..:: duchennemd ::..

Stem cells
Muscle regeneration with differentiating embryonic
stem cells. During the development of an embryo, the precursors
of skeletal muscles appear very early as somites,
mesodermal structures on both sides of the embryonic neural
tube. Under the influence of transcription factors (proteins
that control gene activity – in particular Pax3), the
cells of the somites differentiate (give rise to more specialized
cells) to form, among other structures, the myotome
which develops further to myoblasts, myotubes and finally
muscle fibers. If it were possible to prepare from non-dystrophic
somites those cells which are destined to become
the myotome, they could be multiplied and then used to
regenerate dystrophic muscle cells, because they would
bring with them the intact dystrophin gene.
Prof. Rita Perlingeiro and her team at the University of
Texas Southwestern Medical Center in Dallas were trying
to isolate such early cells somite cells from mouse embryonic
stem cells in cell cultures. They found that in order to
obtain among them the myogenic (muscle forming) cells,
the differentiating embryonic stem cells needed the transcription
factor Pax3, whose gene they could introduce into
the X-chromosome of the stem cells by genetic techniques.
Using flow cytometry (a cell sorting method), the researchers
could isolate myogenic cells from these Pax3-
induced embryonic stem cells that had differentiated for
five days. Cells that had the PDGF-alpha receptor and not
the Flk-1 receptor generated a cell population that produced
only muscle fibers without the risk of cancer formation.
Cells with these properties were multiplied and then
injected locally into the tibialis anterior muscle and into
the blood circulation of mdx mice. New dystrophin appeared
in 11% to 16% of all muscle fibers and was accompanied
by a significant increase in muscle force. Because,
as has been shown in other gene therapy experiments with
mdx mice, not all fibers in a muscle need to contain dystrophin
for a significant therapeutic effect, the results of
this stem cell approach may lead to an effective therapy of
Duchenne dystrophy.
Darabi R, Gehlbach K, Bachoo RM, et al. and Perlingeiro
RCR. Functional skeletal muscle regeneration from
differentiating embryonic stem cells. Nature Medicine,
2008;14;134-143.
Muscle regeneration with muscle stem cells. Stem cells
to be used for a Duchenne therapy should have the following
properties: (1) They must be easy to isolate from human
biological material like muscle tissue; (2) they should
be easy to multiply in the laboratory to amounts necessary
for a systemic treatment of children; (3) it should be possible
to transfer into them “healthy” dystrophin-gene sequences
with viral vectors; (4) systemic delivery into the
blood circulation should be possible; (5) they have to be
able to migrate from the blood circulation into the muscles;
(6) they must give rise to large amounts of functional
muscle cells with dystrophin and with functional satellite
cells inside the dystrophic muscle tissue, and (7) they
should not produce any serious side effects, especially no
cancer. Two types of cells have been identified which
seem to have these properties and which are adult and not
embryonic stem cells: Mesoangioblasts and pericytes,
which are located on the outside of small blood vessels
within muscle tissue from where they can be isolated.
After preliminary positive experiments with mesoangioblasts
on mice which were lacking the protein, alphasarcoglycan,
Prof. Giulio Cossu and his colleagues at the
Stem Cell Institute of the Hospital San Raffaele in Milan
isolated similar cells called pericytes from the walls of
capillaries (small blood vessels) inside human normal and
dystrophic muscle tissue and injected them systemically
into mdx mice whose immune system was inactivated. Before
the dystrophic pericytes were injected, they were
treated with viral vectors containing mini-dystrophin
genes. In both cases, a large number of muscle fibers of
the mdx mice had new dystrophin and their muscle function
was significantly improved.
As the next step towards a human application, Dr.
Cossu’s team treated four dystrophic dogs systemically
with cells from their own muscle tissue (autologous treatment)
into which the gene for human micro-dystrophin
was transferred, and six dogs with cells from healthy dogs
(heterologous treatment) which contained normal dystrophin,
but which required immunesuppression with cyclosporine.
The results were much better after the heterologous
treatment than after the autologous treatment. One
dog which received the cells through a catheter into the
aorta was walking well five months after the final of five
weekly treatments. The other five dogs recovered more
slowly.
The autologous experiments on dogs are now being repeated
with stem cells that contain longer dystrophin gene
sequences. Some control experiments are also performed
to determine the effect of cyclosporin alone and to see
whether the satellite cells on the new muscle fibers are also
functional. After further long-term trials, again with dogs,
and the preparation of the cells under clinical grade conditions,
a clinical phase-I trial with Duchenne patients will
be started to check for safety and appearance of at least
some new dystrophin.
Muscle regeneration with genetically exon-skipped
stem cells. The research teams of Prof. Luis García at the
Généthon Institute in Évry near Paris and Prof. Yvan Torrente
at the Stem Cell Laboratory of the University of Milan
worked together for a new therapy of Duchenne dys9
trophy: they isolated stem cells from muscles of Duchenne
patients, repaired their dystrophin gene with a genetic exon
skipping method, transferred them into mdx mice where
they regenerated their muscle fibers and ameliorated the
dystrophic symptoms significantly.
The following is a very simplified short summary of a
highly complex research strategy which needed many additional
biochemical control and activity experiments, and
also biological tests for muscle function.
The stem cells, mainly satellite cells, were obtained
from muscle bioptic material of Duchenne boys whose
dystrophin gene had a deletion of exons 49 and 50. For
their experiments, the Franco-Italian researchers used only
those about 1% of the cells which contained the marker
protein CD133 in their membranes: These cells had been
shown earlier to be able to repair muscle cells and form
new ones in damaged muscle tissue. The CD133+ cells
were multiplied in cell culture in the laboratory and then
treated with a transforming vector of lentiviruses which
carried in their genetic material genes of two antisense oligoribonucleotides
for the skipping of exon 51 in a similar
way as it has been done (and described on page 5) for the
genetic skipping of exon 23 in mdx mice.
Twenty-four hours before the experiments with twomonth-
old scid/mdx mice, which had neither dystrophin
nor a functioning immune system, the animals were stressed
by a swimming exercise to intensify their muscle degeneration-
regeneration process. Twenty-four of these
mice received 20,000 to 40,000 of the genetically engineered
human stem cells from a Duchenne boy locally into
one tibialis anterior muscle with three injections. Six other
mice were treated systemically by injection of 500,000 of
these stem cells into the femoral artery of one leg. At 21
and 45 days after these local and systemic injections, many
tests were performed to assess the outcome of this complex
genetic therapy.
The results showed a better muscle regeneration, a large
amount of the expected dystrophin in the regenerated fibers
without the amino acids determined by exons 49, 50
and 51, an amelioration of muscle morphology (their structure),
and a significantly restored muscle function.
This technique which is related to the direct genetic
exon-skipping approach as mentioned on page 5 because it
uses the same U-7 system, opens the way to a new strategy
for a Duchenne therapy. However, before clinical trials can
be contemplated, the exact mechanism of exon skipping
with lentiviral vectors will have to be understood completely,
because these viruses with their charge enter the
genetic material of the muscle cells in a random way, and
possibly could disturb other genes or even induce tumors.
Benchaouir R, Merigalli M, Farini A, et al. and García
L, Torrente Y. Restoration of human dystrophin following
transplantation of exon-skipping-engineered DMD patient
stem cells into dystrophic mice. Stem Cell 2007; 1; 646-
657.