Research Updates May-June 2006

Article Highlights:

The latest updates on research as of April 2006

by Quest Staff on May 1, 2006 - 3:05pm

QUEST Vol. 13, No. 3

MDA begins first U.S. Duchenne gene therapy trial

Jerry Mendell injects a boy with DMD.
Andrew Kilbarger of Lancaster, Ohio, received the first injection in MDA’s gene therapy trial for Duchenne muscular dystrophy March 28. Here Andrew, 8, receives comfort from his mom, Julie, while Jerry Mendell marks Andrew’s arm in preparation for the injection. Photo by Columbus Children’s Hospital

The firstU.S.-based human gene therapy trial in children with Duchenne muscular dystrophy (DMD) began in late March at Columbus (Ohio) Children’s Hospital.

This MDA-supported trial is the culmination of years of research funded by the Association, including a $1.6 million grant to Asklepios BioPharmaceutical of Chapel Hill, N.C., and additional grants to molecular geneticist Xiao Xiao at the University of Pittsburgh and Jerry Mendell at Columbus Children’s Research Institute. The trial will test the safety of the procedure.

The very large gene for the muscle protein dystrophin, missing in boys with DMD, has been miniaturized in such a way that it retains its essential functions in muscle cells but can fit inside an apparently safe and effective transport vehicle.

The vehicle, developed principally by Xiao Xiao and R. Jude Samulski, a virologist at the University of North Carolina and Asklepios, is an adeno-associated virus (AAV).

After being inserted into the AAV shells, the genes are injected into the biceps or another suitable muscle in six boys with DMD.

If the results prove the safety of the procedure, the next step will be further tests to see if the therapy is effective in producing dystrophin in muscle cells of boys with Duchenne MD.

The researchers aren’t seeking additional participants.

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Mouse studies bode well for Exon skipping in DMD

Qi Long Lu
Qi Long Lu

MDA grantees Qi Long Lu at Carolinas Medical Centerin Charlotte,N.C., and Stephen Wilton at the University of Western Australia in Perth, were on a research team that recently achieved encouraging results with a strategy known as exon skipping to repair the mutated dystrophin gene in mice with a disease resembling Duchenne muscular dystrophy (DMD).

The investigators, who announced their results in the February issue of Nature Medicine, achieved widespread production of the dystrophin protein, which is absent in DMD-affected muscles, and improvement in muscle function. They injected a morpholino antisense oligonucleotide (AON) compound into a vein.

The morpholino AON causes cells to remove (skip) the mutation in the dystrophin gene and to splice together the surrounding genetic instructions. The exon-skipping technique can be targeted toward several types of mutations seen in DMD.

AONs act at the RNA stage of protein synthesis. In this phase, the processing normally removes some sections, known as introns, and keeps others, known as exons. Therapeutic AONs cause cells to treat mutated exons as if they were introns.

The investigators say three consecutive intravenous injections of a specific AON resulted in dystrophin production at about 30 percent of normal levels in the muscles of the abdomen, upper legs and rib areas. Lower levels were present in the front lower leg muscles and diaphragm, and no dystrophin was detected in the heart.

After seven weekly injections, the mice produced up to 50 percent of the normal level of dystrophin in some leg muscles, and 10 percent to 20 percent of the normal level in their rib, abdominal and other leg muscles.

The dystrophin-producing muscle cells showed restoration of a normal protein cluster at the cell membrane and other signs of cellular normalization. When lower leg muscles were tested, they showed greater force generation than did the same muscles in untreated dystrophin-deficient mice, although it wasn’t as great as that of normal mice.

“Our results show that morpholino-mediated antisense therapy can achieve and maintain therapeutic levels of dystrophin throughout the body musculature and provide a realistic option for the treatment of the majority of DMD,” the authors say in their publication.

Qi Lu said most DMD-affected boys have mutations in the functionally noncritical region of the dystrophin gene, where the tested therapy would work. “It has been estimated that no more than 20 antisense oligos are required to treat the majority of the patients in whom this would be applicable.”

Lu cautioned that much still needs to be done to improve the efficiency of the dystrophin production, especially in the heart muscle, and that the potential of the new AON strategy remains to be tested in clinical trials. Trials in DMD patients are on the drawing board in the United Kingdom. (Go to and enter “Duchenne muscular dystrophy” into the search box.)

An Italian research team published similar results in the March 7 issue of Proceedings of the National Academy of Sciences. The team delivered antisense genes to dystrophin-deficient mice via intravenous injection and also got widespread dystrophin production and significant functional improvement.

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More genes implicated in Charcot-Marie-Tooth

Michael Shy
Michael Shy

Charcot-Marie-Tooth (CMT) disease is associated with mutations in dozens of genes. More genetic causes have been implicated nearly every year since the early 1990s, and two new disease-causing genes have recently been identified.

CMT is a disorder of the peripheral nerves, which run between the spinal cord and the periphery of the body, and allow movement and sensation.

Broadly speaking, CMT is divided into type 1, which results primarily from defects in the insulating coating, myelin, that surrounds nerve fibers (axons); and type 2, which results primarily from defects in the axons themselves. Intermediate types are a mixture of axonal and myelin degeneration.

Since both myelin and nerve fibers contain multiple proteins, it’s not surprising that many genetic flaws can lead to CMT.

In the February issue of Nature Genetics, Albena Jordanova at the University of Antwerp (Belgium) and Sofia (Bulgaria) Medical University, and colleagues, describe three mutations in the gene for the protein YARS in three unrelated families with an intermediate form of CMT known as DI-CMTC. Jordanova has had recent MDA support for CMT research.

YARS belongs to a family of proteins that are instrumental in the translation of final genetic instructions into protein molecules. It may play a special role in nerve fibers.

“What is fascinating about the genetic results, is that the disease is caused by a ‘housekeeping’ gene,” says Florian Thomas, professor of neurology, virology, and microbiology and immunology at Saint Louis University and a member of the study team. “That term is used to describe genes that ‘keep house’ and that have as wide a use in the body as, for instance, Ajax does in the household.

“So the scientific question is, why does this CMT gene cause only a peripheral neuropathy and not total body dysfunction?” Thomas has some ideas but, he says, “The story is not finished.”

In another study, published in the February issue of Annals of Neurology, Stephan Zuchner, at Duke University in Durham, N.C., and colleagues, identified mutations in the gene for the protein mitofusin 2 as a cause of a form of CMT that involves degeneration of the optic nerves as well as the usual losses in movement and sensation.

Known as hereditary motor and sensory neuropathy type 6, this condition causes a severe, early-onset peripheral nerve disorder, with vision impairment later in life. (In this study, six out of 10 patients with vision loss experienced recovery to normal or nearly normal vision levels, which the researchers attribute to probable nerve fiber regeneration.)

The research team included Vincent Timmerman and Jordanova at the University of Antwerp (Belgium), and Michael Shy at Wayne State University in Detroit, all of whom have current or recent MDA funding for CMT research. They studied six families and identified six different CMT-causing mutations in the mitofusin 2 gene.

Mitofusin 2 has previously been found to cause CMT2A, an axonal form of CMT that only rarely affects the optic nerves. The protein is thought to affect the behavior of the mitochondria, the energy-producing parts of cells.

Shy, a neurologist and a professor of molecular medicine and genetics, sees rare and common forms of peripheral nerve disease at the CMT clinic he directs at Wayne State. He has MDA funding to develop gene therapy for CMT.

“My interest came from the genetic and biological side,” he says, “but you learn [about daily care issues] as you go, by seeing people.”

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Protein variant may lead to myasthenia gravis

According to a report in the February issue of Annals of Neurology, a variant form of the gene for the protein PTPN22 may increase susceptibility to at least one type of myasthenia gravis (MG). PTPN22 is thought to have regulatory functions in the immune system.

In MG, antibodies attack Ach receptors, where signals from nerve cells are received.
In MG, antibodies attack Ach receptors, where signals from nerve cells are received.

In MG, an autoimmune disease, the immune system mistakenly attacks muscle cells and interferes with their ability to receive signals from nerve cells. About 90 percent of MG patients make antibodies that attack the muscle cells’ Ach receptors, where chemical nerve signals normally land.

In December, Torkel Vang at the Burnham Institute in La Jolla, Calif., and colleagues reported that the PTPN22 variant may predispose people to autoimmune disease in general (see “Research Updates,” March-April).

Now, Claire Vandiedonck, at INSERM-Paris-Descartes University in France, and colleagues have found a strong association of the variant with one type of MG.

The investigators studied 470 people with MG and 296 without the disease. They categorized the MG patients according to whether or not they had a thymus tumor (thymoma); and whether they were producing antibodies to the protein titin.

Of the 293 people studied who had MG but didn’t have a thymoma or titin antibodies, 80 (13.7 percent) had the suspect PTPN22 variant. In two other MG groups, about 10 percent had it; and in the non-MG group, it was there in only 7.4 percent.

The authors say their finding supports the idea that MG is more than one disease and provides a valuable lead for further investigations.

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Membrane damage added to FSHD picture

Robert Bloch
Robert Bloch

A research group coordinated by Robert Bloch, an MDA grantee at the University of Maryland in Baltimore, has added more pieces to the puzzle of facioscapulohumeral muscular dystrophy (FSHD).

Bloch and colleagues, who published their results in the February issue of Annals of Neurology, found unusual diagonal structures in the membranes of muscle cells taken from people with FSHD, as well as a marked increase in the gap between each cell and its surrounding membrane. Both changes were found in all nine FSHD biceps and deltoid muscle samples (although they weren’t present throughout each sample), with none found in samples from people without FSHD.

The researchers say the abnormalities are likely to interfere with force transmission in the affected muscles. They don’t know if, or how, these abnormalities are related to a recent finding that the FRG1 protein is overproduced in FSHD (see “Research Updates,” March-April) or to other genes in the FSHD region of chromosome 4.

“It remains unclear whether the basic defect in FSHD resides in a [membrane]-associated protein or whether the observed changes at the [membrane] are secondary to a primary defect that lies elsewhere,” the authors say.

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