Scientists
Bullish on Stem Cells for Muscle Repair
Two MDA-supported groups have
recently made great strides in stem cell therapies
for muscle disease.
Stem
Cells Treat MD in Dogs
MDA grantee Giulio Cossu, director of the Stem
Cell Research Institute of the San Raffaele
Scientific Institute of Milan (Italy), was part
of an Italian and French research team that
restored mobility to two dogs and stabilized
function in a third, using stem cells taken
from muscle blood vessels.
Maurilio Sampaolesi, at the San Raffaele Scientific
Institute in Milan and the Institute of Myology
at the University of Pavia (Italy), and colleagues,
isolated mesoangioblasts from canine muscle
biopsy samples and administered them through
an artery into 10 dogs with a disorder resembling
human Duchenne
muscular dystrophy (DMD).
The results, published online Nov. 15 in Nature,
received national media attention because the
technique was effective in a large animal. This
success is considered a major step toward testing
these cells in humans.
Four of the dogs received their own mesoangioblasts,
with an added, functional gene for the protein
dystrophin, which is missing in DMD. Six other
dogs received normal mesoangioblasts from a
healthy donor animal, and three dogs weren't
treated at all.
The dogs that received donor cells responded
the best, with one of that group walking well
at 13 months. (Most dogs with the disease have
difficulty walking by 8 months of age and die
when they're about a year old.)
The dogs were injected three to five times
with 50 million cells at one-month intervals.
Some of the animals were also treated with drugs
to suppress the immune system's potential rejection
of the new cells.
Results were documented biochemically and through
at least two measures of muscle function. There
was no evidence of immune response in any of
the dogs.
The study's authors write that "the work reported
here sets the logical premise for the start
of clinical experimentation that may lead to
an efficacious therapy for Duchenne muscular
dystrophy," also noting that any immune system
suppression needed "might be modest and perhaps
transient."
Two
Stem Cell Types May Be Better Than One
Researchers in the
laboratory of MDA grantee Emanuela Gussoni at
Children's Hospital in Boston have identified
two proteins that counter each other's actions
to regulate the balance between muscle stem cells
and mature muscle fibers. They say further experiments
may improve the success of muscle cell transplant
strategies.
Natasha Frank at Children's, and colleagues,
who published their results online Oct. 2 in
the Journal of Cell Biology, say bone morphogenetic
protein 4 (BMP4) keeps muscle stem cells from
maturing and encourages them to continue to
proliferate, while another protein, gremlin,
coaxes them to mature and stop proliferating.
BMP4 is secreted at high levels by the "side
population" of muscle cells that Gussoni and
her colleagues identified several years ago
as having stem-cell-like qualities. Gremlin
is secreted in abundance in the main population
of muscle cells, which don't have these stemlike
characteristics.
"It seems that cells in skeletal muscle 'talk'
to one another, and they can induce cell division
or differentiation [maturation] by secreting
specific factors," says Gussoni, who adds that
the findings may have some implications for
modifying cell transplantation approaches.
"Perhaps stem cells and more mature cells should
be injected together in mouse models to see
whether engraftment can be improved," she notes.
"Perhaps the stem cells give support to other
cells and may help them function better in muscle
repair."
Gene
Therapy Now in Its Teen Years
Gene therapy - inserting new genes
into cells to treat disease - first became a
reality in the early 1990s. Now past its earliest
years and into its adolescence, the science
has progressed along two major pathways.
The first seeks to define the
best way of delivering genes inside the shell
of a defanged virus, with the precise type of
viral shell dependent on the target tissue.
Viral delivery of genes is highly effective
but carries some risk of eliciting an unwanted
response from the immune system.
The second pathway seeks to perfect
gene transfer to tissues without the use of
viruses, a technique that may be some-what less
effective but is probably safer.
MDA is funding research groups
using both approaches and is supporting a gene
therapy trial using viral delivery in boys with
Duchenne muscular
dystrophy.
An MDA-supported gene therapy
trial in a form of limb-girdle
MD that results from a deficiency of the
alpha-sarcoglycan protein recently received
a go-ahead from the Recombinant DNA Advisory
Committee. This green light is the first step
in the approval process for the gene therapy
trial in the United States.
Viral
Gene Transfer Moves Ahead
The first U.S. human gene therapy trial directed
at Duchenne
muscular dystrophy (DMD),was launched in
March at Columbus (Ohio) Children's Hospital.
The project will soon move to a second stage,
with a $2.5 million grant from MDA, and matching
funds from Asklepios Biopharmaceutical (www.askbio.com)
of Chapel Hill, N.C.
In this second stage, researchers will test
in animals the safety and effectiveness of delivering
miniaturized genes for the dystrophin protein,
encased in the shell of an adeno-associated
virus, via the bloodstream, so that multiple
muscles can be reached.
Results from phase 1 of the clinical trial
are expected this spring. Investigators tested
the safety of injecting the laboratory-engineered
gene therapy compound directly into a single
muscle in six boys with the disease.
The $2.5 million grant to AskBio is the largest
MDA has ever awarded to a for-profit company.
MDA gave AskBio $1.6 million for the first phase
of the trial.
In October, Jon Wolff, an MDA grantee at the
University of Wisconsin-Madison, with that institution
and the biotech company Mirus (www.mirusbio.com),
announced the group now has a European patent
for a method of delivering "naked" genes (not
encased in a viral shell) to muscle tissue via
the bloodstream. The group already has a patent
for this process in the United States.
Mirus' lead gene therapy compound for muscular
dystrophy is being developed with Transgene
(www.transgene.com)
of Strasbourg, France. The announcement says
a clinical trial is planned for 2008.
HDAC
Inhibitor Improves Mice With Forms of MD
MDA grantee Pier Lorenzo Puri,
at the Dulbecco Telethon Institute in Rome,
with colleagues in the United States and Italy,
has found that a type of compound known as an
HDAC (histone deacetylase) inhibitor increases
muscle fiber size and reduces signs of scarring
and inflammation in mice with muscular dystrophy.
The researchers, who published
their findings in the October issue of Nature
Medicine, gave HDAC inhibitors to dystrophin-deficient
and alphasarcoglycan-deficient mice, which are
mouse models of Duchenne
and limb-girdle
MD, respectively.
After trying three HDAC inhibitors,
they selected one called trichostatin A (TSA)
as the best. The investigators think the beneficial
effects of TSA are due to its ability to increase
production of the follistatin protein, which
blocks the growth-limiting muscle protein myostatin.
"These data are so far restricted
to mice," Puri says, "and will not necessarily
extend to humans. However, we have already planned
preclinical studies that will define the suitability
of these treatments in humans."
Anti-Inflammatory
Non-Steroid Drugs Aid DMD-Affected Mice
Mice missing the dystrophin protein
and showing a disease resembling Duchenne
muscular dystrophy (DMD) that received the
anti-inflammatory drug etanercept (Enbrel) were
significantly protected against exercise-related
muscle damage, say researchers at the University
of Western Australia in Crawley.
Stuart Hodgetts and colleagues,
who published their findings in the October
issue of Neuromuscular Disorders, injected etanercept
into adult mice exposed to an exercise wheel
for 48 hours and compared their muscle samples
to those of untreated animals.
Muscle fibers from the upper legs
of the treated mice showed much less inflammation
and better cell survival than did those of the
untreated mice. The investigators cite an earlier
study in which the anti-inflammatory drug in-fliximab
(Remicade) had similar benefits in dystrophin-deficient
mouse muscles.
The authors say that etanercept
and infliximab, both of which are approved in
the United States for other conditions and have
fewer side effects than the corticosteroids
now used in DMD, might be useful "to reduce
the severity of the disease in DMD and other
dystrophies." They caution that "the merit of
using these and other emerging, highly targeted
anti-inflammatory drugs ... remains to be demonstrated."
Mice
With SBMA Answer, Raise Questions
Researchers coordinated by Andrew
Lieberman, an MDA grantee at the University
of Michigan in Ann Arbor, have developed a new
mouse model of spinal-bulbar
muscular atrophy (SBMA, or Kennedy's disease)
that they say will improve understanding of
the human disease.
Their method was distinct from
one employed by MDA grantee Albert La Spada
at the University of Washington-Seattle and
colleagues (see "Research
Updates," September-October 2006), although
both groups used established technologies.
Publishing their results in the
October issue of the Journal of Clinical Investigation,
they report that, contrary to previous assumptions,
SBMA is not only a disease of the muscle-controlling
nerve cells (motor neurons), but it also results
from direct damage to muscle fibers
"Understanding the muscle disease
that occurs in SBMA patients and in our mice
may help us define the processes that lead to
this disease and may point toward new therapeutic
strategies," Lieberman says.
The researchers bred mice with
an expanded section of DNA in the gene for the
androgen (male hormone) receptor on the X chromosome,
the defect known to cause human SBMA. Like men
with SBMA, the mice developed shrunken testicles,decreased
fertility and weakness.
Castrating the mice (thereby removing
all androgen activity) improved their grip strength,
and implanting androgen pellets reversed the
improvement. This told the researchers that
the weakness depends on androgen levels.
La Spada's experiments showed
that SBMA-affected mice that retained some androgen
receptor function fared better than those without
any, which suggested to him that blocking all
androgen activity might not be a good idea.
In addition, he noted later, blocking male hormones
is a "very unappealing therapeutic intervention,"
not only for the more obvious reasons but because
sudden reduction in androgen levels adversely
affects brain function.
La Spada says SBMA results from
two problems: expanded DNA in the androgen receptor
gene, which is toxic when it interacts with
androgens; and a lack of normal androgen receptor
function, which would allow androgens to help
motor neurons withstand stressful conditions.
His mice, which have expanded
androgen receptor DNA inserted on an artificial
chromosome, show muscle shrinkage resulting
from motor neuron damage and have a slowly progressive
disease course, like human SBMA.
Lieberman's mice, which have expanded
androgen receptor DNA inserted into one of their
own chromosomes, show motor neuron damage, as
do SBMA patients, as well as direct muscle damage
(until now, not suspected in patients). Yet,
unlike people with SBMA, they have a more rapid
disease course, die early and show disease in
muscles of the urinary tract.
"It's good to have different models,
because they can provide you with different
windows into what's going on in different aspects
of the disease process, and because they will
be useful in different ways for testing new
treatments," La Spada says.
CLINICAL
TRIALS AND STUDIES
PTC124
Trial Shows Promise in Boys With Duchenne MD
Biotechnology company PTC Therapeutics
of South Plainfield, N.J., which has received some
$1.5 million from MDA for drug development, announced
encouraging results in October from the phase 2
trial of its experimental compound PTC124 in 26
boys with Duchenne
muscular dystrophy (DMD).
PTC124 is designed to coax cells to
ignore, or "read through," a molecular stop signal
known as a premature stop codon. These signals tell
cells to stop processing a gene too soon, before
they've read all the genetic instructions for synthesis
of a protein.
The cause of DMD in about 15 percent
of boys with the disease is a premature stop codon
that cuts short the synthesis of the muscle protein
dystrophin, before a functional dystrophin molecule
is formed.
In this study, six boys took low-dose
PTC124, and 20 took it at a higher dose, for 28
days. The average age in the low-dose group was
10, and in the high-dose group, it was 9.
When muscle biopsy samples were examined,
dystrophin production was found in three of the
six boys who took the lower dose and eight of the
20 who took the higher dose. In the high-dose group,
there were significant decreases in blood levels
of creatine kinase, an enzyme that leaks from damaged
muscle cells into the bloodstream. The drug was
well tolerated and appears safe.
PTC Therapeutics now wants to evaluate
higher dose levels of PTC124 to see whether dystrophin
production can be increased.
As of late November, the company was
seeking regulatory approval to enroll patients at
Children's Hospital of Philadelphia and Cincinnati
Children's Hospital Medical Center.
After the trial criteria are finalized,
information will be provided to families on PTC's
mailing list, which can be joined by filling out
the form under "Contact Us" at www.ptcbio.com.
Information will also be posted on the MDA Web site
at www.mda.org/research/ctrials.aspx
and at www.clinicaltrials.gov.
It can also be obtained from Kerri Donnelly at PTC
Therapeutics at (908) 222-7000, ext. 112, or kdonnelly@ptcbio.com.
Anyone seeking entry into a PTC124
trial must have a documented premature stop codon
mutation. Testing can be obtained through the University
of Utah in Salt Lake City. Contact Kim Hart at (801)
585-1299 or khart@genetics.utah.edu;
or see www.genome.utah.edu/DMD/clinical_test.shtml.
'Exon Skipping' Trial
for DMD Opens in Netherlands
The first trial to test a treatment strategy known
as exon skipping in boys with Duchenne
muscular dystrophy (DMD) is now under way at
Leiden University Medical Center in the Netherlands,
says Gerard Platenburg, CEO of the Dutch biopharmaceutical
company Prosensa (www.prosensa.nl),
the trial's sponsor.
Exon skipping, which has shown promise in rodent
models of DMD, is a technique that encourages cells
to skip over faulty genetic information and construct
a nearly normal protein from the remaining, correct
information. (The parts of genes that supply codes
for a protein's structure are called exons.) It
differs from the stop codon read-through strategy
described in "PTC124 Trial"
because it could potentially be used to counteract
a wider variety of DMD-causing genetic mutations.
The strategy is accomplished through antisense
oligonucleotides (AONs), compounds that stick to
specifically targeted genetic sequences and prevent
cells from using those exons when manufacturing
proteins.
In boys with DMD, the gene for the muscle protein
dystrophin can have a variety of abnormalities,
at least some of which can probably be circumvented
by exon skipping.
The trial, which is fully enrolled, will include
four to six boys with DMD who are between 8 and
16 years old. Each participant will receive a single
intramuscular injection of an experimental AON.
The test AON was developed by Prosensa and builds
on scientific contributions from many sources, including
Judith van Deutekom at Leiden University's Department
of Genetics, a former MDA grantee who developed
this type of therapy in mice.
Platenburg notes that the trial is an "exploratory
study on the efficacy, safety and tolerability of
a single intramuscular dose" of an AON construct
in DMD. If such treatment can restore production
of dystrophin, the trial would provide "proof of
principle" evidence that would encourage researchers
to deliver the AON to the whole body.
"We have all reasons to expect that this trial
will prove the therapeutic potential of our technology
and thus form the basis for a viable cure for this
terrible disease," Platenburg says.
Myodur Kept On
Hold
CepTor, a Hunt Valley, Md., biopharmaceutical company,
announced Oct. 6 that its application to test its
experimental compound Myodur in boys with Duchenne
muscular dystrophy (DMD) remains on hold. Myodur
is designed to interfere with the actions of calpain,
an enzyme that breaks down proteins.
The company says the Food and Drug Administration
asked it to address two remaining issues. These
issues are "straightforward and addressable," says
an Oct. 6 press release from CepTor.
Congenital Myopathies
Study Seeks Participants
MDA grantee Alan Beggs at Children's Hospital in
Boston is conducting an ongoing study of the congenital
myopathies (muscle diseases present from birth),
including centronuclear/myotubular
myopathy, congenital fiber-type
disproportion, multiminicore disease,
nemaline
myopathy, and undefined congenital
myopathies.
The primary goal of the research is to better understand
the genes and proteins (gene products) involved
in these diseases, through family history information
and lab tests. Travel to Boston isn't required.
For details, see www.mda.org/research/ctrials.aspx
or contact Elizabeth Taylor at (617) 919-2169 or
etaylor@enders.tch.harvard.edu.
A team led by Beggs, in collaboration with researchers
in the United Kingdom, Australia and Finland, recently
identified a new gene that can cause nemaline
myopathy when flawed.
Pankaj Agrawal and colleagues, who published their
findings online in October in the American Journal
of Human Genetics, identified a mutation in the
gene for the protein cofilin 2 in two siblings from
a Middle Eastern family whose previous diagnosis
was a nonspecific congenital muscle disease. Like
the other five genes associated with this disease,
the cofilin 2 gene carries instructions for a protein
associated with the inside of the muscle fiber,
in the part where muscle filaments slide over each
other to cause muscle contraction.
Idebenone Shows Safety,
Potential in Friedreich's
The antioxidant idebenone "has the potential to
provide a possible treatment" for Friedreich's
ataxia (FA), according to the U.S. National
Institute of Neurological Disorders and Stroke (NINDS)
of the National Institutes of Health in Bethesda,
Md. A trial of idebenone in FA conducted by NINDS,
in collaboration with Santhera Pharmaceuticals of
Liestal, Switzerland, also showed the drug is safe
and well tolerated.
The six-month, phase 2 trial included 48 participants,
with 12 in each of three dosage groups taking Santhera's
SNT-MC17 idebenone compound and a placebo (inactive
substance) group. There was a statistical trend
toward dose-related improvement in neurological
function, based on the International Cooperative
Ataxia Rating Scale (ICARS), which consists of 19
measurements of sensory and motor skills
Principal investigator Nicholas Di Prospero at
NINDS says in an Oct. 6 press release that his group
is "encouraged to pursue further trials to establish
[idebenone's] positive effect on neurological function
in young [FA] patients."
Blood
Cell Infusions Helpful in MNGIE
Infusions of either mature blood cell fragments
(platelets) or blood stem cells from healthy donors
partially corrected biochemical abnormalities in
three out of four patients with the mitochondrial
disease MNGIE.
In this disease, mutations in the gene for the
thymidine phosphorylase (TP) enzyme severely reduce
its ability to metabolize thymidine and deoxyuridine,
which accumulate to toxic levels and damage mitochondria,
the energy-producing units of cells.
MDA grantee Michio Hirano at Columbia University
in New York was involved in both studies, results
of which were published online Sept. 13 in Neurology.
The researchers reasoned that supplying patients
with TP-producing blood cells from donors might
normalize the chemical environment and reduce damage
to mitochondria. They infused (introduced through
a blood vessel) mature, TP-producing platelets into
a 23-year-old woman and a 16-year-old boy with MNGIE.
In both cases, the infusions briefly increased TP
levels and reduced levels of thymidine and deoxyuridine,
although the patients' symptoms didn't improve.
The infused TP apparently doesn't have to enter
muscle cells to be effective; it only has to be
in the vicinity to lower the po-tentially damaging
compounds.
They next tried infusing stem cells from donors,
with the hope that these cells might permanently
establish themselves in the circulation (engraft)
and continuously produce TP.
When umbilical cord stem cells were infused into
a 21-year-old man with severe MNGIE, they failed
to engraft. But when a 30-year-old woman with MNGIE
received blood stem cells from her healthy brother,
some of them engrafted, and she experi-enced less
abdominal pain, better swallowing, and decreased
numbness in her hands and feet.
The researchers note that direct administration
of stabilized TP protein or perhaps gene therapy
with the TP gene might be more effective treatments
than cell infusions. They added that treatment should
begin as early after diagnosis as possible, before
irreversible damage to the mitochondria occurs.
Sleep Problems
Linked to Impaired Learning in Myotonic MD
A French research group has found that disordered
sleep is common in young people with childhood-onset
type 1 myotonic
dystrophy (MMD1). They define the condition
as showing symptoms between ages 1 and 10, with
normal development during the first year of life
and subsequent failure to thrive, abdominal problems,
mental retardation and weakness.
Maria-Antonia Quera Salva at the Raymond Poincare
Hospital in Garches, and colleagues, who published
their findings in the October issue of Neuromuscular
Disorders, speculated that disordered sleep might
contribute to learning disabilities in MMD-affected
children.
They conducted overnight sleep studies, questionnaire
surveys and other tests in 21 children and young
adults with MMD1 whose average age was 15.
Of the 21 patients, 19 (90 percent) had learning
disabilities, and 17 (81 percent) reported fatigue
and/or sleepiness. Six were found to have sleep
apnea (periodic cessation of breathing during sleep);
eight had abnormal periodic limb movements during
the night; and one person had both.
The researchers say that fatigue and sleepiness
are well documented in adult MMD1 and can severely
impair performance. They recommend oral and upper
airway examinations to look for obvious causes of
sleep apnea that can be treated with surgery or
orthodontic braces. They also advise testing children
with periodic limb movements for blood iron level
abnormalities, and considering modafinil, a drug
that promotes wakefulness, for some patients.
"Whether effective treatment of these abnormalities
improves the learning difficulties seen in these
patients deserves to be investigated," they write.
Infantile-Onset
FSHD Rare But Severe
Facioscapulohumeral
muscular dystrophy (FSHD) symptoms usually begin
in the teen years, with weakness in the muscles
of the face and shoulders and a slow progression
to other muscle groups. But about 4 percent of the
time, FSHD follows a rapidly progressive course
beginning in infancy, even though the baby's parents
may have few or no symptoms themselves.
Lars Klinge and colleagues at University of Newcastle
Upon Tyne in the United Kingdom studied seven infantile-onset
FSHD patients who were between 9 and 25 years old,
finding that only one was still walking; all had
impaired ability to show facial expression (none
could smile); one had abnormal retinal blood vessels;
and two had diminished hearing.
In some cases, a parent of an affected child is
found to have an FSHD mutation in only some of his
or her cells but to have conceived the child with
a sperm or egg cell containing the mutation.
In these families, the parent's disease is masked
by the presence of large numbers of normal muscle
cells, but the child, whose conception begins with
an FSHD-causing mutation that will be replicated
in all of his or her cells, can be severely
affected.
"Our data confirm that infantile FSHD represents
less than 5 percent of the total FSHD population,
but that it is a severe and progressive disease,"
the authors write in the October issue of Neuromuscular
Disorders. They say that the risk to an adult with
classical FSHD of having a child with this severe
form of the disease is "probably low."
Wyeth Analyzing
MYO-029 Results in MD
In November, Wyeth Pharmaceuticals announced that
results of its trial of MYO-029 in adult muscular
dystrophy were being analyzed.
MYO-029, a recombinant human antibody (laboratory-developed
immune system protein), is designed to stick to
and interfere with the activity of myostatin, a
natural protein that prevents skeletal muscle formation.
Studies conducted in mice with muscular dystrophy
contributed to the development of this trial, which
included about 100 adults with Becker,
limb-girdle
or facioscapulohumeral
MD. |
