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Muscle-Derived Stem Cells Survive, Make Dystrophin in 3 Boys With DMD

Jacques Tremblay

Researchers in the Human Genetics Unit of Laval University in Quebec City have announced that three boys, ages 8, 10 and 16, with Duchenne muscular dystrophy (DMD), who received muscle-derived stem cells from their fathers, began producing normal dystrophin in a small percentage of injected muscle fibers and didnt reject the donated cells.

Dystrophin is the protein thats missing from the muscle cell membrane in DMD.

Between 1990 and 1995, MDA sponsored five clinical trials to transplant immature, muscle-derived cells (sometimes called myoblasts) from healthy donors into boys with DMD. None of these trials showed any benefit, with few of the transplanted cells surviving.

The new experiment, conducted by Jacques Tremblay and colleagues, differs from earlier trials of muscle cell transplantation (sometimes called myoblast transfer) on a number of points, the investigators say. In comparison to earlier experiments, larger numbers of muscle cells were injected, the injections were spaced more closely, the donor fathers were matched with their sons on measures of tissue compatibility, and a powerful immunosuppressant, tacrolimus, was given to cell recipients.

The cells were isolated from the fathers arm muscles and subjected to laboratory tests to ensure that they could form muscle either by themselves or by fusing with existing muscle. They were injected into the boys lower leg muscles.

Four weeks after the transplant, biopsies that removed the entire injected area 1 cubic centimeter (0.4 cubic inches) of tissue showed that the first boy was producing normal dystrophin in 9 percent of the fibers in the grafted section; the second produced it in 6.8 percent; and the third in 11 percent.

Because the injected area was very small, the researchers didnt expect to see any functional improvement and didnt test for it.

Tremblay, whose MDA funding supported some of the lab experiments underlying the trial, said he was encouraged by the findings, published online Jan. 14 in Molecular Therapy. He noted, however, that still more cells per area of muscle will have to be injected to get meaningful results.

His plans, if regulatory agencies in Canada approve them, include increasing the number of injections and ultimately transplanting cells into the entire biceps.

"Im confident that this [cell transplantation] is part of the solution" in DMD, Tremblay said.

Myotonic MD Research Makes Good Progress

This fall brought much progress in understanding the molecular basis of both kinds of myotonic muscular dystrophy (MMD) and also saw plans finalized for a new clinical trial in the disease.

Richard Junghans

First, MDA grantee Richard Junghans at Harvard University in Cambridge, Mass., was on a team whose experiments demonstrated the possible role played by a loss of proteins called transcription factors in causing MMD. They published their results in the Dec. 4 online edition of Science Express. (See "Research Updates," January-February.)

Then, on Dec. 12, a team that included MDA-supported Maurice Swanson of the University of Florida in Gainesville published another set of MMD-related experiments in Science.

Of Mice, Men and Muscleblind

Swansons group found that a loss of proteins known as muscleblind may underlie both types of MMD.

Muscleblind protein molecules, which are needed for the development of muscles and eyes (both of which are abnormal in MMD), stick to the abnormally long pieces of the genetic material known as RNA that are produced in MMD, the researchers found, and are thus prevented from performing their normal functions.

Maurice Swanson

When the investigators bred mice in which a muscleblind protein was missing, the mice developed nearly all the symptoms of MMD. The mice demonstrated myotonia (difficulty relaxing muscles), cataracts, flaws in proteins related to heart function and chloride movement, and microscopic abnormalities in skeletal muscle cells.

"Our results suggest that restoration of normal levels of functional muscleblind protein might prove to be a useful therapeutic approach for treating both muscle and eye problems associated with MMD," Swanson said.

Stephen Tapscott of the Fred Hutchinson Cancer Research Center in Seattle, who has had MDA funding to study MMD and is on MDAs Scientific Advisory Committee and Translational Research Advisory Committee, says hes impressed with the transcription factor and muscleblind findings. He finds the muscleblind results particularly intriguing for the pursuit of treatment.

"I look at the transcription factor paper as being compatible with the muscleblind paper but not as definitive," Tapscott said. "The most simple approach [for eventual treatment] would be to prevent muscleblind from interacting with RNA or to increase the expression [production] of muscleblind protein."

In myotonic dystrophy, proteins appear to be trapped by excess RNA.

MDA grantee Laura Ranum, a molecular biologist at the University of Minnesota in Minneapolis, whose group identified the type 2 form of MMD in 2001, said she believes the muscleblind findings "fit nicely" with whats known about both types of MMD.

"I think it will hold up for both kinds of myotonic dystrophy," Ranum said. "Some of the other theories that were proposed earlier were less consistent. The type 2 form didnt really fit into those models."

Ranum noted that Swansons results with mice missing a muscleblind protein and developing more than one feature of MMD "provide quite strong support that muscleblind plays a prominent role in this disease."

IGF1 Ready for Human Testing

While the biologists work out the details of the disease and try to point clinicians toward treatment avenues, investigators at the University of Rochester (N.Y.) Medical Center are preparing to start a clinical trial to test the safety and tolerability of insulin-like growth factor 1 (IGF1) in a small number of adults with myotonic dystrophy (MMD).

The trial is supported by MDA and the National Institutes of Health.

IGF1, a natural substance secreted by the body, has been found to have general muscle-building effects in animal experiments conducted by MDA grantees H. Lee Sweeney at the University of Pennsylvania, Nadia Rosenthal at Massachusetts General Hospital, and others, and in a small human trial in 1995. (For more on IGF1, see "Protein Turns Average Mouse Into Mighty Mouse," April 2001, and "Muscle-Building Gene Protects Against Duchenne MD in Mice," April-May 2002.)

The Rochester group will use an experimental compound developed by Insmed of Glen Allen, Va. (www.insmed.com), which combines IGF1 with another natural substance, IGF binding protein 3 (IGFBP3). The compound, called SomatoKine, is expected be even more effective and perhaps safer than IGF1 alone, says neurologist Charles Thornton, an investigator on the new trial who co-directs the MDA clinic at Rochester.

"The expectation is that using IGF1 this way, in combination with a protein that binds [sticks] to it, will actually increase its effects on muscle, while at the same time reducing potential side effects," Thornton says. He describes the binding protein as "a companion that safeguards IGF1 as it travels through the body and may help in delivering it to muscles."

Study Finds Creatine Ineffective in Type 1 MMD

MDA grantee Mark Tarnopolsky in the Department of Medicine at McMaster University in Hamilton, Ontario, was part of a research team that recently found that the dietary supplement creatine wasnt beneficial in type 1 MMD.

The report, published in the January issue of Muscle & Nerve, found there were no significant differences in muscle function, lean body mass, specific muscle-related tasks, activities of daily living, or pulmonary function in the treated and placebo groups.

The 34 adult participants took either 5 grams of creatine per day for four months or a placebo (inert substance).

The dietary supplement was well tolerated, with no ill effects on the kidneys or liver reported during the study.

Pregnancy in MG: Proceed With Caution

Investigators at the University of Bergen and Haukeland University Hospital in Bergen, Norway, recently examined the medical records of 79 women with myasthenia gravis (MG) who gave birth to 127 babies between 1967 and 2000, and compared them with records from women without MG who gave birth to 1.9 million infants during the same period.

In the Nov. 25 issue of Neurology, they report that mothers with MG were likely to be older than those in the control group (those without MG) and were more likely to have given birth in a university hospital. They found no difference in the average weight, gestational age at birth or death rate of newborns in the two groups. Likewise, birth defects werent significantly higher in the MG group.

Mothers with MG underwent Caesarean delivery 17.6 percent of the time, compared with an 8.6 percent incidence of Caesareans in the control group. Deliveries assisted by forceps or vacuum devices were higher in the MG-affected mothers before 1981 but not after. (Both these rates may reflect general changes in obstetrical practice during the time of the study.)

Premature (before labor) rupture of the membranes surrounding the fetus, which increases the risk of infection or premature delivery, occurred 5.5 percent of the time in women with MG and only 1.7 percent of the time in women without MG.

Serious complications requiring transfer of the newborn to a pediatric intensive care unit occurred in 21.3 percent of babies born to mothers with MG compared to 2 percent of those born to the controls.

Temporary, neonatal MG, which is caused by a transfer across the placenta of MG-causing proteins, occurred in 4 percent of the babies of mothers with MG.

Neurologist Carlayne Jackson, who directs the MDA clinic at the University of Texas Health Science Center in San Antonio, commented on the report in an accompanying summary.

She noted that, while having MG shouldnt prevent a woman from considering pregnancy, parents should take into account the possible complications that MG may cause for both mother and child. She recommends that mothers with MG receive prenatal care from doctors experienced with this disease and that they deliver their babies in hospitals prepared to deal with maternal and fetal complications and those with intensive care units for newborns.

CLINICAL TRIALS AND STUDIES

Study Suggests LGMD Carriers Can Have Weakness

Several members of a German family with a sarcoglycan-deficient form of limb-girdle muscular dystrophy (LGMD) have been found to have muscle weakness themselves, even though carriers of this type of MD havent until now been thought to show disease symptoms.

In this family, seven out of 12 carriers of a mutation in the gene for alpha-sarcoglycan, a protein in the muscle cell membrane, whose absence leads to LGMD, showed signs of upper-body weakness with a winglike appearance of the shoulder blades.

Carriers of recessively inherited diseases like sarcoglycan-deficient LGMD, for which the gene isnt on the X chromosome, have previously been considered "exempt" from disease symptoms. However, this report, published in the November issue of Annals of Neurology, suggests disease manifestations can occur in these carriers after all.

About 20 percent of carriers of Duchenne muscular dystrophy, for which the gene is on the X chromosome, show some muscle-related symptoms.

The investigators say there may be another gene in this LGMD family thats modifying the effects of the single alpha-sarcoglycan mutation in the carriers cells.

Sugar Boost Helps in McArdles Disease

Ingesting a sugar-containing drink 30 to 40 minutes before exercising appears to boost endurance and prevent muscle damage in people with phosphorylase deficiency (McArdles disease), according to a study published in the Dec. 25 issue of the New England Journal of Medicine, in which MDA research grantee Ronald Haller was a co-investigator.

People with McArdles disease cant break apart large glycogen molecules, a process necessary to manufacture the kind of energy needed for exercise.

Earlier research has shown that intravenous infusions of glucose boost exercise tolerance in the disease, presumably by compensating for the block in glycogen metabolism.

Hallers study shows that oral ingestion of sugar (in this case, sucrose) also works.

Haller, who directs the Neuromuscular Center at the University of Texas Southwestern Medical Center in Dallas, along with John Vissing of the Copenhagen Muscle Research Center in Denmark, studied 12 people with McArdles disease. Each participant had a lifelong history of exercise intolerance, with repeated episodes of cramps and myoglobin (a muscle protein) in their urine when they engaged in sudden, vigorous exercise.

Participants drank 660 milliliters (22 ounces) of a caffeine-free soft drink that contained either 75 grams (about 2.6 ounces) of sugar or a taste-equivalent amount of an artificial sweetener. Some 30 to 40 minutes later, they were asked to ride a stationary bicycle for 15 minutes.

The sugar drink resulted in marked improvement in exercise tolerance, lowered participants perceived levels of exertion, and reduced their heart rates considerably compared with the artificially sweetened drink.

Haller noted that his findings conflict with earlier recommendations that patients with McArdles restrict carbohydrates (presumably to improve their ability to use alternate fuel sources, such as fatty acids).

"The results of this study support our view that glucose has a critical role in muscle oxidative metabolism and that the diet of affected patients should contain an adequate amount of carbohydrates," he said. "However, we stress that patients should not consume more calories than they expend."

Genzyme to Study Later-Onset Pompes

The biotechnology company Gen-zyme of Cambridge, Mass., which is conducting two trials to test a laboratory-engineered enzyme in infants and young children with acid maltase deficiency (Pompes disease), will perform an observational study of people with the late-onset form of the disease.

The investigators are interested in observing and testing approximately 60 people with mild to intermediate, late-onset Pompes disease. Participants must be able to walk, with or without assistive devices, and cant require ventilatory support while awake.

Final selection will probably admit U.S. participants who are 8 years old or older, while European participants must be at least 18.

The study will involve pulmonary and muscle function testing so the researchers can better understand disease progression in preparation for a clinical trial.

Later this year, some participants in the observational study may be selected for a clinical trial of a therapeutic agent. Participation in the treatment trial is neither required nor guaranteed for those in the observational study.

For information, send e-mail to Genzyme at medinfo@genzyme.com, or call (800) 745-4447; press 2. See also Genzymes Web sites at www.genzyme.com and www.pompe.com.