• Miniaturized Gene Raises Hopes in Duchenne, Becker
• Duchenne Muscles May Be Starved for Oxygen
• Dystrophin's 'Heartiness' Good for Gene Therapy
• Myotonic MD (MMD) Treatments Appear Nearer As Knowledge Grows
• Antiseptic May Target Mitochondial Disease
• Antioxidant May Help Heart in Friedreich's Ataxia
• Mutant Protein Creates Mess in OPMD
• Heart Problems Differ Among MD's

Xiao Xiao

Miniaturized Gene Raises Hopes in Duchenne, Becker

MDA-supported researchers Xiao Xiao and Juan Li in the Department of Molecular Genetics and Biochemistry of the University of Pittsburgh were on a team that constructed miniaturized versions of the dystrophin gene small enough to fit inside an adeno-associated virus (AAV).

Dystrophin is the protein needed by people with Duchenne or Becker muscular dystrophy, in whom the dystrophin gene is flawed. AAVs are considered among the safest and most effective vehicles for delivering genes to muscle tissue.

In experiments injecting AAVs carrying the highly miniaturized genes into leg muscles of dystrophin-deficient mice, the research team achieved adequate dystrophin production in 40 to 88 percent of muscle fibers. The team published its findings in the Dec. 5 issue of Proceedings of the National Academy of Sciences.

To get the dystrophin gene small enough to fit into an adeno-associated virus, Xiao Xiao and colleagues miniaturized it but preserved its important functions.

In designing the very small dystrophin genes, Xiao's group focused on the midsection, or "rod domain," of the protein. This section is thought to provide some shock absorption and act as a link between the anchored ends of the ropelike dystrophin molecule, Xiao says.

The researchers also removed some of the DNA that codes for the section of dystrophin called the C terminus, which attaches dystrophin to a group of proteins spanning the cell membrane. Previous attempts to miniaturize the gene have left this section intact, making the gene too large to fit inside an AAV.

But, says Xiao, "the C terminus has two domains." The minigene includes the cysteine-rich domain, which is essential for dystrophin to anchor to the membrane complex, but eliminates another, less essential domain, called the actin-binding section.

Xiao's group made three slightly different dystrophin minigenes and found one especially effective. Xiao says he hopes the new minigene can eventually be used in clinical trials for Duchenne or Becker MD.

Duchenne Muscles May Be Starved for Oxygen

A new study suggests that insufficient oxygen to active muscles might contribute to muscle degeneration in boys with Duchenne muscular dystrophy (DMD). The finding has implications for management and treatment of the disease.

In the 1980s, MDA-funded researchers figured out that DMD is caused by loss of the dystrophin protein in muscle cells. Despite the importance of that discovery, it's still not clear why dystrophin is vital to muscle cells.

Because dystrophin is a large protein that connects the inner and outer architecture of muscle cells, researchers have suspected that loss of the protein might lead to structural damage during contraction. But a new study in the December issue of the Proceedings of the National Academy of Sciences indicates that loss of dystrophin indirectly cuts off the muscle cells' blood supply during exercise.

The study's authors, including MDA grantees Susan Iannaccone, James Stull and Gail Thomas of the University of Texas Southwestern Medical Center in Dallas, suggest that dystrophin interacts with another protein found in muscle cells -- neuronal nitric oxide synthase (nNOS). In active muscles, nNOS produces the gas NO, which promotes the dilation of nearby blood vessels, ensuring a steady supply of oxygen. Although it's not clear how, the loss of dystrophin displaces nNOS from its normal location in muscle cells. The result is that nNOS doesn't increase blood flow to muscles during exercise, limiting the muscles' oxygen supply when they need it most.

The study shows oxygen deprivation occurred during a hand grip exercise in muscles of 10 boys with DMD and four children with spinal muscular atrophy. The effect didn't occur in children without muscle disease or in those with limb-girdle muscular dystrophy or polymyositis.

The study has implications for the long-standing debate over whether exercise is helpful or harmful to children with DMD, says Iannaccone, co-director of the MDA clinic at UT Southwestern. "It makes it more likely that exercise is bad for them, but it depends on the kinds of exercise," she says. Further research will be necessary to investigate this issue.

The study's findings will also affect the development of gene therapy and other treatments for DMD. The shortened forms of dystrophin being designed for DMD gene therapy should be tested for their effects on nNOS function, suggests MDA grantee H. Lee Sweeney, who's investigating DMD gene therapy at the University of Penn-sylvania in Philadelphia. Iannaccone says the study also means that "there may be some treatments that are already available on the market that might help kids with DMD," such as drugs that affect blood flow during exercise.

Jeffrey Chamberlain

Dystrophin's 'Heartiness' Good for Gene Therapy

MDA grantees Jeffrey Chamberlain, now in the Department of Neurology of the University of Washington Health Sciences Center in Seattle, and Andrea Amalfitano of the Department of Human Genetics at Duke University Medical Center in Durham, N.C., were part of a team that created a new system to shed light on the potential of gene therapy to treat Duchenne or Becker muscular dystrophy. (Chamberlain recently moved from the University of Michigan's Department of Human Genetics, where this work was conducted.)

The team bred mice carrying a dystrophin gene under the control of a molecular switch that "turned off" production of dystrophin when the animals were given tetracycline, an antibiotic medication. They found that dystrophin persisted in the muscle cells of the mice for at least six months after production was shut off.

"The results suggested that the dystrophin protein may be an especially hearty protein, since the functional abilities of sarcolemma-associated dystrophin to prevent muscle fiber degeneration in vivo [in live animals] exceeded six months," the authors wrote in a paper published in the Oct. 12 issue of Human Molecular Genetics. (The sarcolemma is the muscle cell membrane.)

Results were good in younger mice and difficult to evaluate in older mice. The researchers note that the studies may not have allowed time for the older mice to recover from their long-standing dystrophin deficiency.

"My feeling is that, in the [older] mice, the muscles are indeed being protected, but this protection and functional correction does not result in a 'visible' improvement in the way the muscle looks under a microscope," Chamberlain says. "Further studies are needed to verify this point and to determine the degree of functional protection that results from introduction of various levels of dystrophin into mice at different ages."

Chamberlain says the researchers are much encouraged by the data on the heartiness of the dystrophin protein and the persistence of dystrophin in muscle cells. But, he noted, "additional work needs to be done to determine what degree of correction can be expected from the introduction of dystrophin into muscles that are already dystrophic."

Myotonic MD Treatments Appear Nearer as Knowledge Grows

Stephen J. Tapscott

In summarizing research on myotonic muscular dystrophy (MMD) since its genetic cause was discovered in 1992, Stephen J. Tapscott at the Fred Hutchinson Cancer Research Center in Seattle says new findings point the way to possible interventions in this often baffling disorder. Tapscott, a member of MDA's Scientific Advisory Committee and a past MDA grantee, reported on MMD research status in the Sept. 8 issue of the journal Science.

"The new mouse models [mice bred to show certain characteristics of MMD] will allow us to test different hypotheses about what causes myotonic dystrophy in all its aspects," Tapscott says. "These models may unveil targets for drug therapy."

The most common genetic defect underlying MMD, which was identified in 1992 with MDA support, is in an area of DNA on chromosome 19. An expanded section of DNA made up of chemical structures known as CTG repeats, though it isn't actually part of a gene or a protein made from a gene, causes trouble in nearly every organ of the body. The most prominent myotonic dystrophy symptoms occur in the skeletal and involuntary muscles, heart muscle, the lens of the eye and the central nervous system. The disease may affect sleep and respiratory centers in the brain.

Tapscott's summary reviews research, much of it MDA-supported, on how these diverse and sometimes severe effects may arise from the CTG expansions. He says there are at least three things going wrong in MMD and therefore three possible targets for therapy.

For example, near the CTG repeats on chromosome 19 is the gene for the DMPK protein. The level of DMPK made by cells may be lowered by the expanded repeats, a theory supported by the finding that mice lacking this protein show heart defects remarkably similar to those in people with MMD.

Also near the repeats is a gene for the protein known as SIX5. The ocular lenses of mice lacking adequate SIX5 have cataracts, as do people with MMD.

A third problem in MMD may be the CTG repeats themselves. In cell nuclei, CTG repeats in the DNA are changed to similar compounds called CUG repeats in RNA, the chemical step that follows DNA in each cell nucleus. Studies in mice with extra CUG repeats revealed that these repeats may cause the muscle weakness and inability to relax muscles at will (myotonia) that people with MMD experience. One possibility, says Tapscott, is that the body misreads the CUG repeats and behaves as if the cells harbored a virus.

"If a cell is mistaking CUG repeats for viral RNA and killing the cell, you might be able to target the protein that causes that to happen," Tapscott speculates.

In a broad sense, he says, "it may actually be easier to treat myotonic dystrophy than it will be to treat diseases where information from the DNA is missing, where you're missing a gene or a part of a gene."

Antiseptic May Target Mitochondrial Disease

Certain mitochondrial diseases are currently beyond the reach of gene therapy, but a chemical commonly used as an antiseptic might be the magic bullet for that problem.

A DQAsome is a hollow capsule formed from many bolaform-shaped DQA molecules (highlighted). The capsule might make an ideal gene therapy vector for mitochondrial diseases.

Gene therapy — the delivery of corrective genes to make up for a genetic defect — holds promise for treating most genetic diseases, and in some cases, is already being tested in clinical trials. But some mitochondrial diseases are off-limits to current gene therapy techniques because they're caused by defects in hard-to-reach genes.

Most of our genetic material (DNA) is housed within the nuclei, the control centers found in nearly all our cells. But some DNA is housed in the mitochondria, the tiny powerhouses that provide energy to cells. Defects in mitochondrial DNA can cause energy deficits that lead to the extreme fatigue and weakness characteristic of mitochondrial muscle diseases.

While scientists have achieved success in targeting therapeutic genes to nuclei, it's more challenging to get genes into mitochondria. The problem lies in the gene delivery vehicles, or vectors, says Volkmar Weissig, a biochemist at Northeastern University in Boston.

In gene therapy, a vector's first task is to penetrate the cell's outer membrane, or skin. Once inside the cell, the vector must breach a second membrane surrounding either the nucleus or the mitochondrion, all the while holding on to its genetic payload.

The most commonly used vectors for gene therapy are viruses and synthetic compounds called liposomes, hollow spheres composed of an outer shell of lipid (the same type of chemical that makes up cell membranes). Viruses, says Weissig, naturally infect cells and introduce foreign genes into nuclei, but apparently can't send genes to mitochondria. With a composition similar to cell membranes, liposomes can penetrate cells, but they usually release their DNA into the cellular space shortly afterward.

To make mitochondrial gene therapy feasible, Weissig is using MDA support to custom-design a vector from a lipidlike, antiseptic chemical called dequalinium (DQA). A single unit of DQA looks like a traditional Native American weapon called a bola, and this bolaform shape allows DQA to form liposome-like capsules called DQAsomes. DQAsomes can be filled with DNA, and thus might be ideal weapons against mitochondrial diseases, Weissig suggests. So far, he's shown that the DQAsomes can selectively target DNA to mitochondrial membranes.

"This is basic research" that won't immediately lead to treatment for mitochondrial disease, Weissig says. But it's a significant step in the right direction.

Massimo Pandolfo

Antioxidant May Help Heart in Friedreich's Ataxia

The 1996 identification of the gene that, when defective, causes Friedreich's ataxia (FA) has led to more than four years of intensive research to understand what the gene and its protein product (now called frataxin) normally do — and what goes wrong in FA.

Massimo Pandolfo, a neurologist and MDA grantee now at the University of Montreal's Hospital Center, led the Baylor College of Medicine team that found the frataxin gene and has since been studying FA and its biochemistry.

Recent studies have confirmed early guesses, he says, that frataxin keeps iron levels normal inside structures known as mitochondria, which are cellular energy-producing units. When there isn't enough frataxin, iron builds up in the mitochondria, according to laboratory studies performed mostly in yeast cells. The iron buildup leads not only to a loss of mitochondrial function but to a general buildup of toxic chemicals known as free radicals and to a toxic process known as oxidative stress.

The challenge now, says Pandolfo, is to move from simple organisms, such as yeast, to mice and even humans to see if people with FA also have oxidative stress and iron accumulation in mitochondria. "If we do show this, then can we find medications that would modify this process? And can we find simple, biochemical parameters [indicators] that we can monitor to show that these medications are effective?," he asks.

The antioxidant (anti-free radical) medication idebenone, a close cousin of the widely available coenzyme Q10, actually improved the heart conditions of three French patients with FA in a pilot study reported in the British journal Lancet on Aug. 7, 1999, he notes.

That abnormally big hearts actually got smaller with idebenone treatment is a "remarkable thing" and "very encouraging," says Pandolfo. Idebenone, while not approved for sale in the United States, is slightly different in structure from coenzyme Q10 and may be able to enter the nervous system more easily.

Pandolfo's Canadian research group has just finished a one-year trial of the drug in children with FA and is analyzing the results, while working out the details of FA biochemistry in mice. A small trial of idebenone for FA at the National Institutes of Health in Bethesda, Md., is planned for this spring.

Meanwhile, Pandolfo says, it will be necessary to monitor the effects of antioxidant drugs with clear-cut markers. Research groups have found blood levels of two compounds, malondialdehyde and 8OH2'dG, promising as scorekeepers.

Another line of research has explored the use of iron-binding drugs like desferrioxamine for FA, Pandolfo notes. These compounds, which stick to iron and escort it out of the body, are far more dangerous than coenzyme Q10 and other antioxidants, he says.

Rather than showing an overall excess of iron, FA patients show "very specific accumulations, probably excessive iron in certain intracellular compartments, especially the mitochondria. The total cellular iron is probably not changed," Pandolfo says. "Depleting patients of iron leaves you a very small safety margin. They can start to be anemic, so it can be a rather dangerous way to treat the disease."

Until idebenone is approved in the United States, Pandolfo cautions American patients not to try to get it through the Internet or other sources. "If you buy from a generic source that sells it more like a nutritional supplement, you can't be sure that the manufacturing has been up to standards."

As for coenzyme Q10, Pandolfo says he prefers that patients take drugs under the supervision of a neurologist or as part of a clinical trial. "But," he says, "taking some over-the-counter coQ10 is probably not going to hurt."

Pandolfo's group published results on its biochemical marker in the Dec. 12 issue of Neurology. MDA grantees Pragna Patel at Baylor College of Medicine in Houston and Grazia Isaya at the Mayo Clinic in Rochester, Minn., published their results on the effects of frataxin gene mutations on yeast cells in the Oct. 12 issue of Human Molecular Genetics.

To find out more about the proposed NIH trial of idebenone, contact Kenneth H. Fischbeck, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, at (301) 435-9318 or kf@codon.nih.gov.

Mutant Protein Creates Mess in OPMD

MDA-funded researchers are beginning to unravel the microscopic events that lead to muscle damage in oculopharyngeal muscular dystrophy (OPMD). The inherited condition strikes around age 40, primarily causing weakness in muscles that control the eyes and throat.

In 1998, MDA grantee Guy Rouleau of McGill University in Montreal, Canada, led a team of researchers to establish that OPMD is caused by genetic mutations that insert an abnormal stretch of amino acids into a protein called poly(A) binding protein 2 (PABP2).

In the cell's nucleus, PABP2 normally adds a stabilizing attachment called a poly(A) tail to RNA, an essential intermediate between genes and the proteins they make. Researchers are attempting to determine how the mutations change the function of PABP2 and cause OPMD.

Previously, researchers had observed that OPMD muscles contain intranuclear inclusions -- heaps of cellular debris inside the nucleus. A flurry of recent studies suggests that these inclusions form from clumps of mutant PABP2. In the September issue of Human Molecular Genetics, Rouleau and his colleagues show that the intranuclear inclusions contain an abnormal accumulation of mutant PABP2. MDA grantee David Bear and his colleague Mark Becher, at the University of New Mexico in Albuquerque, independently confirm that finding in November's Annals of Neurology.

In the same issue, Rouleau shows that mutant PABP2 can actually cause nuclear inclusions to form when it's delivered to an isolated cell line. Rouleau also has found evidence that RNA accumulates within the inclusions, suggesting that mutant PABP2 might trap the RNA there and interfere with protein production.

"The mutation makes the protein very sticky," says Bear. Identifying exactly which proteins or RNAs get stuck to mutant PABP2 might yield insights into the formation of inclusions and their effects on muscle cells, he says.

"Formation of the inclusions might be how the cell defends itself against abnormal proteins," says Rouleau. "When the proteins are all compacted together, they might have less damaging effects than if they were spread throughout the nucleus." Nonetheless, as the inclusions grow, they might damage the nucleus, he suggested.

Studying mutant PABP2 is also leading to insights into possible treatments for OPMD. "One possibility is that if you could prevent the expression of the mutant protein, you could then prevent the disease," Rouleau says. The cell line that Rouleau has used in previous studies might be useful for testing possible treatments, he says.

HEART PROBLEMS DIFFER AMONG MDs

While doctors have suspected for some years now that the types of heart problems seen in the various muscular dystrophies differ and respond differently to treatment, molecular biologists have now shed further light on these issues and are helping doctors to plan better therapies.

Coronary Arteries May Be Problem in Some LGMDs

MDA grantee Kevin Campbell at the University of Iowa's Department of Physiology and Biophysics was recently part of a team studying the cardiac effects of the loss of beta-sarcoglycan and delta-sarcoglycan genes in mice. When these genes are flawed in humans, the result is limb-girdle muscular dystrophy types 2E and 2F, respectively.

Campbell's research team showed that, in mice lacking genes for these sarcoglycan proteins, normally located in the muscle-cell membrane in both heart and skeletal muscles, the mice not only sustain some damage to the cardiac muscle itself, but they sustain even more obvious damage to the arteries supplying the heart. The team published its findings in the Jan. 15 issue of the Journal of Clinical Investigation.

When the researchers gave some of the mice a drug that dilates blood vessels by blocking the flow of calcium across membranes, they found that the coronary artery and heart damage didn't occur.

"The most important thing about this is that it may lead to a treatment for the cardiac involvement in patients with limb-girdle dystrophy related to either beta- or delta-sarcoglycan deficiency," Campbell says.

The drug didn't help mice lacking dystrophin, the protein missing in Duchenne and Becker MD. These mice didn't show any abnormalities in their coronary arteries. This finding is consistent with observations that boys with Duchenne and Becker MD likely have a problem in the cardiac muscle itself, not the blood vessels leading to the heart.

The researchers also found that levels of a protein called troponin I in the blood correlate well with damage to heart muscle in the mouse models, just as levels of the protein creatine kinase correlate well with damage to skeletal muscle in both mice and humans with MD. The team suggests that troponin I would leak out of heart muscle and into the bloodstream regardless of whether the heart damage originated in the coronary arteries or in the muscle itself.

They suggest that the troponin I level could be developed as a diagnostic test for cardiac damage in muscular dystrophy. Campbell says the protein is already used to check for heart damage in cases of suspected heart attack (myocardial infarction).

MMD: Heart and Skeletal Muscle Weakness Correlated

An Italian research team has studied 50 people with myotonic muscular dystrophy (MMD) in an effort to develop ways of predicting which patients are likely to need treatment for cardiac problems before they become severe.

The team found that people with MMD whose skeletal muscles were weaker developed more changes in their electrocardiograms (EKGs), tests used to measure the heartbeat abnormalities known as conduction blocks or arrhythmias common in MMD. Electronic pacemakers are often used for treatment, but not always successfully.

The team, led by Giovanni Antonini at the University of Rome, found that damage to the electrical system of the heart, detected by changes in EKGs, showed a strong association with the severity of the MMD as evaluated by a muscular disability rating scale. The findings were published in the Oct. 24 issue of the journal Neurology.

The researchers also found a correlation between the age of onset of the EKG changes and the size of the expanded DNA segment that causes MMD: The longer the repeated DNA segment, the earlier the EKG changes took place.

William Groh, an MDA grantee at the Krannert Institute of Cardiology at Indiana University School of Medicine in Indianapolis, is also studying CTG repeats and cardiac problems in MMD. "The big question," Groh says, "is not whether EKG abnormalities are present — we know they are — but what we need to do to decrease the risk of sudden death or major morbidity [adverse effects]."