrogenesis/osteogenesis using alkaline phosphatase
determinations. Histological analysis of recovered samples was also
performed using toluidine blue, alcian blue and safranin O
staining. Results obtained using the recombinant CDMP-1 produced in
COS-1 cells revealed chondrogenic activity in this in vivo assay.
Significantly, no osteogenic activity was observed in any of the
recovered samples. Osteogenic activity would ordinarily have been
observed if the same procedures had been carried out using
recombinant BMPs. This difference highlighted the unique properties
of recombinant CDMP-1.
[0075] Future in vitro chondrogenic experiments will be performed
to determine the precursor cells responsive to the CDMPs.
Undifferentiated (10T1/2 cells, bone marrow stromal cells,
mesenchymal stem cells) and already committed skeletal cells (limb
bud cells, perichondrial or periosteal cells, fetal epihyseal
chondroblasts, and chondrocytes) will be transfected with the cDNAs
or treated with recombinantly expressed CDMPs to evaluate the stage
of differentiation associated with the chondrogenic activity of the
CDMPs.
[0076] Future in vivo chondrogenic experiments will be directed to
expression of large quantities of CDMP-1 and CDMP-2 by stable
transfectants. We contemplate the use of hybrid expression
constructs in which the pro-region of one BMP family member (for
example BMP-2) is operationally linked to the regions encoding the
mature CDMPs. We also anticipate in vivo assays based on
implantation in other sites, apart from subcutaneous implantation,
which may reveal distinct or superior biological activities of the
CDMPs. For example, we anticipate implantation in the synovial
cavity may have utility in such assays.
[0077] The CDMPs disclosed in the present invention have important
applications in the repair of cartilage defects. We contemplate two
general approaches for this type of therapy. In the first place,
the CDMPs are used as lineage-specific growth factors for the ex
vivo expansion of chondrocytes isolated from a donor who requires
therapeutic intervention. Following expansion, these cells can be
reimplanted into a cartilage lesion in the donor, whereafter repair
of cartilage will take place. In a different scenario, CDMPs are
introduced into a cartilage lesion. For example, a composition
containing an appropriate CDMP or mixture of CDMPs can be implanted
into a lesion for the purpose of stimulating in vivo chondrogenesis
and repair of cartilage. The CDMPs can be combined with any of a
number of suitable carriers. An appropriate carrier can be selected
from the group comprising fibrin glue, cartilage grafts, and
collagens. An implantable mixture can be introduced into the site
of a lesion according to methods familiar to those having ordinary
skill in the art. In one application, we contemplate that
periosteal synovial membrane flap of tissue or inert material can
be impregnated with CDMPs and implanted for cartilage repair.
[0078] Example 10 illustrates one application of the CDMP
preparations described above. Specifically, the following Example
describes the use of CDMPs to facilitate repair of cartilage in the
knee joint.
EXAMPLE 10
Treatment of Deep Knee Defects With Cartilage-Derived Morphogenetic
Proteins
[0079] A young patient having a large defect in the articular
surface of the knee joint is identified. According to standard
surgical procedures, a periosteal flap is obtained from the bone
beneath the joint surface of rib cartilage. The tissue flap is
pre-soaked in an extract containing CDMPs or alternatively in a
solution containing recombinant CDMPs. The periosteal flap treated
in this way is then attached over the lesion in the articular
surface of the knee joint by a sewing procedure, for example using
resolvable material. The joint is then closed. The joint is
injected with a solution containing CDMPs dissolved or suspended in
a pharmacologically acceptable carrier to maintain the chondrogenic
process. Injection is continued until the monitoring physician
indicates repair of the cartilage is complete. The patient notices
markedly less joint pain as the cartilage repair process
progresses. Exam by arthroscopy indicates repair of the lesion
within several weeks following the initial procedure.
[0080] We also contemplate gene therapy protocols based on
expression of CDMP cDNAs or genomic constructs as a way of
facilitating in vivo cartilage repair. Diseases such as
chondromalacia or osteoarthritis are examples for which such gene
therapy protocols are contemplated. Therapy may be achieved by
genetically altering synoviocytes, periosteal cells or chondrocytes
by transfection or infection with recombinant constructs that
direct expression of the CDMPs. Such altered cells can then be
returned to the joint cavity. We contemplate that gene transfer can
be accomplished by retroviral, adenoviral, herpesvirus and adeno
associated virus vectors.
[0081] Both in vivo and ex vivo approaches are anticipated for
continuously delivering CDMPs for the purpose of retarding ongoing
osteoarthritic processes and for promoting cartilage repair and
regeneration. In addition, one might employ inducible promoter
constructs (e.g. under transcriptional control of a dexamethasone
promoter) in gene therapy applications of the present invention. A
combined approach to osteoarthritis therapy may have particular
advantages. For example, CDMP-2 could be continuously expressed to
support the integrity of the articular surface. An inducible
construct could be employed to express CDMP-1 so that
chondrogenesis could be accelerated at the time of more aggressive
destruction.
[0082] The foregoing experimental results and characterization
confirmed the CDMP-1 and CDMP-2 isolates belong to the TGF-.beta.
superfamily. Based on the high percentage identity of their
C-terminal domains, CDMP-1 and CDMP-2 can be classified as members
of a novel subfamily. Although CDMP-1 and CDMP-2 were identified in
two different species (human and bovine), they represent distinct
genes since the sequences of their pro-regions are significantly
divergent.
[0083] Several BMPs have now been implicated in early skeletal
development, including BMPs -2, -4, -5, -7 and CDMP-1 (GDF-5).
Other members, such as BMPs -3, -6, -7 and CDMP-2, may be involved
in later stages of skeletal formation (13, 15). The role of the
BMPs in early development could be chemotactic, mitogenic or
inductive. Their function in later stages of skeletal development
might be promotion of differentiation and maintenance of the
established phenotype. The availability of mouse strains with null
mutations in specific BMP members, such as the short-ear mice
(Bmp5) and the bp mice (Cdmp1/Gdf5), allows analysis of the
specific contributions of the respective members in each of the
stages of skeletal development.
[0084] The absence of expression of both CDMP-1 and CDMP-2 in the
axial skeleton has implications for models of skeletal development.
For example, the bp mice have disturbed limb development but a
normal axial skeleton. This is the first evidence that the
developmental mechanisms and differentiation pathways of the
vertebral bodies are distinct from those of the peripheral skeletal
elements. Further, this indicates the basic form and pattern of the
skeleton are likely to be determined by a number of BMP-like
signaling molecules.
Sequence CWU 1
1
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