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    Home> Publications > QUEST Vol.15 No.2 March/April 2008
Research Updates

European team combines stem cells, gene correction to treat DMD-affected mice

A European group of researchers successfully used a combination of genetic correction and stem cells to treat Duchenne muscular dystrophy (DMD) in mice with the disease. The compound they used for the genetic correction was developed by Judith van Deutekom, then at Leiden (Netherlands) University Medical Center, with MDA support.

DMD results from the loss of the muscle protein dystrophin because of any of a number of genetic errors in the dystrophin gene.

Yvan Torrente at the University of Milan (Italy), Luis Garcia at the Institut de Myologie in Paris, and colleagues, who published their results Dec. 13 in Cell Stem Cell, corrected the genetic error in the DMD-causing gene in cells taken from patients with the disease and then used the corrected cells to treat DMD-affected mice.

The strategy is thought to have potential for treatment of human patients, although there are safety concerns.

In their experiments, the investigators first extracted muscle-generating stem cells known as CD133-positive cells from both muscle tissue and blood in people with DMD.

Research Illustration
Torrente, Garcia and colleagues 1) took muscle and blood samples from people with DMD; 2) extracted stem cells from the samples; 3) corrected the cells’ dystrophin genes by giving them an exon-skipping construct inside a viral shell; and 4) injected the corrected cells into DMD-affected mice.

They then corrected the genetic error in the cells’ dystrophin genes outside the body with a technique known as exon skipping.

This strategy blocks the part of the gene that contains an error, coaxing the cell to skip that part and produce a shortened, but still functional, dystrophin protein from the remaining genetic instructions.

When the researchers injected the genetically corrected stem cells into dystrophin-deficient, DMD-affected mice, either into an artery or into muscle tissue, they saw a significant recovery of muscle form and function and production of dystrophin. (The immune systems of the mice had been altered so that they could tolerate the human cells.)

The muscle-derived CD133-positive cells gave rise to better muscle regeneration than did the blood-derived cells. Injections into muscle and into an artery both led to muscle regeneration, but it was more widespread with the arterial injections.

“Our approach of using exon skipping for the expression of human dystrophin within the DMD CD133-positive cells should allow the use of the patient’s own stem cells, thus minimizing the risk of immunological graft rejection,” the researchers write.

But the main hurdle is that, with present technology, the exon-skipping compounds are delivered to cells using a virus that integrates into chromosomes. If the virus integrates into the wrong place, it could cause cancer or disrupt important genes.

Current MDA grantees who are working on exon skipping for the dystrophin gene are Stephen Wilton at the University of Western Australia in Perth; Howard Michael at the University of Utah in Salt Lake City; Gordon Lutz at Drexel University in Philadelphia; and Carmen Bertoni at Stanford (Calif.) University.

Related human testing

In December, in the journal Cell Transplantation, Torrente and colleagues announced they had transplanted CD133-positive stem cells taken from the muscles of eight boys with DMD back into the same patients from whom they had originally been extracted, with no observed adverse effects. No gene correction was involved.

And a phase 1 trial in the Netherlands recently tested an exon-skipping compound developed by the Dutch company Prosensa in boys with DMD. No stem cells were involved. (See “Results of phase 1 trial”.)

Embryonic stem cells benefit muscles in DMD-affected mice

Injection of modified mouse embryonic stem cells into mice with Duchenne muscular dystrophy (DMD) led to considerable muscle regeneration and a significant increase in muscle force, reports a research team at the University of Texas Southwestern Medical Center in Dallas.

Rita Perlingeiro and colleagues, who published their results online Jan. 20 in Nature Medicine, induced production of a protein called pax3 in mouse embryonic stem cells and then further sorted the cells to select for a pattern of protein production indicating the cells had muscle-restoring potential.

These specially modified and selected cells restored production of dystrophin (the protein missing in DMD) in 11 percent to 16 percent of muscle fibers, whether they were injected into a muscle or into a blood vessel. They persisted for at least four months after injection and produced no tumors.

Researchers say this beneficial effect is similar to that seen with transfer of the dystrophin gene by itself (gene therapy).

Noting that previous experiments showed that not all, or even most, muscle fibers need to produce dystrophin to obtain a therapeutic effect in DMD, researchers say these results are “encouraging for the application of cell therapies to muscular dystrophy.”

Gene ID’d for X-linked SMA

Mutations in a gene known as ubiquitin-activating enzyme E1 (UBE1) have been identified as the underlying cause of a rare form of spinal muscular atrophy (SMA) that affects male infants and has been previously linked to the X chromosome.

Most cases of SMA result from a mutation in the SMN1 gene, which is on chromosome 5. This more common form of SMA affects both sexes and shows varying degrees of severity and age of onset.

The rare, X-linked form closely resembles the most severe, infantile-onset form of chromosome-5 SMA, except that it also affects the joints, which are spared in chromosome-5 SMA.

Lisa Baumbach-Reardon, head of the Neurogenetics Laboratory at the University of Miami (Fla.), who received MDA support for this work, led the study team with Alfons Meindl at Technical University Munich (Germany). They published their findings online in January in the American Journal of Human Genetics.

The UBE1 gene is part of the ubiquitin-proteasome system, a cellular waste disposal mechanism that keeps cells free of improperly formed or otherwise toxic proteins. It’s involved in “tagging” defective proteins with ubiquitin molecules. If UBE1 isn’t functioning properly, the researchers speculate, the cellular waste disposal system might be compromised.

“This study is the culmination of 15 years of investigation, starting with identification of the first families with X-linked SMA, through years of gene-mapping studies to finally, last year, gene discovery and mutation identification,” Baumbach-Reardon said. “It’s been a long road, but we never gave up, because we promised the families who have this devastating illness that, with their participation in our research studies, we would someday identify the causal disease gene.”

MDA grantee receives prestigious Pfizer award

MDA research grantee Paul Gregorevic has received a $1 million award from Pfizer Australia to develop gene-based therapies for muscle disease.

The award, the 2008 Pfizer Australia Research Fellowship, recognizes excellence in Australian biomedical researchers who the company believes will contribute to the development of the country’s standing among the world’s scientific leaders.

Gregorevic, who recently relocated from the University of Washington-Seattle to the Baker Heart Research Institute in Melbourne, Australia, received MDA funding from 2004 to 2007 to develop gene-delivery methods in mouse models of muscular dystrophy. In January, he began receiving MDA support to explore the effects of follistatin on mice with MD.

New studies improve understanding of myotonic dystrophy

Myotonic dystrophy (MMD), a complex disease that results from an expanded and repeated section of DNA on either chromosome 19 (MMD1) or chromosome 3 (MMD2), has long posed a challenge to researchers because of its effects on multiple body systems, its varying degrees of severity and its complex molecular origins.

In both forms of the disease, extra-long strands of genetic material called RNA are made from expanded DNA sections, causing a variety of cellular problems. Recently, scientists have shed light on some of these, particularly in MMD1.

Normal muscle relaxation restored in MMD-affected mice by fixing chloride channels

Researchers at the Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center at the University of Rochester (N.Y.) and the University of Florida in Gainesville report that they’ve used a molecular strategy to restore normal muscle relaxation after contraction in mice with a disease resembling MMD1. The Wellstone Center is supported by the U.S. National Institutes of Health and has also received support from MDA.

Charles Thornton, who co-directs the MDA clinic at the University of Rochester Medical Center, led the research team, which published its results online Nov. 15 in the Journal of Clinical Investigation. Maurice Swanson, an MDA grantee at the University of Florida in Gainesville, was also part of this study.

The genetic defect that underlies MMD1 indirectly causes a variety of muscle and other problems, including myotonia, the inability to relax muscles after using them. The direct cause of the myotonia, however, is abnormally constructed cellular tunnels called chloride channels.

Earlier studies have shown that, in MMD1, the expanded section of DNA on chromosome 19 leads to errors in a process called RNA splicing and thereby to faulty construction of the chloride channel and other proteins.

When the research team injected MMD-affected mice with a synthetic compound that corrects the RNA splicing process, they fixed the chloride channels and relieved the myotonia.

Overproduction of heart protein implicated

Overproduction of a protein crucial for the development of the heart should be added to the growing list of effects of the genetic defect that underlies MMD1, MDA research grantees report.

In recent years, researchers have identified several mechanisms, including defects in RNA splicing (see “Normal muscle relaxation”) that result from this defect.

Now, MDA grantee Mani Mahadevan at the University of Virginia in Charlottesville, and colleagues, who published their results online Dec. 16 in Nature Genetics, have added yet another piece to the MMD1 puzzle.

By conducting experiments in MMD-affected mice, Mahadevan’s group found that overproduction of NKX2-5, a protein essential for normal heart development that also plays a key role in maintaining regular heartbeats, may account for the heart rhythm abnormalities seen in MMD1.

Surprisingly, in mice and humans with MMD1, they also saw NKX2-5 in skeletal muscles, where it’s not normally found at all. They say this too may have deleterious consequences.

“I think this study emphasizes that the effects of the toxic RNA in MMD1 are increasingly complex,” said Mahadevan, who will now conduct MDA-supported research on NKX2-5 and its biochemical targets.

MDA grantee Jack Puymirat at Laval University in Quebec City, and Charles Thornton, co-director of the MDA clinic at the University of Rochester (N.Y.) Medical Center, also contributed to this study.

DMPK may warrant closer look

A deficiency of an enzyme known as DMPK (myotonic dystrophy protein kinase) may have more to do with MMD1 than scientists had previously believed, say Perla Kaliman at the Hospital Clinic of Barcelona (Spain) and colleagues in the November issue of PLoS One.

MMD1 results from an expanded stretch of DNA in the gene for DMPK that keeps this protein’s genetic instructions (RNA) from leaving the nucleus of muscle cells. However, almost everyone with MMD1 has one normal and one abnormal DMPK gene, so it’s expected that they manufacture about half the normal amount of DMPK.

Although a deficiency of DMPK causes some cardiac problems, most experts have thought until recently that lack of DMPK wasn’t a very important contributor to the multiple abnormalities seen in MMD1.

Now, Kaliman and colleagues have shown that when mice are bred to produce no DMPK at all, they develop high blood sugar and higher-than-normal levels of circulating insulin, indicating that they have insulin resistance, as do many MMD1 patients.

Further studies are needed to see whether mice with half the normal amount of DMPK have any insulin abnormalities, but the new experiments, added to the older ones showing cardiac defects from lack of DMPK, suggest that a deficiency of DMPK in MMD1 may be worth a closer look.

Clinical Trials and Studies

Results of phase 1 trial of exon skipping in DMD heralded as ‘proof of concept’

The Dec. 27 issue of the prestigious New England Journal of Medicine featured the fully analyzed and encouraging results of a phase 1 clinical trial of an exon-skipping compound in four boys with Duchenne muscular dystrophy (DMD). Preliminary findings from this trial, conducted in the Netherlands, were announced in May (see “Research Updates,” July-August 2007).

Judith van Deutekom, formerly at Leiden University in the Netherlands and now at Prosensa in Leiden, received MDA funding to work on preclinical development of exon skipping for DMD (see “European team”) and is the first author on this study. The clinical trial was funded by several European agencies and Prosensa, which produced the therapeutic compound.

The exon skipping was achieved by blocking the mutation-containing part of the gene with an antisense oligonucleotide, or AON.

The hoped-for result, which occurred in this trial in all four participants, is the production of smaller-than-normal, but potentially functional, dystrophin protein, which is missing in DMD.

Two of the participants were 10 and 11 years old, and two were 13. None had any detectable dystrophin in muscle biopsy samples prior to treatment, and all had mutations that were potentially correctable by skipping over a part of the dystrophin gene known as exon 51, which Prosensa’s PRO051 AON was designed to block.

Each boy received four injections of PRO051 in a muscle at the front of the lower leg. A biopsy a month later showed dystrophin production that was between 3 percent and 12 percent of a normal level of the protein. No functional changes were observed, but none were expected from this low dose and restricted area of injection. The results are considered “proof of concept,” however, for exon skipping via antisense as a strategy to treat DMD.

The investigators did not observe any problems related to safety of PRO051, although one participant reported pain at the injection site, two reported a few days of flulike symptoms after the injections, and one experienced mild diarrhea for a day.

Importantly, there were no apparent inflammatory or toxic responses to the compound.

In an accompanying editorial, long-time DMD researcher Eric Hoffman, now at Children’s National Medical Center in Washington, describes the promise of the Dutch study, as well as the hurdles yet to be cleared.

Hoffman, who has been awarded several MDA grants, notes that an exon-skipping compound, to be truly useful, would have to be delivered systemically to all muscles, without toxicity, and that several compounds (perhaps dozens) would have to be designed for the hundreds of different dystrophin mutations in boys with DMD.

If each AON compound has to be put through the full gamut of U.S. Food and Drug Administration approval tests, he says, the regulatory hurdles for this treatment strategy will be formidable. Instead, he suggests, perhaps the FDA will consider approving such compounds as a class of drugs, rather than as individual therapeutic agents.

PTC Therapeutics to start longer studies of PTC124

PTC Therapeutics of South Plainfield, N.J., has announced its intention to conduct longer and larger studies of its drug PTC124, which targets a specific type of mutation in the dystrophin gene in boys with Duchenne muscular dystrophy (DMD).

This oral drug, developed by PTC with support from MDA, is designed to coax muscle cells to ignore, or “read through,” a molecular stop signal, known as a premature stop codon or nonsense mutation, that stops dystrophin protein synthesis too early in some 15 percent of people with DMD.

Because PTC124 and exon-skipping compounds require knowledge of the specific DMD-causing mutation, MDA will launch a program this year to assist all interested DMD-affected families in obtaining dystrophin DNA testing.

For more news as it becomes available, see or and the May-June issue of Quest.

Gene transfer appears safe in six boys with DMD

A phase 1, MDA-supported trial in Duchenne muscular dystrophy (DMD) to test the effects of intramuscular injections of genes for the muscle protein dystrophin inside adeno-associated viruses has shown the procedure is safe, the investigators say.

So far, six participants at Nationwide Children’s Hospital and Research Institute (Columbus, Ohio) have received dystrophin injections into an arm muscle.


The contact person for the Philadelphia site in the Santhera SNT-MC17 (idebenone) study in Friedreich’s ataxia (see Research Updates, January-February) is now Lisa Friedman at (267) 426-7538 or Participants must be 8 to 17 years old.

“The patients have been carefully followed for side effects of the treatment, and none has been encountered,” says neurologist Jerry Mendell, director of the Gene Therapy Center and co-director of the MDA clinics at Nationwide Children’s and Ohio State University Hospitals in Columbus, and the clinician on this study. “This is primarily a safety trial, and we can confidently report that safety has been achieved.”

Mendell adds that an additional goal of this study is to lay the groundwork for future gene therapy trials by establishing the ideal dose for treatment. “In this trial, two doses have been tested, and another will be required before completion of the study,” he says, noting that “by all indications,” a higher dose will be safe to administer.

Results are expected when all participants have completed the study.

Amarin will continue testing EN101 in MG

Development of EN101, an experimental drug for myasthenia gravis (MG) that blocks the synthesis of the enzyme acetylcholi-nesterase, will continue, but under the auspices of a new company. In December, the London-based biotechnology firm Amarin acquired Ester Neurosciences, the Israeli company that first developed the drug. (See Research Updates, November-December 2007).

Amarin is now overseeing an ongoing phase 2a trial of EN101, in which three different doses of the drug are being compared to Mestinon, a standard treatment for MG.

Acetylcholinesterase, the target of EN101, is an enzyme that degrades acetylcholine, a carrier of signals from nerve fibers to muscle fibers. Interfering with this enzyme’s action boosts acetylcholine levels, which is a logical way to increase strength and function in MG. Mestinon blocks the acetylcholinesterase enzyme after it’s been synthesized, and EN101 prevents its synthesis.

In December, Amarin said that “interim data suggest EN101 may have superior efficacy, longer duration of action, [and] a more favorable side effect profile and dosing regimen, as compared with Mestinon.”

The company said its focus will be on completing the phase 2a study and laboratory studies in preparation for beginning a phase 2b or phase 2-3 trial.

For more information, see

Children with severe SMA are surviving longer

Survival time in type 1 spinal muscular atrophy (SMA), the most severe form of the disease, is significantly longer in children born between 1995 and 2006 than for those born between 1980 and 1994, say researchers at Columbia University in New York and Indiana University in Indianapolis.

Maryam Oskoui, now at the Montreal Neurological Institute, together with Petra Kaufmann and Darryl De Vivo, both co-directors of the MDA clinic at Columbia, with colleagues there and at IU, analyzed treatment and survival data for 143 children with type 1 SMA from the International SMA Patient Registry. They published their findings in the Nov. 13 issue of Neurology.

The researchers say the improved survival time is due to more widespread use of noninvasive assisted ventilation, cough assist devices, and gastrostomy tube feeding, as well as to a change in attitude about aggressive treatment of children with severe SMA because of increased optimism about future treatments.

The probability of survival to age 2 for children with an SMA1 diagnosis born between 1980 and 1994 was 30.8 percent. For children with this same diagnosis born between 1995 and 2006, the likelihood of surviving to age 2 was 73.9 percent. Surviving to age 4 for the group born between 1980 and 1994 had a probability of 26.2 percent, while for those born between 1995 and 2006, the probability was 65.1 percent.

The authors note that the new findings should affect the way doctors counsel parents of children with SMA1 and should affect the plans parents make for their children, many of whom eventually will start school.

They also note that it will be important to take into account the change in the “natural history” of SMA1 when planning clinical trials in this disease.

“Given the improvement of outcomes over time, it could be misleading to compare trial participants now to untreated patients in the past,” Kaufmann said, adding that the study underscores the need for controlled clinical trials, in which a group taking the test medication is compared to a current group taking a placebo.

Myozyme improves endurance, pulmonary function in late-onset Pompe disease

The Cambridge, Mass., biotechnology company Genzyme has determined that treating patients with late-onset Pompe disease (also known as acid maltase deficiency) with the synthetic enzyme Myozyme for 18 months increased endurance and pulmonary function.

The company announced these results in a Dec. 13, 2007, press release.

The study involved 90 people with Pompe disease who were at least 8 years old, at eight sites in the United States and Europe. Participants received either Myozyme, an enzyme designed to compensate for a deficiency of acid maltase (also known as acid alpha-glucosidase), or a placebo (inactive substance) by intravenous infusion. MDA provided supplemental support for the trial.

After 18 months, participants treated with Myozyme increased the distance they could walk in six minutes by an average of about 30 meters (about 90 feet), while those in the placebo group showed no improvement from their baseline measurement.

A respiratory measurement known as forced vital capacity increased by 1 percent in the Myozyme-treated group, while it declined by approximately 3 percent in the placebo-treated patients.

The safety of Myozyme was found to be similar to that of an intravenously infused placebo.

Genzyme says it’s completing an analysis of this study and intends to present more detailed results at medical meetings and in a scientific journal.

MDA and chest physicians release recommendations for anesthesia care in DMD

MDA, in collaboration with the American College of Chest Physicians, has issued a consensus statement on management of patients with Duchenne muscular dystrophy (DMD) undergoing anesthesia or sedation. The complete statement is published in the December issue of the journal Chest.

Valerie Cwik, MDA’s medical director and vice president of research, along with several MDA clinic directors, pulmonologists and respiratory therapists, participated in its development. They noted that longer survival because of better cardiac and respiratory care in DMD has made it more likely that patients will undergo surgical procedures and more important that guidelines for patient care during and after surgery be developed.

The physicians recommended considering using intravenous, rather than gas, anesthetics; not using depolarizing muscle relaxants, such as succinylcholine; having an intensive care unit for postoperative care; providing respiratory support during anesthesia or sedation; monitoring blood oxygen saturation using a pulse oximeter throughout the procedure; and when possible, monitoring blood or lung carbon dioxide levels.

They also advised consideration of moving the patient from intubation (tube in the trachea) during surgery to noninvasive positive pressure ventilation right after surgery; using extreme caution when administering supplemental oxygen; using manually assisted cough and insufflation-exsufflation assisted cough postoperatively to clear secretions; and obtaining a cardiology consultation and closely monitoring cardiac and fluid status postoperatively.