Research Updates
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| Xiao Xiao |
Miniaturized Gene Raises Hopes in Duchenne, Becker
MDA-supported researchers Xiao Xiao and Juan Li in the Department of Molecular
Genetics and Biochemistry of the University of Pittsburgh were on a team that
constructed miniaturized versions of the dystrophin gene small enough to fit
inside an adeno-associated virus (AAV).
Dystrophin is the protein needed by people with Duchenne or Becker muscular
dystrophy, in whom the dystrophin gene is flawed. AAVs are considered among the
safest and most effective vehicles for delivering genes to muscle tissue.
In experiments injecting AAVs carrying the highly miniaturized genes into leg
muscles of dystrophin-deficient mice, the research team achieved adequate
dystrophin production in 40 to 88 percent of muscle fibers. The team published
its findings in the Dec. 5 issue of Proceedings of the National Academy of
Sciences.
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| To get the dystrophin gene small enough to fit into an adeno-associated virus,
Xiao Xiao and colleagues miniaturized it but preserved its important functions. |
In designing the very small dystrophin genes, Xiao's group focused on the
midsection, or "rod domain," of the protein. This section is thought to provide
some shock absorption and act as a link between the anchored ends of the
ropelike dystrophin molecule, Xiao says.
The researchers also removed some of the DNA that codes for the section of
dystrophin called the C terminus, which attaches dystrophin to a group of
proteins spanning the cell membrane. Previous attempts to miniaturize the gene
have left this section intact, making the gene too large to fit inside an AAV.
But, says Xiao, "the C terminus has two domains." The minigene includes the
cysteine-rich domain, which is essential for dystrophin to anchor to the
membrane complex, but eliminates another, less essential domain, called the
actin-binding section.
Xiao's group made three slightly different dystrophin minigenes and found one
especially effective. Xiao says he hopes the new minigene can eventually be
used in clinical trials for Duchenne or Becker MD.
Duchenne Muscles May Be Starved for Oxygen
A new study suggests that insufficient oxygen to active muscles might contribute
to muscle degeneration in boys with Duchenne muscular dystrophy (DMD). The
finding has implications for management and treatment of the disease.
In the 1980s, MDA-funded researchers figured out that DMD is caused by loss of
the dystrophin protein in muscle cells. Despite the importance of that
discovery, it's still not clear why dystrophin is vital to muscle cells.
Because dystrophin is a large protein that connects the inner and outer
architecture of muscle cells, researchers have suspected that loss of the
protein might lead to structural damage during contraction. But a new study in
the December issue of the Proceedings of the National Academy of Sciences
indicates that loss of dystrophin indirectly cuts off the muscle cells' blood
supply during exercise.
The study's authors, including MDA grantees Susan Iannaccone, James Stull and
Gail Thomas of the University of Texas Southwestern Medical Center in Dallas,
suggest that dystrophin interacts with another protein found in muscle cells --
neuronal nitric oxide synthase (nNOS). In active muscles, nNOS produces the gas
NO, which promotes the dilation of nearby blood vessels, ensuring a steady
supply of oxygen. Although it's not clear how, the loss of dystrophin displaces
nNOS from its normal location in muscle cells. The result is that nNOS doesn't
increase blood flow to muscles during exercise, limiting the muscles' oxygen
supply when they need it most.
The study shows oxygen deprivation occurred during a hand grip exercise in
muscles of 10 boys with DMD and four children with spinal muscular atrophy. The
effect didn't occur in children without muscle disease or in those with
limb-girdle muscular dystrophy or polymyositis.
The study has implications for the long-standing debate over whether exercise is
helpful or harmful to children with DMD, says Iannaccone, co-director of the
MDA clinic at UT Southwestern. "It makes it more likely that exercise is bad
for them, but it depends on the kinds of exercise," she says. Further research
will be necessary to investigate this issue.
The study's findings will also affect the development of gene therapy and other
treatments for DMD. The shortened forms of dystrophin being designed for DMD
gene therapy should be tested for their effects on nNOS function, suggests MDA
grantee H. Lee Sweeney, who's investigating DMD gene therapy at the University
of Penn-sylvania in Philadelphia. Iannaccone says the study also means that
"there may be some treatments that are already available on the market that
might help kids with DMD," such as drugs that affect blood flow during
exercise.
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| Jeffrey Chamberlain |
Dystrophin's 'Heartiness' Good for Gene Therapy
MDA grantees Jeffrey Chamberlain, now in the Department of Neurology of the
University of Washington Health Sciences Center in Seattle, and Andrea
Amalfitano of the Department of Human Genetics at Duke University Medical
Center in Durham, N.C., were part of a team that created a new system to shed
light on the potential of gene therapy to treat Duchenne or Becker muscular
dystrophy. (Chamberlain recently moved from the University of Michigan's
Department of Human Genetics, where this work was conducted.)
The team bred mice carrying a dystrophin gene under the control of a molecular
switch that "turned off" production of dystrophin when the animals were given
tetracycline, an antibiotic medication. They found that dystrophin persisted in
the muscle cells of the mice for at least six months after production was shut
off.
"The results suggested that the dystrophin protein may be an especially hearty
protein, since the functional abilities of sarcolemma-associated dystrophin to
prevent muscle fiber degeneration in vivo [in live animals] exceeded six
months," the authors wrote in a paper published in the Oct. 12 issue of Human
Molecular Genetics. (The sarcolemma is the muscle cell membrane.)
Results were good in younger mice and difficult to evaluate in older mice. The
researchers note that the studies may not have allowed time for the older mice
to recover from their long-standing dystrophin deficiency.
"My feeling is that, in the [older] mice, the muscles are indeed being
protected, but this protection and functional correction does not result in a
'visible' improvement in the way the muscle looks under a microscope,"
Chamberlain says. "Further studies are needed to verify this point and to
determine the degree of functional protection that results from introduction of
various levels of dystrophin into mice at different ages."
Chamberlain says the researchers are much encouraged by the data on the
heartiness of the dystrophin protein and the persistence of dystrophin in
muscle cells. But, he noted, "additional work needs to be done to determine
what degree of correction can be expected from the introduction of dystrophin
into muscles that are already dystrophic."
Myotonic MD Treatments Appear Nearer as Knowledge Grows
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| Stephen J. Tapscott |
In summarizing research on myotonic muscular dystrophy (MMD) since its genetic
cause was discovered in 1992, Stephen J. Tapscott at the Fred Hutchinson Cancer
Research Center in Seattle says new findings point the way to possible
interventions in this often baffling disorder. Tapscott, a member of MDA's
Scientific Advisory Committee and a past MDA grantee, reported on MMD research
status in the Sept. 8 issue of the journal Science.
"The new mouse models [mice bred to show certain characteristics of MMD] will
allow us to test different hypotheses about what causes myotonic dystrophy in
all its aspects," Tapscott says. "These models may unveil targets for drug
therapy."
The most common genetic defect underlying MMD, which was identified in 1992 with
MDA support, is in an area of DNA on chromosome 19. An expanded section of DNA
made up of chemical structures known as CTG repeats, though it isn't actually
part of a gene or a protein made from a gene, causes trouble in nearly every
organ of the body. The most prominent myotonic dystrophy symptoms occur in the
skeletal and involuntary muscles, heart muscle, the lens of the eye and the
central nervous system. The disease may affect sleep and respiratory centers in
the brain.
Tapscott's summary reviews research, much of it MDA-supported, on how these
diverse and sometimes severe effects may arise from the CTG expansions. He says
there are at least three things going wrong in MMD and therefore three possible
targets for therapy.
For example, near the CTG repeats on chromosome 19 is the gene for the DMPK
protein. The level of DMPK made by cells may be lowered by the expanded
repeats, a theory supported by the finding that mice lacking this protein show
heart defects remarkably similar to those in people with MMD.
Also near the repeats is a gene for the protein known as SIX5. The ocular lenses
of mice lacking adequate SIX5 have cataracts, as do people with MMD.
A third problem in MMD may be the CTG repeats themselves. In cell nuclei, CTG
repeats in the DNA are changed to similar compounds called CUG repeats in RNA,
the chemical step that follows DNA in each cell nucleus. Studies in mice with
extra CUG repeats revealed that these repeats may cause the muscle weakness and
inability to relax muscles at will (myotonia) that people with MMD experience.
One possibility, says Tapscott, is that the body misreads the CUG repeats and
behaves as if the cells harbored a virus.
"If a cell is mistaking CUG repeats for viral RNA and killing the cell, you
might be able to target the protein that causes that to happen," Tapscott
speculates.
In a broad sense, he says, "it may actually be easier to treat myotonic
dystrophy than it will be to treat diseases where information from the DNA is
missing, where you're missing a gene or a part of a gene."
Antiseptic May Target Mitochondrial Disease
Certain mitochondrial diseases are currently beyond the reach of gene therapy,
but a chemical commonly used as an antiseptic might be the magic bullet for
that problem.
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| A DQAsome is a hollow capsule formed from many bolaform-shaped DQA molecules
(highlighted). The capsule might make an ideal gene therapy vector for
mitochondrial diseases. |
Gene therapy — the delivery of corrective genes to make up for a genetic defect
— holds promise for treating most genetic diseases, and in some cases, is
already being tested in clinical trials. But some mitochondrial diseases are
off-limits to current gene therapy techniques because they're caused by defects
in hard-to-reach genes.
Most of our genetic material (DNA) is housed within the nuclei, the control
centers found in nearly all our cells. But some DNA is housed in the
mitochondria, the tiny powerhouses that provide energy to cells. Defects in
mitochondrial DNA can cause energy deficits that lead to the extreme fatigue
and weakness characteristic of mitochondrial muscle diseases.
While scientists have achieved success in targeting therapeutic genes to nuclei,
it's more challenging to get genes into mitochondria. The problem lies in the
gene delivery vehicles, or vectors, says Volkmar Weissig, a biochemist at
Northeastern University in Boston.
In gene therapy, a vector's first task is to penetrate the cell's outer
membrane, or skin. Once inside the cell, the vector must breach a second
membrane surrounding either the nucleus or the mitochondrion, all the while
holding on to its genetic payload.
The most commonly used vectors for gene therapy are viruses and synthetic
compounds called liposomes, hollow spheres composed of an outer shell of lipid
(the same type of chemical that makes up cell membranes). Viruses, says
Weissig, naturally infect cells and introduce foreign genes into nuclei, but
apparently can't send genes to mitochondria. With a composition similar to cell
membranes, liposomes can penetrate cells, but they usually release their DNA
into the cellular space shortly afterward.
To make mitochondrial gene therapy feasible, Weissig is using MDA support to
custom-design a vector from a lipidlike, antiseptic chemical called dequalinium
(DQA). A single unit of DQA looks like a traditional Native American weapon
called a bola, and this bolaform shape allows DQA to form liposome-like
capsules called DQAsomes. DQAsomes can be filled with DNA, and thus might be
ideal weapons against mitochondrial diseases, Weissig suggests. So far, he's
shown that the DQAsomes can selectively target DNA to mitochondrial membranes.
"This is basic research" that won't immediately lead to treatment for
mitochondrial disease, Weissig says. But it's a significant step in the right
direction.
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| Massimo Pandolfo |
Antioxidant May Help Heart in Friedreich's Ataxia
The 1996 identification of the gene that, when defective, causes Friedreich's
ataxia (FA) has led to more than four years of intensive research to understand
what the gene and its protein product (now called frataxin) normally do — and
what goes wrong in FA.
Massimo Pandolfo, a neurologist and MDA grantee now at the University of
Montreal's Hospital Center, led the Baylor College of Medicine team that found
the frataxin gene and has since been studying FA and its biochemistry.
Recent studies have confirmed early guesses, he says, that frataxin keeps iron
levels normal inside structures known as mitochondria, which are cellular
energy-producing units. When there isn't enough frataxin, iron builds up in the
mitochondria, according to laboratory studies performed mostly in yeast cells.
The iron buildup leads not only to a loss of mitochondrial function but to a
general buildup of toxic chemicals known as free radicals and to a toxic
process known as oxidative stress.
The challenge now, says Pandolfo, is to move from simple organisms, such as
yeast, to mice and even humans to see if people with FA also have oxidative
stress and iron accumulation in mitochondria. "If we do show this, then can we
find medications that would modify this process? And can we find simple,
biochemical parameters [indicators] that we can monitor to show that these
medications are effective?," he asks.
The antioxidant (anti-free radical) medication idebenone, a close cousin of the
widely available coenzyme Q10, actually improved the heart conditions of three
French patients with FA in a pilot study reported in the British journal Lancet
on Aug. 7, 1999, he notes.
That abnormally big hearts actually got smaller with idebenone treatment is a
"remarkable thing" and "very encouraging," says Pandolfo. Idebenone, while not
approved for sale in the United States, is slightly different in structure from
coenzyme Q10 and may be able to enter the nervous system more easily.
Pandolfo's Canadian research group has just finished a one-year trial of the
drug in children with FA and is analyzing the results, while working out the
details of FA biochemistry in mice. A small trial of idebenone for FA at the
National Institutes of Health in Bethesda, Md., is planned for this spring.
Meanwhile, Pandolfo says, it will be necessary to monitor the effects of
antioxidant drugs with clear-cut markers. Research groups have found blood
levels of two compounds, malondialdehyde and 8OH2'dG, promising as
scorekeepers.
Another line of research has explored the use of iron-binding drugs like
desferrioxamine for FA, Pandolfo notes. These compounds, which stick to iron
and escort it out of the body, are far more dangerous than coenzyme Q10 and
other antioxidants, he says.
Rather than showing an overall excess of iron, FA patients show "very specific
accumulations, probably excessive iron in certain intracellular compartments,
especially the mitochondria. The total cellular iron is probably not changed,"
Pandolfo says. "Depleting patients of iron leaves you a very small safety
margin. They can start to be anemic, so it can be a rather dangerous way to
treat the disease."
Until idebenone is approved in the United States, Pandolfo cautions American
patients not to try to get it through the Internet or other sources. "If you
buy from a generic source that sells it more like a nutritional supplement, you
can't be sure that the manufacturing has been up to standards."
As for coenzyme Q10, Pandolfo says he prefers that patients take drugs under the
supervision of a neurologist or as part of a clinical trial. "But," he says,
"taking some over-the-counter coQ10 is probably not going to hurt."
Pandolfo's group published results on its biochemical marker in the Dec. 12
issue of Neurology. MDA grantees Pragna Patel at Baylor College of Medicine in
Houston and Grazia Isaya at the Mayo Clinic in Rochester, Minn., published
their results on the effects of frataxin gene mutations on yeast cells in the
Oct. 12 issue of Human Molecular Genetics.
To find out more about the proposed NIH trial of idebenone, contact Kenneth H.
Fischbeck, Neurogenetics Branch, National Institute of Neurological Disorders
and Stroke, at (301) 435-9318 or kf@codon.nih.gov.
Mutant Protein Creates Mess in OPMD
MDA-funded researchers are beginning to unravel the microscopic events that lead
to muscle damage in oculopharyngeal muscular dystrophy (OPMD). The inherited
condition strikes around age 40, primarily causing weakness in muscles that
control the eyes and throat.
In 1998, MDA grantee Guy Rouleau of McGill University in Montreal, Canada, led a
team of researchers to establish that OPMD is caused by genetic mutations that
insert an abnormal stretch of amino acids into a protein called poly(A) binding
protein 2 (PABP2).
In the cell's nucleus, PABP2 normally adds a stabilizing attachment called a
poly(A) tail to RNA, an essential intermediate between genes and the proteins
they make. Researchers are attempting to determine how the mutations change the
function of PABP2 and cause OPMD.
Previously, researchers had observed that OPMD muscles contain intranuclear
inclusions -- heaps of cellular debris inside the nucleus. A flurry of recent
studies suggests that these inclusions form from clumps of mutant PABP2. In the
September issue of Human Molecular Genetics, Rouleau and his colleagues show
that the intranuclear inclusions contain an abnormal accumulation of mutant
PABP2. MDA grantee David Bear and his colleague Mark Becher, at the University
of New Mexico in Albuquerque, independently confirm that finding in November's
Annals of Neurology.
In the same issue, Rouleau shows that mutant PABP2 can actually cause nuclear
inclusions to form when it's delivered to an isolated cell line. Rouleau also
has found evidence that RNA accumulates within the inclusions, suggesting that
mutant PABP2 might trap the RNA there and interfere with protein production.
"The mutation makes the protein very sticky," says Bear. Identifying exactly
which proteins or RNAs get stuck to mutant PABP2 might yield insights into the
formation of inclusions and their effects on muscle cells, he says.
"Formation of the inclusions might be how the cell defends itself against
abnormal proteins," says Rouleau. "When the proteins are all compacted
together, they might have less damaging effects than if they were spread
throughout the nucleus." Nonetheless, as the inclusions grow, they might damage
the nucleus, he suggested.
Studying mutant PABP2 is also leading to insights into possible treatments for
OPMD. "One possibility is that if you could prevent the expression of the
mutant protein, you could then prevent the disease," Rouleau says. The cell
line that Rouleau has used in previous studies might be useful for testing
possible treatments, he says.
HEART PROBLEMS DIFFER AMONG MDs
While doctors have suspected for some years now that the types of heart problems
seen in the various muscular dystrophies differ and respond differently to
treatment, molecular biologists have now shed further light on these issues and
are helping doctors to plan better therapies.
Coronary Arteries May Be Problem in Some LGMDs
MDA grantee Kevin Campbell at the University of Iowa's Department of Physiology
and Biophysics was recently part of a team studying the cardiac effects of the
loss of beta-sarcoglycan and delta-sarcoglycan genes in mice. When these genes
are flawed in humans, the result is limb-girdle muscular dystrophy types 2E and
2F, respectively.
Campbell's research team showed that, in mice lacking genes for these
sarcoglycan proteins, normally located in the muscle-cell membrane in both
heart and skeletal muscles, the mice not only sustain some damage to the
cardiac muscle itself, but they sustain even more obvious damage to the
arteries supplying the heart. The team published its findings in the Jan. 15
issue of the Journal of Clinical Investigation.
When the researchers gave some of the mice a drug that dilates blood vessels by
blocking the flow of calcium across membranes, they found that the coronary
artery and heart damage didn't occur.
"The most important thing about this is that it may lead to a treatment for the
cardiac involvement in patients with limb-girdle dystrophy related to either
beta- or delta-sarcoglycan deficiency," Campbell says.
The drug didn't help mice lacking dystrophin, the protein missing in Duchenne
and Becker MD. These mice didn't show any abnormalities in their coronary
arteries. This finding is consistent with observations that boys with Duchenne
and Becker MD likely have a problem in the cardiac muscle itself, not the blood
vessels leading to the heart.
The researchers also found that levels of a protein called troponin I in the
blood correlate well with damage to heart muscle in the mouse models, just as
levels of the protein creatine kinase correlate well with damage to skeletal
muscle in both mice and humans with MD. The team suggests that troponin I would
leak out of heart muscle and into the bloodstream regardless of whether the
heart damage originated in the coronary arteries or in the muscle itself.
They suggest that the troponin I level could be developed as a diagnostic test
for cardiac damage in muscular dystrophy. Campbell says the protein is already
used to check for heart damage in cases of suspected heart attack (myocardial
infarction).
MMD: Heart and Skeletal Muscle Weakness Correlated
An Italian research team has studied 50 people with myotonic muscular dystrophy
(MMD) in an effort to develop ways of predicting which patients are likely to
need treatment for cardiac problems before they become severe.
The team found that people with MMD whose skeletal muscles were weaker developed
more changes in their electrocardiograms (EKGs), tests used to measure the
heartbeat abnormalities known as conduction blocks or arrhythmias common in
MMD. Electronic pacemakers are often used for treatment, but not always
successfully.
The team, led by Giovanni Antonini at the University of Rome, found that damage
to the electrical system of the heart, detected by changes in EKGs, showed a
strong association with the severity of the MMD as evaluated by a muscular
disability rating scale. The findings were published in the Oct. 24 issue of
the journal Neurology.
The researchers also found a correlation between the age of onset of the EKG
changes and the size of the expanded DNA segment that causes MMD: The longer
the repeated DNA segment, the earlier the EKG changes took place.
William Groh, an MDA grantee at the Krannert Institute of Cardiology at Indiana
University School of Medicine in Indianapolis, is also studying CTG repeats and
cardiac problems in MMD. "The big question," Groh says, "is not whether EKG
abnormalities are present — we know they are — but what we need to do to
decrease the risk of sudden death or major morbidity [adverse effects]."  |
