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Research Updates

Research Updates

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Freeing MBNL1 protein from toxic RNA helped mice with MMD1-like disease

Researchers at the University of Rochester (N.Y.) Wellstone Muscular Dystrophy Cooperative Research Center have identified a compound that has the potential to be developed into a treatment for type 1 myotonic dystrophy (MMD1, or DM1).

Freeing MBNL1
In type 1 MMD, extra-long pieces of RNA form hairpin-like structures, which stick to and trap MBNL1 protein molecules. Thornton and colleagues believe their CAG25 molecule stuck to the abnormal RNA, opening up the “hairpin” and releasing the trapped MBNL1 protein.

The compound, dubbed CAG25, is an “antisense oligonucleotide,” a type of construct that’s used to block RNA, a close chemical relative of DNA.

In the current experiments, the antisense oligonucleotide was given to mice with a disease resembling human MMD1. The researchers wanted to see whether it would counteract any of the effects of MMD1 by sticking to abnormally long RNA and freeing a protein called MBNL1 that would otherwise become trapped in it.

Charles Thornton, who co-directs the MDA clinic at the University of Rochester Medical Center, coordinated the research team, which published its findings July 17, 2009, in the journal Science.
The Wellstone Center at the University of Rochester has had funding from MDA and the National Institutes of Health.

The hypothesis Thornton’s research group set out to test was whether releasing protein molecules stuck to long strands of RNA would allow the proteins to resume their normal activities and improve symptoms in mice with an MMD1-like disease.

These mice were given injections of CAG25 into one leg muscle and an inactive substance into the same muscle on the other leg. Researchers interpreting the results didn’t know which legs had received CAG25. The CAG25-treated legs showed improvements in myotonia, the inability to relax muscles, which is a hallmark of myotonic dystrophy.

“What we have now is proof of concept that this general approach for treating myotonic dystrophy is potentially effective,” Thornton said, noting that results of the new study should encourage researchers to improve and refine the strategy.

Masking unwanted instructions coaxed synthesis of needed protein in SMA-affected cells

Scientists at three U.S. institutions have used a very small synthetic molecule to correct the genetic defect in cells taken from a person with spinal muscular atrophy (SMA).

Full-Length and Short SMN
Genetic information moves from its storage form as DNA to a set of instructions known as RNA, from which protein molecules are made. Most of the RNA instructions from SMN1 genes, which are missing in SMA, tell the cell to make full-length SMN protein. Most of the instructions from SMN2 genes, which are present in SMA, tell the cell to make short SMN protein. Antisense to mask some of the instructions in the SMN2 RNA can cause synthesis of full-length SMN from the SMN2 gene.

The multicenter research team, which published results in the July-August-September 2009 issue of RNA Biology, was coordinated by MDA grantee Ravindra Singh at Iowa State University in Ames. The team also included MDA-supported Laxman Gangwani at the Medical College of Georgia in Augusta.

The molecule the researchers developed is called an “antisense oligonucleotide,” a type of compound that can cause cells to skip over erroneous genetic instructions. The compound is being tried experimentally in a number of genetic diseases to block the effects of abnormal genetic material (see “Freeing MBNL1 protein”).

In SMA, the compound is being used to mask genetic instructions that, when present, result in the synthesis of a short, nonfunctional SMN protein. A full-length SMN protein is needed to treat this disease.

Antisense oligonucleotides have been used this way previously in SMA, but the molecules have been larger. The new, smaller version has potential advantages for both safety and effectiveness, the researchers say.

Three-protein membrane repair cluster ID’d

Scientists in the United States and Japan have identified a three-protein cluster that reseals damaged muscle-fiber membranes. The findings, published June 5, 2009, in the Journal of Biological Chemistry, could have implications for development of treatments for muscular dystrophies.

Muscle fiber membrane
Defects in the muscle-fiber membrane underlie many muscular dystrophies. The newly identified “repair complex” consisting of mitsugumin 53, caveolin 3 and dysferlin provides a new therapeutic target.

In experiments using mouse muscle fibers, the investigators determined that mitsugumin 53 (MG53), a protein they announced in January as contributing to muscle-fiber repair, works closely with two other proteins, dysferlin and caveolin 3.

Scientists have known for a few years that dysferlin is involved in muscle-fiber membrane repair and that mu-tations of the gene for dysferlin or caveolin 3 can cause limb-girdle muscular dystrophy (LGMD). They’ve also known that mutations of dysferlin can cause Miyoshi myopathy, a form of distal muscular dystrophy. Now it appears that these three proteins — dysferlin, caveolin 3 and MG53 — form a cluster (complex) that repairs damaged membranes. Targeting the molecular functions of this cluster provides a new and promising avenue for therapeutic research, the researchers say.

Such research is especially important for muscular dystrophies in which membrane damage plays a major role, such as Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), some types of limb-girdle muscular dystrophy (LGMD) and possibly some types of congenital muscular dystrophy (CMD).

WNT7a protein boosts muscle repair

In experiments in mice, Michael Rudnicki, an MDA grantee at the Sprott Center for Stem Cell Research at Ottawa Hospital Research Institute (OHRI), and colleagues, found the WNT7a protein stimulates muscle repair by causing proliferation (an increase in number) of “satellite stem cells.” They say the protein probably operates similarly in humans. The findings were published June 5, 2009, in the journal Cell Stem Cell.

Satellite cells
Satellite cells are located near muscle fibers in mice and humans and stay in a dormant state until called upon for repair work. The WNT7a protein causes them to proliferate.

Satellite cells are located near mature muscle fibers in mice and humans and stay in a dormant state until called upon for repair work. In earlier experiments, Rudnicki found that some satellite cells function as stem cells and maintain overall numbers of satellite cells. He distinguished these from other satellite cells, which are in various stages on the road to becoming muscle tissue.

In muscular dystrophy, satellite cells are believed to become depleted quickly because tissue damage places great demands on them for repairs. Enhancing their numbers could slow the process of muscle degeneration, even in the face of disease.

“In muscle degenerative diseases, one of the big problems is thought to be that the muscles run out of repair cells,” says Paul Muhlrad, a research program coordinator at MDA. “Rudnicki’s laboratory has figured out the biochemical pathways the body uses to maintain the supply.”

When the OHRI researchers injected genes for the WNT7a protein into muscle fibers in mice, they saw an increase in satellite stem cell numbers and enhanced muscle regeneration compared to what they saw in fibers that weren’t treated this way.

“The identification of satellite stem cells and the pathways that regulate their function is an important advance in our knowledge,” Rudnicki said. “We believe that this discovery points the way forward toward the development of new drugs that will stimulate muscle repair.”

Cardiac actin can substitute for skeletal-muscle actin

A protein present in skeletal muscles during fetal development and in the heart after birth apparently can compensate for a similar protein that’s missing in a small percentage of patients with the muscle disease known as nemaline myopathy.

MDA research grantee Nigel Laing at the University of Western Australia in Perth was part of a multinational team of scientists who published their findings May 25, 2009, in the Journal of Cell Biology.

When the investigators bred mice missing the gene for the skeletal-muscle alpha-actin protein but with extra cardiac-muscle alpha-actin protein, they found the cardiac actin compensated well for the loss of the skeletal-muscle actin.

The findings open up the possibility of developing a treatment for some patients with human nemaline myopathy by increasing their own production of cardiac actin or giving them cardiac actin protein or genes.

Nemaline myopathy results from defects in a number of different muscle protein genes, all of which have to do with the muscle’s contractile filaments. These filaments slide over each other during muscle contraction in both cardiac and skeletal muscle tissue. Actin is a major filament component.

In its severest form, nemaline myopathy results in death in early infancy. In its less severe forms, affected children attain motor milestones slowly and may weaken further at puberty.

Previously, researchers had found mice bred not to produce any skeletal-muscle actin died by 9 days of age. In contrast, in this latest study, mice bred to produce extra cardiac-muscle actin but no skeletal-muscle actin survived into old age and had virtually normal muscle function.

These mice had grip strength and motor activity equal to that of healthy mice, and their muscles displayed a normal appearance, even under an electron microscope.

The researchers say their results show cardiac actin can effectively replace skeletal-muscle actin in muscles after birth, at least in mice and possibly in humans.

Previously, Laing said, it’s been shown that higher cardiac actin levels in patients without skeletal-muscle actin correlate with higher levels of function. The present results might indicate that increasing the level of cardiac actin even more in these patients would improve their motor abilities, he said.

The researchers caution that these experiments only showed compensation for a deficiency of the skeletal-muscle actin protein, not an abnormality in the protein. Patients with abnormalities in the skeletal-muscle alpha-actin gene that result in abnormalities of the actin protein, rather than deficiency, might not be helped by extra cardiac actin.

“Our results show that cardiac actin can work remarkably well in skeletal muscle,” Laing said. “This means that cardiac actin is a valid target for developing therapies for skeletal-muscle actin disease. However, we have a long way to go to be able to apply this to human patients. We have to find ways to increase cardiac actin in the muscles of human patients. That could take a long time, although we remain hopeful.”

Anti-scarring protein helps DMD mice

A protein called osteopontin has been implicated as a cause of some of the detrimental inflammation and scarring (“fibrosis”) of muscle tissue that takes place in Duchenne muscular dystrophy (DMD).

Eliminating osteopontin was beneficial to mice with a DMD-like disease, and the researchers concluded that reducing osteopontin should be investigated as a possible therapy for DMD.

Sylvia Vetrone at the University of California-Los Angeles (UCLA) and colleagues published their findings online May 18, 2009, in the Journal of Clinical Investigation.

The eight-person study team was coordinated by Melissa Spencer at UCLA and included Carrie Miceli, a UCLA immunologist whose contribution Spencer called “hugely significant.” Also on the team was Eric Hoffman, who has MDA support for related work at Children’s National Medical Center in Washington.

Osteopontin plays a role in promoting tissue damage in autoimmune diseases, disorders in which the immune system mistakenly attacks the body’s own tissues, the investigators note.

Although DMD is a genetic disease whose underlying cause is the loss of the muscle protein dystrophin, it shares some features with autoimmune disorders, such as inflammatory tissue changes. The inflammatory changes are believed to be secondary to the loss of dystrophin.

The investigators in this study found elevated osteopontin levels in muscle biopsy samples from people with DMD and in the blood and muscles of dystrophin-deficient mice with a disease resembling human DMD. Elevation of osteopontin correlated with progression of the disease process in the mice.

To see the effects of eliminating osteopontin, the researchers bred mice lacking both osteopontin and dystrophin. In these dystrophin-deficient, osteopontin-deficient mice, they saw fewer immune-system cells and more regulatory cells known to dampen the immune response than they saw in the dystrophin-deficient mice. The mice missing both dystrophin and osteopontin also showed lower levels of a protein known to cause fibrosis than the mice missing only dystrophin.

Dystrophin-deficient, osteopontin-deficient mice were stronger than dystrophin-deficient mice when they were tested at 4 and 8 weeks of age.

Although they didn’t maintain this strength advantage at 6 months, their diaphragm and heart muscles did show less scarring than those of the dystrophin-deficient mice at the age of 6 months. (Spencer said studies are under way to test diaphragm and heart function in these mice.)

The researchers interpreted these early findings to mean that osteopontin promotes inflammation and contributes to the deposition of scar tissue in dystrophic muscles.

They say their studies suggest that blocking osteopontin “may be a promising therapeutic target for reducing inflammation and fibrosis in individuals with DMD.” They note that further studies should be designed to find ways of reducing osteopontin in muscle tissue and to better understand the relationship among osteopontin, regulatory cells and the dystrophic process.

Neurotrophin 3 genes strengthened mice with CMT1A-like disease

Zarife Sahenk at Nationwide Children’s Hospital and Ohio State University in Columbus, and colleagues, has found mice with a disease resembling type 1A Charcot-Marie-Tooth disease (CMT1A) benefited from a transfer of genes for the neurotrophin 3 protein. CMT1A is caused by a duplication of the PMP22 gene.

Jerry Mendell, who has received many MDA research grants and co-directs the MDA clinic at Nationwide Children’s, was part of the study team, as was Brian Kaspar, who has received MDA support at Nationwide.

The researchers injected the leg muscles of CMT1A mice with either neurotrophin 3 genes inside shells made from adeno-associated viruses, or with a sham injection. The legs injected with the genes showed better grip strength and had more normal-looking nerve fibers.

The investigators concluded that neutrophin 3 gene therapy is a promising avenue for treatment development in human CMT1A.

These findings were reported at the 2009 American Academy of Neurology meeting, which was held recently in Seattle.

Webcast available for families with CNM

Molecular geneticist Alan Beggs
Molecular geneticist Alan Beggs has had MDA support to study centronuclear and other myopathies at Children’s Hospital in Boston.

Families affected by muscle diseases known as centronuclear myopathies, including myotubular myopathy, gathered for a conference in Houston July 24-26, 2009. The organizers have made the conference proceedings available as a Webcast at

Among the speakers are Alan Beggs, a molecular geneticist at Children’s Hospital in Boston, who has had MDA support to study centronuclear and other congenital myopathies; and Susan Iannaccone, a pediatric neurologist at Children’s Medical Center in Dallas who has received MDA support for neuromuscular disease research and directs the MDA clinic at her institution.

Recent advances have shown that the course of disease in centronuclear myopathies is highly variable, with myotubular myopathy being the most severe form. (See “Taking a Closer Look at Myoubular Myopathy,” Quest, September-October 2007.)


Idebenone not effective in 70 children with FA

On May 19, 2009, Santhera Pharmaceuticals ( reported that a phase 3 trial of its idebenone compound Catena showed the drug was not associated with a statistically significant benefit in 70 children between 8 and 17 years old with Friedreich’s ataxia (FA) who took it for six months. The drug appeared safe and well tolerated at doses up to 2,250 milligrams per day.

Santhera’s press release says there’s an ongoing phase 3 trial of Catena now being conducted in Europe that has a different design from the U.S. study and for which results are expected in 2010.

The European trial is a year long, and includes 232 participants, predominantly adults. Santhera says if the results of this European trial are positive, they will form the basis of filings for regulatory approval in the United States and Europe.

Idebenone is believed to improve energy production in cellular structures called mitochondria. Earlier studies showed the drug was safe and well tolerated in Friedreich’s ataxia and that there was a statistical trend toward dose-related improvement in neurological function associated with its use.

Santhera’s press release quotes Sue Perlman, clinical professor of neurology at the University of California-Los Angeles and one of the two study investigators on the U.S. trial, as saying, “I still strongly support the disease-modifying effect of Catena in Friedreich’s ataxia. I believe it slows the progression of the neurological and cardiac aspects of this condition over time.”

She strongly recommended that patients continue to take the drug in the open-label extension study so that the investigators can gather as much longer-term data as possible.

Jury still out on vitamin C for CMT1A

A recent trial has shown inconclusive results from various doses of vitamin C (ascorbic acid) in patients with the peripheral nerve disease type 1A Charcot-Marie-Tooth disease (CMT1A), and the jury remains out on this form of treatment, says Michael Shy, an MDA grantee at Wayne State University in Detroit.

Shy, a neurologist, is conducting a trial of high-dose vitamin C in CMT with neurologist Richard Lewis, also an MDA grantee at Wayne State. That trial is now closed, and results are under analysis.

According to results published in the June 2009 issue of Lancet Neurology, a one-year trial of high-dose vitamin C in 81 children ages 2 to 16 with CMT1A in Australia showed the treatment was safe and well tolerated but not effective.

However, some of the mildly affected patients in that trial appeared to perform better on relatively low doses of vitamin C than children taking a placebo. (Their story was highlighted on Australian television on May 28, 2009. See the Webcast at For most patients, the results were neutral.

Shy expressed concerns about the design of the study in a commentary in the same issue of Lancet Neurology in which the results were reported.

Shy says the Australian study was conducted in growing children, which makes the analysis particularly difficult to interpret. (The MDA-supported study includes adolescents and adults ages 13 to 70.)

“Kids grow, and they grow at different rates at different ages,” he says. “There’s this whole field of how to evaluate children with neuromuscular disease that’s evolving. We’re in the process of creating ways of evaluating children with CMT, through a study that’s in part funded by another MDA grant. One of the aims is to develop a CMT pediatric scoring system.”

He says the outcome measure the Australian researchers used was based on an electrodiagnostic measurement called nerve conduction velocity (the speed at which nerve impulses travel), values for which have been shown not to correlate with impairment in movement or sensation in children with CMT.

The Australian study used varying doses of vitamin C in the children, but they “never got to a high dose.”

“Kids are an important group,” says Shy, “but they need to be studied in a more complete and carefully designed manner.”

Other studies now under way, Shy says, may show there isn’t much effect of 1 gram of vitamin C per day on CMT. However, he says, most of these studies are limited by small numbers of participants and short durations.

Shy and Lewis note that results of a French study, presented at a meeting in Antwerp, Belgium, in July 2009, suggest that a trial of 3 grams of vitamin C per day in people with CMT is “looking positive” but that this trial also may not have been designed to yield a definitive answer.

“The bottom line is that the definitive study is going to be the MDA-supported CMT1A study,” Shy says, noting that he and Lewis agree. “It’s 4 grams a day, which is a high dose, and is powered [has enough participants and lasts long enough] to give a meaningful result.”

Iplex shows limited benefit in MMD1; development on hold

The drug Iplex, developed by the Richmond, Va., biopharmaceutical company Insmed (, did not improve muscle function, strength or endurance in a phase 2 trial in type 1 myotonic dystrophy (MMD1, or DM1), the company announced June 25, 2009.

No conclusions were reached about the effect of Iplex on cognitive function, gastrointestinal function or pain, because of a limited number of trial participants with problems in these areas.

However, measurements of insulin sensitivity — the ability of cells to respond to insulin — did show improvements in trial participants. Insulin sensitivity was assessed by serum levels of glucose and insulin, as well as cholesterol and triglycerides.

MDA helped support the clinical trial, which involved 69 adults with MMD1 who were randomly assigned to receive either Iplex or a placebo for six months. Neither participants nor investigators knew who received the drug until after all data had been collected.

On July 27, Insmed announced it would analyze the available data on Iplex for MMD1 and the limited data available related to the drug’s usage in amyotrophic lateral sclerosis (ALS) before deciding whether to proceed with development of this compound for either disease.

Iplex is a combination of a protein called insulin-like growth factor 1 (IGF1) and IGF binding protein 3. It was approved in the United States in 2005 for treatment of children with growth failure due to severe deficiency of IGF1. (Insmed no longer markets Iplex for this purpose in the United States after losing a patent dispute.)

People with LGMD2B, Miyoshi myopathy sought for patient registry

The Jain Foundation, dedicated to research in diseases that result from a deficiency of the muscle protein dysferlin, is seeking people with type 2B limb-girdle muscular dystrophy (LGMD2B) or a distal muscular dystrophy called Miyoshi myopathy for its database (registry) of patient-submitted information.

LGMD2B and Miyoshi myopathy are caused by mutations in the dysferlin gene that lead to a deficiency of the dysferlin protein. A free mutation analysis (DNA diagnosis) is offered to registrants who meet the necessary criteria.

For more information, go to and click on Patient Registration; or contact Esther Hwang at (425) 882-1440 or

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