STEPS TAKEN TOWARD UTROPHIN THERAPY FOR DUCHENNE

MDA researchers have made strides in developing an alternative strategy for replacing the function of the missing dystrophin protein in people with Duchenne muscular dystrophy (DMD). MDA grantees Bernard Jasmin of the University of Ottawa in Canada and Kay Davies of the University of Oxford in the United Kingdom found that the compound heregulin may stimulate muscle cells grown in the laboratory to produce more utrophin, a naturally occurring protein that may be able to substitute for dystrophin.

The researchers hope that, by taking advantage of utrophin, it may be possible to avoid some of the pitfalls that have slowed progress in developing gene therapy to replace dystrophin itself.

The progressive muscle weakness that occurs in DMD is due to the loss of dystrophin, which is important for maintaining the structural integrity of the muscle cells. One proposed therapeutic strategy is to use a disabled virus to deliver the DNA instructions for making dystrophin to the muscle cells. Armed with the correct instructions, the cells theoretically could begin producing the missing protein and grow stronger.

A major obstacle to this type of gene therapy is the possiblity that the immune system may attack both the delivery virus and the newly made dystrophin. (People with DMD typically have little or no dystrophin in their tissues, so their immune systems may not recognize dystrophin as a "friendly" molecule.) While MDA researchers continue to iron out the problems associated with supplying dystrophin via gene therapy, alternatives are being explored as well.

Utrophin is a promising substitute for dystrophin because its DNA and protein sequences are similar to those of dystrophin, and it binds to many of the same proteins that dystrophin likes to bind to. MDA-funded studies in animals have demonstrated that utrophin can do the same job as dystrophin if enough utrophin can get into the regions of the muscle membrane normally occupied by dystrophin. Further, utrophin is already in the body and is accepted by the immune system.

In studies of the cellular pathways that control the distribution and production of utrophin in muscle cells, Jasmin and Davies discovered that adding a compound called heregulin to muscle cells growing in petri dishes can stimulate them to produce more utrophin than they normally would. Heregulin is produced by both the nerve endings and the muscle cells. These findings were published in the March issue of the Proceedings of the National Academy of Sciences.

Earlier research showed that adding a compound called agrin, which is normally made by the nerve cell endings, also stimulated utrophin production. Identification of compounds that can increase the production of utrophin could potentially lead to a drug treatment for DMD.

MDA GRANTEES PIONEER MULTI-MUSCLE GENE TRANSFER TECHNIQUE

MDA-funded researchers Hansell Stedman and James Wilson at the University of Pennsylvania in Philadelphia are developing a technique that could eventually allow the transfer of therapeutic genes to many muscles with one injection.

Considered essential for the practical use of gene therapy to treat people with neuromuscular disorders, the technique -- if it proves feasible in humans -- should allow researchers to treat even deep muscles, such as the heart, with a single injection of a gene-carrying virus into the bloodstream.

Preliminary experiments in a hamster model of limb-girdle muscular dystrophy (LGMD), published in the April issue of Nature Medicine, demonstrate what Stedman calls a "proof of concept," meaning the researchers have shown that the technique is possible. However, many problems of practicality and safety must be addressed before the technique could be used in humans.

Until now, attempts to supply therapeutic genes to muscle cells have consisted of injecting the gene-carrying virus, or vector, directly into the muscle tissue. Although vectors delivered by this method in non-human studies have led individual muscle cells to make correct versions of defective proteins, usually only cells within a few millimeters of the injection site are treated (a region smaller than the size of a pencil eraser for each injection).

Stedman reasoned that the best way to effectively deliver a therapeutic vector to all of the muscles would be to take advantage of the body's own mechanism for delivering materials to the muscles: the bloodstream. Unfortunately, unlike most drugs, viral vectors are too large to pass freely across the walls of the blood vessels under normal circumstances.

To circumvent this problem, Stedman used a combination of the chemicals histamine and paperavine to make tiny temporary holes in the blood vessels of the animals so that the vector could pass out of the bloodstream.

"We're basically creating a temporary sieve," Stedman explains, "to allow the virus to leave the bloodstream and be taken up by the muscle cells."

Using this technique, Stedman was able to deliver a vector carrying the delta-sarcoglycan gene, which is defective in one form of LGMD, to the muscles of the entire hindlimb of delta-sarcoglycan deficient hamsters, with a single injection.

Unfortunately, the combination of chemicals used to promote the passage of the gene therapy vector from the blood vessels can trigger a life-threatening response similar to a severe allergic reaction. In the animal experiments, the technique could only be applied when a limb was isolated from the rest of the bloodstream with tourniquets because it is technically difficult to protect such small animals from shock.

Stedman, who is a surgeon, speculates that someday this technique could be performed in humans in an intensive care unit with the appropriate facilities to nurse people through the critical period when the chemicals are administered. In the meantime, Stedman is conducting experiments to establish the safest way to control and rapidly reverse the effects of temporary vascular leak.

Although the MDA-sponsored stage-one clinical trials for gene therapy in humans with LGMD will be restricted to intramuscular injections, MDA researchers are setting the stage for the future by tackling tough gene therapy problems now.

GENETIC BASIS FOUND FOR DOMINANT EMERY-DREIFUSS MD

A group of French, Canadian and Italian researchers led by Giselle Bonne of INSERM, France's National Institute of Health and Medical Research, have discovered the genetic cause of the autosomal dominant form of Emery-Dreifuss muscular dystrophy (EDMD). EDMD is a rare form of muscular dystrophy that causes slowly progressive muscle weakness, tendon contractures and potentially life-threatening heart problems.

At least three patterns of inheritance for EDMD are known: X-linked, autosomal dominant and autosomal recessive. In 1994, researchers discovered that mutations in the gene that codes for a protein called emerin are responsible for the more common X-linked form of the disease. (In X-linked inheritance, the gene that causes the disorder is carried on the X chromosome and is usually passed from a carrier mother to a son, who gets the disease.)

Now, the team led by Bonne has discovered the genetic defect responsible for a less common form of EDMD that arises when a single copy of the mutation is passed on to a child from either parent (autosomal dominant inheritance). A study of six families with autosomal dominant EDMD revealed that affected members all had mutations in the gene that codes for the protein lamin. This discovery was reported in the March issue of Nature Genetics.

Like emerin, which is missing or defective in X-linked EDMD, lamin is normally found in the membrane that surrounds the nuclei of muscle cells. Researchers aren't yet sure how the loss or dysfunction of these nuclear membrane proteins causes the symptoms of EDMD.

CAUSE FOUND FOR FORM OF NEMALINE MYOPATHY

MDA-funded research has led to the discovery that defects in a gene that codes for the protein nebulin are linked to the most common form of the congenital neuromuscular disorder known as nemaline myopathy.

Nemaline myopathy typically causes widespread weakness in the trunk and limb muscles and sometimes in facial muscles. More severe forms of the disease can lead to death from respiratory failure in the first few years of life. Muscle biopsies from people with nemaline myopathy reveal the presence of many abnormal rod-like particles in the muscle.

The disorder can be inherited in either an autosomal dominant fashion (caused by one copy of a defective gene contributed by either parent) or in an autosomal recessive pattern (two copies of the defective gene must be present to cause the disease, one contributed from each parent). Previously, researchers had linked defects in a gene for alpha-tropomyosin, a protein found in the contractile sliding filaments of muscle cells, to the less typical autosomal dominant form of the disease.

Now, MDA grantee Alan Beggs of Children's Hospital and Harvard Medical School, both in Boston, has pinpointed defects in a gene that produces nebulin as the cause of the more common autosomal recessive form of the disease. Nebulin is a large protein that's also found in the sliding filaments of muscle cells and may be required to help assemble these contractile elements.

In the future, researchers will try to discover whether defects in particular regions of the nebulin gene cause more or less severe forms of the disease -- information that may provide a basis for better genetic counseling.

The researchers' findings were published in the March issue of the Proceedings of the National Academy of Sciences.

FSHD STUDY SEEKS TWINS, OTHERS

MDA grantees Rabi Tawil and Denise Figlewicz at the University of Rochester (N.Y.) are looking for people with facioscapulohumeral muscular dystrophy (FSHD) to participate in a research study to determine the molecular cause of the disorder. The study involves one visit to Rochester to obtain blood and muscle samples (biopsies). Of particular interest are identical twins, only one of whom is affected by FSHD.

Contact FSHD study coordinator Lynn Cos at (716) 275-7680, or email lcos@mail.neurology.rochester.edu.

WEB SITE GIVES GENETIC, BIOCHEMICAL TEST SITES

Need a mitochondrial DNA mutation screen? Looking for a laboratory to calculate your free-versus-bound carnitine ratios? Try the World-Wide Biochemical Genetics Test List at biochemgen.ucsd.edu.

The free World Wide Web site, maintained by the University of California-San Diego, contains a database of laboratories that perform biochemical and genetic testing for many neuromuscular and other disorders. Multiple search parameters allow users to search by disease, test or laboratory to find information on specific tests. The database is particularly useful for those with metabolic or mitochondrial muscle disorders.

RESEARCHERS FIND DEFECTS CAUSED BY CALPAIN DEFICIENCY

Limb-girdle muscular dystrophy (LGMD) is a group of disorders that primarily cause weakness of the shoulder and pelvic regions. A subtype of LGMD called LGMD2A is caused by defects in the gene for a muscle protein called calpain 3.

Unlike other known proteins that cause muscular dystrophies when defective or missing, calpain 3 isn't a structural protein, but an enzyme. Whereas structural proteins contribute to the stability of cells, like steel girders in a building, enzymes help alter other molecules in the cell. Until recently, it wasn't known how a deficiency in calpain 3 could lead to the symptoms of LGMD.

New research from Gerard Lefranc of the National Center for Scientific Research in France shows that calpain 3 probably plays an important role in the way muscle cells deal with stress. When calpain 3 is present, it degrades a second protein that blocks self-preservation signals within the cell. When there is little calpain 3 in the muscle cell, the blocking protein builds up and the cell can't defend itself against stress. The result is that individual nuclei within a muscle fiber start to die, even though the muscle fiber doesn't appear to be damaged structurally.

Lefranc reports that dead nuclei aren't the most prominent feature of other muscular dystrophies examined, including Duchenne, Becker, facioscapulohumeral, and alpha- and gamma-sarcoglycan-deficient LGMD, suggesting that the mechanism of muscle injury in LGMD2A is unique among the muscular dystrophies. This finding points the way to developing treatments for LGMD2A designed to compensate for the lack of calpain 3 by upregulating the cell's ability to make protective proteins, or blocking the pathway by which nuclear death occurs.

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