U.S. patent application number 11/223856 was filed with the patent office on 2006-03-30 for methods and compositions for enhancing cartilage repair.
This patent application is currently assigned to Yissum Research Development Company of the Hebrew University of Jerusalem. Invention is credited to Dan Gazit, Gerhard Gross, Andrea Hoffmann, Gadi Pelled, Gadi Turgeman, Yoram Zilberman.
Application Number | 20060069053 11/223856 |
Document ID | / |
Family ID | 30118728 |
Filed Date | 2006-03-30 |
United States Patent
Application |
20060069053 |
Kind Code |
A1 |
Gazit; Dan ; et al. |
March 30, 2006 |
Methods and compositions for enhancing cartilage repair
Abstract
The invention relates to methods of enhancing repair of a
cartilage and/or inducing formation of a cartilage by administering
a cell, which expresses a factor of the T-box family, which
includes inter-alia the brachyury. In another embodiment, the
invention relates to an engineered cell, which is transfected with
a vector comprising a nucleic acid sequence encoding a factor of
the T-box family, thereby expressing a factor of the T-box family.
In another embodiment, the invention relates to compositions
comprising a vector, which comprises a nucleic acid sequence
encoding a factor of the T-box family and in another embodiment the
composition-comprising cell that expresses a factor of the T-box
family, which includes inter-alia the brachyury.
Inventors: |
Gazit; Dan; (Jerusalem,
IL) ; Zilberman; Yoram; (Jerusalem, IL) ;
Turgeman; Gadi; (Jerusalem, IL) ; Pelled; Gadi;
(Rishon-LeZion, IL) ; Gross; Gerhard;
(Braunschweig, DE) ; Hoffmann; Andrea; (Hannover,
DE) |
Correspondence
Address: |
Martin Moynihan;PRTSI, Inc.
P.O. Box 16446
Arlington
VA
22215
US
|
Assignee: |
Yissum Research Development Company
of the Hebrew University of Jerusalem
Jerusalem
IL
Gesellschaft Fuer Biotechnologische Forschung mbH - GBF
Braunschweig
DE
|
Family ID: |
30118728 |
Appl. No.: |
11/223856 |
Filed: |
September 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10298215 |
Nov 18, 2002 |
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11223856 |
Sep 12, 2005 |
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10067980 |
Feb 8, 2002 |
6849255 |
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10298215 |
Nov 18, 2002 |
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09376276 |
Aug 18, 1999 |
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10067980 |
Feb 8, 2002 |
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Current U.S.
Class: |
514/44R ;
424/93.21 |
Current CPC
Class: |
C12N 2510/00 20130101;
C12N 5/0655 20130101; A61K 35/12 20130101 |
Class at
Publication: |
514/044 ;
424/093.21 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 18, 1998 |
DE |
19837438.0 |
Claims
1-10. (canceled)
11. A method of enhancing repair of a cartilage in the body
comprising the step of administrating a recombinant vector which
comprises a nucleic acid encoding a factor of the T-box family to
the cartilage of a subject, thereby enhancing repair of the
cartilage.
12. The method of claim 11, wherein said factor of the T-box is
brachyury.
13. The method of claim 11, wherein said method further comprises
administering a recombinant vector which comprises a nucleic acid
encoding a factor which upregulates the expression of the T-box
transcription factor.
14. The method of claim 13, wherein said factor which upregulates
the expression of the factor of the T-box is FGF or BMP2.
15. A method of inducing formation of a cartilage in the body
comprising the step of administrating a recombinant vector which
comprises a nucleic acid encoding a factor of the T-box family to
the cartilage of a subject, thereby inducing formation of the
cartilage.
16. The method of claim 15, wherein said factor of the T-box is
brachyury.
17. The method of claim 15, wherein said method further comprises
administering a recombinant vector which comprises a nucleic acid
encoding a factor which upregulates the expression of the T-box
transcription factor.
18. The method of claim 17, wherein said factor which upregulates
the expression of the factor of the T-box is FGF or BMP2.
19. A method of inducing chondrocyte differentiation comprising the
step of administering of a recombinant vector which comprises a
nucleic acid encoding a factor of the T-box family, thereby
inducing chondrocyte formation.
20. The method of claim 19, wherein said factor of the T-box is
brachyury.
21. The method of claim 19, wherein said method further comprises
administering a recombinant vector which comprises a nucleic acid
encoding a factor which upregulates the expression of the T-box
transcription factor.
22. The method of claim 19, wherein said factor which upregulates
the expression of the factor of the T-box is FGF or BMP2.
23-48. (canceled)
49. A composition comprising at least one recombinant vector which
comprises a nucleic acid sequence encoding at least one factor of
the T-box family and a pharmaceutically acceptable carrier.
50. The composition of claim 49, wherein said composition is a
pharmaceutically composition.
51. The composition of claim 49, wherein said factor of the T-box
is brachyury.
52. The composition of claim 49, wherein said method further
comprises administering a recombinant vector which comprises a
nucleic acid encoding a factor which upregulates the expression of
the T-box transcription factor.
53. The composition of claim 52, wherein said factor which
upregulates the expression of the factor of the T-box is FGF or
BMP2.
54-66. (canceled)
Description
[0001] This application is a Continuation-in Part Application of
U.S. Ser. No. 10/067,980, filed Feb. 8, 2002, which claims the
benefit of U.S. Ser. No. 09/376, 276, filed Aug. 2, 1999, now
abandoned, which claims priority from DE Application No.
198.37.438.0, filed Aug. 18, 1998, which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The invention provides methods of enhancing repair of a
cartilage and/or inducing formation of a cartilage by contacting a
cell, which expresses at least one factor of the T-box family. In
another embodiment, the invention provides an engineered cell,
which is transfected with a vector comprising at least one nucleic
acid sequence encoding a factor of the T-box family, thereby
expressing at least one factor of the T-box family. This invention
provides a composition comprising a vector, which comprises at
least one nucleic acid sequence encoding a factor of the T-box
family.
BACKGROUND OF THE INVENTION
[0003] The meniscus, fibrocartilaginous tissue found within the
knee joint, is responsible for shock absorption, load transmission,
and stability within the knee joint. According to the National
Center for Health Statistics, over 600,000 surgeries each year are
the result of complications with the meniscus. The meniscus has the
intrinsic ability to heal itself; unfortunately, this property is
limited only to the vascular portions of the tissue. For damage
outside of these areas and overall degeneration of the tissue,
methods need to be developed that will assist the meniscus in
healing itself Sweigart M A Tissue Eng 7(2), 111-29 (April
2001);
[0004] Degeneration of articular cartilage in osteoarthritis is a
serious medical problem caused by arthritis, both rheumatoid and
osteoarthritis. Drugs are given to control the pain and to keep the
swelling down, but the cartilage continues to be destroyed.
Eventually, the joint must be replaced. It is still unknown why
cartilage does not heal and no solutions to this problem are known
Mankin, N. E. J. Med. 331(14), 940-941 (October 1994). Soon after
superficial injury, chondrocytes adjacent to the injured surfaces
show a brief burst of mitotic activity associated with an increase
in glycosaminoglycan and collagen synthesis. Despite these attempts
at repair, there is no appreciable increase in the bulk of
cartilage matrix and the self-repair process is usually ineffective
in healing the defects.
[0005] Osteochondral, or full-thickness, cartilage defects expand
into the subchondral bone. Such defects arise after the detachment
of osteochondritic dissecting flaps, fractured osteochondral
fragments, or from chronic wear of degenerative articular
cartilage. Osteochondral defects depend on the extrinsic mechanism
for repair. Extrinsic healing relies on mesenchymal elements from
subchondral bone to participate in the formation of new connective
tissue. This fibrous tissue may or may not undergo metaplastic
changes to form fibrocartilage. Even if fibrocartilage is formed,
it does not display the same biochemical composition or mechanical
properties of normal articular cartilage or subchondral bone and
degenerates with use, Furukawa, et al., J. Bone Joint Surg. 62A, 79
(1980); Coletti, et al., J. Bone Joint Surg. 54A, 147 (1972);
Buckwalter, et al., "Articular cartilage: composition, structure,
response to injury and methods of facilitating repair", in
Articular Cartilage and Knee Joint Function: Basic Science and
Arthroscopy, Ewing J E, Ed., (New York, Raven Press, 1990), 19.
[0006] Injection of dissociated chondrocytes directly into the site
of the defect has also been described as a means for forming new
cartilage, as reported by Brittberg, et al., N. E. J. Med. 31,
889-895 (October 1994). Cartilage was harvested from minor
load-bearing regions on the upper medial femoral condyle of the
damaged knee, cultured, and implanted two to three weeks after
harvesting.
[0007] Moreover, if the defect includes a part of the underlying
bone, this is not corrected by the use of chondrocytes. The bone is
required to support the new cartilage.
[0008] Cartilage grafts are also needed in plastic surgery like in
rhinoplasty, and the reconstruction of ears.
[0009] The possibility of using stem cells was also examined. Stem
cells are cells which are not terminally differentiated, which can
divide without limit, and divide to yield cells that are either
stem cells or which irreversibly differentiate to yield a new type
of cell. Unfortunately, there is no known specific inducer of the
mesenchymal stem cells that yields only cartilage.
[0010] In vitro studies in which differentiation is achieved using
different bioactive factors or molecules, yields differentiation of
the cells to cartilage which eventually calcified and turned into
bone.
[0011] Thus, there is a need to have a method and composition for
the formation or repair of a cartilage or a bone. In another
embodiment, it will be highly advantageous to have a cell, which
can divide and form a cartilage or a bone tissue.
SUMMARY OF THE INVENTION
[0012] In one embodiment the invention provides a method of
enhancing repair of a cartilage comprising the step of
administering to a subject an effective amount of a cell which
expresses at least one factor of the T-box family, thereby
enhancing repair of the cartilage.
[0013] In another embodiment the invention provides a method of
inducing formation of a cartilage comprising the step of
administering to a subject an effective amount of a cell which
expresses at least one factor of the T-box family, thereby inducing
formation of the cartilage.
[0014] In another embodiment the invention provides a method of
enhancing repair of a cartilage in the body comprising the step of
administrating a recombinant vector which comprises a nucleic acid
encoding a factor of the T-box family to the cartilage of a
subject, thereby enhancing repair of the cartilage.
[0015] In another embodiment the invention provides a method of
inducing formation of a cartilage in the body comprising the step
of administrating a recombinant vector which comprises a nucleic
acid encoding a factor of the T-box family to the cartilage of a
subject, thereby inducing formation of the cartilage.
[0016] In another embodiment the invention provides a method of
inducing chondrocyte differentiation comprising the step of
administering of a recombinant vector, which comprises a nucleic
acid encoding a factor of the T-box family, thereby inducing
chondrocyte formation.
[0017] In another embodiment the invention provides a method of
repairing or forming a cartilage in a subject in need comprising
the steps of: obtaining a cell from of the subject; transfecting
said cell with a recombinant vector comprising a nucleic acid
sequence encoding a factor of the T-box family, so as to obtain an
engineered cell which expresses a factor of the T-box family; and
administering said engineered cell to the subject.
[0018] In another embodiment the invention provides a method for
the production of transplantable cartilage matrix, the method
comprising the steps of: obtaining a cell; transfecting said cell
with a recombinant vector comprising a nucleic acid sequence
encoding a factor of the T-box family, so as to obtain an
engineered cell which expresses a factor of the T-box family; and
culturing said cell with the cell-associated matrix for a time
effective for allowing formation of a transplantable cartilage
matrix.
[0019] In another embodiment the invention provides an engineered
cell, which expresses a factor of the T-box family.
[0020] In another embodiment the invention provides an implant
device comprising at least one engineered cell which expresses a
factor of the T-box family and a pharmaceutically acceptable
carrier.
[0021] In another embodiment the invention provides a composition
comprising an engineered cell which expresses a factor of the T-box
family and a pharmaceutically acceptable carrier.
[0022] In another embodiment the invention provides a composition
comprising at least one recombinant vector which comprises a
nucleic acid sequence encoding at least one factor of the T-box
family and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1. FGFR3 mediates chondrocytic differentiation in
mesenchymal stem cell line C3H10T1/2. FIG. 1a shows the RT-PCR
analyses of BMP2-dependent expression of FGF- and
PTH/PTHrP-receptors in mesenchymal stem cell line C3H10T1/2 in the
presence or absence of recombinantlly expressed BMP2. FIG. 1b shows
the effect of cyclohexamide pretreatment of C3H10T1/2 cells.
Cycloheximide pre-treatment of C3H10T/1/2 cells does not prevent
BMP-induction of the FGFR3 gene. Cells were mock-treated (control)
or were treated with BMP2 (50 ng/ml). FIG. 1c demonstrates western
immunoblotting for the detection of BMP2-dependent FGFR3 and FGFR2
expression in cellular extracts of C3H10T1/2 lines. FIG. 1d left
panel: western immunoblotting to demonstrate the forced expression
of FGFR3 in mesenchymal progenitors C3H10T1/2; right panel: the
recombinant expression of FGFR3 in C3H10T1/2 leads to enhanced
levels of activated MAP-kinases pERK-1 and pERK-2 during
cultivation. Cell lysates were prepared 0 (=confluence) and 4 days
post-confluence. FIG. 1e demonstrates that the forced expression of
FGFR3 in parental C3H10T1/2 cells is sufficient for the induction
of the chondrogenic lineage.
[0024] FIG. 2. The T-box transcription factor Brachyury mediates
chondrogenic differentiation in MSCs in vitro and ectopically in
vivo. FIG. 2a upper panel: schematic representation of Brachyury
according to Kispert et al., 1995 (1995). FIG. 2a lower panel:
western immunoblotting of recombinant HA-tagged Brachyury (aa
1-436) in cellular extracts of C3H10T1/2 (C3H10T1/2-Brachyury) with
HA-antibody SC-805 (Santa Cruz) Brachyury has been constitutively
expressed under the control of the murine PGK-promoter. Expression
of Brachyury is indicated (triangle). Molecular weight marker (M)
shown is ovalbumin (43 kDa). FIG. 2b shows the histological
characterization of C3H10T1/2-Brachyury cells in culture. Upper
panel: at day 4 post-confluency cells develop alkaline-phosphates
positive osteoblast-like cells. Lower right panel: Alcian Blue
histology of C3H10T1/2 cells stably expressing Brachyury indicative
for secreted proteoglycans and efficient differentiation into the
chondrogenic lineage. Lower left panel:
Collagen-immunohistochemistry of C3H10T1/2-Brachyury cells in
culture 7 days post-confluency. FIG. 2c shows the RT-PCR analysis
of the expression of chondrogenic and osteogenic marker genes in
C3H10T1/2 cells recombinantlly-expressing Brachyury. FIG. 2d shows
the forced expression of the T-box factor Brachyury in C3H10T1/2
cells which leads to differentiation into chondrocytes and
cartilage development at murine ectopic sites after intramuscular
transplantation.
[0025] FIG. 3. Dominant-negative Brachyury (dnBrachyury; T-box
domain) blocks BMP2-mediated chondrogenic development in C3H10T1/2
MSCs in vitro and ectopically in vivo. FIG. 3a shows that
Brachyury's T-box domain interferes with the transcriptional
activity of full-length Brachyury. FIG. 3b shows expression of
dnBrachyury (T-box domain) in C3H10T1/2-BMP2 during cultivation
(day 0; cellular confluence) The T-box domain (aa 1-229) has been
subcloned and HA-tagged in expression vector pMT7T3 and
constitutively expressed in C3H10T1/2-BMP2 cells. The
recombinantlly expressed T-box domain (dnBrachyury) is indicated
(triangle). FIG. 3c demonstrates RT-PCR experiments with
osteo-/chondrogenic marker genes show that T-box domain
(dnBrachyury) expression in C3H10T1/2-BMP2 cells interferes with
the BMP2 dependent of FGFR2 but not FGFR3 expression. FIG. 3d shows
that dnBrachyury (T-box) interferes with BMP2-mediated FGFR2
expression as analyzed by western immunoblotting with antiFGFR3 and
antiFGFR2 antibodies as described FIG. 1. FIG. 3e shows the forced
expression of the dominant-negative acting T-box domain in
C3H10T1/2-BMP2 cells interferes with BMP-2 mediated
osteo-/chondrogenic development.
[0026] FIG. 4. Dominant-negative FGFR3 (dnFGFR3) interferes with
osteo-/chondrogenic development, with FGFR2- and with
Brachyury-expression in C3H10T1/2-BMP2. FIG. 4a shows that forced
expression of dnFGFR3 in C3H10T1/2-BMP2 cells interferes with BMP-2
mediated development of alkaline phosphatase positive and Alcian
Blue positive chondrocyte-like cells, respectively. FIG. 4b shows
that dnFGFR3 interferes with BMP2-dependent FGFR2 and Brachyury but
not with FGFR3 expression in C3H10T1/2-BMP2 cells.
[0027] FIG. 5. FGFR3 and Brachyury are involved in an auto
regulatory loop. FIG. 5a shows RT-PCR analyses of FGFR3 and
Brachyury mRNA levels in mesenchymal progenitors C3H10T1/2
expressing recombinant FGFR3 (C3H10T1/2-FGFR3) or Brachyury
(C3H10T1/2-Brachyury). FIG. 5b shows that Smad1-signaling is not
sufficient for Brachyury and FGFR3 but for osteocalcin expression.
RT-PCR analyses of FGFR3 and Brachyury mRNA levels in mesenchymal
progenitors C3H10T1/2 expressing the biologically active Smad1-MH2
domain (C3H10T1/2-Smad1-MH2).
[0028] FIG. 6. Brachyury is expressed at skeletal sites during late
murine embryonic development (18.5 dpc). Comparative expression
analysis of murine Brachyury (Bra), Collagen 1a1 (Col 1a1) and
Collagen 2a1 (Col 2a1) in embryonic development 18.5 dpc. a,
Intervertebral discs development. Consecutive sagittal (a-g) and
transversal (h-j) sections of 18.5 dpc mouse embryos were
hybridized with riboprobes as indicated. Expression of Brachyury is
enhanced in the nucleus pulposus (a, d), Col 1a1 in the outer
annulus (arrowheads in b, e), and Col 2a1 in the cartilage
primordium of the vertebrae (c,f). No signals are obtained using
RNase pre-incubated sections (g). With transversal sections at the
level of the upper lumbar vertebra expression of Brachyury is in
addition detectable in distinct cells of the neural arch (h)
whereas Col 1a1 is expressed in the outer annulus (i) and Col 2a1
in the cartilage primordium (j). b, Limb bud development.
Consecutive transversal sections of a 18.5 dpc mouse hind limb at
the level of the metatarsals hybridized with riboprobes as
indicated. Expression of Brachyury is evident in distinct
chondrogenic cells of the forming metatarsal bones (a), better
visible with higher magnification (b,c). In contrast Col 1a1 is
expressed in the outer periosteal layer (d-f) and Col 2a1
expression is enhanced in differentiating chondrocytes (g-i). As it
was evident for the intervertebral disc formation expression of
Brachyury is only evident in chondrocyte-like cells that do not
express-Col 2a1. ch, chondrocytes; cp, cartilage primordium; mta,
metatarsal; np, nucleus pulposus; oa, outer annulus; pl, periosteal
layer; sk, skin bar, 100 .quadrature.m
[0029] FIG. 7. Model of BMP2-dependent osteo-/chondrogenic
development in mesenchymal stem cells.
[0030] FIG. 8. Human adult MSCs (3.times.106) were transfected with
30 ug of Brachyury plasmid. 24 hours post transfection, RNA was
isolated and RT-PCR was performed using specific primers to the
Brachyury cDNA. 20 ul of the PCR reaction sample were loaded and
electrophoresed in 2% Agarose gel. The gel image demonstrated
positive Brachyury expression (Positive control-Brachyury
plasmid).
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0031] In one embodiment, the invention relates to methods of
enhancing repair of a cartilage and/or inducing formation of a
cartilage by administering a cell, which expresses a factor of the
T-box family, which includes inter-alia the brachyury. In another
embodiment, the invention relates to an engineered cell, which is
transfected with a vector comprising a nucleic acid sequence
encoding a factor of the T-box family, thereby expressing a factor
of the T-box family. In another embodiment, the invention relates
to compositions comprising a vector, which comprises a nucleic acid
sequence encoding a factor of the T-box family and in another
embodiment the composition-comprising cell that expresses a factor
of the T-box family, which includes the brachyury.
[0032] The term "cartilage" refers hereinabove to a specialized
type of dense connective tissue consisting of cells embedded in a
matrix. There are several kinds of cartilage. Translucent cartilage
having a homogeneous matrix containing collagenous fibers is found
in articular cartilage, in costal cartilages, in the septum of the
nose, in larynx and trachea. Articular cartilage is hyaline
cartilage covering the articular surfaces of bones. Costal
cartilage connects the true ribs and the sternum. Fibrous cartilage
contains collagen fibers. Yellow cartilage is a network of elastic
fibers holding cartilage cells which is primarily found in the
epiglottis, the external ear, and the auditory tube. Cartilage is
tissue made up of extracellular matrix primarily comprised of the
organic compounds collagen, hyaluronic acid (a proteoglycan), and
chondrocyte cells, which are responsible for cartilage production.
Collagen, hyaluronic acid and water entrapped within these organic
matrix elements yield the unique elastic properties and strength of
cartilage.
[0033] As used herein, "hyaline cartilage" refers to the connective
tissue covering the joint surface. By way of example only, hyaline
cartilage includes, but is not limited to, articular cartilage,
costal cartilage, and nose cartilage.
[0034] As used herein, the term "enhancing cartilage repair" refers
to healing and for regeneration of cartilage injuries, tears,
deformities or defects, and prophylactic use in preventing damage
to cartilaginous tissue.
[0035] As used herein, the term "inducing formation" refers to the
use in cartilage renewal or regeneration so as to ameliorate
conditions of cartilage, degeneration, depletion or damage such as
might be caused by aging, genetic or infectious disease, accident
or any other cause, in humans, livestock, domestic animals or any
other animal species. In another embodiment the formation of a
cartilage is required for cartilage development in livestock,
domestic animals or any other animal species in order to achieve
increased growth for commercial or any other purpose. In another
embodiment the formation of a cartilage is required in plastic
surgeries, such as without being limited facial reconstruction in
order to obtain a stabilized shape.
[0036] In one embodiment there is provided a recombinant vector
comprising a nucleic acid sequence encoding a factor of the T-box
family.
[0037] The term "T-box family" defined as a family of transcription
factors that share the T-box, a 200 amino acid DNA-binding domain
(T-box, aa 1-229). The T-box family has been identified in both
vertebrates and in vertebrates and plays a key role in embryonic
development. The T- box family further includes variant and
fragments of the T-box family transcription factors.
[0038] The T-Box gene family can be said to consist of several
generic entities: T, Tbr-1, Tbx1-9, 11, 12, 17 and T2 and many
species has been shown to contain orthologs. Several mouse T-Box
genes have been reported; mu-T, mu-Tbr1 (identified in a
subtractive hybridization screen for genes specifically involved in
regulating forebrain development (Bulfone et al. (1995) Neuron
15:63-78), mu-Tbx1-6, mm-Tbx13 (Wattler et at., Genomics 48:24-33),
and mm-Tbx14 (Wattler et al. (1998) Genomics 48:24-33, 1998). There
are four Xenopus genes (Xbra, x-eomes, x-ET and x-VegT (Zhang et
al. (1996) Development 122:4119-4129; Smith et al. (1995) Semin Dev
Biol 6:405-410; Lustig et al. (1996) Development 122:4001-4012;
Stennard et al. (1996) Development 122:4179-4188; Horb et al.
(1997) Development 124:1689-1698; Ryan et al. (1996) Cell
87:989-1000). Human orthologs for six of eight mouse genes have
been identified. Hu-T (Edwards et al. (1996) Genome Res 6:226-233;
Morrison et al. (1996) Hum Mol Genet 5:669-674) and hu-TBRI
(Bulfone et al. (1995) Neuron 15:63-78) were found by homology with
the mouse orthologs. Hu-TBX2 was isolated independently by two
groups from embryonic kidney cDNA libraries (Campbell et al. (1995)
Genomics 28:255-260; Law et al. (1995) Mamm Genome 6:267-277).
Hu-TBX1, hu-TBX3, and hu-TBX5 were found during investigations
aimed at uncovering the genetic basis of human developmental
dysmorphic syndromes and were recognized as orthologs of the mouse
genes by sequence homology (Li et al. (1997) Nat Genet 15:21-29;
Basson et al. (1997) Nat Genet 15:30-35; Chieffo el al.(1997)
Genome 43:267-277).
[0039] There is currently only a handful of known mutations in
T-Box genes. Spontaneous mutations in hu-TBX3 (Bamshad et al.
(1997) Nat Genet 16:311-315) and hu-TBX5 (Li et al. (1997) Nat
Genet 15:21-29; Basson et al. (1997) Nat Genet 15:30-35) have been
reported. These mutations at T-Box genes play a role in several
human autosomal, dominant developmental syndromes: Ulnar-Mammary
syndrome and Holt-Oram syndrome. Ulnar-Mammary syndrome is
characterized by limb defects, abnormalities of apocrine glands
such as the absence of breasts, axillary hair and perspiration,
dental abnormalities such as ectopic, hypoplastic and absent canine
teeth, and genital abnormalities such as micropenis, shawl scrotum
and imperforate hymen. Holt-Oram syndrome is characterized by
cardiac septal defects and preaxial radial ray abnormalities of the
forelimbs (Li el al. (1997) Nat Genet 15:21-29; Basson et al.
(1997) Nat Genet 15:30-35; Bamshad et al. (1997) Nat Genet
16:311-315). Mutations in the 5' end of TBX5 lead to substantial
cardiovascular malformations and relatively mild skeletal defects
while mutations in the 3' end of the gene cause severe skeletal
malformation and have less effect on cardiac development (McCarthy,
M (1998) Lancet 351(9115):1564; Basson, C. T. et al (1997) Nature
Genetics 15:30-35). A better understanding of the role which T-Box
transcription factors play in embryogenesis, organogenesis and
organ regeneration has been recently recognized. T-Box related
genes have been found in many species, making up a large group of
T-Box transcription factors which are highly conserved in their
DNA-binding capacity but may be highly divergent in the
non-DNA-binding regions. There are common features which define the
family, as well as specific differences that define individual
members. Phylogenetic analysis suggests that the genome of most
animal species will have at least five T-Box genes (related to
mu-Tbx2, mu-Tbx, mu-Tbx1, mu-T, and mu-Tbr1). There are at least 16
distinct members in 11 different animal groups that have been
reported and human orthologs of six of the eight mouse genes have
already been identified.
[0040] In another embodiment, the vector comprising a nucleic acid
which encodes for Brachyury, or T, which refers hereinabove to the
founder factor of the T-box family.
[0041] The immediate BMP2-dependent upregulation of FGFR3 in MSCs
(C3H10T1/2) and the inherent capacity of this receptor to initiate
chondrogenic development in these cells prompted a screen for
FGFR3-regulated transcription factors. The chondrogenic potential
of Brachyury after recombinant expression in wild-type C3H10T1/2
cells has been shown, by the use of a subtractive screening method,
exemplified in Example 1 that, among the transcription factors
tested, the T-box transcription factor Brachyury was upregulated in
FGFR3-expressing C3H10T1/2 cells (see also FIG. 5a).
[0042] As used herein, the term "nucleic acid" refers to
polynucleotides or to ologonucleotides such as deoxyribonucleic
acid (DNA), and, where appropriate, ribonucleic acid (RNA) or
mimetics thereof. The term should also be understood to include, as
equivalents, analogs of either RNA or DNA made from nucleotide
analogs, and, as applicable to the embodiment being described,
single (sense or antisense) and double-stranded polynucleotides.
This term includes oligonucleotides composed of naturally occurring
nucleobases, sugars and covalent internucleoside (backbone)
linkages as well as oligonucleotides having non-naturally-occurring
portions which function similarly. Such modified or substituted
oligonucleotides are often preferred over native forms because of
desirable properties such as, for example, enhanced cellular
uptake, enhanced affinity for nucleic acid target and increased
stability in the presence of nucleases.
[0043] The terms "protein", "polypeptide" and "peptide" are used
interchangeably herein when referring to a gene product.
[0044] The vector molecule can be any molecule capable of being
delivered and maintained within the target cell or tissue such that
the gene encoding the product of interest can be stably expressed.
The vector molecule preferably utilized in the present invention is
either a viral or retroviral vector molecule or a plasmid DNA
non-viral molecule. This method preferably includes introducing the
gene encoding the product into the cell of the mammalian connective
tissue for a therapeutic or prophylactic use. Unlike previous
pharmacological efforts, the methods of the present invention
employ gene therapy to address the chronic debilitating effects of
joint pathologies. The viral vectors used in the methods of the
present invention can be selected form the group consisting of (a)
a retroviral vector, such as MFG or pLJ; (b) an adeno-associated
virus; (c) an adenovirus; and (d) a herpes virus, including but not
limited to herepes simplex 1 or herpes simples 2 or (e) lentivirus.
Alternatively, a non-viral vector, such as a DNA plasmid vector,
can be used. Any DNA plasmid vector known to one of ordinary skill
in the art capable of stable maintenance within the targeted cell
or tissue upon delivery, regardless of the method of delivery
utilized is within the scope of the present invention. Non-viral
means for introducing the gene encoding for the product into the
target cell are also within the scope of the present invention.
Such non-viral means can be selected from the group consisting of
(a) at least one liposome, (b) Ca3 (PO4) 2, (c) electroporation,
(d) DEAE-dextran, and (e) injection of naked DNA.
[0045] As will be appreciated by one skilled in the art, a fragment
or derivative of a nucleic acid sequence or gene that encodes for a
protein or peptide can still function in the same manner as the
entire, wild type gene or sequence. Likewise, forms of nucleic acid
sequences can have variations as compared with the wild type
sequence, while the sequence still encodes a protein or peptide, or
fragments thereof, that retain their wild type function despite
these variations. Proteins, protein fragments, peptides, or
derivatives also can experience deviations from the wild type from
which still functioning in the same manner as the wild type form.
Similarly, derivatives of the genes and products of interest used
in the present invention will have the same biological effect on
the host as the non-derivatized forms. Examples of such derivatives
include but are not limited to dimerized or oligomerized forms of
the genes or proteins, as wells as the genes or proteins modified
by the addition of an immunoglobulin (Ig) group. Biologically
active derivatives and fragments of the genes, DNA sequences,
peptides and proteins of the present invention are therefore also
within the scope of this invention. In addition, any nucleic acid,
which is cis acting and integrated upstream to an endogenous factor
of the T-box family nucleic acid sequence, is relevant to the
present invention.
[0046] The term "cis-acting" is used to describe a genetic region
that serves as an attachment site for DNA-binding proteins (e.g.
enhancers, operators and promoters) thereby affecting the activity
of genes on the same chromosome.
[0047] It was shown that Brachyury expression is upregulated by
certain factor such as BMP2 and/or FGF3 (see FIGS. 4 and 5). Thus,
in another embodiment, the vector of the invention further
comprises a nucleic acid, which encodes to a protein, which
activated the BMP signaling pathway. In another embodiment, the
protein, which activated the BMP signaling pathway, is a member of
the BMP family. In another embodiment the BMP is a BMP2. In another
embodiment the vector further comprising a nucleic acid for
fibroblast growth factor namely FGF-3.
[0048] The term "protein which activates BMP mediated signaling
pathway" is defined hereinabove as a protein that can activate the
BMP receptors, or the signaling cascade down stream of the receptor
to elicit BMP specific cellular response. Examples, without being
limited are members of the BMP family, such as the BMP proteins
BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, disclosed for
instance in U.S. Pat. Nos. 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; and BMP-10 or BMP-11, disclosed in co-pending patent
applications, Ser. No. 08/061,695 presently abandoned, a
continuation-in-part of which has issued as U.S. Pat. No.
5,637,480, and 08/061,464 presently abandoned, a
continuation-in-part of which has issued as U.S. Pat. No. 5,639,638
filed on May 12, 1993.
[0049] In another embodiment the vector may include also nucleic
acids encoding other therapeutically useful agents including MP52,
epidermal growth factor (EGF), fibroblast growth factor (FGF),
platelet derived growth factor (PDGF), transforming growth factors
(TGF-.alpha. and TGF-.beta.), and fibroblast growth factor-4
(FGF-4), parathyroid hormone (PTH), leukemia inhibitory factor
(LIF/HILDA/DIA), insulin-like growth factors (IGF-I and IGF-II). In
another embodiment, the vector comprises nucleic acid encoding an
anti-inflammatory agent such as IL1 receptor antagonist, or IL4 or
IL10 agonist.
[0050] In another embodiment the cell of the invention further
expresses therapeutically useful agents including MP52, epidermal
growth factor (EGF), fibroblast growth factor (FGF), platelet
derived growth factor (PDGF), transforming growth factors
(TGF-.alpha. and TGF-.beta.), and fibroblast growth factor-4
(FGF-4), parathyroid hormone (PTH), leukemia inhibitory factor
(LIF/HILDA/DIA), insulin-like growth factors (IGF-I and IGF-II). In
another embodiment, the vector comprises nucleic acid encoding an
anti-inflammatory agent such as IL1 receptor antagonist, or IL4 or
IL10 agonist.
[0051] In another embodiment, there is provided an engineered cell,
which expresses at least one factor of the T-box family.
[0052] The term "engineered cell" is defined hereinabove to a cell
or to a tissue, which had been genetically modified and is
expressing a factor of the T-box family or in another embodiment,
increased amounts of the factor of the T-box family or in another
embodiment express Brachury. The term "increased amount of the
factor or the at least one factor of the T-box family refers
hereinabove to at least 10 times more than normal.
[0053] In one embodiment, the cell of the invention is a mammalian
cell. In another embodiment, it is a mesenchymal stem cell, in
another embodiment it is a progenitor cell, in another embodiment
it is a cell derived from a cartilage. In another embodiment the
cell can be derived from a fibroblast cell line, a mesenchymal cell
line, a chondrocyte cell line, an osteoblast cell line, or an
osteocyte cell line. The fibroblast cell line may be a human
foreskin fibroblast cell line or NIH 3T3 cell line. In another
embodiment the cell of the invention is a synovial cell or a
synoviocyte. Synoviocytes are found in joint spaces adjacent to
cartilage have an important role in cartilage metabolism.
Synoviocytes produce metallo-proteinases, such as collagenases that
are capable of breaking-down cartilage
[0054] Stem cells are defined as cells which are not terminally
differentiated, which can divide without limit, and divides to
yield cells that are either stem cells or which irreversibly
differentiate to yield a new type of cell. Those stem cells which
give rise to a single type of cell are call unipotent cells; those
which give rise to many cell types are called pluripotent cells.
Chondro/osteoprogenitor cells, which are bipotent with the ability
to differentiate into cartilage or bone, were isolated from bone
marrow (for example, as described by Owen, J. Cell Sci. Suppl. 10,
63-76 (1988) and in U.S. Pat. No. 5,226,914 to Caplan, et al.).
[0055] It is important to note that mesenchymal stem cells and
progenitors can be isolated from different source tissues, skin,
bone marrow, muscle, and liver. In addition any cell type with stem
cell properties or demonstrating differentiation plasticity for
example without limitation, SP cells from the source of bone
marrow, muscle, spleen or any other tissue.
[0056] Chondrogenic cells useful in the practice of the invention
may be isolated from essentially any tissue containing chondrogenic
cells. As used herein, the term "chondrogenic cell" is understood
to mean any cell which, when exposed to appropriate stimuli, may
differentiate into a cell capable of producing and secreting
components characteristic of cartilage tissue. The chondrogenic
cells may be isolated directly from pre-existing cartilage tissue,
for example, hyaline cartilage, elastic cartilage, or
fibrocartilage. Specifically, chondrogenic cells may be isolated
from articular cartilage (from either weight-bearing or
non-weight-bearing joints), costal cartilage, nasal cartilage,
auricular cartilage, 30 tracheal cartilage, epiglottic cartilage,
thyroid cartilage, arytenoid cartilage and cricoid cartilage. The
cell from the cartilage can be derived from another animal, or
another subject or in another embodiment; the cell of the cartilage
or the bone can be derived from the subject in need.
[0057] In another embodiment, the cell further expresses at least
one protein, which activates BMP mediated signaling pathway or a
FGF protein.
[0058] The expression of the at least one factor of the T-box
family in combination with either and BMP or FGF or both in the
cell can be due to the presence of two or more different vectors
(trans vectors) or due to the expression of one vector which
comprises two or more different nucleic acid sequences, which
encode for the at least one member of the T-box family, the FGF and
for at least one protein which activates the BMP mediated signaling
pathway. It was shown that Brachyury's DNA-binding domain without
the associated regulatory domains (aa 230-436) should
dominant-negatively (dn) interfere with endogenous
Brachyury-mediated events in C3H10T1/2-BMP2 cells see FIG. 3b and
example 3.
[0059] In another embodiment the invention provides complex tissue
engineering. This term refers to engineering a cell with different
nucleic acid sequences, wherein each sequence encodes to a specific
pathway of differentiation. As such, the cell of the invention can
be engineered to differentiate to an osteoblast as well as to a
chondrocyte.
[0060] In another embodiment there is provided a composition
comprising recombinant vector comprising a nucleic acid sequence
encoding the a factor of the T-box family and a pharmaceutically
acceptable carrier. It should be noted that the term "a nucleic
acid sequence encoding the a factor of the T-box family" refers
hereinabove to "at least one nucleic acid sequence encoding the at
least one factor of the T-box family". Similarly the term "a cell"
refers to "at least one cell".
[0061] In another embodiment, there is provided a composition
comprising at least one engineered cell, wherein said engineered
cell expresses least one factor of the T-box family at least one
protein and a pharmaceutically acceptable carrier.
[0062] In another embodiment the composition can be a
pharmaceutical composition.
[0063] Compositions of the invention may further comprise
additional proteins, such as additional factors. These compositions
may be used to induce the formation or repair of cartilage
tissue.
[0064] The compositions of the invention may comprise, also BMP-12
or VL-1 (BMP-13), other therapeutically useful agents including
MP52, epidermal growth factor (EGF), fibroblast growth factor
(FGF), platelet derived growth factor (PDGF), transforming growth
factors (TGF-.quadrature. and TGF-.quadrature.), and fibroblast
growth factor-4 (FGF-4), 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. N another embodiment the
composition comprises anti-inflammatory agents such as IL1 receptor
antagonists, or IL4 or IL 10 agonists.
[0065] In another embodiment, there is provided an implant device
for transplantation in a subject in need comprising an engineered
cell which expresses a factor of the T-box family and a
pharmaceutically acceptable carrier. Cartilage implants are often
used in reconstructive or plastic surgery such as rhinoplasty.
[0066] The preparation and formulation of such
pharmaceutically/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 factor of the T-box family due to high
homology between species.
[0067] Particularly domestic animals and thoroughbred horses in
addition to humans are desired patients for such treatment with the
compositions of the present invention.
[0068] The therapeutic method includes administering the
composition topically, systemically, or locally as an injectable
and/or implant or device. When administered, the therapeutic
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 tissue damage. Therapeutically
useful agents other than the proteins, which may also optionally be
included in the composition, as described above, may alternatively
or additionally, be administered simultaneously or sequentially
with the composition in the methods of the invention. In addition,
the compositions of the present invention may be used in
conjunction with presently available treatments for cartilage
injury such as cartilage allograft or autograft, in order to
enhance or accelerate the healing potential of the or graft. For
example, the, allograft or autograft may be soaked in the
compositions of the present invention prior to implantation. It may
also be possible to incorporate the protein or composition of the
invention onto suture materials, for example, by freeze-drying.
[0069] The compositions may include an appropriate matrix and/or
sequestering agent as a carrier. For instance, the matrix may
support the composition or provide a surface for cartilage-like
tissue formation. The matrix may provide slow release of the
protein and/or the appropriate environment for presentation
thereof. The sequestering agent may be a substance, which aids in
ease of administration through injection or other means, or may
slow the migration of protein from the site of application.
[0070] The choice of a carrier material is based on
biocompatibility, biodegradability, mechanical properties, cosmetic
appearance and interface properties. The particular application of
the compositions will define the appropriate formulation. Potential
matrices for the compositions may be biodegradable and chemically
defined. Further matrices are comprised of pure proteins or
extracellular matrix components. Other potential matrices are
non-biodegradable and chemically defined. Preferred matrices
include collagen-based materials, including sponges, such as
Helistat.RTM. (Integra LifeSciences, Plainsboro, N.J.), or collagen
in an injectable form, as well as sequestering agents, which may be
biodegradable, for example hyalouronic acid derived. Biodegradable
materials, such as cellulose films, or surgical meshes, may also
serve as matrices. Such materials could be sutured into an injury
site, or wrapped around the tendon/ligament.
[0071] Another preferred class of carrier are polymeric matrices,
including polymers of poly (lactic acid), poly(glycolic acid) and
copolymers of lactic acid and glycolic acid. These matrices may be
in the form of a sponge, or in the form of porous particles, and
may also include a sequestering agent. Suitable polymer matrices
are described, for example, in W093/00050, the disclosure of which
is incorporated herein by reference.
[0072] Preferred families of sequestering agents include blood,
fibrin clot and/or cellulosic materials such as alkylcelluloses
(including hydroxyalkylcelluloses), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most
preferred being cationic salts of carboxymethylcellulose (CMC).
Other preferred sequestering agents include hyaluronic acid, sodium
alginate, poly (ethylene glycol), polyoxyethylene oxide,
carboxyvinyl polymer and poly(vinyl alcohol). The amount of
sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt
% based on total formulation weight, which represents the amount
necessary to prevent desorption of the protein from the polymer
matrix and to provide appropriate handling of the composition, yet
not so much that the progenitor cells are prevented from
infiltrating the matrix, thereby providing the protein the
opportunity to assist the activity of the progenitor cells.
[0073] Additional optional components useful in the practice of the
subject application include, e.g. cryogenic protectors such as
mannitol, sucrose, lactose, glucose, or glycine (to protect the
protein from degradation during lyophilization), antimicrobial
preservatives such as methyl and propyl parabens and benzyl
alcohol; antioxidants such as EDTA, citrate and BHT (butylated
hydroxytoluene); and surfactants sifdch as poly(sorbates) and
poly(oxyethylenes); etc.
[0074] As described above, the compositions and the devices of the
invention may be employed in methods for enhancing cartilage repair
or for inducing cartilage formation. These methods, according to
the invention, entail administering to a patient needing such
tissue repair; a cell expresses at least one factor of the T-box
family or in another embodiment a composition comprising an
effective amount of vector comprising a nucleic acid encoding a
factor of the T-box family.
[0075] In another embodiment, as described before, the composition
or the cell may comprise also a vector comprising a nucleic acid
encoding FGF and/or a factor of the BMP family.
[0076] Preferably the DNA molecule or protein may be injected
directly into cartilage tissue such as without limitation nasal
cartilage, articular cartilage etc. Therefore, the compounds of the
invention may be utilized as a therapeutic agent in regard to
treatment of cartilage or bone damage caused by disease or aging or
by physical stress such as occurs through injury or repetitive
strain, e.g. "tennis elbow" and similar complaints. The therapeutic
agent of the invention may also be utilized as part of a suitable
drug delivery system to a particular tissue that may be
targeted.
[0077] Other therapeutic applications for the compounds of the
invention may include the following: 1. Use in cartilage and/or
bone renewal, regeneration or repair so as to ameliorate conditions
of cartilage and/or bone breakage, degeneration, depletion or
damage such as might be caused by aging, genetic or infectious
disease, wear and tear, physical stress (for example, in athletes
or manual laborers), accident or any other cause, in humans,
livestock, domestic animals or any other animal species; 2.
Stimulation of skeletal development in livestock, domestic animals
or any other animal species in order to achieve increased growth
for commercial or any other purpose; 3. Treatment of neoplasia or
hyperplasia of bone or cartilage, in humans, livestock, domestic
animals or any other animal species; 4. Suppression of growth of
skeletal components in livestock, domestic animals or any other
animal species in order to achieve decreased growth for commercial
or any other purposes e.g. by the use of anti sense molecules to
the factor of the T-box family; and 5. Alteration of the quality or
quantity of cartilage and/or bone for any other purpose in any
animal species including humans.
[0078] Thus, according to clauses 4 and 5 the invention can be
serve also for suppressing cartilage formation, by the use of an
antagonist to Brachyury or to other factors of the T-box family.
The antagonistic effect of dominant negative Brachyury is
exemplified in Example 3. The term "antagonist" refers to a
molecule which, when bound to the epitope, decreases the amount or
the duration of the effect of the biological or immunological
activity of epitope. Antagonists may include proteins, nucleic
acids, carbohydrates, antibodies, anti sense or any other
molecules, which decrease the effect of Brachyury or to other
factors of the T-box family on cartilage formation. Such a
treatment of suppression is relevant in the treatment of malignancy
of the cartilage, for example without limitation in chondroma and
chondrasarcoma. In another embodiment the antagonist is a dominant
negative factor of the T-box family. In another embodiment the
antagonist is a dominant negative Brachyury.
[0079] The term "dominant negative Brachyury refers hereinabove to
Brachyury DNA binding domain (T-box, aa 1-229) without the
associated regulatory domains (aa 230-436).
[0080] In another embodiment the invention provides a method of
inducing chondrocyte differentiation comprising the step of
administering of a recombinant vector, which comprises a nucleic
acid encoding a factor of the T-box family, thereby inducing
chondrocyte formation. Example 2 and FIG. 2 clearly demonstrates
that forced expression of the T-box factor Brachyury leads to
chondrogenic development in C3H10T1/2 mesenchymal stem cells.
Please note that C3H10T1/2 mesenchymal stem cells line. C3H10T1/2
stem cell line resembles human MSCs by many features including
differentiation multi potentiality, high proliferation capacity and
similar response to growth factors and cytokines such as BMPs.
[0081] Example 3 further strengthen the relation between Brachyury
and chondrogenic development, by demonstrating that dominant
negative Brachyury interferes with BMP2 dependent chondrogenic
development in mesenchymal cells.
[0082] In another embodiment the invention relates to a method for
the production of transplantable cartilage matrix, the method
comprising the steps of: obtaining a cell; transfecting said cell
with a recombinant vector comprising a nucleic acid sequence
encoding a factor of the T-box family, so as to obtain an
engineered cell which expresses a factor of the T-box family; and
culturing said cell with the cell-associated matrix for a time
effective for allowing formation of a transplantable cartilage
matrix. The above method will enable the production of a cartilage
matrix, which will be transplanted to a subject in need when
required.
[0083] In another embodiment, there is provided a method of
treating a subject by ex-vivo implantation of at least one cell
comprising the following steps: obtaining at least one cell from
the subject; transfecting the cell with a nucleic acid which
encodes at least one factor of the T-box family, so as to obtain an
cell which express at least one factor of the T-box family
activated cell; and administering said activated cell to the
subject.
[0084] Optionally, the enriched stem cells are then expanded ex
vivo by culturing them in the presence of agents that stimulate
proliferation of stem cells. The culturing step can be for example
in a bioreactor, which enables three dimensional growth of the
cells. The enriched and optionally expanded stem cells are then
infected with a vector, that expresses the at least one factor of
the T-box family gene. Optionally, the vector may also carry an
expressed selectable marker, in which case successfully transduced
cells may be selected for the presence of the selectable marker.
The transduced and optionally selected stem cells are then returned
to the patient defective connective tissue and allowed to engraft
themselves into the bone marrow.
[0085] One ex vivo method of enhancing repair and/or inducing
formation disclosed throughout this specification comprises
initially generating a recombinant viral or plasmid vector which
contains a DNA sequence encoding a protein or biologically active
fragment thereof. This recombinant vector is then used to infect or
transfect a population of in vitro cultured connective tissue
cells, resulting in a population of connective cells containing the
vector. These connective tissue cells are then transplanted to a
target joint space of a mammalian host, effecting subsequent
expression of the protein or protein fragment within the joint
space. Expression of this DNA sequence of interest is useful in
substantially reducing at least one deleterious joint pathology
associated with a connective tissue disorder.
[0086] It will be understood by the artisan of ordinary skill, that
the source of cells for treating a human patient is the patient's
own connective tissue cells, such as autologous fibroblast cells.
In another embodiment the source of cells can be allogenic cells,
which were treated so as to reduce immune response.
[0087] As used herein, a "promoter" can be any sequence of DNA that
is active, and controls transcription in a eucaryotic cell. The
promoter may be active in either or both eucaryotic and procaryotic
cells. In another embodiment, the promoter is active in mammalian
cells. The promoter may be constitutively expressed or inducible.
In another embodiment, the promoter is inducible. In another
embodiment, the promoter is inducible by an external stimulus. In
another embodiment, the promoter is inducible by hormones or
metals. Still more in another embodiment, the promoter is inducible
by heavy metals. In another embodiment, the promoter is a
metallothionein gene promoter. In another embodiment the promoter
is inducible by antibiotics such as tetracycline. In another
embodiment the promoter is inducible by a tissue specific promoter.
Likewise, "enhancer elements", which also control transcription,
can be inserted into the DNA vector construct, and used with the
construct of the present invention to enhance the expression of the
gene of interest.
[0088] In another embodiment there provided ex vivo and in vivo
techniques for delivery of a DNA sequence of interest to the
connective tissue cells of the mammalian host. The ex vivo
technique involves culture of target connective tissue cells, in
vitro transfection of the DNA sequence, DNA vector or other
delivery vehicle of interest into the connective tissue cells,
followed by transplantation of the modified connective tissue cells
to the target joint of the mammalian host, so as to effect in vivo
expression of the gene product of interest.
[0089] Alternatively, an allograft, (e.g., cartilage grown in vitro
from cartilage tissue removed from the patient) may be implanted by
attaching a periosteum membrane (harvested, e.g., from the
patient's tibia), to the bone surface and injecting the allograft
beneath the membrane.
[0090] As used herein, the term "transfection" means the
introduction of a nucleic acid, e.g., an expression vector, into a
recipient cell by nucleic acid-mediated gene transfer.
[0091] Alternatively, the gene encoding the product of interest can
be associated with liposomes and injected directly into the host,
such as in the area of the joint, where the liposomes fuse with
target cells, resulting in an in vivo gene transfer to the
connective tissue. In another embodiment, the gene encoding the
product of interest is introduced into the area of the joint as
naked DNA. The naked DNA enters the target cells, resulting in an
in vivo gene transfer to the cells.
[0092] The dosage of the treatment, which is the amount of the
cells which express the at least one factor of the T-box family or
in another embodiment the amount of the composition or the device
which contain the vector comprising the nucleic acid encoding the
same in the in vivo and in the ex-vivo treatment the dosage regimen
will be determined by the attending physician considering various
factors which modify the action of the composition, e.g., amount of
bone or cartilage tissue desired to be formed, the site of the bone
or cartilage damage, the condition of the damaged cartilage or
bone, 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
additional 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.
[0093] Progress can be monitored by periodic assessment of
cartilage formation, and/or repair. The progress can be monitored
by methods known in the art, for example, X-rays (CT), ultra-sound,
MRI, arthroscopy and histomorphometric determinations.
[0094] In another embodiment, as is exemplified in Example 1 the
invention provides a method of screening candidate nucleic acid
sequence which is involved in the early stages of cartilage
development, said method comprising the step of: obtaining a cell;
transfecting said cell with a vector comprising a nucleic acid
sequence encoding to FGFR3; obtaining mRNA from said cell;
synthesizing cDNA from said mRNA; amplifying said cDNA-hybrid, so
as to obtain an amplified product; detecting said amplified
product; and comparing said amplified products from said sample to
amplified products derived from known samples thereby identifying
candidate nucleic acid sequence which is involved in the early
stages of cartilage development.
[0095] The term "involved in the early stages of cartilage
development" refers hereinabove to any gene, which is either
upregulated on downregulated during the stage of differentiation
into a cartilage cell. Such genes will enable development of drugs
which will ether enhance or suppress cartilage formation or
repair.
[0096] The step of "synthesizing" refer to step of building cDNA
complementary to the mRNA template. As refer hereinabove and in the
claims section, the step of "amplifying" refer to the selective
replication of a cDNA in greater number than usual. As refer herein
above and in the claims section, the step of "separating" refer to
the step of separation of the products using for example, gel
electrophoresis. As refer hereinabove and in the claims section,
the step of "detecting" refer to the step of noticing, which is
done, for example by visualization of the amplified product's
bands. As refer hereinabove and in the claims section, the step of
"comparing" refers to the step of searching for differences between
the amplified products derived from the at least two samples. The
term "RNA" refers to an oligonucleic in which the sugar is ribose,
as opposed to deoxyribose in DNA. RNA is intended to include any
nucleic acid, which can be entrapped by ribosomes and translated
into protein. The term "mRNA" refers to messenger RNA.
[0097] RNA can be extracted from cells or tissues according to
methods known in the art. In a preferred embodiment, RNA can be
extracted from monolayers of mammalian cells grown in tissue
culture, cells in suspension or from mammalian tissue. RNA can be
extracted from such sources by, e.g., treating the cells with
proteinase K in the presence of SDS. In another embodiment, RNA is
extracted by organic solvents. In yet another embodiment, RNA is
extracted by differential precipitation to separate high molecular
weight RNA from other nucleic acids. RNA can also be extracted from
a specific cellular compartment, e.g., nucleus or the cytoplasm. In
such methods, the nucleus is either isolated for purification of
RNA therefrom, or the nucleus is discarded for purification of
cytoplasmic RNA. Further details regarding these and other RNA
extraction protocols are set forth, e.g., in Molecular Cloning A
Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis
(Cold Spring Harbor Laboratory Press: 1989).
EXAMPLES
Experimental Procedures
[0098] DNA constructs and Transient Transfections
[0099] For the assessment of the transcriptional activity a dimmer
of the double-stranded oligonucleotide of the Brachyury binding
element (BBE) AATTFCACACCTAGGTGTGAAATT (Kispert et al., 1995) was
incorporated in the BamHI site before the HSV thymidine kinase
minimal promoter fused to the cloramphenicol acetyltransferase
(CAT)-reporter of pBLCAT5 (Boshart et al., 1992) to give reporter
plasmid pBBE-CAT5. 20 h before transfection, human embryonic kidney
HEK293T cells were plated at a density of 1.times.104/cm2 in 6-well
plates and allowed to grow under normal culture conditions. For
co-transfection experiments, 250 ng per well of Brachyury
expression vector and 250, 500 or 750 ng of the expression vector
encoding dnBrachyury. Empty vector was added to adjust the amount
of expression plasmids at 1 ug/ml. 260 ng of BBE-CAT reporter
(pBBE-CAT5) was added in the presence of 140 ng of RSV-lacZ vector
using the DOSPER procedure (see below). Cells were allowed to
incubate for 48 h. Then, cells were collected and b-galactosidase
assays were performed with the chemiluminescent b-gal reporter gene
assay (Roche Diagnostics, Mannheim, Germany) and CAT-assays were
carried out with the CAT ELISA kit (Roche Diagnostics, Mannheim,
Germany). b-gal assay results were used to normalize the CAT assay
results for transfection efficiency. All DNA transfection
experiments were repeated at least three times in triplicate.
[0100] Cell Culture and permanent Transfections
[0101] Human embryonic kidney cells HEK293T and murine C3H10T1/2
progenitor cells were routinely cultured in tissue culture flasks
in Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated FCS, 0.2 mM L-glutamine, and antibiotics (50
units/ml penicillin, 50 mg/ml streptomycin). Cells were transfected
using DOSPER according to the manufacturer's protocol (Roche
Diagnostics, Mannheim, Germany). C3H10T1/2 cells which
recombinantlly express BMP2 (C3H10T1/2-BMP2) cells were obtained by
co-transfection with pSV2pac followed by selection with puromycin
(2.5 ug/ml). FGFR3, Brachyury and T-box domain were PCR-amplified
and cloned into expression vectors pMT7T3 and pMT7T3-pgk vectors
which are under the control of the LTR of the myeloproliferative
virus or of the murine phosphoglycerate kinase promoter-1,
respectively (Ahrens et al., 1993). The integrity of the constructs
was confirmed by sequencing. HA-tags were carboxyterminally added
to full-length Brachyury and Brachyury's T-box domain by PCR with
primers encoding the respective peptide sequence. Stable expression
of the DNA binding T-box domain (aa 1-229) and of the
dominant-negative human FGFR3 without the cytoplasmatic tyrosine
kinase domains (aa 1-414) in the C3H10T1/2-BMP2 background was done
by co-transfection with pAG60, conferring resistance to G418 (750
ug/ml). Individual clones were picked, propagated, and tested for
recombinant FGFR3, dnFGFR3, Brachyury or T-box domain (dnBrachyury)
expression by RT-PCR (see below). Selected cell clones were
subcultivated in the presence of puromycine or puromycine/G418 and
the selective pressure was maintained during subsequent
manipulations. C3H10T1/2 cells were cultured in DMEM containing 10%
fetal bovine serum. The features of C3H10T1/2-BMP2 cells have been
described (Ahrens et al., 1993; Hollnagel et al., 1997; Bachner et
al., 1998). For the assessment of in vitro osteo-/chondrogenic
development, cells were plated at a density of 5-7.5.times.103
cells/cm2. After reaching confluence (arbitrarily termed day 0)
ascorbic acid (50 ug/ml) and 10 mM b-glycerophosphate were added as
specified by Owen et al., 1990 (1990).
[0102] BMP2 inductions
[0103] For BMP2-stimulation studies, C3H10T1/2 cells were plated at
a density of 1.times.104 per cm.sup.2 in a 9-cm culture dish. After
48 h cells were washed 3.times. with PBS and then cells were
starved for 24 h in DMEM without serum. Before induction the medium
was replaced with fresh DMEM without serum. Cells were then treated
for the indicated times using recombinant BMP2 from E. coli (50
ng/ml). Cycloheximide (50 ug/ml) treatment started 30 min prior to
the addition of BMP2.
[0104] RNA preparation and RT-PCR
[0105] Total cellular RNAs were prepared by TriReagentLS according
to the manufacturer's protocol (Molecular Research Center Inc.).
Five ug of total RNA was reverse transcribed and cDNA aliquots were
subjected to PCR. RT-PCR was normalized by the transcriptional
levels of HPRT. The HPRT-specific 5' and 3' primers were
GCTGGTGAAAAGGACCTCT and AAGTAGATGGCCACAGGACT, respectively. The
following 5' and 3' primers were used to evaluate
osteo/chondrogenic differentiation: SEQ ID. No. 3: collagen 1a1:
GCCCTGCCTGCTTCGTG, SEQ ID. No. 4: CGTAAGTTGGAATGGTTTTT; collagen
2a1: SEQ ID. No. 5: CCTGTCTGCTTCTTGTAAAAC, SEQ ID. No. 6:
AGCATCTGTAGGGGTCTTCT; SEQ ID. No. 7: osteocalcin:
GCAGACCTAGCAGACACCAT, SEQ ID. No. 8: GAGCTGCTGTGACATCCATAC;
PTH/PTHrP-receptor: SEQ ID. No. 9: GTTGCCATCATATACTGTTTCTGC, SEQ
ID. No. 10: GGCTTCTTGGTCCATCTGTCC; FGFR3: SEQ ID. No. 11:
CCTGCGCAGTCCCCCAAAGAAG; SEQ ID. No. 12: CTGCAGGCATCAAAGGAGTAGT;
FGFR2: SEQ ID. No. 13: TTGGAGGATGGGCCGGTGTGGTG, SEQ ID. No. 14:
GCGCTTCATCTGCCTGGTCTTG. The primer pairs for Brachyury and Sox9
have been described in (Johansson and Wiles, 1995) and (Zehentner
et al., 1999), respectively. Vector-borne transcripts for Brachyury
were evaluated with nested primers sets with either vector specific
5'- or 3'-primers: SEQ ID. No. 15: TTAGTCTTTTTGTCTTTTATTTCA; SEQ
ID. No. 16: GATCGAAGCTCAATTAACCCTCAC.
[0106] Western blotting
[0107] Recombinant cells from petri dishes (13.6 cm diameter) were
harvested at different time points before (day B2), at (day 0) and
after (days 2, 4, 7) confluence. Lysis was in RIPA buffer (1% (v/v)
nonidet P-40, 0.1% SDS (w/v), 0.5% sodium deoxycholate in PBS,
containing 100 ug/ml PMSF, 2 ug/ml aprotinin, and 1 mM Na3VO4).
Lysates were centrifuged (30 min, 10.000 g, 4 C) and the
supernatants were stored at -70 C until analysis. Protein
concentration of the lysates was determined using coomassie
brilliant blue. Protein was precipitated with ethanol, resuspended
in reducing (containing DTT) or non-reducing sample buffer and
subjected to SDS-gel electrophoresis in 12.5%T polyacrylamide gels
(20 ug/lane). Proteins were transferred to nitrocellulose membranes
by semidry-blotting. Protein transfer was checked by staining of
the membranes with Ponceau S. After blocking, membranes were
incubated incubated overnight at 4 C with a polyclonal antibody to
the HA-tag (SC-805, Santa Cruz Biotechnology, Santa Cruz, Calif.)
diluted 1:200 in blocking solution. FGFR3 and FGFR2 antibodies were
from Santa Cruz Biotechnology (#SC-123, #SC-122; Santa Cruz,
Calif.). The secondary antibody (Dianova, Hamburg) was applied at
1:5000 in blocking solution for 2 h at room temperature. Color
development was performed with 4-chloro-1-naphthol and H2O2.
[0108] Histological Methods and Verification of Cellular
Phenotypes
[0109] Osteoblasts exhibit stellate morphology displaying high
levels of alkaline phosphatase, which was visualized by cellular
staining with SIGMA FAST BCIP/NBT (Sigma, St. Louis, Mo.).
Proteoglycan secreting chondrocytes were identified by staining
with Alcian Blue at pH 2.5 and staining with Safranin O (Sigma, St.
Louis, Mo.). For collagen-immunohistochemistry cells were washed
with PBS and fixed with methanol for 15 min at -20 C by methanol.
Primary antibodies were diluted with 1% goat serum in PBS.
Monoclonal anti-collagen II antibodies (Quartett Immunodiagnostika,
Berlin, Germany, # 031502101) were diluted 1:50 and monoclonal
anti-collagen X antibodies (Quartett Immunodiagnostika, Berlin,
Germany, # 031501005) 1:10, respectively. Incubation was for 1 hour
at room temperature followed by staining with Zymed HistoStain SP
kit (Zymed Laboratories Inc., San Francisco, Calif.) applying the
manufacturer's protocol. A positive signal is indicated by a red
color precipitate of AEC (aminnoethylcarbazole).
[0110] In Vivo Transplantation
[0111] Before in vivo transplantation, aliquots of 2-3.times.106
cells were mounted on individual type I collagen sponges (Colastat7
#CP-3n, Vitaphore Corp., 2.times.2.times.4 mm.) and transplanted
into the abdominal muscle of female nude mice (4-8 weeks old).
Before transplantation animals were anaesthetized with
ketamine-xylazine mixture 30 ul/per mouse i.p. and injected i.p.
with 5 mg/mouse of Cefamzolin (Cefamezin7, TEVA). Skin was swabbed
with chlorhexidine gluconate 0.5%, cut in the middle abdominal
area, an intramuscular pocket was formed in a rectal abdominal
muscle and filled with the collagen sponge containing cells. Skin
was sutured with surgical clips. For the detection of engrafted
C3H10T1/2 cells the mice were sacrificed 10 days and at 20 days
after transplantation. Operated transplants were fixed in 4%
paraformaldehyde cryoprotected with 5% sucrose overnight, embedded,
and frozen. Sections were prepared with a cryostat (Bright, model
OTF) and stained with H&E, Alcian Blue and Safranin O.
[0112] RNA-In Situ-Hybridization
[0113] Embryos were isolated from pregnant NMRI mice at day 18.5
post conceptionem (dpc). The embryos were fixed overnight with 4%
paraformaldehyde in PBS at 4 C. 10 um cryosections were mounted on
aminopropyltriethoxysilane coated slides and non-radioactive RNA-in
situ-hybridizations were done as described (Bachner et al., 1998)
and by following the instructions of the manufacturer (Roche,
Mannheim). In short: For hybridization sense- and antisense RNA
probes from a 1.8 kb murine Brachyury cDNA was used. For the
generation of collagen 1a1 or collagen 2a1 the vector pMT7T3 was
used harbouring specific probes (Metsaranta et al., 1991).
Hybridization was performed with 0.5-2 ug denatured riboprobe/ml)
over night at 65 C in a humid chamber. For digoxygenin
(DIG)-detection slides were blocked in 5.times. SSC, 0.1% Triton,
20% FCS for 30 minutes following two washes with DIG-buffer 1 (100
mM Tris, 150 mM NaCl, pH 7.6) for 10 minutes. Slides were incubated
in anti-DIG-alkaline phosphatase coupled antibodies diluted 1:500
in DIG-buffer 1 over night in a humid chamber. Slides were washes
with 0.1% Triton in DIG-buffer 1 for 2 hours with several changes
of the washing solution and equilibrated in DIG-buffer 2 (100 mM
Tris, 100 mM NaCl, 50 mM MgCl2). Detection was performed using
BM-purple substrate (Roche, Mannheim) in DIG-buffer 2 with 1 mM
Levamisole for 1-6 hours depending on the probe. Reaction was
stopped in TE-buffer and slides were incubated in 3%
paraformaldehyde in PBS for 3 minutes, in 0.1 M glycine in PBS for
3 minutes and washed three times in PBS for 3 minutes. Slides were
counterstained with 0.5% methylenegreen in PBS for 1 minute,
dehydrated in graded alcohol series, air-dried and mounted with
Eukitt.
EXPERIMENTAL RESULTS
Example 1
[0114] BMP2-dependent Chondrogenic Development in C3H10T1/2 MSCs
Involves FGF-Receptor 3
[0115] During a substractive screen for BMP-regulated genes in
recombinant BMP2-expressing C3H10T1/2 (C3H10T1/2-BMP2) cells
upregulation of the Fibroblast Growth Factor Receptors 3 and 2
(FGFR3, FGFR2) was noted at both the transcriptional and protein
levels (FIG. 1a, c, respectively). These two receptor types exhibit
different induction kinetics. FGFR3 is upregulated during early
stages of cultivation in the stable C3H10T1/2-BMP2 line while FGFR2
shows a delayed response (FIG. 1a, c). The fast upregulation of
FGFR3 seems to be due to an immediate response to BMP2 since
exogenously-added BMP2 mediated FGFR3 transcription in wild-type
C3H10T1/2 cells in the presence of cycloheximide (FIG. 1b). In
contrast to FGFR3 and FGFR2 is FGFR1 constitutivelly expressed in
wild type and C3H10T1/2-BMP2 cells (FIG. 1a) while FGFR4 does not
show any significant rates of expression. Since FGFs and their
receptors are crucial modulators of chondrogenic development, an
assessment of whether the immediate BMP2-dependent upregulation of
FGFR3 in C3H10T1/2 is involved in the onset of chondrogenic
differentiation was conducted. Indeed the results demonstrated that
forced expression of the wild-type FGFR3 (FGFR3WT) was sufficient
for the development of morphologically distinct chondrocytes in
C3H10T1/2-FGFR3WT cells (FIG. 1d, e). Moreover, the constitutively
active mutant FGFR3 (Ach, G380R) possesses the same capacity. The
forced expression of FGFR3WT in MSCs stimulates MAPK signaling in
these cells as documented by enhanced levels of ERK1 and ERK2
phosphorylation (right panels in FIG. 1d), leads to the development
of histologically distinct chondrocytes and induces or increases
expression of chondrogenic marker genes such as collagen 2a1, the
PTH/PTHrP receptor and transcription factor Sox9 (FIG. 1e).
[0116] The immediate BMP2-dependent upregulation of FGFR3 in MSCs
(C3H10T1/2) and the inherent capacity of this receptor to initiate
chondrogenic development in these cells prompted a screen for
FGFR3-regulated transcription factors. It was observed that among
the transcription factors tested, the T-box transcription factor
Brachyury was upregulated in FGFR3-expressing C3H10T1/2 cells (see
also FIG. 5a). Thereupon, the chondrogenic potential that Brachyury
possesses after recombinant expression in wild-type C3H10T1/2 cells
(see below) was demonstrated.
Example 2
[0117] Forced Expression of the T-box Factor Brachyury Leads to
Chondrogenic Development in C3H10T1/2 Mesenchymal Stem Cells
[0118] Brachyury has originally been described as the first member
of a family of transcription factors that harbors a T-box as the
DNA-binding domain. In order to assess that the FGFR3-dependent
upregulation of the T-box factor Brachyury in C3H10T1/2 plays a
role in chondrogenesis Brachyury cDNA was expressed under the
control of the murine phosphoglycerate kinase-1 (PGK-1) in
mesenchymal stem cell line C3H10T1/2 to allow moderate expression
levels of Brachyury (C3H10T1/2-Brachyury). The recombinant
expression of Brachyury cDNA under the control of the murine
phosphoglycerate kinase-1 (PGK-1) in MSCs (FIG. 2a) gave rise to
efficient chondrogenic differentiation resulting in alkaline
phosphatase positive cells (beginning at day 4) and Alcian Blue
positive chondrocyte-like cells (at day 10 post-confluence; FIG.
2b). Three individual C3H10T1/2 clones were investigated in regard
to their chondrogenic potential and gave similar results.
Immunohistochemistry confirms the presence of the
chondrocyte-specific collagen 2 but not of collagen X which is
typical for late stages of chondrocytic differentiation
(hypertophic chondrocytes) (FIG. 2b, left panel). Major marker
genes of chondrogenic and, also, of osteogenic development show a
transient (collagen 2a1, PTH/PTHrP-receptor) or permanent
upregulation (osteocalcin gene and the chondrogenic transcription
factor Sox9) in C3H10T1/2-Brachyury in comparison with C3H10T1/2
cells which were stably transfected with an empty expression vector
(FIG. 2c). Although, the induction of the osteocalcin gene
indicates an osteogenic potential for C3H10T1/2-Brachyury, ectopic
transplantation of these cells in murine intramuscular sites
results exclusively in the massive formation of proliferating
chondrocytes and cartilage (FIG. 2d). These ectopic
transplantations have been performed three times and in all cases
these transplants developed chondrocytes and cartilage. After both
10 and 20 days transplants exhibit a histological presence of
proteoglycans (Alcian Blue, Safranin O) while bony elements or
mineralized particles are not observed (FIG. 2d). After 20 days the
ectopic implants show areas of extensive extracellular matrix
production as visualized by histological analyses (FIG. 2d). The
use of stronger viral promoters such as the LTR of the
myeloproliferative virus (MATERIALS and METHODS) resulted in
increased cellular proliferation without the apparent formation of
histologically distinct mesenchymal cell types.
Example 3
[0119] Dominant-negative Brachyury Interferes with BMP2-dependent
Chondrogenic Development in MSCs.
[0120] A partial nuclear localization signal (NLS) which has been
attributed to the T-box domain should allow a substantial nuclear
accumulation (Kispert et al., 1995). The dominant-negative nature
of the T-box domain was confirmed in DNA co-transfection assays
performed in HEK293 T cells. This cell line does not express
Brachyury. Exogenous Brachyury transactivated a construct
containing two copies of the consensus Brachyury binding element
(BBE) oligonucleotide fused to a minimal HSV thymidine kinase
(TK)-minimal promoter-CAT chimeric gene, pBBE(2.times.)-CAT5 (FIG.
3a). Indeed, co-transfection of pBBE(2.times.)-CAT5 with a
recombinant Brachyury-expressing vector resulted in a 25-fold
activation, whereas an empty expression vector had no effect (FIG.
3a). Co-transfection of full-length Brachyury (Brachyury wt) with
increasing amounts of an expression vector expressing the T-box
domain (dnBrachyury) (1:1, 1:2, 1:3) led to a clear decrease in CAT
(7-fold). Exogenous dnBrachyury alone transactivated
pBBE(2.times.)-CAT5 (BBE) only 3-fold.
[0121] The forced expression of the HA-tagged T-box domain
(dnBrachyury) is observed throughout in vitro cultivation (FIG. 3b)
and strongly interfered with the BMP2-mediated formation of
alkaline phosphatase positive osteoblast-like and Alcian Blue
chondrocyte-like cells in vitro (FIG. 3e). In vivo, in ectopic
transplantations of C3H10T1/2-BMP2 in intramuscular sites,
dnBrachyury allowed the development of connective tissue only (FIG.
3e). In addition, the chondrocyte-specific collagen 2a1 mRNA levels
are more sensitive to the presence of dnBrachyury than mRNA levels
of the distinct osteogenic marker osteocalcin. The latter is hardly
affected, consistent with the idea that Brachyury possesses a
predominant chondrogenic capacity in this particular cell type.
Interestingly, the BMP2-mediated transcriptional upregulation of
FGFR3 in C3H10T1/2 is not obstructed by dnBrachyury indicating that
the immediate BMP2-mediated FGFR3 induction is independent of
Brachyury or other T-Box factors (FIG. 3c, d). However,
FGFR2-expression that bits a delayed response in C3H10T1/2-BMP2
cells (FIGS. 1 and 3) displays a high sensitivity to dnBrachyury.
BMP-mediated FGFR2 expression is almost completely suppressed by
the dominant-negative acting T-box domain (FIG. 3c, d). This may
indicate that that the presence of FGFR2 seems necessary for the
osteo-/chondrogenic differentiation in this mesenchymal progenitor
line (FIG. 3c, d).
[0122] Furthermore, this suggests a hierarchy of FGFR-mediated
signaling for chondrogenic development. FGFR3-dependent signaling
is induced at first by BMP2 and as a consequence, FGFR2 mediated
signaling becomes active. Such a model is proposed in FIG. 7. This
model predicts that a forced expression of dominant-negative FGFR3
would interfere with BMP2-mediated chondrogenesis and with FGFR2
and Brachyury expression. Indeed, an FGFR3-variant without the
cytoplasmatic tyrosine-kinase domains downregulates BMP2-dependent
mRNA expression levels of FGFR2 and Brachyury (FIG. 4b) and
interferes with the histological manifestation of alkaline
phosphatase or Alcian Blue positive chondrocyte-like cells (FIG.
4a).
Example 4
[0123] FGFR3 and the T-box Factor Brachyury Are Involved in an
Autoregulatory Loop for Chondrogenic Development in C3H10T1/2
Progenitors
[0124] During amphibian gastrulation, mesodermal Brachyury is
involved in an autoregulatory loop with FGF that is present in the
embryo (1998). In C3H10T1/2 cells several FGF genes tested (FGF2,
4, and 9) were not Brachyury- or FGFR3-regulated and, therefore,
are unlikely members of such a loop. However, a loop seems to exist
between FGFR3 and Brachyury since forced expression of either one
lead to the induction of the other one in C3H10T1/2 (FIG. 5a).
These experiments indicate that after BMP2-mediated initiation of
the chondrogenic lineage, the chondrogenic differentiation may
advance for some time in a BMP2-independent fashion maintained by
the autoregulatory loop between FGFRs and FGF-regulated
transcription factors such as the T-box factor Brachyury.
[0125] In a preceding study it was shown that BMP-mediated R-Smad
signaling alone is not sufficient for cartilage development in
C3H10T1/2 cells. Thereby, forced expression of Smad1 or the
biologically active Smad1-MH2 domain is able to mimic BMP2-mediated
onset of osteogenic differentiation (Takeuchi et al., 2000).
However, in contrast to osteogenic marker genes such as the
osteocalcin gene, Smad1-MH2 domain-signaling is not sufficient to
mimic BMP2-dependent FGFR3- and the concomitant Brachyury-gene
induction (FIG. 5b). Other BMP-activated R-Smads such as Smad5 and
Smad8 are also unable to mediate or to mimic BMP2-dependent
FGFR3-induction in C3H10T1/2 cells indicating
R-Smad-MH2-independent pathways for FGFR3 induction or,
alternatively, cooperative activities of R-Smads with other
transcription factors (Mazars et al., 2000).
Example 5
[0126] The T-box Factor Brachyury Is Expressed in Maturing
Cartilage During Murine Embryonic Development
[0127] Brachyury is expressed at high levels early in vertebrate
embryonic development and is involved in gastrulation and in the
dose-dependent determination of mesodermal cell fates. After
gastrulation, Brachyury-expression is downregulated and persists in
the notochord to the end of embryogenesis (Kispert and Herrmann,
1994). Comparative mRNA expression analysis of murine Brachyury
(Bra), collagen 1a1 (col 1a1) and collagen 2a1 (col 2a1) in
skeletal development (18.5 dpc) indicates that Brachyury is
expressed at significant levels in cartilage forming cells of the
intervertebral disks and in limb bud development (FIG. 6).
Expression of Brachyury is enhanced in intervertebral disc
development in the nucleus pulposus in 18.5 dpc mouse embryos (FIG.
6A, a, d) confirming earlier reports (Wilkinson et al., 1990).
Collagen 1a1 is expressed in the outer annulus (arrowheads in FIG.
6A b, e), and collagen 2a1 in the cartilage primordium of the
vertebrae (FIG. 6A, c, f). In transversal sections made at the
level of the upper lumbar vertebra, expression of Brachyury is in
addition detectable in distinct chondrogenic cells of the neural
arch (FIG. 6A, h) whereas collagen 1a1 expression is maintained in
the outer annulus (FIG. 6A, i), as is collagen 2a1 in the cartilage
primordium (FIG. 6A, j). In murine limb bud development (18.5 dpc;
hind limb) expression of Brachyury is evident in distinct
chondrogenic cells of the forming metatarsal bones (FIG. 6B, a-c).
In contrast, collagen 1a1 is expressed in the outer periosteal
layer (FIG. 6B, d-f) and collagen 2a1 expression is enhanced in
differentiating chondrocytes (FIG. 6B, g-i). Interestingly, like in
intervertebral disc formation, the expression of Brachyury is only
evident in chondrocyte-like cells that do not express Col 2a1
indicating that Brachyury expression is upregulated in chondrogenic
cells before or after collagen 2 expressions.
Example 6
[0128] The T-box Factor Brachyury Is Expressed in Human Adult
Mesenchymal Cells following transfection with Brachyury
plasmid.
[0129] Cells isolation:
[0130] Human Adult Mesenchymal Stem Cells (hAMSCs) were isolated
from explants of human bone marrow surgical waste and expanded in
vitro. Isolation of hMSCs was performed as follows: 10 ml marrow
aspirates were collected into a tube with 6000 U heparin, washed
with PBS, and recovered cells were collected by centrifugation at
900 g. Collected cells were then loaded onto Percoll solution
(density 1.073 g/ml). Cell separation was accomplished by
centrifugation at 1100 g (30 min at 20 uC). Nucleated cells
collected were washed twice with PBS and then cultured in 100 mm
culture plates.
[0131] Tissue culture:
[0132] Cells were cultured in low glucose, low bicarbonate DMEM
medium (Beit Haemek)+10% fetal calf serum (Biet Haemek), the
environmental conditions were of 5% CO2 and 370 C.
[0133] Cells transfection:
[0134] 3.times.10.sup.6 hAMSCs were transfected with 30 ug of the
Brachyury plasmid using the Amaxa Nucleofector.TM. technology and
in accordance with the manufacturer's preliminary protocol for
hAMSCs. Briefly, the harvested cells were aliquoted in
5.times.10.sup.5 cells, recovered by centrifugation, and
re-suspended in 100 .mu.l of Amaxa's nucleofection solution. Five
microgram of DNA plasmid were added to the suspended cells, mixed
well and transferred to electroporation cuvette provided by the
Amaxa nucleofection kit. The electroporation was performed using 5
different programs (U28, C12, C17, E14, G22) that basically differ
in the intensity and the length of the electric pulse. Immediately
after the electroporation, the cells were transferred into 6-well
plates, containing 4 ml complete growth medium equilibrated to 37
C, 5% CO2, and incubated at 37.degree. C. in 5% CO.sub.2 atmosphere
for 24 hours.
[0135] Detection of gene expression:
[0136] 24 hours post transfection RNA was isolated from the cells
using the Trizol reagent and protocol provided by the manufacturer
(Life Sciences). 2 ug of RNA were transformed into cDNA by Reverse
Transcriptase (RT) reaction. PCR was then performed using specific
primers to the Brachyury cDNA. 20 ul of the PCR reaction sample
were loaded into a 2% Agarose gel stained with Etidium Bromide. The
gel analysis demonstrated a band matching the expected amplified
region in the Brachyury cDNA (see FIG. 8).
Sequence CWU 1
1
17 1 19 DNA Artificial sequence Single strand DNA oligonucleotide 1
gctggtgaaa aggacctct 19 2 20 DNA Artificial sequence Single strand
DNA oligonucleotide 2 aagtagatgg ccacaggact 20 3 17 DNA Artificial
sequence Single strand DNA oligonucleotide 3 gccctgcctg cttcgtg 17
4 20 DNA Artificial sequence Single strand DNA oligonucleotide 4
cgtaagttgg aatggttttt 20 5 21 DNA Artificial sequence Single strand
DNA oligonucleotide 5 cctgtctgct tcttgtaaaa c 21 6 20 DNA
Artificial sequence Single strand DNA oligonucleotide 6 agcatctgta
ggggtcttct 20 7 20 DNA Artificial sequence Single strand DNA
oligonucleotide 7 gcagacctag cagacaccat 20 8 21 DNA Artificial
sequence Single strand DNA oligonucleotide 8 gagctgctgt gacatccata
c 21 9 24 DNA Artificial sequence Single strand DNA oligonucleotide
9 gttgccatca tatactgttt ctgc 24 10 21 DNA Artificial sequence
Single strand DNA oligonucleotide 10 ggcttcttgg tccatctgtc c 21 11
22 DNA Artificial sequence Single strand DNA oligonucleotide 11
cctgcgcagt cccccaaaga ag 22 12 22 DNA Artificial sequence Single
strand DNA oligonucleotide 12 ctgcaggcat caaaggagta gt 22 13 23 DNA
Artificial sequence Single strand DNA oligonucleotide 13 ttggaggatg
ggccggtgtg gtg 23 14 22 DNA Artificial sequence Single strand DNA
oligonucleotide 14 gcgcttcatc tgcctggtct tg 22 15 24 DNA Artificial
sequence Single strand DNA oligonucleotide 15 ttagtctttt tgtcttttat
ttca 24 16 24 DNA Artificial sequence Single strand DNA
oligonucleotide 16 gatcgaagct caattaaccc tcac 24 17 24 DNA
Artificial sequence Double Stranded DNA oligonucleotide having
sequence of Brachyury binding element 17 aatttcacac ctaggtgtga aatt
24
* * * * *