RxPG News : Muscular Dystrophies

Duchenne muscular dystrophy - also a disease of stem cells

Fri, 10 Dec 2010 08:26:15 PST

( from http://www.rxpgnews.com ) Researchers have long known that the devastating disease called Duchenne muscular dystrophy (DMD) is caused by a single mutation in a gene called dystrophin. The protein encoded by that gene is critical for the integrity of muscle; without it, they are easily damaged. But new findings in mice reported online in the journal Cell on December 9th by researchers at Stanford suggest that disease symptoms, including progressive muscle weakening leading to respiratory failure, only set in when skeletal muscle stem cells can no longer keep up with the needed repairs.

"This is not just a disease of dystrophin deficiency" said Helen Blau of Stanford University School of Medicine, who led the study. "It's also a disease of stem cells." That means that successful treatments would likely need to target muscle stem cells, not just muscle fibers, she says.

"These findings are critical for thinking about how to treat the disease and when," added Jason Pomerantz, the study's co-corresponding author who is now at the University of California, San Francisco. "It predicts any treatment designed solely to build muscle or enhance muscle function without replenishing the stem cell compartment is likely to fail and may even accelerate the decline. It's like pushing the gas pedal to the floor when there is no reserve."

The new study also answers a long-standing puzzle in the field that has stymied basic studies in search of potential treatments or treatment strategies: Mice carrying the same dystrophin mutation found in human patients show only mild symptoms of the disease.

"It has been a mystery for the past 25 years that mice with the genetic defect show minimal or no symptoms," Blau said, "and consequently there has been no mouse model in which to study the pathophysiology of the disease or potential treatments." People thought maybe it was because mice are smaller or don't live as long, but there was no real explanation. That is, until now.

The new findings attribute the discrepancy between the mouse and human symptoms to a characteristic of chromosomes. Regions of repetitive DNA found at the tips of chromosomes, known as telomeres, are longer in mice than they are in humans. Blau and her team have found that mice with the dystrophin mutation and another that leads them to have shortened telomeres have severe symptoms of the disease that worsen with age just as they do in human patients.

Telomeres protect chromosomes from deterioration and they tend to get shorter each time a cell divides. When telomeres become critically shortened, it triggers events that lead cells to die. The longer telomeres normally found in mice apparently give their muscle stem cells greater staying power and a greater capacity to repair the damage caused by the deficiency of dystrophin.

"Mice with shorter telomeres show all the parameters of the disease," Blau said. The animals won't run on a treadmill, their strength is really diminished, and their diaphragms (the muscle needed to breathe) are reduced to the point that they are "thin remnants, or strips of tissue." This muscle weakening paralleled a decline in the regenerative capacity of their muscle stem cells.

"There is continuous damage due to the loss of dystrophin," Pomerantz explained. "When the stem cell reserve is depleted, the symptoms emerge. The mice are spinning their wheels in a cycle of damage, repair, damage, repair, until the ability to repair gives out. In these mice [with shortened telomeres], it gives out earlier."

When the researchers isolated and transplanted healthy muscle stem cells into the sick mice, it alleviated symptoms of the disease.

The mice are now the first tractable model system for studying the disease, and that should come as good news to families affected by this form of muscular dystrophy, the researchers say.

"Our new mouse model changed the way we were thinking about the pathophysiology of the disease," said Foteini Mourkioti of Stanford who is the co-first author on the paper. "We now understand that muscle stem cells are an essential component of this dystrophin-deficient disease and we can now start thinking of more precise ways to treat Duchenne muscular dystrophy."

Treatments intended to restore muscle will likely work only temporarily or not at all. In fact, they are likely to exacerbate the problem by exhausting muscle stem cells more rapidly. Timing will also be key.

"Therapeutic strategies aimed at intervening early in DMD patients, in the first years of their life, are more likely to have a better outcome as they would act before this end-stage tissue failure is reached," said Alessandra Sacco, the study's first author who is now at the Sanford-Burnham Medical Research Institute.

Sarcospan may help in Duchenne muscular dystrophy

Mon, 03 Nov 2008 00:01:09 PST

( from http://www.rxpgnews.com ) The overlooked and undervalued protein, sarcospan, just got its moment in the spotlight. Peter et al. now show that adding it to muscle cells might ameliorate the most severe form of muscular dystrophy.

In Duchenne muscular dystrophy (DMD), the mutated dystrophin protein fails to anchor correctly to its membrane glycoprotein complex. And without this anchoring, muscle cells experience severe contraction-induced damage. Sarcospan is part of the anchoring complex, but because mice without sarcospan don't seem any worse for its absence, it hasn't received much attention. Sarcospan's structure, however, suggests it might help stabilize the membrane complex, so the authors decided to test the effects of increasing sarcospan expression in a DMD mouse model.

The increase did not improve the dystrophin–glycoprotein interaction, but instead, the team was surprised to find sarcospan coaxed a dystrophin relative called utrophin to spread out on the muscle membrane. Utrophin is normally restricted to the neuromuscular junction, where it serves a role similar to that of dystrophin.

The extra sarcospan prompted higher levels of utrophin in the cell, but not by increasing its expression. Sarcospan instead stabilized extrajunctional utrophin complexes, which normally form early in development and then disappear after the first few weeks of life.

Mouse muscle cells were protected by sarcospan, but the true importance of this discovery will lie in its potential for human therapeutics, specifically gene therapy. In that regard, sarcospan's small gene size is significant—at 600 bp, it is easily packaged into the safest viral vectors, unlike either dystrophin or utrophin, which are about 700 times larger and require more immunogenic vectors.

Trichostatin A (TSA) Can Counteract Muscular Dystrophy in Mice

Thu, 05 Oct 2006 01:08:00 PST

( from http://www.rxpgnews.com ) Scientists at the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) and other institutions have demonstrated for the first time that a single drug can rebuild damaged muscle in two strains of mice that develop diseases comparable to two human forms of muscular dystrophy. This advance, which is reported online in Nature Medicine, is the latest from a research collaboration that began several years ago by the teams of Vittorio Sartorelli, M.D., at NIAMS and Pier Lorenzo Puri, M.D., Ph.D., now at Dulbecco Telethon Institute (DTI) in Rome, Italy and The Burnham Institute in La Jolla, Calif.

The scientists tested trichostatin A (TSA), an inhibitor of the enzyme deacetylase, in two mouse models of muscular dystrophy (MD): one that naturally develops a disease similar to Duchenne muscular dystrophy in humans, the other genetically altered to develop a form of dystrophy similar to the human limb-girdle muscular dystrophy. At 45 to 90 days of age, the muscles of the MD mice showed much fibrous tissue and infiltration of inflammatory cells. Unlike healthy mice, the mice with MD were unable to either run on a treadmill or swim. MD mice given TSA daily for two to three months, however, were virtually indistinguishable from healthy mice, and biophysical studies showed virtually no difference between the muscle strength of the mice with MD given the deacetylase inhibitor and healthy mice.

This is the first example of using a drug to counteract muscular dystrophy in mouse models, says Dr. Sartorelli. Yet he points out that the drug is only promoting muscle regeneration it is not curing the defect that causes muscle deterioration. Further studies are needed to determine how long the drug works and if it works in larger animals with bigger muscles, such as dogs, before such drugs can be tested in people.

The finding has its roots in several of the groups earlier advances, the first of which was reported in 2002 in the Proceedings of the National Academy of Sciences 1. The scientists found that treating muscle cells with deacetylase inhibitors caused the cells to grow larger and differentiate better, says Dr. Sartorelli, the group leader of the Muscle Gene Expression Group in NIAMS Laboratory of Muscle Biology. The next advance, published two years later in the journal Developmental Cell 2, was the discovery that the inhibitor worked by changing gene expression, causing some genes to be upregulated, or make more protein, and others to be downregulated, or make less protein. Among the genes positively regulated by the inhibitors was a gene for a key protein called follistatin.

It was known that follistatin had a role in muscle development, so by understanding normal muscle development we knew that follistatin would block the activity of another protein called myostatin, says Dr. Sartorelli. If you block myostatin, you get big muscles.

One way of inactivating myostatin is to upregulate follistatin. Basically, what follistatin does is to prevent myostatin from working, says Dr. Sartorelli. When his group treated the cells with deacetylase inhibitors, they saw that the cells became large and that follistatin was overexpressed. However, when the group treated the cells with the inhibitors and then used other agents to block follistatin, the cells didnt become bigger, showing that one of the most important pathways the inhibitors use to create bigger muscles involves the activation of follistatin. If you didnt have follistatin anymore, these drugs didnt work, he says.

Moreover, Drs. Sartorellis and Puris groups were able to show that in normal animals, follistatin is upregulated when muscle is damaged. When the researchers induced muscle damage and then gave the inhibitors, follistatin was even more expressed, as were two proteins that reflect increased muscle regeneration.

Valproate effective in adult spinal muscular atrophy (SMA)

Sat, 24 Jun 2006 03:00:00 PST

( from http://www.rxpgnews.com ) An epilepsy drug that has been on the market for decades can ease the symptoms of adult sufferers with a genetic disorder that seriously weakens muscles.

Scientists at Washington University School of Medicine in St. Louis retrospectively reviewed results from off-label use of the drug valproate to treat seven adult spinal muscular atrophy (SMA) patients. Clinicians offered the drug to patients on the basis of research conducted elsewhere that showed the drug increased levels of a key protein in cell cultures.

"The treatment has been fairly successful," says lead author Chris Weihl, M.D., Ph.D., a postdoctoral fellow in neurology. "The drug appeared to be well-tolerated and increased the strength of the patients who took it."

The study, now available online, will appear in the August 8 issue of Neurology.

Weihl notes that a larger, prospective trial is needed to firmly establish valproate as a treatment of choice for sufferers of this type of SMA.

Such trials are already underway elsewhere in pediatric patients who suffer from a different type of SMA that begins earlier in life. Weihl and his colleagues are concerned that valproate may not work as well in those patients. They wanted to make sure that researchers did not discard the possibility that valproate could help older sufferers even if the trials in pediatric patients went poorly.

"Based on what we know of the unique genetics of this disease, there was reason to think that this drug could be more helpful to patients who develop SMA later in life," Weihl says.

Patients with all forms of SMA, which affects approximately one of every 6,000 babies born in the U.S., are missing the SMN1 gene, which makes the survival motor neuron (SMN) protein. This progressively weakens the muscles, leading to difficulty in walking, eating, clearing the air passageway, and other essential functions.

Based on when the symptoms of SMA first manifest, physicians divide SMA into four subtypes. SMA I, for example, strikes very young children, causing weakness in the womb, preventing children from ever walking and typically resulting in death at an early age. Patients with SMA IV, in contrast, don't develop weakness until adulthood. The seven patients studied were either SMA III or SMA IV, and ranged in age from 17 to 54.

Differences in age of SMA onset have been directly linked to a second human gene that also makes the SMN protein. That gene, SMN2, isn't as efficient at making the SMN protein as SMN1. Patients who develop SMA early in life have only one copy of the SMN2 gene in their DNA, leaving them with very low levels of the SMN protein. Patients who get the disorder later in life have more copies of the SMN2 gene, increasing the amount of SMN protein made in their cells and delaying onset.

"Because we have learned so much about SMA over the last decade, there's been a big push at NIH to cure this disease," Weihl says. "The search has been on to find a treatment that can increase the amount of SMN2 protein synthesized by SMN2 genes. This rapid bench-to-bedside transition for valproate is a good example of the kind of progress that is encouraged both by NIH and the University's Biomed 21 initiative."

In addition to its use as an epilepsy treatment, valproate, which is sold under the brand name Depakote, has been used to treat bipolar disorder, migraine headaches and other neurological conditions. The drug's effects include increasing the number of times protein-building instructions are read from genes, which is the first step in creating copies of proteins like SMN.

As patients took the drug, clinicians regularly gave them a series of strength tests. When Weihl reviewed the data from those tests, he found patient strength had increased significantly over the course of eight to 15 months of treatment with the drug.

According to Weihl, simply increasing the strength of an SMA patient's cough might enable them to clear their lungs better and reduce incidence of pneumonia, the most common killer of patients with SMA III and IV.

Valproate's side effects can include weight gain, hair loss and acne. One patient stopped taking the drug because she was concerned about weight gain.

"Adding weight can be a problem in patients who are already weak, and it's certainly a legitimate reason to stop taking the drug, but overall we didn't see significant weight gains in patients taking the drug," Weihl says.

Weihl and his colleagues are continuing to follow the seven patients reviewed in the study, who are still taking a daily maintenance dose of the drug.

Muscular dystrophy - early cardiac screening shows better outcomes

Sun, 30 Oct 2005 15:02:00 PST

( from http://www.rxpgnews.com ) Early diagnosis and treatment of heart disease may lead to longer life in Duchenne and Becker muscular dystrophy patients, say experts at Baylor College of Medicine (BCM) and Texas Children's Hospital in Houston in a report that appeared online in the journal Circulation.

Cardiac disease, particularly dilated cardiomyopathy and heart failure, is the major cause of mortality in patients with muscular dystrophy and is present in most boys with Duchenne muscular dystrophy and approximately 70 percent of those with Becker muscular dystrophy. These are the two common forms of muscular dystrophy caused by defects in a gene called dystrophin.

"It should be the standard of care for all newly diagnosed Duchenne and Becker muscular dystrophy patients to be referred to a cardiologist for screening, probably by 10 years of age or earlier," says Dr. Jeffrey A. Towbin, professor of pediatrics at BCM and chief of pediatric cardiology at Texas Children's Hospital.

Towbin and his group studied 69 boys with DMD and BMD. After the first abnormal echocardiogram, which occurred at 14-15 years, 31 boys were started on ACE inhibitor or beta blocker therapy. During the follow-up two patients remained stable with their dilated cardiomyopathy, eight showed improvement and 19 normalized both heart size and function.

"This study also helped us realize that while some dystrophin-gene mutations are predictors of early onset cardiac abnormalities, others may actually protect against early development of these abnormalities," says Towbin.

Cardiac symptoms typically appear late in the course of cardiomyopathy, in part because affected individuals are usually wheelchairchair bound and often physically inactive. Heart disease progresses quickly, leading to premature death, often before 25 years of age.

Gene therapy success for congenital muscular dystrophy (CMD)

Tue, 16 Aug 2005 19:46:00 PST

( from http://www.rxpgnews.com ) Researchers from the University of Pittsburgh report the first study to achieve success with gene therapy for the treatment of congenital muscular dystrophy (CMD) in mice, demonstrating that the formidable scientific challenges that have cast doubt on gene therapy ever being feasible for children with muscular dystrophy can be overcome. Moreover, their results, published in this week's online edition of the Proceedings of the National Academy of Sciences (PNAS), indicate that a single treatment can have expansive reach to muscles throughout the body and significantly increase survival.

CMD is a group of some 20 inherited muscular dystrophies characterized by progressive and severe muscle wasting and weakness first noticed soon after birth. No effective treatments exist and children usually die quite young.

Despite gene therapy being among the most vigorously studied approaches for muscular dystrophy, it has been beset with uniquely difficult hurdles. The genes to replace those that are defective in CMD are larger than most, so it has not been possible to apply the same methods successfully used for delivering other types of genes. And because CMD affects all muscles, an organ that accounts for 40 percent of body weight, gene therapy can only have real therapeutic benefit if it is able to reverse genetic defects in every cell of the body's 600 muscle groups.

By using a miniature gene, similar in function to the one defective in CMD, and applying a newly developed method for "systemic" gene delivery, the Pitt researchers have shown that gene therapy for muscular dystrophy is both feasible and effective in a mouse model of especially profound disease. Using this approach, the team, led by Xiao Xiao, Ph.D., associate professor of orthopaedic surgery and molecular genetics and biochemistry at the University of Pittsburgh School of Medicine, report that treated mice had physiological improvements in the muscles of the heart, diaphragm, abdomen and legs; and they grew faster, were physically more active and lived four times as long as untreated animals.

"While we have much farther to go until we can say gene therapy will work in children, we have shown here a glimmer of hope by presenting the first evidence of a successful gene therapy approach that improved both the general health and longevity in mice with congenital muscular dystrophy," said Dr. Xiao.

The most common form of CMD, and also one of the most severe, is due to a genetic mutation of laminin alpha-2, a protein that is essential for maintaining the structures that surround muscle cells and is an integral link in the chain of proteins that regulate the cell's normal contraction and relaxation. If the protein is defective, or is lacking, this outside scaffold, called the extra-cellular matrix, disintegrates, and the muscle cells become vulnerable to damage.

Simply replacing the defective gene with a good laminin alpha-2 gene is not possible because its size makes it impossible for researchers to get it to squeeze inside viral vectors disarmed viruses that are used to shuttle genes into cells. But the team found a good stand-in in a similar protein called agrin that when miniaturized could be inserted inside an adeno-associated virus (AAV) vector. Dr. Xiao's laboratory is known for its work developing this vector, which they have previously shown is the most efficient means for delivering genes to muscle cells.

In the current study, the authors show that two strains of AAV, AAV-1 and AAV-2, were effective in transferring the mini-agrin gene to cells in two mouse models. The AAV-1 vector was given by systemic delivery a single infusion into the abdominal cavity a method the authors only recently described and which they used for the first time in this study to transfer a therapeutic gene. The AAV-2 vector was delivered locally, given by intramuscular injection to different muscles of the leg. With both approaches, muscle cells were able to assimilate and copy the genetic instructions for making mini-agrin. Once produced, the mini-agrin protein functionally took the place of the laminin alpha-2 protein by binding to the key proteins on either end, thus restoring the cell's outside scaffolding and reestablishing the missing link to key structures inside the cell.

Clearly, the authors are most excited about the impressive results achieved in their experiments using systemic gene delivery, which proved there could be significant therapeutic improvements and even be life-saving. Yet they say their results are far from ideal and more work lies ahead.

"It's probably not realistic to expect that we can achieve complete success using the mini-agrin gene, which while somewhat similar, is structurally unrelated to laminin alpha-2. Unless we address the underlying cause of congenital muscular dystrophy we're not likely to be able to completely arrest or cure CMD," added Chungping Qiao, M.D., Ph.D., the study's first author and a research associate fellow in Dr. Xiao's lab.

Future directions for research include finding a way to engineer the laminin alpha-2 gene. For this study, the authors chose to use the mini-agrin gene because researchers from the University of Basel, Switzerland, had already demonstrated it could improve the symptoms of muscular dystrophy in a transgenic mouse model, which has little clinical relevance. The Pitt researchers might also explore approaches that combine genes that promote both muscle and nerve growth, as well as focus on improving the AAV vectors.