Note: Descriptions are shown in the official language in which they were submitted.
CA 02357403 2001-09-14
FIELD OF THE INVENTION
The present invention pertains to the field of Pax-encoding vectors and more
particularly to
vectors comprising sequences that encode Pax7, Pax3, and/or biologically
active variants or
fragments thereof, and their use to induce differentiation of adult
pluripotent stem cells to
produce myoblasts.
BACKGROUND
Myoblasts are precursor cells of the mesoderm that are destined for
myogenesis. The
determined myoblasts are capable of recognising and spontaneously fusing with
other
myoblasts leading to the production of a differentiated myotube. The
multinucleated myotube
no longer divides or synthesises DNA but produces muscle proteins in large
quantity. These
include constituents of the contractile apparatus and specialised cell-surface
components
essential to neuromuscular transmission.
Eventually, the differentiated muscle cell exhibits characteristic striations
and rhythmic
contractions. A further step in this pathway is maturation; the contractile
apparatus and
muscle at different stages of development contain distinct isoforms of muscle
proteins such as
myosin and actin, encoded by different members of multigene families.
Myoblasts have the potential for being used in a variety of ways. For example,
the myoblasts
may serve as vehicles for cell therapy, where one or more genes may be
introduced into the
myoblasts to provide a product of interest. In order to find wide utility in
therapeutic
applications, however, it will be necessary to develop methods for the
sustained production by
myoblasts of the product of interest.
Myoblasts are thought to be capable of repairing damaged or injured myofibers
(Mauro, A., J.
Biophys. Biochem. Cytol., 9: 493-495 (1961); Bischoff, R., in Mvology, Engel,
A. G. and
Franzini-Armstrong, C., Eds., New York: McGraw Hill, pp. 97-119,1994; and
Grounds, M.,
Adv. Exp. Med. Biol., 280: 101-104 (1990)). Because myoblasts are thought to
be capable of
repairing damaged or injured myofibers, the technique of myoblast transfer
(myoblast
transplantation) has been proposed as a potential therapy or cure for muscular
diseases,
including Duchenne muscular dystropy (DMD).
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Myoblast transfer involves injecting myoblast cells into the muscle of a
mammal, particularly
a human patient, requiring treatment. Although developed muscle fibres are not
regenerative,
the myoblasts are capable of a limited amount of proliferation, thus
increasing the number of
muscle cells at the location of myoblast infusion. Myoblasts so transferred
into mature muscle
tissue will proliferate and differentiate into mature muscle fibres. This
process involves the
fusion of mononucleated myoeenic cells (myoblasts) to form a multinucleated
syncytium
(myofiber or myotube). Thus, it has been proposed that muscle tissue which has
been
compromised either by disease or trauma may be supplemented by the transfer of
myoblasts
into the compromised tissue.
Moreover, cell cultures are widely used as in vitro models for studying the
events involved
during ih vivo cellular or tissular development. For example, muscular
development events
can be reproduced during the rnyogenic differentiation of stem cell cultures.
Accordingly,
permanent mammalian cell cultures, especially human myogenic cell cultures,
would be of
considerable value for providing useful tools for dissecting the molecular and
biochemical
cellular events, for identifying and testing new drugs for muscular diseases,
such as
dystrophies, for the study of myogenesis, etc.
The "paired-box" family of transcription factors is intimately involved in the
control of
embryonic development. Different members of the Pax-family of transcription
factors appear
to regulate the development and differentiation of diverse cell lineages
during embryogenesis
(see Table 1) (Mansouri et al., 1999; Mansouri et al., 1994; Noll, 1993;
Strachan and Read,
1994). Pax7 and the closely related Pax3 gene belong to a paralogous subgroup
of Pax genes
based on similar protein structures and partially overlapping expression
patterns during mouse
embryogenesis (Goulding et al., 1991; Jostes et al., 1990). Interestingly, the
closely related
Pax3 gene plays an essential role in regulating the developmental program of
MyoD-
dependent migratory myoblasts during embryogenesis (Maroto et al., 1997;
Tajbakhsh et al.,
1997).
Pax7 and Pax3 proteins bind identical sequence-specific DNA elements
suggesting that they
regulate similar sets of target genes (Schafer et al., 1994). Furthermore,
increased expression
and gain-of-function mutations in both Pax3 and Pax7 are associated with the
development of
alveolar rhabdomyosarcomas indicating that both molecules regulate similar
activities in
myogenic cells (Bennicelli et al., 1999). However, Pax7 but not Pax3 is
expressed in adult
human primary myoblasts (Schafer et al., 1994). Interestingly, differential
expression of
alternatively spliced Pax7 transcripts correlates with muscle regenerative
efficiency in
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CA 02357403 2001-09-14
different strains of mice (Kay et al., 1998; Kay et al., 1997; Kay et al.,
1995; Kay and Ziman,
1999) .
This background information is provided for the purpose of making known
information
believed by the applicant to be of possible relevance to the present
invention. No admission is
necessarily intended, nor should be construed, that any of the preceding
information
constitutes prior art against the present invention.
SUMMARY OF THE INVENTION
An object of the present invention is to provide Pax7-encoding vectors and use
thereof. In
accordance with an aspect of the present invention, there is provided a vector
comprising an
expression cassette comprising a sequence encoding a Pax protein, wherein the
Pax protein is
selected from the groups consisting of: Pax7; Pax3; an active variant of Pax
7; an active
variant of Pax 3; an active fragment of Pax 7; and an active fragment of Pax
7, and wherein
the Pax protein can induce myogenic differentiation of adult pluripotent stem
cells..
In accordance with another aspect of the invention, there is provided a method
of
differentiating adult pluripotent stem cells to produce myoblasts comprising
the step of
transforming or infecting the stem cells with a vector comprising an
expression cassette
comprising a sequence encoding a Pax protein, wherein the lPax protein is
selected from the
groups consisting of: Pax7; Pax3; an active variant of Pax 7; an active
variant of Pax 3; an
active fragment of Pax 7; and an active fragment of Pax 7.
In accordance with another aspect of the invention, there is provided use of
myoblasts
produced according to the methods described herein for transplantation in a
mammal in need
of such therapy.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 demonstrates that Pax7 is expressed specifically in proliferating
myoblasts. (A)
Pax7 was expressed at high levels in proliferating wild-type myoblasts (Wt-Mb)
and MyoD-
deficient cells (MyoD ~ Mb) cells and down regulated in response to
differentiation
conditions (Wt-D and MyoD'~ D). (B) Expression of Pax7 was specific to
myogenic cells
with low levels detected in C2C12 myoblasts. (C) Pax7 was not detected in RNA
from a
panel of tissues.
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Figure 2 depicts the expression of Pax7 in muscle satellite cells. (A) In situ
hybridisation
revealed that Pax7 mRNA was expressed at a frequency and location consistent
with specific
expression in satellite cells and myogenic precursor cells. (B) Pax7
expression was associated
with PI positive nuclei (40X magnification). (C,D) High magnification (200X)
of Pax7
expressing cell in wild type muscle was characteristic of a satellite cell
residing beneath the
basal lamina. (E,F) Increased numbers of cells expressed Pax7 in regenerating
mdx muscle
(40X). Black and white arrowheads indicate cells stained positive for Pax7
mRNA, and PI
positive nuclei respectively. (PI: propidium iodide).
Figure 3 demonstrates that Pax7 ~ mice exhibit skeletal muscle deficiency. (A)
Seven-day-
old Pax7 mutant animals were approximately one-half the weight of wild type
animals and
had splayed hind limbs and an abnormal gait. (B,C) Hematoxylin-Eosin (HE)
stained tibialis
anterior muscle sections (40X) revealed a normal histological appearance of
(C) Pax7 mutant
muscle but fibre diameter was reduced 1.5 fold as compared to (B) wild type
muscle. (D,E)
The diaphragm of (E) mutant animals shown here in cross-section was
significantly thinner
than in (D) wild type animals (40X).
Figure 4 depicts the absence of rnyoblasts in cultures derived from Pax~~
muscle. (A-J)
Primary cell cultures were analysed by (A,F) phase microscopy; and
immunocytochemistry
with (B,G) anti-desmin and (D,I) anti-c-Met antibodies. (C,E,H,J) Cells
stained with
antibodies were counter-stained with Hoechst 33342 to show all nuclei. Black
arrowheads
depict satellite cell derived myoblasts in (A). White arrowheads indicate
immunoreactive cells
and corresponding nuclei in (B-E).
Figure 5 depicts the complete ablation of satellite cells in Pax7 ~ muscle. (A-
D)
Transmission electron micrographs of 7-10 day old Pax7+~~~ and (E,F) Pax7 ~
muscle. (A,C)
Satellite cells (SC) are readily identified in Pax7+~+ muscle (7500X). (B,D)
High
magnification of satellite cells clearly revealed the plasma membrane (black
arrowheads)
separating the satellite cell from its adjacent myofiber, the continuous basal
lamina
surrounding the satellite cell and myofiber and the heterochromatic appearance
of the nucleus
(20 000X). (E,F) Myonuclei (fiber nuclei) (MN) but not satellite cells were
present in Pax7
mutant muscles. Other ultrastructural differences were not detected.
Figure 6 demonstrates the enhanced hematopoietic potential of Pax~~ muscle-
derived
pluripotent stem cells. (A-D) FACS analysis of hoechst stained muscle-derived
cells
demonstrated approximately equal numbers of verapamil sensitive side-
population (SP) cells
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in both (A,B) Pax7+~+ and (C,D) Pax7 ~ muscles. (E) Myosin heavy chain
positive muscle
colonies predominate in stem cell medium/methylcellulose cultures of Pax7+~+
muscle cells.
(F) Pax7 ~ muscle cells have increased hematopoietic potential and generate
granulocyte and
monocyte colonies verified by (G,H) Ly-6G immunoreactivity. (I) Colony forming
assay of
muscle cells cultured in stem cell medium/methylcellulose over a period of two
weeks
demonstrated almost a 10-fold increased hematopoietic potential of Pax7 mutant
stem cells.
Other cells represent both fibroblasts and adipocytes.
Figure 7 is a schematic representation of the role of Pax7 in the
specification of satellite cells.
Muscle-derived pluripotent stem cells primarily give rise to myoblasts when
cultured in stem
cell medium. By contrast, Pax7 ~ muscle stem cells exhibit almost a 10-fold
increase in
propensity towards hematopoietic differentiation and are incapable of forming
adult
myoblasts. These data therefore implicate Pax7 in regulating the specification
of adult muscle
satellite cells by restricting the fate of pluripotent stem cells. Taken
together, these
experiments suggest the following hypothesis. Pluripotent stem cells (msc)
within muscle
represent the progenitors of sublaminar satellite cells that are specified
following induction of
Pax7. Satellite cells are subsequently activated in response to physiological
stimuli to
generate daughter myogenic precursor cells (mpc) prior to terminal
differentiation into new or
previously existing fibres.
Figure 8 provides a demonstration of myogenic specification of SP cells.
Fractionated SP cells
infected with Ad-empty control virus (mock) and Ad-Pax7 virus (Ad-Pax7d) were
analysed
for expression of desmin and counter-stained with DAPI to show all nuclei.
Figure 9 depicts the structure of an exemplary adenovirus-Pax7. Pax7 is
expressed under the
control of the murine CMV promoter (mCMV). The SV40 poly A (SVpA) sequence is
downstream of the cDNA.
Figure 10 depicts western analysis of Ad-Pax7 infected Cells. C2C12 myoblasts
or lOTl/2
fibroblasts were infected with either Ad-Pax7 or Ad-empty. Western analysis
indicates that
Pax7 protein is expressed at high levels from the recombinant Ad-Pax7 virus.
C2C12
myoblasts expressed low-levels of endogenous Pax7.
Figure 11 provides a demonstration of myogenic specification of SP cells.
Fractionated SP
cells infected with Ad-empty control virus (A,B) and Ad-Pax7 virus (C-H) were
analysed for
expression of desmin (A,C,E,G) and counter-stained with DAPI to show all
nuclei (B,D;F,H).
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Figure 12 depicts induction of Myf5lacZ by Pax7. Muscle-derived cells from
MyfSnlacZ
transgenic mice were infected with Ad empty (A,B) or Ad-Prxx7 (C-F).
Expression of Pax7
resulted in up-regulation of MyfSnLacz indicating entry into the myogenic
differentiation
program.
Figure 13 depicts the amino acid sequence of a human Pax7 protein (NCBI
Accession number
NM 002584).
Figure 14 depicts the amino acid sequence of variants of the human Pax7
protein (A NCBI
Accession number NP 002575; B NCBI Accession number NM 013645).
Figure 15 depicts the amino acid sequence of a long splice form of human Pax7
protein
(NCBI Accession number 578502).
Figure 16 depicts the amino acid sequence of a human Pax7 protein (NCBI
Accession number
CAA16432).
Figure 17 depicts the amino acid sequence of a fragment of a human Pax7
protein (NCBI
Accession number 550115).
Figure 18 depicts the amino acid sequence of a chicken Pax 7 protein (NCBI
Accession
number BAA23005).
Figure 19 depicts the amino acid sequence of a human Pax3 protein (NCBI
Accession number
P23760)
Figure 20 depicts the amino acid sequence of a human Pax3A protein (NCBI
Accession
number NP 000429).
Figure 21 depicts the amino acid sequence of a human Pax3B protein (NCBI
Accession
number NP 039230).
Figure 22 depicts the amino acid sequence of a human Pax3 protein (NCBI
Accession number
AAA03628).
Figure 23 depicts the amino acid sequence of a mouse Pax3 protein (NCBI
Accession number
NP 032807).
CA 02357403 2001-09-14
Figure 24 depicts the amino acid sequence of a chicken Pax3 protein (NCBI
Accession
number AH004319)
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art to which
this invention
belongs.
Characterisation and Preparation of Pax-Encoding Vectors
One embodiment of the present invention provides a vector comprising an
expressible
sequence encoding Pax7, Pax3 or an active variant or fragment thereof.
Gene sequences encoding Pax7 and Pax3 are known and a worker skilled in the
art would
readily appreciate that these sequences can be obtained from publicly
available databases, for
example, GenBank. For example, NCBI Accession number AL021528 provides the
sequence
of a human Pax7 gene. Provided herein are non-limiting examples of amino acid
sequences
that can be expressed by the Pax-encoding vectors of the present invention
(see Figures 13
through 24).
Nucleic acids comprising a sequence that encodes Pax7, Pax3, or an active
variant or fragment
thereof can be cloned into a vector using standard techniques that are well
known to workers
skilled in the art. The Pax-encoding vectors of the present invention
facilitate the expression
of Pax7, Pax3 or an active variant or fragment thereof such that the expressed
protein can
induce differentiation of adult pluripotent stem cells. A variety of vectors
suitable for use in
the preparation of the Pax-encoding vectors of the present invention are known
in the art.
These vectors must be replicable and viable in the stem cells to be
differentiated. The vector
used in the preparation of the Pax-encoding vector of the present invention
may be, for
example, in the form of chromosomal, nonchromosomal and synthetic DNA
sequences, e.g.,
derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast
plasmids; vectors
derived from combinations of plasmids and phage DNA, viral DNA such as
vaccinia,
adenovirus, fowl pox virus, and pseudorabies.
Viral based systems provide the advantage of being able to introduce
relatively high levels of
a heterologous nucleic acid into a variety of cells. Additionally, such
viruses can introduce
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CA 02357403 2001-09-14
heterologous DNA into nondividing cells. Suitable viral vectors for
preparation of the Pax-
encoding vector of the present invention for use in mammalian cells are well
known in the art.
These viral vectors include, for example, Herpes simplex virus vectors (U.S.
Pat. No.
5,501,979), Vaccinia virus vectors (U.S. Pat. No. 5,506,138), Cytomegalovirus
vectors (U.S.
Pat. No. 5,561,063), Modified Moloney murine leukemia virus vectors (U.S. Pat.
No.
5,693,508), adenovirus vectors (U.S. Pat. Nos. 5,700,470 and 5,731,172), adeno-
associated
virus vectors (U.S. Pat. No. 5,604,090), constitutive and regulatable
retrovirus vectors (U.S.
Pat. Nos. 4,405,712; 4,650,764 and 5,739,018, respectively), papilloma virus
vectors (U.S.
Pat. Nos. 5,674,703 and 5,719,054), and the like.
In one embodiment of the present invention, adenovirus-Pax7 vectors are
employed to induce
specification of stem cells in culture. Any of the Pax-encoding vectors
described herein may
be employed to induce specification or differentiation of adult pluripotent
stem cells.
As used herein, "retroviral vector" refers to the well known gene transfer
plasmids that have
an expression cassette encoding an heterologous gene residing between two
retroviral LTRs.
Retroviral vectors typically contain appropriate packaging signals that enable
the retroviral
vector, or RNA transcribed using the retroviral vector as a template, to be
packaged into a
viral virion in an appropriate packaging cell line (see, for example, U.S.
Pat. No. 4,650,764).
Suitable retroviral vectors for use herein are described, for example, in U.S.
Pat. No.
5,252,479, and in WIPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO
92/05266 and WO 92/14829, incorporated herein by reference, which provide a
description of
methods for efficiently introducing nucleic acids into human cells using such
retroviral
vectors. Other retroviral vectors include, for example, the MMTV vectors (U.S.
Pat. No.
5,646,013), vectors described supra, and the like.
In the preparation of the Pax-encoding vectors of the present invention the
nucleic acid
sequence encoding the Pax protein is placed under the control of a suitable
promoter. Suitable
promoters which may be employed include, but are not limited to, adenoviral
promoters, such
as the adenoviral major late promoter; or hetorologous promoters, such as the
cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)
promoter; inducible
promoters, such as the MMT promoter, the metallothionein promoter; heat shock
promoters;
the albumin promoter; the ApoAI promoter; human globin promoters; viral
thymidine kinase
promoters, such as the Herpes Simplex'thymidine kinase promoter; retroviral
LTRs (including
the modified retroviral LTRs hereinabove described); the (1-actin promoter;
and human growth
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hormone promoters. The promoter also may be the native promoter which controls
the genes
encoding the Pax proteins.
In accordance with one embodiment of the present invention the Pax-encoding
vectors may
contain additional sequences that encode heterologous biologically active
proteins and/or
polypeptides. For example, a Pax-encoding vector of the present invention may
additionally
express a therapeutic protein, such as a growth or trophic factor (e. g.,
GDNF, neurturin,
BDNF, bFGF, NT-3, TGF-P), a transcription factor (e. g., Nurr-1), or an
immunosuppressant
and operably linked to a suitable promoter. The expression of such a
therapeutic protein may
be beneficial in order to enhance the survival of cell transplants or increase
the therapeutic
potential of the cells following transplant. For example, the vectors can be
introduced into
pluripotent stem cells that are capable of differentiating as muscle cells
prior to transplantation
into Duschenne patients.
Isolation and Culture of Stem Cells
Methods of cell isolation and culture are described in numerous publications
known to the art,
for example "Culture of Animal Cells: A Manual of Basic Technique", 4th Ed.
(R.I. Freshney,
2000), and "Current Protocols in Cell Biology" (Wiley & Sons (eds), 2000).
Useful naive stem cells include adult pluripotential stem cells, which may be
isolated from
bone marrow using conventional methodologies, (see, for example, Faradji et
al., (1988) Vox
Sang., 55 (3):133-138 or Broxmeyer et al., (1989) PNAS 86:3828-3832), as well
as naive
stem cells obtained from blood.
Mesenchymal stem cells (MSCs) are the formative pluripotent blast or embryonic-
like cells
found in bone marrow, blood, dermis, and periosteum that are capable of
differentiating into
specific types of mesenchymal or connective tissues including adipose,
osseous, cartilaginous,
elastic, muscular, and fibrous connective tissues ( US Pat. No. 5736396). The
specific
differentiation pathway which these cells enter depends upon various
influences from
mechanical influences and/or endogenous bioactive factors, such as growth
factors, cytokines,
and/or local microenvironmental conditions established by host tissues.
Although these cells
are normally present at very low frequencies in bone marrow, a process for
isolating,
purifying, and mitotically expanding the population of these cells in tissue
culture is reported
in Caplan et al. U.S. Pat. Nos. 5,197,985 and 5,226,914 and 5,736,396. Factors
which have
myogenic inductive activity on human MSCs are present in several classes of
molecules,
CA 02357403 2001-09-14
especially cytidine analogs, such as 5-azacytidine and 5-aza--2'-
deoxycytidine. The effect of
these modulating factors on human MSCs is disclosed in Caplan et al. U.S. Pat.
No 5,736,396.
Suitable solid tissue from which cells can be obtained includes any organ or
tissue from adult,
mammalian tissue. Any mammalian tissue or organ can be used in this invention,
including
but not limited to those obtained from mice, cattle, sheep, goat, pigs, dogs,
rats, rabbits, and
primates (including human). Specific examples of suitable solid tissues
include skeletal
muscle, brain and central nervous system tissue from which neurons and other
supporting
cells are derived, skin derived from cultured keratinocytes, germ cells or
embryonic stem cells
or cells from other organs (liver, pancreas, spleen, kidney, thyroid, etc.).
Stem cells and
progenitor cells isolated from any other solid organ are also amenable
candidates for
culturing. Stem cells isolated from solid tissues (the exception to solid
tissue is whole blood,
including blood, plasma and bone marrow) which were previously unidentified in
the
literature are also within the scope of this invention.
In adult skeletal muscle, the progenitor cell is referred to as a satellite
cell. Normally, satellite
cells are dormant, but when muscle is traumatized, these cells divide and
differentiate, and so
are the source of regenerated skeletal muscle. Methods of isolating,
identifying, culturing and
differentiating satellite cells are well known to those of skill in the art.
For example, in U.S.
Pat. No. 5,328,695, (1994) Lucas et al. describe a myogenic protein isolate
from mammalian
(chick) bone that stimulates lineage commitment and differentiation of
skeletal muscle stem
cells.
It is understood that the initial medium for isolating stems/progenitors, the
medium for
proliferation of these cells, and the medium for differentiation of these
cells can be the same
or different. The medium can be supplemented with a variety of growth factors,
cytokines,
serum, etc. As a general principle, when the goal of culturing is to keep
cells dividing, serum
is added to the medium in relatively large quantities (10-20% by volume).
Specific purified
growth factors or cocktails of multiple growth factors can also be added or
sometimes used in
lieu of serum. As a general principle, when the goal of culturing is to
reinforce differentiation,
serum with its mitogens is generally limited (about 1-2% by volume). Specific
factors or
hormones that promote differentiation and/or promote cell cycle arrest can
also be used.
Examples of suitable growth factors are basic fibroblast growth factor (bFGF),
vascular
endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming
growth
factors (TGF.alpha. and TGF.beta.), platelet derived growth factors (PDGF's),
hepatocyte
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CA 02357403 2001-09-14
growth factor (HGF), insulin-like growth factor (IGF), insulin, erythropoietin
(EPO), and
colony stimulating factor (CSF). Examples of suitable hormone medium additives
are
estrogen, progesterone or glucocorticoids such as dexamethasone. Examples of
cytokine
medium additives are interferons, interleukins, or tumor necrosis factor-
.alpha. (TNF.alpha).
Following differentiation, the specific differentiated cell types are
identified by a variety of
means including fluorescence activated cell sorting (FACS), protein-conjugated
magnetic
bead separation, morphologic criteria, specific gene expression patterns
(using RT-PCR), or
specific antibody staining. The gene products expressed between two or more
given
differentiated cell types will vary. For example, following diifferentiation
of skeletal muscle
satellite cells, the transcription factors myf5, MyoD, myogenin, and mrf4 are
expressed. It is
understood that developmental pathways often involve more than one step or
stage for
differentiation and any of these steps or stages may be affected by variations
in culture
conditions.
Use of the Pax-Encoding Vectors
One embodiment of the present invention provides a method of inducing myogenic
differentiation of adult pluripotent stem cells comprising the step of
contacting the stem cells
with the Pax-encoding vector under conditions that allow expression of the Pax
protein, Pax7,
Pax3 or an active variant or fragment thereof. This method optionally includes
the step of
first obtaining and culturing the stem cells from various sources as described
herein.
In a related embodiment of the present invention the Pax-encoding vector is
used in
combination with one or more separate expression vectors that express a
molecule that can,
for example, aid in the induction of differentiation or improve the
therapeutic potential of the
myoblasts that are generated.
The differentiated cells that result from the method of the present invention
have various uses,
including but not limited to their use as a source material for
transplantation in the treatment
of muscle disease or disorder in animals, including humans. Additionally, the
differentiated
cells can be used as a research tool and as part of diagnostic assays.
The present invention further relates to a pharmaceutical composition
comprising at least one
myoblast prepared using the method of the present invention. According to one
embodiment,
said myoblast comprised in said pharmaceutical composition is encapsulated.
Cell
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CA 02357403 2001-09-14
encapsulation methodology has been previously described which allows
transplantation of
encapsulated cells in treatment of Parkinson's disease (Tresco et al., 1992,
ASAIO J. 38, 17-
23) or Amyotrophic lateral sclerosis (Aebischer et al., 1996, Hurn. Gene Ther.
7, 851-860).
According to said specific embodiment, cells are encapsulated by compounds
which form a
microporous membrane, and said encapsulated cells can further be implanted in
vivo.
Capsules, for example approximately 1 cm in length containing the cells of
interest may be
prepared employing a hollow microporous membrane fabricated from poly-ether-
sulfone
(PES) (Akzo Nobel Faser AG, Wuppertal, Germany; D glon. et al, 1996, Hum. Gene
Ther. 7,
2135-2146). This membrane has a molecular weight cutoff greater than 1,000,000
Da, which
permits the free passage of proteins and nutrients between tlhe capsule
interior and exterior,
while preventing the contact of transplanted cells with host cells. The
entrapped cells may be
implanted by intradermal, subdermal, intravenous, intramuscular, intranasal,
intracerebral,
intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural,
intracoronary or
intratumoral ways.
In a further embodiment, the invention concerns the use of at least one
myoblast cell
generated, and eventually modified, as described above for the preparation of
a composition
for administration into a human tissue. In a preferred embodiment the prepared
composition in
accordance with the use claimed in the present invention is in a form for
administration into a
vertebrate tissue. These tissues include those of muscle, skin, nose, lung,
liver, spleen, bone
marrow, thymus, heart, lymph, bone, cartilage, pancreas, kidney, gall bladder,
stomach,
intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,
connective tissue, blood,
tumor etc. The administration may be made by intradermal, subdermal,
intravenous,
intramuscular, intranasal, intracerebral, intratracheal, intraarterial,
intraperitoneal, intravesical,
intrapleural, intracoronary or intratumoral injection, with a syringe or other
devices.
Moreover, myoblast cells are found to migrate from the original site of
administration to other
sites, particularly injured sites, e.g. degenerating foci. This migration
phenomenom permits
the treatment of injured sites by injecting myoblasts into the patient in
need, particularly in
tissue, usually muscle tissue, proximal to the injuries, although injection
into the circulation or
at a distal site may also be possible. By employing genetically engineered
myoblasts one may
provide for directed application of products of interest to the injured
region. Usually, cell
injection will be about 104 to 107 cells (modified or not) per cm3 of muscle
tissue to be treated.
In this particular case, the composition according to the invention may also
comprise a
phrmaceutically acceptable injectable carrier. The carrier is preferably
isotonic, hypotonic or
weakly hypertonic and has a relatively low ionic strength, such as provided by
a sucrose
13
CA 02357403 2001-09-14
solution. It includes any relevant solvent, aqueous or partly aqueous liquid
carrier comprising
sterile, pyrogen-free water, dispersion media, coatings, and/or equivalents.
The pH of the
pharmaceutical preparation is suitably adjusted and buffered.
In a further aspect, the invention relates to a diagnostic kit comprising at
least one myoblast
cell generated according to the invention useful for in vitro assessment of
muscular cellular
toxicity or damages of candidate or commercially available pharmaceutical
molecules (pre-
clinical assays) or for in vitro screening of new drugs. In course of said
applications, cell lines
generated from Duchenne Muscular Dystrophy patient would be preferred. The
cultured
myoblasts may also serve as a tool to analyse physiopathology of muscular
diseases.
Myoblasts prepared using the methods of the present invention can be used for
delivery of a
muscle protein to the circulation of a mammal. A muscle protein, as used
herein, refers to a
protein which, when defective or absent in a mammal, is responsible for a
particular muscle
disease or disorder. Muscle proteins include dystrophin ,calpain-3,
sarcoglycan complex
members (e. g., a-sarcoglycan, P-sarcoglycan, y-sarcoglycan and 5-sarcoglycan)
and laminin
0 : 2- chain. The term circulation is meant to refer to blood circulation. The
term blood refers
to the "circulating tissue" of the body, the fluid and its suspended formed
elements that are
circulated through the heart, arteries, capillaries and veins.
In the method for delivery of a muscle protein to the circulation of a mammal,
an effective
amount of purified donor myoblasts is transplanted into a mammal in need of
such treatment
(also referred to as a recipient or a recipient mammal). As used herein,
"donor" refers to a
mammal that is the natural source of the stem cells that are transformed using
the viral vectors
of the present invention into myoblasts. Preferably, the donor is a healthy
mammal (e. g., a
mammal that is not suffering from a muscle disease or disorder). In a
particular embodiment,
the donor and recipient are matched for immunocompatibility.
Preferably, the donor and the recipient are matched for their compatibility
for the major
histocompatibility complex (MHC) (human leukocyte antigen (HLA))-class I (e.
g., loci A, B,
C) and-class II (e. g., loci DR, DQ, DRW) antigens.
Immunocompatibility between donor and recipient are determined according to
methods
generally known in the art (see, e. g., Charron, D. J., Curr. Opin. Hematol.,
3: 416-422 (1996);
Goldman, J., Curr. Opin. Hematol., 5: 417-418 (1998); and Boisjoly, H. M. et
al.,
14
CA 02357403 2001-09-14
Opthalmology, 93: 1290-1297 (1986)). In an embodiment of particular interest,
the recipient a
human patient.
As used herein, muscle diseases and disorders include, but are not limited to,
recessive or
inherited myopathies, such as, but not limited to, muscular dystrophies.
Muscular dystrophies are genetic diseases characterized by progressive
weakness and
degeneration of the skeletal or voluntary muscles which control movement. The
muscles of
the heart and some other involuntary muscles are also affected in some forms
of muscular
dystrophy. The histologic picture shows variation in fiber size, muscle cell
necrosis and
regeneration, and often proliferation of connective and adipose tissue.
Muscular dystrophies
are described in the art and include Duchenne muscular dystrophy (DMD), Becker
muscular
dystrophy (BMD), myotonic dystrophy (also known as Steinert's disease), limb-
girdle
muscular dystrophies, facioscapulohumeral muscular dystrophy (FSH), congenital
muscular
dystrophies, oculopharyngeal muscular dystrophy (OPMD), distal muscular
dystrophies and
Emery-Dreifuss muscular dystrophy. See, e. g., Hoffman et al., N. Engl. J.
Med., 318. 1363-
1368 (1988); Bonnemann, C. G. et al., Curr. Opin. Ped., 8: 569-582 (1996);
Worton, R.,
Science, 270: 755-756 (1995); Funakoshi, M. et al., Neuromuscul. Disord., 9
(2): 108-114
(1999); Lim, L. E. and Campbell, K. P., Cure. Opin. Neurol., 11 (5): 443-452
(1998); Voit, T.,
Brain Dev., 20 (2): 65-74 (1998); Brown, R. H., Annu. Rev. Med., 48: 457-466
(1997);
Fisher, J. and Upadhyaya, M., Neuromuscul. Disord., 7 (1): 55-62 (1997).
Two major types of muscular dystrophy, DMD and BMD, are allelic, lethal
degenerative
muscle diseases. DMD results from mutations in the dystrophin gene on the X-
chromosome
(Hoffman et al., N. Engl. J. Med., 318. 1363-1368 (1988)), which usually
result in the absence
of dystrophin, a cytoskeletal protein in skeletal and cardiac muscle. BMD is
the result of
mutations in the same gene (Hoffman et al., N. Engl. J. Med., 318 : 1363-1368
(1988)), but
dystrophin is usually expressed in muscle but at a reduced level and/or as a
shorter, internally
deleted form, resulting in a milder phenotype.
Thus, the present invention also provides a method of treating a muscle
disease or disorder in
a mammal in need thereof comprising administering an effective amount of
myoblasts to the
mammal. In a particular embodiment, the invention relates to a method of
treating a muscular
dystrophy in a mammal in need thereof comprising administering an effective
amount of
myoblasts to the mammal. In another embodiment, the invention relates to a
method of
treating DMD in a mammal in need thereof comprising administering an effective
amount of
"'-~~ "-'~-' w's~r~-sec, ,~aa.es .~.rc. . . .. sws .---..--..---- . ~..~..-a-
..-.
CA 02357403 2001-09-14
myoblasts to the mammal. In a third embodiment, the invention relates to a
method of treating
BMD in a mammal in need thereof comprising administering an effective amount
of
myoblasts to the mammal. In the latter two embodiments, a proportion of the
administered
myoblasts can fuse with DMD or BMD host muscle fibres, contributing dystrophin-
competent
myonuclei to the host fibres (mosaic fibres). The expression of normal
dystrophin genes in
such fibres can generate sufficient dystrophin in some segments to confer a
normal phenotype
to these muscle fibre segments.
The invention also relates to a method of treating a limb-girdle muscular
dystrophy in a
mammal in need thereof comprising administering an effective amount of
myoblasts to the
mammal.
Myoblasts prepared in accordance with the methods of the present invention can
also be used
in gene therapy, a utility enhanced by the ability of the myoblasts to
proliferate and fuse.
Myoblasts can be genetically altered by one of several means known in the art
to comprise
functional genes which may be defective or lacking in a mammal requiring such
therapy. The
recombinant myoblasts can then be transferred to a mammal, wherein they will
multiply and
fuse and, additionally, express recombinant genes. Using this technique, a
missing or
defective gene in a mammal's muscular system can be supplemented or replaced
by infusion
of genetically altered myoblasts. Gene therapy using myoblasts can also be
applied in
providing essential gene products through secretion from muscle tissue to the
bloodstream
(circulation). Because myoblasts are capable of contributing progeny
comprising recombinant
genes to multiple, multinucleated myofibres during the course of normal
muscular
development.
To gain a better understanding of the invention described herein, the
following examples are
set forth. It should be understood that these examples are for illustrative
purposes only.
Therefore, they should not limit the scope of this invention in any way.
16
CA 02357403 2001-09-14
EXAMPLES
Materials and Methods
Molecular cloning of Pax7 and expression analysis
RDA was performed as described by Hubank and Schatz, 1994. Satellite cell
derived
myoblast cDNA was subtracted twice against mouse embryonic fibroblast (MEF)
cDNA
(1:100; 1:400) and once against skeletal muscle cDNA (1:400) to generate the
final difference
products. The full-length mouse cDNA for Pax7 was isolated by screening an
adult mouse
skeletal muscle library (Clontech) using the RDA clone as a probe (Maniatis et
al., 1982).
Total RNA was extracted as previously described (Chomczynski and Sacchi,
1987). Northern
Analysis of 20 p,g of total RNA from tissue or cell cultures was performed as
per Maniatis et
al., 1982. In situ hybridisation for Pax7 mRNA was performed as described
elsewhere
(Braissant and Wahli, 1998). Sections were counter-stained with 100 p,g/mL
Propidium Iodide
(Sigma) in PBS for 10 minutes at room temperature. Three different Pax7
sequences from the
full-length cDNA were used as cRNA probes: Pax7-Sall: nts 150-1600; dpi-7 nts
4200-4700;
Pax7-Clal: nts 515-1500.
Myoblast and Stem Cell Culture
Primary muscle cultures were isolated as per Sabourin et al., 1999 . Primary
MEFs were
isolated from 13.5-day-old Balb/c mouse embryos (Robertson, 1987). Single
muscle fibers
were isolated from hind limb skeletal muscles as described previously
(Cornelison and Wold,
1997). Individual fibers were cultured in methocult GF M3434 containing 15%
FBS, 1%
BSA, 10-4M 2-Mercaptoethanol, 10 p,g/mL pancreatic insulin, 200 p,g/mL
Transferrin, 50
nglmL SCF, 10 ng/mL IL-3, 10 ng/mL IL-6 and 3 units/mL EPO (Stem Cell
Technologies)
for 48 hr-10 days.
For hematopoietic colony forming assays, cell suspensions were derived from
skeletal muscle
by digestion in 0.4% collagenase Type A (Roche)/DMEM for 1.5 hr at
37°C, filtered (74 pm
Costar Netwell) and resuspended at 100 cells/pl in 10% horse serum/DMEM.
10,000 cells
were cultured in 3 mL of methocult (Stem Cell Technologies) for 14 days.
17
CA 02357403 2001-09-14
Fluorescence Activated Cell Sorting (FACS~
Hoechst staining and FACS analysis was essentially performed as described
previously
(Goodell et al., 1996). FACS was performed on a Becton-Dickinson FacStar flow
cytometer
equipped with dual lasers. Hoechst dye was excited at 350 nm and its
fluorescence was
measured at two wavelengths using a 424BP44 filter (Blue emission) and a 650LP
filter (Red
emission). A 640 DMSP mirror was used to separate wavelengths.
Immunocytochemistry and Electron Microscopy
Primary cell cultures or colonies picked from methocult medium were fixed and
stained as
described elsewhere (Sabourin et al., 1999) using anti-c-Met SP260 (Santa
Cruz); anti-desmin
DE-U-10 (DAKO), anti-mouse Ly-6G (clone RB6-8C5) (Pharmingen); anti-mouse
Integrin
aM (M1/70) (Pharmingen) and MF20 mAb (anti-Myosin Heavy Chain).
Gastrocnemius muscle was prepared for transmission electron microscopy by
overnight
fixation at 4°C in 2% gluteraldehyde/0.1 M Cacodylate (pH 7.4) and
processed using standard
procedures as described elsewhere (Kablar, 1995). Randomly chosen fields were
viewed with
a Jeol 1200EX Biosystem TEM. Diaphragm and tibialis anterior muscles were
prepared for
HE staining as described elsewhere (Bancroft and Stevens, 1.990).
EXAMPLE I: Identification of Genes Expressed in Satellite Cell Derived
Myoblasts
Muscle satellite cells represent a distinct lineage of myogenic progenitors
responsible for the
postnatal growth, repair and maintenance of skeletal muscle (reviewed by Seale
and Rudnicki,
2000). At birth, satellite cells account for about 30% of sublarninar muscle
nuclei in mice
followed by a decrease to less than 5% in a 2 month old adult (Bischoff,
1994). This decline
in satellite cell nuclei reflects the fusion of satellite cells during the
postnatal growth of
skeletal muscle (Gibson and Schultz, 1983). Satellite cells were originally
defined on the basis
of their unique position in mature skeletal muscle and are closely juxtaposed
to the surface of
myofibers such that the basal lamina surrounding the satellite cell and its
associated myofiber
is continuous (Bischoff, 1994).
In mice over 2 months of age, satellite cells in resting skeletal muscle are
mitotically
quiescent and are activated in response to diverse stimuli including
stretching, exercise,
injury, and electrical stimulation (Appell et al., 1988; Rosenblatt et al.,
1994; Schultz et al.,
18
CA 02357403 2001-09-14
1985; reviewed by Bischoff, 1994). The descendents of activated satellite
cells, called
myogenic precursor cells (mpc), undergo multiple rounds of cell division prior
to fusion with
new or existing myofibers. The total number of quiescent satellite cells in
adult muscle
remains constant over repeated cycles of degeneration and regeneration,
suggesting that the
steady state satellite cell population is maintained by self-renewal (Gibson
and Schultz, 1983;
Schultz and Jaryszak, 1985; Morlet et al., 1989). Therefore, satellite cells
have been suggested
to form a population of monopotential stem cells that are distinct from their
daughter
myogenic precursor cells as defined by biological and biochemical criteria
(Bischoff, 1994;
Grounds and Yablonka-Reuveni, 1993).
Satellite cells clearly represent the progenitors of the myogenic cells that
give rise to the
majority of the nuclei within adult skeletal muscle. However recent studies
have identified a
population of pluripotential stem cells, also called side-population (SP)
cells in adult skeletal
muscle. Muscle-derived SP cells are readily isolated by fluorescence activated
cell sorting
(FACS) on the basis of Hoechst dye exclusion (Gussoni et al., 1999; Jackson et
al., 1999).
Purified SP cells derived from muscle exhibit the capacity to differentiate
into all major blood
lineages following tail vein injection into lethally irradiated mice (Jackson
et al., 1999). Of
particular significance is the observation that transplanted SP cells isolated
from bone marrow
or muscle actively participate in myogenic regeneration. However only muscle-
derived SP
cells appear to give rise to myogenic satellite cells (Gussoni et al., 1999).
In addition, SP cells
convert to desmin-expressing myoblasts following exposure to appropriate cell
culture
conditions (Gussoni et al., 1999). However, whether SP cells are equivalent to
satellite cells,
are progenitors for satellite cells or alternatively represent an entirely
independent cell
population has remained unclear.
The gene expression profile of quiescent satellite cells and their activated
progeny is largely
unknown. Quiescent satellite cells express the c-met receptor (receptor for
HGF) and M-
cadherin protein (Cornelison and Wold, 1997; Irintchev et al.., 1994).
Activated satellite cells
up regulate MyoD orMyfS prior to entering S-phase (Cornelison and Wold, 1997).
Proliferating myogenic precursor cells, the daughter cells of satellite cells,
express desmin,
MyfS, MyoD and other myoblast specific markers (Cornelison and Wold, 1997;
George-
Weinstein et al., 1993). Nevertheless, the paucity of cell-lineage specific
markers has been a
significant impediment to understanding the relationship between satellite
cells and their
progeny.
19
CA 02357403 2001-09-14
Based on our poor understanding of molecular events responsible for satellite
cell
development and activation, a PCR based subtractive hybridisation approach
(Hubank and
Schatz, 1994) was used to identify tissue-specific genes expressed in the
satellite cell
myogenic lineage. Results from this analysis identified several myoblast-
specific genes
potentially involved in satellite cell function. Pax7 was selected for further
analysis based on
the established role of the closely related Pax3 protein in regulating the
developmental
program of embryonic myoblasts (Tajbakhsh et al., 1997; Maroto et al., 1997).
To gain insight into the developmental program responsible for the
differentiation and
activation of skeletal muscle satellite cells, representational difference
analysis of cDNAs
(RDA) (Hubank and Schatz, 1994) was employed to identify genes expressed
specifically in
satellite cell derived myoblasts. This analysis resulted in the identification
of 17 distinct
products corresponding to 12 known and 5 potentially novel genes by searching
GenBank
(NCBI) using the FASTA program (unpublished). RDA clone dpi-7 encoded a
fragment from
within the Pax7 mRNA. Pax7 is a member of the paired-box family of
transcription factors
that play important regulatory roles in the development of diverse cell
lineages (Mansouri,
1999). Therefore, a full-length 4.3-kb Pax7 cDNA was isolated from an adult
mouse skeletal
muscle cDNA library (Clontech) to facilitate further analyses (NCBI Accession
Number:
AF254422).
EXAMPLE II: Pax7 is Specifically Expressed in Proliferating Myoblasts
Detailed expression analysis of the distribution of Pax7 mRNA was conducted by
Northern
analysis (Figure 1). These analyses demonstrated that Pax7 was expressed
exclusively in
proliferating primary myoblasts, with comparable levels of expression in both
wild type and
MyoD ~ cultures (Figure 1A). However, Pax7 mRNA was down regulated following
myogenic differentiation (Figure 1A). Furthermore, Pax7 was not expressed at
detectable
levels in a variety of non-muscle cell lines (Figure 1B). Rather, Pax7was
strictly expressed in
myogenic cells including low levels in proliferating C2C12 mouse myoblasts,
which are a
continuous cell line originally derived from satellite cells (Figure 1B). In
addition, Pax7
mRNA was not detectable in 20 p.g of total RNA from several adult mouse tissue
samples
(Figure 1C). Analysis of polyA+ RNA from select mouse tissues revealed
expression of Pax7
at low levels only in adult skeletal muscle (not shown). Therefore, in adult
mice Pax7
expression appears specific to the satellite cell myogenic lineage.
CA 02357403 2001-09-14
EXAMPLE III: Pax7 is Expressed in Satellite Cells
To localise Pax7 mRNA in skeletal muscle in situ hybridisation was performed
on fresh
frozen sections of tibialis anterior and gastrocnemius muscles from wild type
(Balb/c), MyoD-
mdx and compound mutant mdxMyoD ~ animals. Interestingly, Pax7 mRNA was
associated with a subset of nuclei in discrete peripheral locations within
undamaged wild type
(wt) (Figure 2A,C) and MyoD-~ (not shown) skeletal muscle. Propidium-Iodide
(PI) staining
was used to identify all nuclei within skeletal muscle thereby allowing for
the enumeration of
Pax7 positive cells (Figure 2B,D,F). The in situ hybridization was repeated on
muscle
sections from three independent mice using three separate sequences as anti-
sense cRNA
probes to verify the expression patterns described. Approximately 5% of muscle
nuclei
(including satellite cell nuclei and myonuclei) were associated with Pax7
expression in adult
wild type muscle. By contrast, the number of Pax7 positive cells increased to
22% in MyoD
muscle. The increased expression of Pax7 in MyoD ~ muscle strongly supports
the notion that
Pax7 is expressed in satellite cells as previous work has revealed that MyoD-
deficient muscle
contains increased numbers of satellite cells (Megeney et al., 1996). At high
magnification
(200X), Pax7 appeared to be expressed in cells residing beneath the basal
lamina of wild type
muscle fibers in positions characteristic for quiescent satellite cells
(Figure 2C).
To determine whether Pax7 was up regulated in regenerating skeletal muscle, 3-
week-old mdx
and compound mutant mdxMyoDW skeletal muscle was analyzed by in situ
hybridization.
Due to lack of dystrophin protein, mdx muscle undergoes repeated cycles of
muscle
degeneration and regeneration (Sicinski et al., 1989). As predicted, based on
high levels of
expression in cultured satellite cell derived myoblasts, Pax7 was widely
expressed in
regenerating areas of mdx and md~MyoD-~ skeletal muscle (Figure 2E). Centrally
located
nuclei within muscle fibers of mdx (Figure 2E), MyoD-~ (not shown) and mdxMyoD
~ (not
shown) muscle were also associated with Pax7 expression, suggesting that
recently activated
and fusing myogenic precursors express Pax7. Lastly, a similar distribution of
immunoreactive nuclei was observed in muscle sections stained with anti-Pax7
antibody
(Developmental Studies Hybridoma Bank). Taken together, the expression
analysis supports
the notion that Pax7 is expressed within the satellite cell lineage.
Therefore, these results raise
the hypothesis that Pax7 is required for the ontogeny or function of muscle
satellite cells.
21
CA 02357403 2001-09-14
EXAMPLE IV: Skeletal Muscle Deficiency in Pax7 Mutant Animals
To evaluate possible roles for Pax7 in the formation or function of satellite
cells, we examined
skeletal muscle from mice carrying a targeted null mutation in Pax7 (Mansouri
et al., 1996).
Mice deficient for Pax7 express muscle-specific markers including MyoD and
MyfS in a
normal spatial and temporal pattern within the developing myotome (Mansouri et
al., 1996).
However, Pax7 ~ mice were significantly smaller than their wild type and
heterozygous
counterparts (Figure 3A). The body weight of Pax7~ mice at 7 days of age was
50% reduced
in comparison to wild type littermates (N=20). This weight differential
increased with age
such that at two weeks of age, mutant animals were about 33% the weight of
wild type
littermates. As previously reported, Pax7 mutant animals failed to thrive and
usually died
within two weeks after birth (Mansouri et al., 1996). In addition, we observed
that mutant
mice exhibited muscle weakness characterized by an abnormal gait and splayed
hind limbs
(not shown). Light microscopic analysis of hematoxylin-eosin (HE) stained
lower hind limb
skeletal muscle (below the knee) of one-week-old wild type (Figure 3B) and
Pax7 ~ (Figure
3C) animals revealed a 1.5-fold reduced diameter of Pax7 mutant fibres (N=100
fibres).
However, the overall organisation of muscle fibres was not affected. Moreover,
the diaphragm
from 7-day-old Pax7 ~ mice (Figure 3E) was notably thinner than that from
their wild type
littermates (Figure 3D). Therefore, the markedly decreased muscle mass and
reduced fibre
calibre of Pax7 mutant muscle suggested that the postnatal growth phase of
skeletal muscle
normally mediated by satellite cells was deficient in the absence of Pax7.
EXAMPLE V: Absence of Satellite Cell Derived Myoblasts from Pax?~ Muscle
To gain insight into satellite cell function in Pax7 mutant mice, primary
cells were cultured
directly from the muscle of 7-10 day old wild type mice and Pax7~ littermates
in five
independent experiments. After two days in culture, many bursts of satellite
cell derived
myoblasts were readily identified in wild type primary cultures based on
morphological
criteria (Figure 4A) and immunocytochemistry using both anti-desmin and anti-c-
Met
antibodies that mark satellite cell derived myoblasts (Figure 4B-E).
Strikingly, no myoblasts
were identified in mutant cultures, which instead were uniformly composed of
fibroblasts and
adipocytes as identified by morphological, and immunochemical criteria (Figure
4F-J).
To further investigate whether myogenic cells were present in postnatal Pax7
mutant muscle,
individual muscle fibres from 7-10 day old wild type mice and Pax7 ~
littermates were
isolated in five independent experiments and cultured in methylcellulose stem-
cell medium.
22
CA 02357403 2001-09-14
Methylcellulose stem-cell medium readily promotes the activation, migration
and
proliferation of satellite cells associated with muscle fibres (Atsushi
Asakura and Michael A.
Rudnicki, unpublished observation). After 48 and 72 hours in culture,
satellite cells associated
with wild type fibres generated distinct bursts of desmin-expressing myogenic
cells. By
contrast, Pax7 mutant muscle fibres did not give rise to any mononuclear
cells. Following two
weeks in culture, large colonies of fully contractile myosin heavy chain (MHC)
expressing
myotubes were present in cultures of wild type but not Pax7~~ fibres (not
shown). Therefore,
these results suggest that satellite cells do not exist, or alternatively fail
to proliferate in the
absence of Pax7.
EXAMPLE VI: Complete Ablation of Satellite Cells in PaxT' Muscle
To determine whether or not satellite cells were present in mutant animals,
transmission
electron microscopy (TEM) was used to analyse skeletal muscle from wild type
and Pax7
mice. Biopsies from gastrocnemius muscle of three 7-10 day old wild type mice
and mutant
littermates were analysed by TEM. For each sample, 100 peripheral sublaminar
nuclei were
analyzed and identified as either satellite cell or myofiber nuclei. Criteria
for the identification
of satellite cells consisted of: a plasma membrane separating the satellite
cell from its adjacent
muscle fibre, an overlying basal lamina continuous with the satellite cell and
associated fibre,
and the characteristic heterochromatic appearance of the nucleus (reviewed in
Bischoff, 1994).
Satellite cells were readily identified in wild type muscle and comprised 25%
of peripheral
sublaminar nuclei (N=300) (Figure 5A-D). By contrast, satellite cells could
not be identified
in over 300 sublaminar nuclei examined from mutant muscles (Figure SE,F).
Furthermore,
satellite cells were not found in muscle from E18 embryos (18 days post-
coitum) (not shown).
Therefore, in the absence of Pax7, complete ablation of muscle satellite cells
was observed.
The failure of muscle satellite cells to form in Pax7 ~ muscle thus
unequivocally establishes
an essential role for Pax7 in the ontogeny of the satellite cell lineage.
EXAMPLE VII: Muscle-Derived SP Cells are Present in Pax7 Mutant Muscle
To investigate the relationship between satellite cells and muscle-derived
pluripotent stem
cells, fluorescence activated cell sorting (FACS) analysis of cells isolated
from wild type and
Pax7 ~ muscle was performed. Recent work has identified a population of
pluripotent stem
cells (also called side-population (SP) cells) in skeletal muscle as defined
by Hoechst 33342
23
CA 02357403 2001-09-14
dye exclusion (Gussoni et al., 1999; Jackson et al., 1999). Cell suspensions
isolated directly
from one-week-old skeletal muscle were stained with Hoechst dye in the
presence or absence
of verapamil. The SP cell population is sensitive to verapamil, which is
thought to prevent dye
efflux through the inhibition of mdr (mufti-drug resistant)-like proteins
(Goodell et al., 1996;
Goodell et al., 1997). Based on results from three independent trials with six
7-10 day old
Pax7 ~ and wild type animals, the proportion of muscle SP cells was unaffected
by the
absence of Pax7 (Figure 6A-D). The relative proportion of SP cells in wild
type (1.8%)
(Figure 6A) versus Pax7 mutant muscle (1.5%) (Figure 6C) did not differ
significantly. Taken
together, these data indicate that muscle satellite cells are either a
population distinct from
muscle SP cells, or alternatively represent only a small subpopulation of
muscle SP cells.
EXAMPLE VIII: Stem Cells Derived From PaxT~' Exhibit Markedly Increased
I~ematopoietic Potential
To characterise the differentiation potential of Pax7 deficient stem cells,
dissociated muscle
cells from 7-10 day old Pax7 ~ and wild type animals were assayed for colony
formation in
methylcellulose stem cell medium, which allows the growth of muscle as well as
hematopoietic colonies (Atsushi Asakura and Michael A. Rudnicki, unpublished).
Seven
independent experiments were analysed in which 10,000 cells from both wild
type and Pax~
muscle were cultured. Hematopoietic colonies included granulocytic and
monocytic cells
and were present in both wild type and mutant cultures based on
immunoreactivity with Ly-
6G (Figure 6G,H) and Integrin aM chain (not shown). Ly-6G is a cell surface
antigen, which is
expressed exclusively in granulocyte and monocyte lineages (Fleming et al.,
1993). Integrin
aM chain, also known as MAC-1 is expressed on granulocytes, macrophages and
Natural
Killer Cells (Leenen et al., 1994). ~%Vild type cultures were predominantly
composed of
contractile muscle colonies reactive with antibody to Myosin Heavy Chain
(Figure 6E). By
contrast, Pax~~ cultures exhibited a markedly increased potential for
hematopoietic
differentiation (Figure 6F) and generated about 10 times the number of
hematopoietic
colonies as compared to wild type cultures (Figure 6I). To rule out the
possibility that the
presence of differentiating muscle cells was inhibiting hematopoietic
differentiation in wild
type cultures, mixed cultures of Pax7~ and wild type cells were analysed (not
shown).
Results from these experiments showed that hematopoietic colony formation was
not
adversely affected by differentiating myocytes.
The colony forming assays summarised in Figure 6I depict the average number of
hematopoietic, skeletal myocyte and other (e.g. fibroblast, adipocyte)
colonies from 7
24
CA 02357403 2001-09-14
independent isolations performed in triplicate. Therefore, stem cells isolated
from muscle
lacking Pax7 exhibited a strongly increased propensity towards hematopoietic
differentiation
and were incapable of forming adult myoblasts. Importantly, highly purified SP
cells from
wild type muscle convert to myoblasts under the appropriate culture conditions
(Gussoni et
S al., 1999). Taken together, these results suggest the hypothesis that
induction of Pax7 in
pluripotent muscle-derived stem cells directs the specification of satellite
cells through
restriction of developmental potential (Figure 7).
EXAMPLE IX: Generation of Recombinant Adenovirus-Pax7
In order to demonstrate the ability of Pax? to induce myogenic specification
of muscle-
derived stem cells, exogenous Pax? was expressed in fractionated SP cells and
muscle-derived
cells using recombinant Adenovirus vectors. Adenovirus was selected as the
vector for gene
delivery in this application due to its transient high level expression in
replicating cells (i.e.
does not integrate into host cell genome), its ability to infect a wide range
of cell types
including quiescent cells arid its potential to be grown to high titres,
required for in vivo
applications. For these experiments, the full-length coding sequence for Pax7
was cloned
downstream of the murine CMV promoter in the adenoviral shuttle vector, pDC516
(Microbix) using EcoRl and Sall restriction sites (Figure 9). Recombinant,
replication-
defective adenovirus type 5 (El deficient) was generated by co-transfection of
pDC516-Pax?
and the plasmid containing the adenoviral genome, pBHG~E 1 into permissive 293
cells (Ng
et al., 1999). Recombinant Ad-Pax7 viral plaques were picked and expanded by
serial
passages in 293 cells, which permits the growth and reproduction of virus. The
structure of
recombinant Ad-Pax7 virus was verified by restriction digest analysis. To
confirm that Pax?
protein was appropriately expressed from the adenovirus, Aa'-Pax7 and Ad-empty
(i.e. no
transgene) were used to infect C2C12 myoblasts as well as lOT'~Zfibroblasts.
Adherent cells
were infected with crude viral preparations for 1 hour at room temperature.
Expression of
Pax7 in infected cells was assessed 1-day post infection by western blot
analysis of cell
lysates using an antibody reactive to Pax? (Developmental Studies Hybridoma
Bank) (Figure
10). The results of western analysis indicate that Pax7 is expressed at
relatively high levels in
infected cells. High-titre viral stocks (~10'zpfu/ml) were subsequently
prepared and purified
using cesium chloride gradients and dialysis against tissue-culture grade PBS.
CA 02357403 2001-09-14
EXAMPLE X: Isolation and Infection of SP cells
Fluorescence activated cell sorting (FACS) was used to isolate SP cells from
skeletal muscle
of 2 month old wild type mice. Hind limb muscles were dissected from bones and
connective
tissues and subsequently digested with 3% collagenase B (Roche)/2.4 U/ml
dispase II (Roche)
to disperse mononuclear cells. Cells were separated from undigested tissue,
fibers and debris
by filtration through 74 pxu nytex filters (Costar). Suspensions were spun
down and
resuspended in muscle stem cell medium (Ham's F-10 nutrient mixture (Life
Technologies);
20% FCS; 5% chicken embryo extract (Life Technologies)) and plated on plastic
10 cm
tissue-culture dishes overnight (10-14 hours). The following day, adherent
cells were
collected by trypsinization and combined with suspension cells (i.e. non-
adherent), spun-
down and suspended in 2% FCSIDMEM at a concentration of 2 x 106 cells/ml.
Hoechst
33342 staining was carried out as previously described (Goodell et al., 1996).
Specifically,
Hoechst 33342 (Sigma) was added to cell suspensions to a final concentration
of 5 p.g/ml with
or without the addition of 50 pM verapamil (Sigma) and incubated for 90 min.
at 37°C.
Following Hoechst staining, cells were spun and suspended at 2 million
cells/ml in Hank's
balanced salt solution (Life Technologies) supplemented with 2% FCS and 2
p.g/ml
Propidium Iodide (Sigma). FAGS analysis was subsequently carried out on a
Becton-
Dickinson FACStar-Plus equipped with dual lasers. The SP fraction was
visualised as a well-
defined, distinct cell population, which stains weakly with Hoechst dye (in
far red >670 nrn
and blue 450 nm) due to the active efflux of dye by multi-drug resistance
(mdr)-type proteins
on the surface of SP cells. In order to confirm the presence of the SP and
establish appropriate
sorting gates, verapamil was used to inhibit rndr-protein activity, resulting
in loss of SP cells
(i.e. cells from the SP fraction shifted into the main population (MP)). 1 x
104 purified SP
cells were sorted from a starting population of approximately 5 x 106 muscle-
derived cells.
Purified SP cells were spun down at 1000 rpm and resuspended in 50 p1 of PBS,
divided into
2 tubes (5000 cells/tube) for immediate infection with 2.5 x 105 viral
particles (multiplicity of
infection = 50) of Ad-Pax7 or Ad-empty (no transgene) . SP cells were
maintained in
suspension at 37°C/5% C02 during 1 hour infection. After infection, 1
ml of myoblast growth
medium consisting of Ham's F-10 Nutrient mixture (Life Technologies)
supplemented with
20% FCS and 2.5 ng/ml bFGF (R&D systems) was added to cultures. Infected SP
cells were
plated in wells of 12 well dishes previously coated with 0.1% rat-tail
collagen (Roche) and
thereafter maintained in myoblast growth medium for 7 additional days with
medium
exchanged every two days.
26
CA 02357403 2001-09-14
To assess the myogenic conversion of SP cells, immunohistochemistry with
antibody reactive
to the muscle specific intermediate filament protein, desmin was performed.
Importantly the
SP fraction of cells from muscle does not contain satellite cells or desmin
positive myoblasts
(A. Asakura, unpublished data). For staining, infected SP cultures were fixed
with 4%
paraformaldehyde and permeabilised with 0.3% Triton-X100. Anti-desmin antibody
(Clone
D33; Dako) was used at a dilution of 11200 and detected using fluorescein
conjugated anti-
rnouse IgG (Chemicon). Significantly, desmin expression was observed in cell
cultures
infected with Ad-Pax7 (Figure 11). By contrast, no desmin reactive cells were
observed in
cells of cultures infected with Ad-empty. These results indicate that some
proportion of
muscle-derived SP cells have the capacity to undergo myogenic conversion
following
exposure to exogenous Pax7.
EXAMPLE XI: Isolation and Infection of MyfSnlacZ Muscle Cells
Mononuclear cells were obtained from the hind limb skeletal muscle of 2 month
old
MyfSnlacz mice as described above. The LacZ gene is expressed under the
control of the MyfS
locus in these mice. Expression of Myf5nlacZ is observed in. cells, which are
committed to the
muscle lineage thus providing a useful lineage marker for myogenic cells
(Tajbakhsh et al.,
1996). MyfSnlacZ is not expressed in muscle-derived SP cells (A. Asakura,
unpublished
data) however satellite cells and myogenic precursor cells in adult muscle
express this
transgene (Tajbakhsh et al., 1996). Following isolation, MyfShlacZ muscle
derived cells
were suspended in muscle stem cell medium composed of Ham's F-10 nutrient
mixture (Life
Technologies) supplemented with 20% FCS; 5% chicken embryo extract (Life
Technologies);
antibiotics and fungizone and plated onto plastic tissue culture dishes. The
muscle cultures
were grown for 5 days under these conditions with the medium exchanged after 1
and 3 days.
These culture conditions have been used previously to grow muscle cells with
bone-marrow
repopulating activity (Jackson et al., 1999). Furthermore, satellite cells and
myoblasts do not
adhere to plastic and fail to thrive under these conditions (unpublished
observations). These
muscle-derived cell cultures were subsequently infected with Ad Pax7 and Ad-
empty at a
multiplicity of infection of 50. Specifically, 1 x 105 cells were infected
with 5x106 viral
particles of either Ad Pax7 or Ad-empty. Adherent cells on 60 mm tissue
culture plates were
infected with 1 mL of PBS/virus for 1 hour at 37°-C/5% C02. Following
infection, 5 mL of
myoblast growth medium was added to cultures. Cultures were maintained in
myoblast
growth medium for an additional 7 days. To assess expression of MyfnlacZ in Ad
Pax7 and
Ad empty infected cultures, cells were fixed with 4% paraformaldehyde for X-
Gal staining as
27
CA 02357403 2001-09-14
described previously (Asakura et al., 1995). Interestingly, a large number of
cells infected
with Ad-Pax7 up regulated expression of MyfSnlacZ (Figure 12). By contrast,
MyfnlacZ
expressing cells were rarely observed in Ad-empty infected cultures likely a
result of
contaminating myoblasts. These results suggest that Pax7 expression is
sufficient to induce a
S subset of competent stem cells to enter into the myogenic differentiation
program.
The invention being thus described, it will be obvious that the same may be
varied in many
ways. Such variations are not to be regarded as a departure from the spirit
and scope of the
invention, and all such modifications as would be obvious to one skilled in
the art are
intended to be included within the scope of the following claims.
28
CA 02357403 2001-09-14
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