Tackling DMD on Many Fronts
by Margaret Wahl
The underlying cause of Duchenne muscular dystrophy (DMD) can be any of a large number of mutations in the gene for the dystrophin protein. One strategy now being tested for overcoming this problem is gene therapy, the insertion of new dystrophin genes. Another is the transplantation of cells that give rise to muscle. Other strategies include changing the way cells interpret the genetic information that’s already there; reducing some of the secondary effects of dystrophin deficiency; and compensating for the loss of dystrophin.
20th Anniversary
of DMD Gene ID Marked in Ottawa
More than 40 speakers and some 150 registrants convened in
Ottawa, Ontario, May 3-5 to celebrate the 20th anniversary of the identification of the dystrophin gene and protein.
Nearly all presenters and many of the registrants were current or former MDA grantees, and many acknowledged MDA as the funding source that allowed them to start their careers. MDA, along with other organizations and companies, also contributed funding to the conference itself.
Among the presentations were updates on:
-
gene and cell transfer strategies
- how muscle stem or precursor cells become muscle cells
- stop codon read-through, a strategy in which cells are coaxed to ignore abnormal stop signals in the dystrophin gene
-
stimulation of utrophin, a protein very similar to dystrophin that can at least partially compensate for its absence
- exon skipping, a technique in which error-containing sections of the dystrophin gene are masked, allowing production of functional dystrophin protein molecules.
Gertjan van Ommen at Leiden University in the Netherlands presented some exciting new data. He announced that four DMD-affected boys in the Netherlands who were given an exon skipping compound targeted to their genetic errors all began producing what appears to be functional dystrophin.
Van Ommen, with colleagues at Leiden University and at Prosensa, a Dutch biotech company, gave each of the 10-to-13-year-old boys a single injection of an exon skipping compound into a front lower leg muscle. All were missing sections of the dystrophin gene lying between exons 48 and 52.
Twenty-eight days after injection, biopsies of the boys showed strong and even dystrophin distribution in the injected area. There were no adverse effects except mild injection site pain.
The Dutch group hopes to start systemic injections of the exon skipping construct later this year.
For several years, MDA has supported Judith van Deutekom at Leiden University and Stephen Wilton (see “Team Finds 300,” ) at the University of Western Australia in Perth for development of exon skipping. The skipping is accomplished with “antisense” compounds, which keep cells from obeying (making “sense” of) targeted genetic instructions.
PTC124 Shows
Benefit in Mice
Scientists say data from mice that received PTC124, an experimental drug already in clinical trials in boys with DMD, confirm the “proof of principle” on which the drug’s action is based.
The investigators, including MDA grantee H. Lee Sweeney of the University of Pennsylvania, published their results online April 22 in the journal Nature.
PTC124, developed by PTC Therapeutics of South Plainfield, N.J., with funding from MDA, is a drug that causes cells to ignore stop codons that are abnormally (“prematurely”) placed in the dystrophin gene. About 15 percent of DMD-affected patients have stop codon mutations.
Dystrophin-deficient, DMD-affected mice with premature stop codons that received PTC124 by a combination of oral feeding and/or injections were stronger and showed less contraction-related muscle damage than is characteristic of these mice.
Blood levels of the enzyme creatine kinase, which leaks out of damaged muscle cells, dropped significantly compared to those of untreated mice, indicating many more muscle cells remained intact.
Tests showed the treated mice produced about 20 percent to 25 percent of the normal level of full-length dystrophin, which was in its normal place in the muscle fiber membrane.
In dystrophin-deficient mice, exercise normally leads to muscle fiber damage and loss of strength. However, that didn’t happen in the PTC124-treated mice.
“We could not detect the changes in force due to damage caused by exercise that we typically find in animals with this disease,” Sweeney said.
Of equal importance, the investigators found no evidence of “off-target” effects from PTC124. Cells appeared to read properly positioned stop codons in other genes normally. No toxicity of any kind was observed.
Experimental Compound Shows Promise for SBMA
A compound known as ASC-J9 has shown promising effects in treating spinal
bulbar muscular atrophy (SBMA), also called Kennedy’s disease, in mice with the disorder. Mice treated with ASC-J9 dissolved in corn oil and injected into the abdomen every other day showed better motor and sexual function and longer survival than mice treated with a corn oil solution alone. The treatment was effective whether it was given before the onset of symptoms or long after.
SBMA, which affects males almost exclusively, is a disease in which muscle-controlling nerve cells (motor neurons) in the spinal cord and brain stem degenerate. Feminization and impairment of fertility and sexual function may also occur.
The root cause of the disease is an expanded section of DNA in the gene for the androgen receptor, a protein that normally transports male hormones (androgens) inside cells.
The expanded DNA leads to an expanded, sticky androgen receptor protein that forms clumps in the nuclei of nerve and muscle cells. Trapped inside the clumps are a number of proteins that would otherwise regulate various cellular functions.
Zhiming Yang at the University of Rochester (N.Y.) Medical Center, and colleagues, who published their results in the March issue of Nature Medicine, say they believe ASC-J9 disrupts this abnormal clumping.
Diane Merry at Thomas Jefferson University in Philadelphia, who’s had MDA support for SBMA research, was part of the research team. |
Team Finds 300 Exon Skipping Compounds
Stephen Wilton at the University of Western Australia in Perth, and colleagues, have developed with MDA support some 300 compounds that can coax cells into skipping over errors in the dystrophin gene. The majority, he says, have therapeutic potential for treating DMD.
“This is the first report showing that every targeted dystrophin exon can be removed [skipped],” Wilton said. Exons are regions of a gene that contain portions of the code, or recipe, for a protein, and Wilton and others will target those exons that contain errors in the dystrophin code.
In the experiments conducted by Wilton’s group, results for which were published online Feb. 6 in Molecular Therapy, the researchers found that more than 50 percent of dystrophin exons can be efficiently targeted and removed. Wilson said work is continuing to enhance skipping across the entire gene.
Some children with DMD are missing large sections of dystrophin DNA, or carry a defect in a crucial part of the gene, and exon skipping probably won’t help them, Wilton says. However, he notes, the majority of boys with DMD have small errors in the DNA that either stop dystrophin synthesis too early or cause the cell to interpret the genetic instructions incorrectly. For an estimated 80 percent of these boys, exon skipping theoretically would allow the error-containing genetic region to be spliced out (skipped) and nearly normal dystrophin protein to be made.
Preliminary results of an exon skipping clinical trial in the Netherlands, under the auspices of Prosensa of Leiden and Leiden University Medical Center, has already produced tantalizing results (see “20th Anniversary” ).
A trial to test the safety of a compound designed by the Wilton lab that targets exon 51 is scheduled to begin in nine boys with DMD later this year in London. “The challenge will be to extend the trials to address other amenable dystrophin mutations as soon as possible,” Wilton said.
Blocking Inflammation
A research group headed by MDA grantee Denis Guttridge at Ohio State University Medical Center in Columbus has significantly added to knowledge of how inflammation and inhibition of muscle formation are involved in DMD. Their data support the validity of existing therapies, such as corticosteroids, and suggest additional pathways to pursue in new therapy development.
Swarnali Acharyya at Ohio State, with colleagues there and at several other institutions, published their findings online March 22 in the Journal of Clinical Investigation.
They report that a signaling pathway called IKK/NF-kappa-B, which markedly increases the inflammatory response of the immune system and inhibits muscle regeneration, is abnormally active in DMD-affected, dystrophin-deficient mice.
When the investigators treated the mice with NBD, a compound that blocks the activation of the IKK/NF-kappa-B pathway, the animals showed significantly less inflammation and more regeneration of muscle tissue. Their diaphragm muscles showed better force development than those of mice treated with an inactive form of this compound.
Corticosteroid drugs, such as prednisone, also interfere with inflammation by blocking NF-kappa-B. These medications, which help maintain muscle function in patients with DMD, have been in use in this disease for several years, although neither their mechanism nor their proper dosage level has been clear. (A multinational clinical trial, slated to begin soon, will answer some of the questions surrounding their use.)
“We’re excited about these findings, because we believe they add to our understanding of how this signaling pathway functions in muscle disorders such as DMD,” Guttridge said. “Based on our genetic and pharmacological data, it suggests that NF-kappa-B acts in both immune cells and muscle cells to regulate the dystrophic process. We believe this would make NF-kappa-B an attractive therapeutic target. Presumably, if you can inhibit its activity in both cell types, you should be able to block inflammation and at the same time stimulate new muscle growth.”
Guttridge says evidence from their work and other published studies demonstrates that NBD isn’t toxic in mice but that follow-up studies will be needed to confirm this before human trials can be contemplated.
Improving Response to Exercise
Raising levels of a protein called PGC-1-alpha, part of a complex system that changes the way nerves and muscles interact in response to exercise, could be a new therapeutic avenue in treating DMD and perhaps other neuromuscular disorders, say MDA-supported researchers in the April 1 issue of Genes & Development.
MDA grantee Christoph Handschin at the University of Zurich (Switzerland), and colleagues, say DMD-affected mice given extra genes for PGC-1-alpha fared much better than their untreated counterparts. (Handschin was at the Dana-Farber Cancer Institute in Boston when he did this work.)
The investigators say rapid increases in PGC-1-alpha levels in response to exercise, and decreases in response to inactivity, suggest that this protein normally helps muscles and nerves adapt to changing circumstances. Their experiments indicate that it does this by switching genes on and off at the neuromuscular junctions, places where nerve and muscle fibers meet. Higher levels of PGC-1-alpha correlate with higher levels of proteins that help form each neuromuscular junction.
Mice with DMD that were bred with extra PGC-1-alpha genes performed significantly better when required to run on a treadmill, and their muscles showed far less evidence of damage than those of their untreated counterparts when examined under the microscope. Blood levels of creatine kinase, an enzyme that leaks from damaged muscle cells, were significantly reduced in the treated mice.
The researchers write that increasing PGC-1-alpha levels is a “promising and novel strategy for the treatment of DMD, the related Becker muscular dystrophy and other neuromuscular diseases.”
Female Muscle Stem
Cells Work Better
Stem cells taken from the muscles of healthy mice and transplanted into the muscles of mice with a disease resembling Duchenne muscular dystrophy (DMD) are more effective at causing muscle regeneration if they’re taken from female donors instead of male donors, say investigators at Children’s Hospital of Pittsburgh, the University of Pittsburgh and the University of California-Los Angeles.
Researchers in the laboratory of Johnny Huard at the University of Pittsburgh, who published their findings in the April 9 issue of the Journal of Cell Biology, say they think the difference may be caused by “innate sex-related differences in the cells’ stress responses.” Huard received MDA support for this work.
Their experiments, they say, show that the superior regenerative abilities of the female-derived stem cells probably isn’t directly related to hormonal factors or to a difference in the immune response to male versus female cells.
Instead, they say, it seems to be related to the female-derived stem cells’ propensity for staying immature and proliferating for the first three days or so after transplantation, while the male-derived cells tend to mature (differentiate) immediately into muscle cells.
After three days, they say, the environment in which the new cells find themselves becomes more hospitable, with less inflammation and more oxygen available. The female cells, many of which will have survived and proliferated during the early phase of transplantation, can then differentiate, fusing with muscle fibers and causing them to regenerate.
But by this time many of the male cells will have already matured in the more hostile circumstances and been killed by cellular defenses, leading to the regeneration of a smaller number of muscle fibers.
The new findings, Huard said, “may shed light on the conflicting results in the literature on stem cells, since in many instances the gender of the [recipients] and the donor are not even characterized.” |
|
Canadian Team Will Pursue Cell
Transplants in Men with DMD, BMD
Jacques Tremblay at Laval University in Quebec City says he has obtained permission from Health Canada to conduct a clinical trial to see whether transplantation of muscle precursor cells (MPCs) can improve the strength of an arm muscle in 10 men with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) who are at least 18 years old.
Tremblay, who received MDA support to develop laboratory procedures associated with MPCs in the late 1990s and in 2004, has previously shown that dystrophin (the protein needed in DMD) was produced in the muscle fibers of eight out of nine patients who received transplantations of this type of cell.
The new trial will use a technique his lab developed called high-density cell transplantation, which allows millions of cells per square centimeter to be injected at a time.
In January, in the journal Neuromuscular Disorders, Tremblay’s group reported they’d used the high-density technique successfully in a 26-year-old man with advanced DMD, who tolerated the procedure well and may have benefited from it.
They used cells that were isolated from an arm muscle of the patient’s father and then coaxed in the lab toward becoming muscle, but they weren’t fully mature. They then injected 67.5 million MPCs per square centimeter into a lower leg muscle, 55 million per square centimeter into the area below the thumb, and 25 million per square centimeter into the biceps muscle.
The patient, who was given the immunosuppressant drug tacrolimus (Prograf) to prevent rejection of the cells, said he found the injections were more tolerable than the average dental procedure.
A year and a half after the transplant, some 35 percent of the injected muscle fibers in the leg were making dystrophin. In the biceps, where there was more scar tissue, there were only a few fibers left, two of which were making dystrophin.
The only functional improvement the team saw was a significant increase in the patient’s ability to move his thumb. They say this may have been a placebo effect [a favorable response to a treatment because the patient believes in it], since the subject knew he was receiving MPCs.
The new trial, Tremblay notes, will be “blinded,” meaning one arm will get MPCs and the other a salt solution. Neither the investigators nor the participants will know which arm got which preparation.
Tremblay says progress in his lab can be followed at http://jptremblaylab.crchul.ulaval.ca.
NIH Opens Two
New SMA Trials
Two National Institutes of Health-sponsored, multicenter studies of sodium phenylbutyrate in spinal muscular atrophy (SMA) have opened. One will enroll up to 30 babies with type 1 SMA who are 2 months to 2 years old, and the other will enroll up to 30 participants with type 2 or 3 SMA who are 2 to 12 years old.
Preliminary evidence from laboratory studies and a pilot trial suggests that sodium phenylbutyrate may increase levels of the SMN protein, which is needed but deficient in SMA. For details, contact Barbara Driver at (301) 738-3698 or BarbaraDriver@westat.com.
IVIG Treatment Benefits Those with Worsening MG
A clinical trial to test the effectiveness of intravenous immunoglobulins (IVIG) in treating myasthenia gravis (MG) has provided what the investigators say is the “first reliable evidence for the effectiveness of IVIG in patients with MG and worsening weakness.”
In MG, the immune system mistakenly attacks components of the neuromuscular (nerve-to-muscle) signal transmission system, resulting in fluctuating weakness that can be severe. It’s generally treated with drugs that prolong the action of a chemical carrier of nerve signals and/or with those that suppress the immune system.
Two other treatments, less often used, are plasmapheresis, which removes the offending immunoglobulins from the blood; and, somewhat paradoxically, immunoglobulin infusion (IVIG), which adds pooled immunoglobulins from donors. The added immunoglobulins are thought to redirect the immune system.
Lorne Zinman at Sunnybrook Health Sciences Center in Toronto, and colleagues, who published their findings in the March 13 issue of Neurology, randomly assigned 51 adults with MG and worsening weakness to receive a two-day infusion of either IVIG or an inactive solution (placebo).
Two weeks later, 25 percent of the IVIG group showed improvement, compared with 6 percent of the placebo group. None of those who received IVIG worsened, while 4 percent of those on placebo did.
Functional changes were relatively small. They were the most meaningful in those with moderate to severe MG, compared to those with mild symptoms. Onset of the effect was rapid compared with the usual response to medications.
There were no serious side effects or adverse events.
Researchers Share New
Findings at AAN Meeting
The 59th annual meeting of the American Academy of Neurology (AAN) took place April 28-May 5 in Boston and included several presentations relevant to people with neuromuscular disorders. Summaries of a few of them follow. (For a complete report, see “What’s New.”)
Alemtuzumab Shows Promise in IBM
An immunosuppressant medication called alemtuzumab (Campath), approved to treat a form of chronic leukemia, may have promise in treating inclusion-body myositis (IBM), a muscle disease that’s characterized by both inflammation and degeneration.
Marinos Dalakas at the National Institutes of Health, and colleagues, gave alemtuzumab to 13 IBM patients for four days at 0.3 milligrams per kilogram per day and saw an average strength increase of 8 percent four months later and 4 percent six months after treatment. Six of the 13 participants experienced strength increases averaging 16 percent and reported improvement in activities of daily living. The other seven declined by 6 percent.
High-Dose Vitamin C Not Helpful
In 12 People With CMT1A
High-dose ascorbic acid (vitamin C) was neither well tolerated nor beneficial in a small study in people with type 1A Charcot-Marie-Tooth disease (CMT1A).
In a one-year study of 12 people with CMT1A who received 5 grams per day of ascorbic acid, six tolerated the dose for the whole year, five developed gastrointestinal side effects that required lowering the dose, and one discontinued the medication because of general malaise. No significant differences in symptoms or electrophysiological measurements before and after treatment were detected in anyone.
Drug Combination Didn’t Help in MG
Adding the immunosuppressant drug mycophenolate mofetil (CellCept) to prednisone, a more commonly used immunosuppressant, didn’t improve strength, activities of daily living or any other measurement in people with myasthenia gravis (MG) after three months of treatment. Eighty people with MG, a disorder in which the immune system mistakenly attacks the nerve-muscle junction, took prednisone at 20 milligrams a day, plus either 2.5 grams a day of mycophenolate mofetil or a look-alike placebo (inert substance).
The investigators speculated that the lack of difference in the two groups could be due to a greater-than-predicted benefit from the prednisone alone or the short duration of the study.
Anti-Androgen Drug May Improve Strength in SBMA
A drug that reduces levels of the male hormone testosterone (an androgen) has shown promise in the treatment of spinal-bulbar muscular atrophy (SBMA, or Kennedy’s disease), in which a mutation in the gene for the androgen receptor protein causes progressive weakness. In this study from Japan, four patients received injections of goserelin (Zoladex) every three months. Three of the four improved in either strength or speed of nerve signal conduction or both.
The investigators said a larger study was needed to confirm the effectiveness of this drug. |
|
