Research Updates

• Putting Mini-Dystophins to the Test
• Developing Viral Vectors
• Rescuing the Diaphragm with Gene Therapy
• Heart and Breathing Muscles Involved in some LGMD
• Stem Cells Repair Heart Damage in Mice
• Second Gene Found in Myotonic Dystrophy
• Cardiac Exams Suggested for DMD and Becker Carriers
• Vitamin Deficiencies May Worsen SMA
• Walker-Warburg Syndrome and MEB Not Genetically Related
• New Genes Linked to PEO
• Protein Involved in Heart Formation Found
• Two Mitochondrial Disorders Studies Still Open - MELAS and MERRF

MDA Grantees Make Advances in Gene Therapy for DMD

At the American Society of Gene Therapy's fourth annual meeting, held in June in Seattle, several MDA-supported researchers described progress toward gene therapy for Duchenne muscular dystrophy (DMD). The researchers are working on several tactics to get muscles in the body to take up the dystrophin gene, which is defective in DMD. Along those lines, some of the most exciting topics reported at the meeting were:

Xiao Xiao

Jeffrey Chamberlain

Mini-dystrophins retain enough function to protect limb muscles and respiratory muscles in mice with DMD, according to reports from two different research groups. MDA grantees Xiao Xiao and Jeffrey Chamberlain designed these shortened versions of the dystrophin gene to fit inside the tiny adeno-associated virus (AAV) — considered one of the safest, most effective vectors (vehicles) for delivering therapeutic genes (see "Research Updates," Quest, vol. 8, no. 1).

But Xiao and Chamberlain needed to be sure that mini-dystrophins had the essential parts of the dystrophin gene. Xiao, at the University of Pittsburgh, tested several mini-dystrophins by using AAV to deliver them to the limb muscles of mice with DMD. At the ASGT meeting, he reported that the mini-dystrophins not only prevented wasting, but also largely preserved strength in the treated limb muscles.

Chamberlain, at the University of Washington in Seattle, showed that when mice with DMD are genetically engineered to produce mini-dystrophin, they remain largely free of muscle wasting, even in the diaphragm (a muscle in the chest that controls breathing).

MDA grantees Dongsheng Duan and John Engelhardt from the University of Iowa in Iowa City reported that they've designed an artificial version of AAV that's more potent than the AAV normally used in gene therapy studies. By replacing proteins found in the coat (covering) around AAV, they increased the virus's efficiency for delivering genes to muscle by over 200-fold.

Adeno-Associated Viruses	Adenoviruses	Naked DNA
Three different gene therapy methods have now been successfully used to restore dystrophin expression — and prevent muscle wasting — in mice with DMD. The adeno-associated virus can ferry mini-dystrophin to limb muscles, and the larger adenovirus can do the same thing with the full-length gene. Naked DNA — the dystrophin gene enclosed within a circle of free-floating bacterial DNA — can be injected into the blood and forced into the diaphragm.

Chamberlain reported progress with another viral vector that holds promise for DMD gene therapy, the adenovirus. This virus is highly attracted to muscle and large enough to accommodate the entire dystrophin gene, but it also can trigger a potentially dangerous immune reaction. To make a safe version of adenovirus, Chamberlain genetically deleted proteins in the virus that set off the immune system. These "gutted" adeno-viruses, he found, can safely and effectively deliver dystrophin to limb muscles — and preserve limb muscle strength — in mice with DMD.

MDA grantee Leaf Huang from the University of Pittsburgh has devised a gene therapy method to save the vital diaphragm muscle from the effects of DMD. In experiments on DMD mice, he injected "naked" dystrophin DNA (the gene without the protection of a virus) into a tail vein and surgically clamped a large vein that siphons blood from the diaphragm. The clamp, applied for just a few seconds, created enough pressure to force the DNA into the diaphragm before it could be degraded in the blood.

After treatment, the diaphragm showed long-lasting expression of dystrophin, and little sign of degeneration. With some slight modifications, Huang said this new procedure could someday be used to save people with DMD from respiratory failure.

Heart and Breathing Muscles Involved in Some Limb-Girdle MDs

Cardiac or respiratory impairment can occur in sarcoglycan-related limb-girdle muscular dystrophies (LGMD types 2C, 2D, 2E and 2F) and are worthy of the attention of physicians and patients, says a study in the March issue of Neuromuscular Disorders.

Cardiomyopathy — the degeneration of the cardiac muscle tissue — occurs often in neuromuscular disorders. The heart's muscle layer (myocardium) can be thickened or thinned, and either change can lead to defective pumping action. Defining the problem and determining how the heart muscle could be coaxed toward regeneration are the goals of much current research. For more on heart involvement in neuromuscular disorders, see "The Heart is a Muscle Too, Part 1" & "The Heart is a Muscle Too, Part 2"

The sarcoglycan-deficient LGMDs are forms of the disease caused by mutations in any of four genes — those for alpha-, beta-, gamma- or delta-sarcoglycan. The sarcoglycan proteins are normally present in cardiac and skeletal muscle cells.

Luisa Politano of the Section of Cardiomyology and Medical Genetics of the Second Naples University in Italy, and colleagues, studied 20 patients with sarcoglycan-deficient LGMDs. They found heart function was normal in five of 16 patients who underwent cardiac evaluations. The other 11 patients had varying signs of cardiomyopathy (cardiac muscle problem). There was no correlation between the amount of muscle involvement in the skeletal and cardiac muscles.

Of the 17 people who underwent respiratory evaluations, the researchers found that four had normal respiratory function; six had mild respiratory impairment; five had moderate impairment; and two had severe impairment.

Politano said her department checks muscular dystrophy patients for cardiac and respiratory, as well as skeletal, muscle problems at three-month intervals, and conducts detailed cardiac and respiratory examinations at least twice yearly for those with sarcoglycan-deficient LGMDs. Cardiac drugs and assisted ventilation are recommended for MD-associated cardiac and respiratory muscle weakness, she said.

Stem Cells Repair Heart Damage in Mice

MDA grantee Margaret Goodell at Baylor College of Medicine's Center for Cell and Gene Therapy in Houston recently led a team that demonstrated a possible new approach to treating damaged heart muscle.

The team studied mice exposed to conditions that simulated a heart attack (a blockage in the heart's blood supply) and then gave some of the mice an infusion of stem cells from the bone marrow of donor mice. The stem cells were selected for special characteristics that suggested they'd be able to generate new heart tissue.

The stem cells contributed to the growth of new cardiac blood vessels and cardiac muscle tissue, leading the researchers to announce in the June issue of the Journal of Clinical Investigation that the finding might eventually result in a therapy for treatment of damaged hearts.

Margaret Goodell

In these experiments, the researchers destroyed with high-dose radiation the bone marrow of the mice that received the infusions, a technique unlikely to be used in humans. The high-dose radiation may have contributed to the effectiveness of the treatment, however, so a substitute for its effects may have to be found.

Even with the radiation, Goodell emphasizes, the number of donor cells that actually became heart cells was very small, and much more work needs to be done before such techniques can be considered an option for human patients. Still, the team considers the work an encouraging first step that could eventually apply to the heart damage seen in some neuromuscular disorders. In genetic disorders, the patients' own stem cells could possibly be modified via gene therapy before being reinfused; or, donated cells without genetic defects could be used.

Second Gene Found for Myotonic Dystrophy

Since 1992, when a genetic flaw on chromosome 19 was found to underlie most cases of myotonic dystrophy (MMD), there have been patients whose genetic test for the disease was negative but who appeared to have the disorder — puzzling scientists and families alike.

Now, MDA-sponsored investigators Laura Ranum and John Day of the Institute of Human Genetics at the University of Minnesota in Minneapolis appear to have at least partially solved the mystery. Ranum is a molecular biologist, and Day is a neurologist who directs the MDA clinic at Fairview University Medical Center.

In 1998, Ranum and Day found that there's a second form of MMD for which a defect is located on chromosome 3. In the Aug. 3 issue of the prestigious journal Science, the team announced that the flaw for this form of MMD is located inside a specific gene on chromosome 3 and that it consists of an expanded, repeated section of DNA.

An expanded section of DNA on chromosome 19 underlies the first identified form of MMD. Scientists are already working to understand the molecular steps that lead from these DNA expansions to the signs and symptoms of chromosome 19 MMD.

To identify the newly pinpointed genetic flaw, the Minnesota group worked closely with scientists in Texas and Germany. The team studied about 20 Minnesota families and about 200 families in Germany in which myotonic dystrophy symptoms appeared but whose chromosome 19 DNA tests were negative.

Laura Ranum (front left) and John Day (front right), with their laboratory colleagues at the University of Minnesota

In both forms of the disease, those affected have progressive muscle weakness; difficulty relaxing muscles after use (myotonia); cataracts; insensitivity to insulin and other hormonal abnormalities; and cardiac problems. Other organs can also be involved in both forms.

The researchers hope a DNA test for the chromosome 3 MMD (officially called DM2, as opposed to DM1 for the chromosome 19 form) will soon be widely available and that the new findings will shed light on the causes of both disorders — a step that may ultimately lead to treatment.

Editor's Note: The standard medical abbreviation for myotonic dystrophy is DM. MDA uses MMD for myotonic dystrophy to avoid confusion with the abbreviation for dermatomyositis, which is also DM.

Cardiac Exams Suggested for Duchenne and Becker MD Carriers

Heart problems can occur in female carriers of Duchenne and Becker muscular dystrophies (DMD and BMD), but serious ones may be less common than some previous studies have suggested, says a report in the March issue of Neuromuscular Disorders. The report confirms previous studies in finding no correlation between skeletal muscle weakness and cardiac involvement.

The new study, led by Lucy Grain of the Department of Paediatric Cardiology at John Radcliffe Hospital in Oxford, England, examined 56 female carriers of DMD or BMD and 35 women who weren't carriers (a control group).

The investigators found that 18 percent of the carriers (10 women) and 6 percent of the control group (two women) had cardiac abnormalities. This difference was considered important.

However, the investigators noted that most of those with cardiac changes had measurements on electrocardiograms and echocardiograms just outside the normal ranges, and that only one woman had symptoms, which were mild. They also noted that the study didn't measure progression over time.

The study authors recommend electrocardiograms and echocardiograms for DMD and BMD carriers at approximately five-year intervals, particularly in older women. More frequent and/or detailed examinations should be undertaken if any abnormalities are found, they said.

Vitamin Deficiencies May Worsen SMA

A new study suggests that people with spinal muscular atrophy (SMA) should be sure their diets are providing adequate levels of the vitamins folate and B12.

SMA is caused by genetic deficiencies of the SMN (survival motor neuron) protein. The study shows that the activity of SMN depends on folate and vitamin B12, which means that insufficient levels of the two vitamins might magnify the effects of SMN deficiency and hasten the course of SMA.

Gideon Dreyfuss

"It would be advisable for [SMA] patients to consult their doctors to see that they're getting at least the recommended daily dose of these vitamins, but we're not suggesting that people rush out and take them," said the study's lead scientist, Gideon Dreyfuss, a molecular biologist at the University of Pennsylvania in Philadelphia.

Dreyfuss had previously shown that SMN is required for putting the finishing touches on RNA — an essential intermediate between DNA (the material that makes up genes) and protein.

To perform its job, SMN has to work with several other proteins, and, in his latest study, Dreyfuss found that SMN is more likely to recognize those proteins when they carry a chemical "tag" called a methyl group. Methylation — the process of sticking a methyl group onto a protein — requires folate and vitamin B12. So the two vitamins are probably critical to SMN's normal function, and could be especially important in someone with SMA, Dreyfuss said.

For his study, which appeared in the May 25 issue of Molecular Cell, Dreyfuss examined laboratory cell lines and biochemically isolated proteins — not people with SMA.

Dreyfuss also said it's possible that, for some people, using folate and vitamin B12 as nutritional supplements might help ameliorate the muscle weakness and wasting caused by SMA. He and other researchers are planning a clinical trial to determine whether dietary intake of folate and vitamin B12 actually influence the course of SMA.

Walker-Warburg Syndrome and MEB Not Genetically Related

A study in the April 24 issue of Neurology shows that two forms of congenital muscular dystrophy (CMD) — Walker-Warburg syndrome (WWS) and muscle-eye-brain disease (MEB) — are genetically distinct, despite speculation until now that they might result from defects in the same gene. A region of chromosome 1 was found linked to MEB in 1999; the precise gene involved hasn't yet been found.

Both WWS and MEB involve infancy-onset muscular dystrophy and brain abnormalities, but MEB is less severe than WWS.

A multinational group led by Bru Cormand at the Pompeu Fabra University in Barcelona, Spain, was also on the team that linked MEB to chromosome 1, and conducted the new study. Jerry Mendell, an MDA grantee and MDA clinic co-director at Ohio State University in Columbus, wrote an accompanying editorial praising recent progress in sorting out the congenital forms of MD and noting an urgent need to reclassify these disorders in the light of new knowledge.

New Genes Linked to PEO

Scientists have long been perplexed by a syndrome known as progressive external ophthalmoplegia (PEO), but they've now identified a handful of culprit genes linked to the disorder.

PEO refers to a gradual paralysis of eye movements, caused by weakness in muscles surrounding the eyes. It's often one of many symptoms of mitochondrial myopathy — a type of disorder caused by defects in the tiny cellular energy factories called mitochondria. But in some families, PEO stands out as a distinct inheritable syndrome.

This type of PEO, called autosomal dominant PEO (adPEO), is clearly caused by defects in the most abundant type of genetic material — DNA that's packed into the cell control center known as the nucleus. But it's also associated with widespread deletions (missing pieces) of DNA housed within the mitochondria themselves.

For years, scientists have suspected that defects in certain nuclear genes might somehow destabilize mitochondrial DNA, leading to adPEO. It turns out they're right: To date, they've discovered three nuclear genes linked to adPEO, and all of them appear to specifically control the repair and replication of mitochondrial DNA.

One adPEO-linked gene was identified last year. Called ANT1, it's needed to import building blocks for DNA into the mitochondria. Two new genes, called Twinkle and POLG, were reported in the July issue of Nature Genetics by independent European research groups. Twinkle encodes a "helicase" that unwinds tightly wrapped strands of DNA, and POLG encodes a "polymerase" that extends new strands of DNA.

Studying those three genes and their functions will lead to a better understanding of the disease process underlying adPEO and other inherited mitochondrial disorders, scientists say.

Protein Involved in Heart Formation Found

MDA grantee Da-Zhi Wang in the Department of Molecular Biology at the University of Texas Southwestern Medical Center in Dallas was part of a team that identified a protein that turns on cardiac genes essential to the formation of heart muscle cells. The researchers have named the protein myocardin.

The finding, which is published in the June 29 issue of Cell, may have implications for repair of damaged hearts in neuromuscular diseases.

Two Mitochondrial Disorders Studies Still Open

Two studies on mitochondrial disorders are taking place at the Mitochondrial Research Center of Columbia University in New York. Both studies are under the direction of Darryl C. DeVivo, professor of pediatrics and neurology, who co-directs the MDA clinic at Columbia-Presbyterian Medical Center.

To find out more about either of the following studies, contact:

DCA for MELAS Trial

The researchers are conducting a three-year clinical trial to evaluate the safety and effectiveness of dichloroacetate (DCA) in older children and adults with MELAS (mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes) that stems from a genetic flaw known as the 3243 point mutation. Previous research has shown that lactic acidosis — a high level of lactate in the blood — is associated with neurological impairment in people with MELAS. DCA is a medication that lowers blood lactate levels.

The trial opened last year and is expected to close in August 2004.

Participants must be older than 6 years; have the full list of MELAS symptoms; and have a confirmed 3243 point mutation. Patients who don't know their genetic defect can enter the natural history study (see next item) to be tested.

Natural History Study in Point Mutations

This study will investigate clinical syndromes (clusters of symptoms) associated with the genetic defects that underlie two mitochondrial disorders — MELAS and MERRF (myoclonus epilepsy with ragged red fibers). Specifically, MELAS stemming from the 3243 point mutation and MERRF stemming from the 8344 point mutation will be studied.

Multiple organs are involved in these disorders, but the nervous system is particularly affected. The researchers are engaged in a long-term evaluation of patients and their maternal relatives who have the same mitochondrial genetic defects but who have minimal or no symptoms.

They'll address questions regarding the natural history of the diseases, their pathophysiology (the things that go wrong in the body), disease management and prognosis.

The study can test patients to determine whether they have one of the mutations being studied.