Research Updates July-August 2003

Article Highlights:

Updates on research news as of June 2003

by Quest Staff on July 1, 2003 - 4:30pm

In this issue: A protein called dysferlin may hold the answer to repairing muscle in MD ** Cardiac problems seen in Becker and Duchenne MD after childhood ** Fukuyama MD is found in a child not of Japanese descent ** and more...

Protein stimulates muscle repair, holds potential for treating MD

MDA-funded researchers have discovered that a protein called dysferlin plays an essential role in repairing muscle fibers that have been ripped and torn by repeated contraction.

Kevin Campbell
Kevin Campbell
When the membrane surrounding a muscle cell breaks open, vesicles (tiny spheres of membrane) can accumulate at the break and form a patch. Dysferlin is essential in this process.

"Once we learn more about this protein and others involved in muscle repair, we may find a way to enhance the repair process so that it's helpful in treating muscular dystrophy," said lead researcher Kevin Campbell, a professor of physiology at the University of Iowa in Iowa City and an investigator of the Howard Hughes Medical Institute.

Two rare types of muscular dystrophy, limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), a form of distal MD, are caused by a genetic deficiency of dysferlin. The protein was identified in 1998, but until now, its normal role in muscle has been a mystery.

Campbell and his research associate Dimple Bansal probed dysferlin's functions by creating mice with a genetic deficiency of the protein. Their study is the cover article in the May 8 issue of Nature.

Campbell and Bansal examined the muscles of the dysferlin-deficient mice and found signs of muscular dystrophy, including disruptions in the plasma membrane (the surface around muscle fibers and other cells).

Closer examination revealed that dysferlin is needed to patch those disruptions. Previous studies have shown that damaged plasma membranes can be resealed by vesicles (tiny spheres of membrane that home to the site of damage and fuse together to form a patch). The researchers observed an accumulation of such vesicles just inside the plasma membrane in muscle fibers of dysferlin-deficient mice. This is the first evidence of such a patching mechanism in muscle cells, Campbell said.

In collaboration with Paul McNeil from the Medical College of Georgia in Augusta, Campbell's group performed experiments that showed dysferlin mobilizes to damaged areas of plasma membrane in normal muscle fibers, and that when dysferlin-deficient muscle fibers are punctured with a laser beam, they're slow to seal the disrupted membrane.

In humans with LGMD2B and MM, "normal wear-and-tear to muscles caused by contraction is probably not repaired efficiently," Campbell said. "With time, the damage accumulates, and eventually leads to muscle degeneration and weakness." People with these diseases shouldn't fear exercise, but should probably avoid eccentric (lengthening) contractions, he said.

Dysferlin levels appear to be increased in muscle biopsies from boys with Duchenne muscular dystrophy, he said, suggesting that the muscle is trying to repair itself. To determine whether boosting dysferlin levels can slow Duchenne MD, he plans to genetically engineer mice with the disease to overproduce dysferlin.

Other proteins related to dysferlin might be used to compensate for its loss in LGMD2B and MM.

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'Naked DNA' looks safe for Duchenne, Becker MDs

French researchers say the 'naked DNA' method of gene transfer looks safe and promising for treatment of Duchenne and Becker muscular dystrophies (DMD and BMD).

In a recently completed study, researchers injected naked DNA containing the gene for dystrophin — the muscle protein defective in DMD and BMD — into an arm muscle in nine boys with either disease.

Naked (or plasmid) DNA is genetic material delivered without the use of viruses, which are effective at ferrying genes into cells but can provoke a potentially dangerous immune response. The technology is based partly on the work of Jon Wolff, an MDA grantee at the University of Wisconsin in Madison.

Scientists at Transgene, a company based in Strasbourg, France, headed the new study. They announced their results in June at a meeting of the American Society of Gene Therapy in Washington.

The nine boys were 15 years of age or older; six received a single injection of dystrophin DNA and three received two injections. Three weeks later, muscle biopsies revealed some dystrophin protein in the injected areas of half the boys who received one injection and of all the boys who received two injections. None of the boys appeared to have an immune response to dystrophin DNA or protein, and none experienced muscle damage from the injections.

In collaboration with Wolff and Mirus Corp., also in Madison, the French researchers plan another study in boys with DMD or BMD to determine if dystrophin DNA injected into the blood can make its way to muscle fibers.

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European study finds creatine beneficial in Duchenne, Becker MDs

Researchers in Belgium and France recently completed a study of 15 boys with Duchenne and Becker muscular dystrophies (DMD and BMD) and found that this dietary supplement slowed the progression of joint stiffness, improved strength and increased resistance to fatigue.

In addition, creatine appeared to increase bone density by 3 percent in those participants not using wheelchairs. No adverse effects of creatine were noted.

Magali Louis and colleagues published these results in the May issue of Muscle & Nerve. They gave eight boys 3 grams a day of oral creatine for three months, while seven boys with similar characteristics received a placebo (inactive substance). After two months during which no treatments were given (" washout" period), the groups were switched, so that everyone got creatine for one three-month period.

Although creatine's ability to increase muscle energy production is perhaps its most established function, these researchers and others suspect it may also have other muscle-promoting characteristics.

The authors of this study say the creatine may have worked through a signaling pathway in the cells that affected both muscles and bones. They also say participants who took creatine may have been more active, which is good for bone density.

A recent study of creatine in type 2 myotonic muscular dystrophy suggested that it may help with strength and pain relief in that condition.

MDA-supported studies of creatine in muscular dystrophy are being conducted in the United States, Canada and elsewhere. These trials are now closed, and data are being analyzed.

Creatine is also being studied in amyotrophic lateral sclerosis.

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Muscles of dogs given utrophin show benefit

Dogs with Duchenne muscular dystrophy (DMD) that received a miniaturized version of the gene for the muscle protein utrophin showed some benefit, but they required medication to suppress their immune systems, researchers say.

MDA grantees Kay Davies from the University of Oxford in England and George Karpati at Montreal Neurological Institute in Quebec were among those who performed the animal studies and published their results in the early May issue of Gene Therapy.

Kay Davies George Karpati
Kay Davies George Karpati

Utrophin has been considered a good substitute for dystrophin, the protein missing in humans and animals with DMD, because of its strong similarity to dystrophin and its theoretically better profile with respect to the body's ever-alert immune system.

Because those with DMD lack the dystrophin protein, their immune systems are likely to consider it "not self" and attack it. Utrophin, which those with DMD make naturally, would be considered "self" and not be attacked, experts have theorized.

In these experiments, dogs did require the immunosuppressant cyclosporine to benefit from the utrophin gene transfer, but the researchers say the immunologic problem may have been caused by the adenovirus used to insert the utrophin genes and not by the utrophin itself.

They conclude that using adeno-associated virus (AAV), which they say is less likely to provoke the immune system, would be a logical next step in utrophin studies.

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Scientists closing in on deleting excess DNA in myotonic dystrophy

Stephen Testa Jack Puymirat
Stephen Testa Jack Puymirat

A series of advances in targeting and deleting the excess DNA that leads to type 1 myotonic dystrophy (MMD1) has given hope to scientists and families fighting this and other diseases caused by excess DNA and its close chemical relative, RNA. (DNA forms the " instruction sheet" for RNA, which then becomes the guide for protein production.)

In November, investigators in the laboratory of Stephen Testa at the University of Kentucky were able to snip out the elongated tract of RNA in MMD-affected cells, using molecular scissors called ribozymes.

Then, in April, MDA grantee Jack Puymirat at Laval University in Quebec was on a team that destroyed excess RNA in MMD-affected cells using a strategy called antisense, which targets the elongated genetic material for destruction. His group showed that cells that got the antisense treatment became mature muscle cells in a more normal way and reacted more normally to insulin and sugar than did untreated cells.

In the May issue of Molecular Therapy, Puymirat's team published further studies, this time using ribozymes to break apart extra-long RNA that gets stuck in the cell nucleus in cells with the MMD mutation. They again found that, compared to untreated cells, the ribozyme-targeted cells functioned better with regard to sugar uptake and that they harbored fewer clumps of stuck RNA. These clumps, investigators believe, may cause much of the biochemical disruption underlying MMD.

Puymirat says human trials using antisense or a ribozyme could be possible in three to five years.

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Cardiac problems in DMD, BMD carriers seem to start after childhood

It's been known for many years that female carriers of Duchenne (DMD) and Becker muscular dystrophies (BMD) sometimes have symptoms themselves, particularly related to heart function. It's also been believed that these cardiac problems usually occur in adult life, if they arise at all.

To test this assumption, Melinda Nolan of Sydney Children's Hospital in Australia studied 23 girls between 6 and 15 years old who were carriers of DMD or BMD and had no symptoms.

Each girl had a physical exam, electrocardiogram and an echocardiogram, and the assumption was supported: No abnormalities were detected.

The researchers, who published their results in the February issue of Neuromuscular Disorders, conclude that it probably isn't necessary to subject symptom-free girls who may be DMD or BMD carriers to cardiac testing or even to carrier testing before their teenage years.

In another study, presented at the spring meeting of the American Academy of Neurology, pediatric neuromuscular disease specialist Katherine Mathews presented preliminary data from her multidisciplinary research team about adult carriers of DMD and BMD.

Mathews, who directs the MDA clinic at the University of Iowa Hospitals and Clinics in Iowa City, and her colleagues compared 12 apparently healthy carriers of DMD or BMD whose average age was 37, to nine noncarrier women with an average age of 43.

They found that the carrier women had significantly more abnormalities in cardiac function following exercise than did the control (noncarrier) group. They also had lower exercise tolerance, higher peak heart rates, and less blood pumped from the heart at rest and after exercise than did the noncarriers.

Mathews suggested that screening and in some cases treatment for cardiac dysfunction in adult carriers of DMD and BMD might improve their quality of life.

For more information about female carriers of DMD and BMD, see "But Girls Don't Get Duchenne — Or Do They?" Quest, December 1998.

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Improved treatments, tests for MG on the way

Myasthenia gravis (MG) is treatable with medication in most cases, but sometimes even the most tried-and-true medications don't seem to work or they have intolerable side effects. Donald Sanders, director of the MDA clinic at Duke University in Durham, N.C., recently completed two studies aimed at better treatment options for MG.

Sanders is investigating the possibility of treating MG with CellCept (mycophenolate mofetil), a drug originally developed to prevent immune rejection of transplanted organs. In a pilot trial, eight out of 12 MG patients improved after taking CellCept for several months.

At the spring meeting of the American Academy of Neurology, Sanders reported that, of 92 MG patients taking CellCept, improvement was seen in 67. Five people experienced complete remission.

"[CellCept] works in the majority of MG patients," he said. "Its advantages over other immunosuppressants are that it has a more rapid onset and fewer side effects."

In another study presented at the meeting, Sanders collaborated with Angela Vincent at the University of Oxford in England to examine patients with "seronegative" MG — those who have no ACh receptor antibodies detectable in their blood. About 15 percent of all MG cases are seronegative.

Out of 35 seronegative patients, Sanders and Vincent identified 10 with antibodies to MuSK, a protein that helps organize ACh receptors (AChR) on the muscle cell surface. Most of the MuSK-positive patients had a distribution of weakness different from that seen in AChR-positive MG. More remarkably, cholinesterase inhibitors (a common MG therapy) were ineffective in three of the patients, and removal of an immune system gland called the thymus (a widely practiced surgical treatment for AChR-positive MG) was ineffective in six.

Detection of MuSK antibodies "is going to be a major clinical tool in evaluating patients with seronegative MG" and might be useful for selecting effective treatments, Sanders said. Athena Diagnostics, a company in Worcester, Mass., is expected to come out with a commercially available test for the antibodies this year, he said.

For more on MG research and treatments, see "Managing Myasthenia" (May-June 2003).

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Hormone blocker works against SBMA in mice

A drug commonly used to treat prostate cancer might make an effective treatment against spinal-bulbar muscular atrophy (SBMA).

SBMA, also known as Kennedy's disease, arises from an expanded DNA tract in the gene for the androgen receptor, a protein that enables cells to respond to testosterone and other masculinizing hormones. Shortening of the same DNA tract has been linked to prostate cancer.

Drugs that block the actions of testosterone (produced by the testes) are effective against prostate cancer. So, after finding that castration reduces the symptoms of SBMA in mice, Gen Sobue at the Nagoya University Graduate School of Medicine in Japan reasoned that such drugs might also work against SBMA (see "Research Updates," December 2002).

In a report published online by Nature Medicine on May 28, Sobue and his colleagues found that the prostate cancer drug leuprorelin — made by Japanese-based Takeda Pharmaceuticals — decreased muscle wasting, and increased the mobility and lifespan of mice with SBMA. It also prevented the androgen receptor from entering the cell nucleus, believed to be a critical event in SBMA.

Another prostate cancer drug, flutamide, didn't block the receptor's entry into the nucleus, and didn't improve symptoms in the mice.

In prostate cancer patients, leuprorelin can cause reduced sex drive, impotence, hot flashes, osteoporosis and fatigue; in mice with SBMA, it causes infertility.

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New trials support idebenone use in Friedreich's

European researchers have completed two clinical trials that support using the antioxidant idebenone to treat cardiac problems in Friedreich's ataxia (FA).

FA is caused by a deficiency of the frataxin protein, which helps protect cells from oxidative stress. Hypertrophic cardiomyopathy — a thickening of the heart's muscular walls — is common in FA, and can lead to heart failure.

Recent trials in France have shown that idebenone can reduce the size of the heart in people with FA, but the trials have been criticized because they didn't compare idebenone to a placebo (an inert substance) or demonstrate any improvement in heart function (see "Friedreich's Ataxia," October-November 2002).

Results of the new trials were reported in the May 28 issue of Neurology.

In a yearlong trial in Italy, 14 people with FA received idebenone while 14 others received a placebo. The idebenone group began to show significant decreases in heart wall thickness after six months, but in the placebo group, those measures increased or stayed about the same.

Another yearlong trial in Belgium didn't involve a placebo, but found small improvements in heart wall thickness in eight people with FA. A technique called strain rate imaging showed that the treatment also reduced strain on the heart.

Kenneth Fischbeck of the National Institute of Neurological Disorders and Stroke called the results " encouraging." Though available in Europe, idebenone isn't yet approved by the U.S. Food and Drug Administration (FDA).

Fischbeck is leading a dose-escalation trial of idebenone against FA, in hopes of securing FDA approval. "We plan to determine whether high-dose treatment results in neurological as well as a cardiac benefit," he said.

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Fukuyama MD found in non-Japanese child

A Turkish infant with no known Japanese ancestry was recently found to have the autosomal recessive disease known as Fukuyama congenital muscular dystrophy, a disorder that so far has been seen only in people of Japanese origin. It results from mutations (changes) in the gene for the protein known as fukutin, located on chromosome 9.

The child, who was very severely affected and lived only 10 days, had a different fukutin mutation than that found in virtually all Japanese patients.

Fatma Silan and colleagues, who published their results in the March issue of Annals of Neurology, say this finding implies a need for considering fukutin mutations even in non-Japanese patients, and caution that the correct diagnosis can be important for genetic counseling.

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