Research Updates

Weekend Prednisone to Be Tested in DMD

In children with Duchenne muscular dystrophy (DMD), the corticosteroid drugs prednisone (widely available in the United States) and deflazacort (only available outside the United States) have been shown to prolong walking ability and maintain respiratory function for longer than placebos (inert substances) in several clinical trials since the 1970s. However, these drugs often extract a high price in side effects.

Recent evidence gathered from American and European studies and presented at November’s National MDA Clinic Directors Conference suggests that different dosage schedules of prednisone may maximize the desired effects while minimizing the side effects.

Now, pediatric neuromuscular disease specialist Diana Escolar at Children’s National Medical Center in Washington is joining with other investigators to test moderate-dose, daily prednisone compared with high-dose, weekend-only prednisone in boys with DMD. The study, which began in January, is supported by MDA and the National Institutes of Health.

Participants must have a clear diagnosis of DMD; be between 4 and 11 years old; be able to walk without assistance; never have been exposed to corticosteroid medications; and have stayed away from certain dietary supplements or herbal medications for at least three months prior to the trial. Study details and requirements will be further discussed at the time of application.

Other study sites include Washington University in St. Louis and Texas Scottish Rite Hospital for Children in Dallas.

For more information, contact CINRG study coordinator Erik Henricson at (202) 884-3813 or ehenricson@cnmcresearch.org, or visit www.cnmcresearch.org/cinrg/index.jsp.

Blocking Myostatin Proves Beneficial in DMD Mice

New research shows that using a drug or small molecule to inhibit myostatin — a secreted protein that inhibits muscle growth — might one day be an effective treatment for Duchenne muscular dystrophy.

In a new study published online in November in the Annals of Neurology, MDA grantee Kathryn Wagner of Johns Hopkins University in Baltimore shows that loss of the myostatin gene increases muscle mass and strength in mice with DMD.

In another study published in the Nov. 28 issue of Nature, Tejvir S. Khurana, of the University of Pennsylvania and Pennsylvania Muscle Institute in Philadelphia, reports that treatment with a myostatin-blocking antibody has similar effects on mice with DMD. Antibodies are the Y-shaped missiles that immune cells use to attack bacteria and other foreign substances.

For her study, Wagner and Hopkins geneticist Se-Jin Lee bred a strain of mutant mice lacking the myostatin gene with mice with DMD, and found that the offspring had significantly larger, stronger muscles than mice with DMD and with an intact myostatin gene. The double-mutant mice also had less fibrosis (the replacement of muscle by fat and connective tissue).

For his study, Khurana collaborated with Wyeth Pharmaceuticals of Cam-bridge, Mass., whose scientists developed the antimyostatin antibody. Khurana gave mice with DMD weekly injections of the antibody into the abdominal cavity, before they showed signs of the disease. After three months, they had increased muscle mass and strength in their leg muscles, and less degeneration and fibrosis of their respiratory muscles, when compared to untreated mice. They also had lower blood levels of creatine kinase (CK), a protein that leaks out of damaged muscle. (For reasons that aren’t clear, in Wagner’s study, deleting the myostatin gene didn’t lower CK levels in the mice.)

“There’s a real possibility for moving forward in clinical trials with [myostatin

Mice lacking the myostatin gene (left) are bigger and more muscular than mice with the gene.
inhibition],” Wagner said. “There are other ways to inhibit myostatin that might be pursued, but antibodies are the ones furthest down the pipeline.”

But Khurana said the research is years away from clinical trials, noting that the myostatin-blocking antibodies he used were made in mouse cells to target mouse myostatin. Specific human antibodies would have to be made before proceeding to clinical trials, he said.

He also cautioned that long-term inhibition of myostatin might not be safe for youngsters with DMD, since the protein regulates muscle progenitor cells (immature cells that form muscle).

“There’s a very real risk that if you block myostatin in humans in an unregulated and unmonitored fashion, you could deplete the cells at an accelerated rate and actually worsen the [muscular dystrophy],” he said.

Khurana’s group plans to address this issue in further studies of the mice, and to test myostatin blockade in dogs with DMD — a high benchmark for assessing effectiveness and safety since the dogs are larger and have a more severe disease than the mice. He also plans to test the strategy in animal models of limb-girdle muscular dystrophy.

Wagner plans to “switch off” the myostatin gene in mice with DMD at different time points to determine if the treatment is still effective at late stages of the disease.

Spinal Muscular Atrophy: Progress in Research and Care

Creatine Under Study

Eighty children and adolescents with spinal muscular atrophy (SMA) will be part of a study directed by MDA grantee Susan Iannaccone, a pediatric neurologist at Texas Scottish Rite Hospital for Children in Dallas. The multicenter study will enroll participants until March 1.

Susan Iannaccone

In 2000, with support from the National Institutes of Health, Iannaccone and colleagues began developing precise methods to measure strength, function and quality of life in SMA, with support from MDA for specialized measuring equipment. Team members described their methods at the National MDA Clinic Directors Conference in November.

Now, the investigators are studying whether or not the dietary supplement creatine may benefit children with SMA. Creatine purportedly increases energy production in cells, particularly those of the muscles and nervous system.

For more information, contact Karen Rabb, project coordinator in Dallas, at (800) 421-1121, ext. 7829, or karen.rabb@tsrh.org.

Vigilance With Feeding, Respiratory Care Urged

At the MDA clinic directors conference, guest speaker Peter Schochet, a pediatric pulmonary specialist at Children’s Medical Center of Dallas, emphasized the need to vigilantly support nutrition and maintain respiratory function in children with SMA.

He noted that children with SMA can have “maladaptive feeding patterns,” including overeating out of depression, boredom or not enough physical activity for their intake; or undereating, because of fear of inhaling food into the lungs or choking on it. Schochet said that, in the case of undereating, a gastrostomy tube may make family mealtimes more pleasant and help the child take in needed calories.

Schochet also stressed the importance of combining mucus clearance by manual or mechanical methods with ventilation strategies to treat the respiratory impairment associated with severe SMA.

Prompt treatment and prevention of infections is important as well, he noted, recommending vaccinations against pneumococcal pneumonia and other infectious agents.

New Function Attributed to SMN Protein

A study in the Nov. 29 issue of Science holds new insights into the function of survival motor neuron (SMN), the protein deficient in SMA. The findings could help researchers understand how the disease kills muscle-controlling nerve cells (motor neurons).

SMN plays a critical role in processing RNA — the intermediate between DNA and proteins. Some parts of RNA don’t contain any protein-coding information, and those parts are removed in a cut-and-paste process called splicing.

Previous studies have shown that SMN helps assemble a component of the splicing machinery, called an snRNP. But under some conditions, snRNPs can assemble without SMN, leading researchers to wonder why SMN is needed at all.

The new study shows that SMN controls the specificity of snRNP assembly and that in its absence, abnormal snRNPs can form. The authors, led by Gideon Dreyfuss of the Howard Hughes Medical Institute and the University of Pennsylvania in Phil-adelphia, suggest that these abnormal snRNPs might form in people with SMA and interfere with RNA splicing in their motor neurons.

Ultrasound Might Help in Gene Therapy

MDA-funded researchers have developed a new gene therapy approach that uses ultrasound to improve the uptake of genetic material into muscle cells.

Getting a therapeutic gene into muscle is a tricky business (see “From Steroids to Stem Cells.”). In the most widely tested approach, the DNA is packaged into a virus capable of infecting muscle cells. Unfortunately, many viruses trigger an immune response that can destroy the therapeutic gene.

Other, stealthier viruses, like the adeno-associated virus, are too small to accommodate large genes like the one encoding dystrophin, the protein missing in Duchenne muscular dystrophy.

To avoid these problems, George Karpati and Basil Petrof, of McGill University in Montreal, have been experimenting with another gene delivery vehicle called a plasmid or naked DNA, a piece of circular DNA derived from a bacterium. Plasmids can evade the immune system, and some are large enough to hold the dystrophin gene. Unfortunately, they don’t penetrate muscle cells as well as viruses do.

In the November issue of Molecular Therapy, Karpati and Petrof describe how they combined plasmid delivery with ultrasound — a technique commonly used in medicine to produce images (sonograms) of blood vessels, organs and, of course, developing babies. Ultrasound works by bouncing soundwaves off solid or fluid-filled tissues, and though it’s considered harmless, it can create small pores in the membranes that surround cells. Karpati and Petrof reasoned that plasmids might be able to slip through these pores.

In experiments on mice, they injected plasmids carrying a bacterial gene into a leg muscle, and then applied ultrasound over the muscle. In mice with DMD, the ultrasound (combined with a chemical used to enhance sonograms) increased levels of the bacterial gene by about 22-fold compared to plasmid alone. Importantly, the ultrasound didn’t appear to damage the muscle.

Karpati and Petrof say that ultrasound could be used to improve intramuscular gene delivery. And, because it can create small breaches in blood vessels, it might also hold promise for systemic gene delivery.

IVIG Future Uncertain in Myasthenia Gravis

MDA researchers who recently studied the use of intravenous immunoglobulins (IVIG) in myasthenia gravis (MG) in 15 people say they can’t tell whether the treatment is beneficial in the disease.

IVIG treatment consists of infusing immunoglobulins, which are proteins made by the immune system, into the bloodstreams of those with immunologic disorders. The reason the treatment sometimes works in diseases such as MG, in which the immune system mistakenly attacks the body’s own tissues, isn’t clear, but the proteins appear to “confuse” the immune system.

The multicenter, MDA-supported study was terminated early, the researchers say, because they were unable to obtain enough IVIG to treat an adequate number of participants. They needed 88 patients to make the study statistically significant but only had 15, the researchers write in the October issue of Muscle & Nerve.

Earlier studies had suggested that treatment with IVIG might have benefit in MG and is probably safe. In this study, two patients developed severe headaches, but the treatment was otherwise well tolerated.

Researchers Snip Out Genetic Flaw in Type 1 Myotonic Dystrophy

Using a laboratory-engineered ribozyme, Stephen Testa and colleagues have repaired the genetic defect that underlies type 1 myotonic dystrophy.

Investigators in the laboratory of MDA-supported Stephen Testa, in the Department of Chemistry at the University of Kentucky in Lexington, have corrected the genetic flaw that underlies type 1 myotonic dystrophy (MMD1) in cells that carry the flaw. They published their results in the Nov. 27 online edition of the journal Biochemistry.

Using a laboratory-engineered chemical based on the biological substances known as ribozymes, the scientists were

Stephen Testa
able to home in on an elongated segment of the genetic material RNA (the flaw that leads to MMD1) and remove it from cells grown in containers. The reaction was very specific, raising hopes that it could one day be given to people with MMD without interfering with normal RNA or other cellular material.

The scientists say the treatment wouldn’t be permanent, since MMD-affected cells would continue to produce elongated RNA molecules that would then need to be shortened.

“Although many questions need to be addressed, the results so far are extremely encouraging,” Testa said. “We have shown, for the first time, the potential to develop biomolecules that can simply excise genetic mutations that lead to diseases.”