QUEST Volume 11, Number 5, SEPTEMBER/OCTOBER 2004

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Chamberlain Team Achieves Widespread Dystrophin Delivery

Delivery of the gene for dystrophin, the protein needed in Duchenne muscular dystrophy (DMD), throughout the skeletal muscles of DMD-affected mice, has been achieved after a single injection of genes into a tail vein, researchers at the University of Washington in Seattle say.

The research team, which included MDA grantee Jeffrey Chamberlain as principal investigator, published its results in the August issue of Nature Medicine. The development may suggest an answer to a long-standing obstacle in gene therapy for muscle diseases: how to deliver a gene without injecting single muscles.

"We now have obtained a proof of principle that it is possible to deliver new genes bodywide to all the muscles of an adult animal," Chamberlain says.

Some modifications in standard gene transfer procedures apparently made widespread delivery possible. First, the researchers used a new, more effective viral vector, or delivery system, called type 6 adeno-associated virus, or AAV6.

Second, they also injected the mice with vascular endothelial growth factor (VEGF). This compound makes blood vessel walls more permeable, allowing the gene-carrying vector to move across them into muscle tissue.

Third, the researchers experimented with various types of promoters, or molecular "on switches," which tell a cell to start making protein from a gene's instructions.

They found that one particular switch, known to turn on genes only inside muscle cells, was safe in that it didn't elicit an unwanted immune response to the injections. But it failed to turn on dystrophin production in the diaphragm or heart.

Other promoters can turn on production in more cell types, including the diaphragm and heart, but may be more likely to arouse the immune system. Chamberlain says the team is seeking a promoter that activates production in all targeted muscles without causing the immune system to do battle.

The gene-treated mice had muscles that were more resistant to injury, and their blood creatine kinase levels were 50 percent below those of untreated mice, indicating a reduction in muscle damage.

The mice continued to produce dystrophin for at least eight weeks after treatment with the dystrophin gene and the more restricted promoter. Chamberlain notes that even longer persistence has been observed since these experiments were completed.

The next goal is to test the AAV6 vector for safety in humans, he says.

'LARGE' Protein Corrects CMD Cells

A protein known as LARGE may have the capacity to restore normal structure and function to cells in several forms of muscular dystrophy, says an MDA-supported research group that published its findings in the July issue of Nature Medicine.

Kevin Campbell

MDA grantees Rita Barresi, Steven Moore and Kevin Campbell, a Howard Hughes investigator, all at the University of Iowa in Iowa City, were on the study team, which also included researchers from Canada, Sweden and Japan.

The investigators found that LARGE, an enzyme that attaches sugar molecules to proteins (called a glycosyltransferase), corrects the molecular defect in several muscular dystrophies in which the attachment of sugars (glycosylation) to a protein in the cell membrane is faulty.

The muscular dystrophies that result from these glycosylation defects include several forms of congenital MD Fukuyama MD, muscle-eye-brain disease, Walker-Warburg syndrome and types 1C and 1D. In several of these, both muscle and brain cells are affected.

Another disorder, type 2I limb-girdle MD, is also caused by this type of glycosylation defect.

In each case, an enzyme that's responsible for attaching sugar molecules to alpha-dystroglycan, a cell membrane protein, is missing or abnormal. The result is less than adequate attachment of the sugars, which leads to serious cell damage.

Most of the other muscular dystrophies, Campbell notes, are caused by defects in proteins that form parts of the cells physical structure. It's harder to replace or compensate for those, he says.

"When you have enzymatic activity, you don't have to produce a lot of the protein; a little will probably do," Campbell noted.

A. In normal muscle cells, clusters of proteins are nestled in the cell membrane and connect to the inside of the cell via the dystrophin protein and to the outside of the cell via the alpha-dystroglycan and laminin proteins. Sugar molecules on alpha-dystroglycan allow laminin to attach to it and to the basal lamina, a supporting structure that surrounds each muscle cell. B. In cells affected by a glycosylation disorder, there aren't enough sugar molecules on alpha-dystroglycan to allow laminin to attach to it. Without laminin, the basal lamina is disrupted. C. When LARGE was added to normal or abnormal cells, it increased the number of sugar molecules on alpha-dystroglycan. Laminin attached normally, even in the abnormal cells, and the structure of the basal lamina was restored.

The researchers studied mice that had a defect in the gene for LARGE. Giving the mice a working version of that gene via a viral delivery system returned the biochemistry, structure and function of their muscle fibers nearly to normal, the team founds.

They then added the LARGE gene to cells from people with Fukuyama MD, muscle-eye-brain disease and Walker-Warburg syndrome, and found that adequate numbers of sugar molecules were attached to the alpha-dystroglycan protein. Further study of some of the cells revealed that the laminin protein, which must attach to the sugars on alpha-dystroglycan and can't "dock" without them, was properly attached.

The authors call LARGE "an attractive target for the design of therapies intended to manipulate alpha-dystroglycan glycosylation." They were pleasantly surprised that the protein worked in cells affected by a variety of genetic defects, not just defects in LARGE itself.

"We're looking at testing different compounds in cells to see if we can increase LARGE's activity," Campbell says. He notes that the only people who lack functional LARGE are those with type 1D CMD. They have the LARGE protein but apparently not enough of it to compensate for their other enzymatic defects.

That's good news, he says, because it means adding LARGE is unlikely to generate an undesirable immune response.

Campbell's team is pursuing several strategies, including a gene delivery system for LARGE that could potentially be used in people.

Loss of Myostatin Builds Muscle in Child

A boy born without the ability to produce the protein myostatin has given scientists important clues that could lead to development of new treatments for a variety of muscle-wasting diseases, suggests a case study in the June 24 issue of the New England Journal of Medicine.

Markus Schuelke of Charite-University Medical Center in Berlin and colleagues, including MDA grantee Kathryn Wagner at Johns Hopkins University in Baltimore, describe a 4-year-old boy who was born with a mutation in both copies of the gene for myostatin, resulting in a complete loss of the myostatin protein. The child is extremely well-muscled and strong (able to hold up two 6.6-pound weights in his outstretched arms) and has no apparent ill effects from the abnormality.

Natural defects in the myostatin gene have been identified previously in animals, including Belgian Blue cattle that appear to be "double-muscled," but this child is the first human in which the mutation has been found.

Myostatin normally acts to slow muscle growth, and its absence allows for unusually large muscles. Researchers have long wondered if blocking myostatin might represent a useful approach to treating muscular dystrophy, and this report has boosted support for this strategy.

Mice lacking the myostatin gene (left) are bigger and more muscular than mice with the gene.

Wagner's research has shown that the loss of the myostatin gene leads to much milder disease in mice bred to have a disease like Duchenne muscular dystrophy. And MDA grantee Tejvir Khurana of the University of Pennsylvania in Philadelphia demonstrated that the effects of DMD in mice can be reduced by administering antibodies (proteins produced by the immune system) that block myostatin. (See "Research Updates," February 2003.)

MDA is funding Wagners group to further explore the potential for developing a muscular dystrophy therapy based on blocking myostatin.

Clinical Trials and Studies

MDA Plans Network to Facilitate Trials of MD Treatments

Richard Moxley

Some 35 neuromuscular disease experts, biotechnology industry representatives and government officials gathered in Tucson, Ariz., near MDA's national headquarters in June to lay plans for a large-scale network of institutions that will test potential treatments in muscular dystrophy, particularly the Duchenne form.

The network, which will likely allow for centralized data collection and sharing, is being developed in anticipation of an increased number of trials of experimental therapies. Such a network could also help researchers learn how to better manage medical complications of these diseases, including breathing and heart problems.

Susan Iannaccone

"MDA investigators are working hard to translate new findings in the lab into therapies that will benefit those we serve," MDA Director of Research Development Sharon Hesterlee says. "We want to make sure they have all the tools they need to accomplish these goals."

Susan Iannaccone, a co-director of MDA's clinic at the University of Texas Southwestern Medical Center in Dallas, and Richard Moxley, director of the Neuromuscular Disease Center at the University of Rochester (N.Y.) Medical Center, co-chaired the conference.

New Jersey Company Testing Drug for DMD Mutations

PTC Therapeutics of South Plainfield, N.J., announced in July that it was beginning a phase 1 trial to evaluate the safety of its experimental compound, called PTC124, in healthy volunteers. If all goes well, the company says, it will test the drug in people with Duchenne muscular dystrophy (DMD) in 2005.

PTC124, like the antibiotic gentamicin, appears to permit cellular machinery to "read through" mutations that stop production of dystrophin protein molecules before they're completed. Some 15 percent of boys with DMD are thought to have this type of mutation.

The company says the drug differs in chemical structure from gentamicin and is likely to pose fewer risks.

H. Lee Sweeney

H. Lee Sweeney, an MDA grantee at the University of Pennsylvania in Philadelphia, who's studying stop codon read-through in DMD, is "extremely optimistic" about PTC124.

"It looks like a real drug, and it looks like it's going to work, at least in some of the kids," he says.

Sweeney is a member of MDA's Translational Research Advisory Committee and serves on PTC Therapeutics Scientific Advisory Board.

The "read-through" strategy may be superior to gene therapy for this type of mutation because it doesn't pose the challenges of delivering genes to muscle cells, alter recipients genes or employ viruses.

(For information about a study of gentamicin in DMD, contact Cheryl Wall at Ohio State University at (614) 293-9016 or wall.49@osu.edu.)

PTC124 seems to allow cells to "read through" stop signals (stop codons) and make full-length dystrophin molecules.

 

Genzyme's Pompe's Studies Progress

Three studies in acid maltase deficiency (AMD, or Pompe's disease) being conducted by Genzyme of Cambridge, Mass., with support from MDA, are progressing on schedule, the company says.

In AMD, the acid maltase (also known as acid alpha-glucosidase) enzyme is missing or deficient. Genzyme has designed a replacement enzyme called Myozyme.

Two studies of Myozyme in infantile-onset AMD have reached full enrollment, as has a study designed to observe the course of late-onset AMD. Some participants in the latter study will have the opportunity to enroll in a treatment study expected to begin in mid-2005.

In addition to these studies, Genzyme is making Myozyme available to babies with infantile (within the first year) onset of Pompe's disease who meet several specific criteria. This expanded access program is described at www.clinicaltrials.gov, a Web site of the National Institutes of Health.

Recruitment for an expanded access program in late-onset Pompe's disease has been suspended after the number of potential participants exceeded Genzyme's resources. The company hopes to resume enrollment at some point. Those already enrolled will continue to receive the investigational enzyme.

Genzyme encourages physicians who have patients of any age with AMD to participate in a company-sponsored disease registry. For information, go to www.Pomperegistry.com or www.pompe.com.

U.S. residents can contact Genzyme at medinfo@genzyme.com, (800) 745-4447 or (617) 768-9000. Europeans can e-mail eumedinfo@genzyme.com or call 31-35-699-1499.

Two Drugs Being Tested in Youngsters With SMA

Results of a small pilot trial of riluzole (brand name Rilutek) in spinal muscular atrophy (SMA) suggest that the glutamate inhibitor might confer a survival benefit.

A larger trial of riluzole in type 1 SMA is being conducted by the clinical trials group AmSMART (American SMA Randomized Trials), and funded by the National Institutes of Health. The study is open-label, meaning theres no placebo group.

Participants must be less than 2 years old at enrollment, have type 1 SMA, be unable to sit alone for more than 10 seconds when placed, and meet other study criteria. Study sites are in Palo Alto, Calif.; Rochester and St. Paul, Minn.; St. Louis; Cincinnati; Portland, Ore.; Philadelphia; Dallas; Salt Lake City; Richmond, Va.; and Toronto.

For details, contact Karen Rabb, AmSMART project coordinator, at (800) 421-1121, ext. 7829, or karen.rabb@tsrh.org.

Riluzole is prescribed in amyotrophic lateral sclerosis, and has been shown to increase survival modestly. In both SMA and ALS, muscles weaken when motor neurons (muscle-controlling nerve cells) die.

In another avenue, Stanford University in Stanford, Calif., is testing hydroxyurea in SMA. In lab experiments, the drug has been shown to increase the production of working SMN, the protein thats needed in SMA.

One trial involves infants with type 1 SMA whose symptoms began before age 6 months; the other is for children 16 months to 10 years old with either type 2 or type 3 SMA. Some participants will receive the drug, and others will receive a placebo.

For information on both trials, contact Tony Trela, study coordinator, at (650) 498-7658 or sma@stanfordmed.org.

Utah Project Seeks Rare Dystrophin Flaws

The Utah Dystrophinopathy Project is attempting to define precise genetic changes (mutations) in the gene for the dystrophin protein that underlie Duchenne and Becker muscular dystrophies in boys and young men who don't have the more common mutations seen in these disorders.

Neurologist and neurogeneticist Kevin Flanigan at the University of Utah in Salt Lake City heads the project, which has funding from the National Institutes of Health. Using a method they described in the April 2003 issue of the American Journal of Human Genetics, Flanigan and colleagues will directly sequence the entire dystrophin gene of each participant.

Direct sequencing allows detection of less common types of dystrophin gene mutations, such as premature stop codons, missense mutations, and small insertions and deletions that alter how the cell "reads" the gene to form a protein. (Missing or inadequate dystrophin is the usual cause of DMD and BMD.)

Theres no cost to families to participate in the study.

The researchers will also try to correlate the dystrophin mutations with disease severity. Participants in this part of the study will need to visit one of three study centers, in Salt Lake City, St. Louis or Columbus, Ohio.

Details can be found at dystrophy.genetics.utah.edu. Or contact study coordinator Kim Hart at khart@genetics.utah.edu or (801) 585-1299.

Is There a Test for My Disease?

Want to know if there's a genetic (DNA) or protein-based test for your disorder? Chances are, if the test exists, you'll find it in one of these two places.

Athena Diagnostics in Worcester, Mass., is a commercial laboratory that maintains a Web site with a searchable database at www.athenadiagnostics.com.

The National Institutes of Health in Bethesda, Md., in conjunction with the University of Washington in Seattle, operates a testing database at www.genetests.org, which lists tests available all over the world, including those only available on a research basis. Click on "Laboratory Directory" and search by disease. "Educational Materials" has a helpful summary of genetic testing.