Donald S. Wood (left) and Louis M. Kunkel chaired the conference.

The plan reflects significant progress since MDA's first stem cell workshop, in 2000, when scientists were wrestling with the basic properties of muscle-forming stem cells.

At this year's workshop many of the same scientists discussed their attempts at bone marrow transplantation in animals with Duchenne MD. They came up with the "to-do" list with the help of neurologists and transplant specialists.

Bone marrow transplantation is a well-established treatment for disorders of the blood or the immune system (both of which are derived from bone marrow). But before a patient receives a bone marrow transplant, the benefits of the procedure have to be weighed against risks of infection and graft-versus-host disease, in which immune cells in the transplant mount a potentially fatal attack against the recipient's body.

At present, using bone marrow transplantation for MD "has the potential to do more harm than good," said Louis M. Kunkel of Children's Hospital in Boston, who co-chaired the workshop with MDA Director of Science Technology Donald S. Wood.

At the close of the workshop, the assembled clinicians and scientists agreed that before bone marrow transplantation can enter clinical trials for MD, it must show therapeutic effects in animals with the disease.

Muscle-Building Gene Protects Against Duchenne MD in Mice

The MDA-funded team that used a muscle-building gene to create a breed of superstrong mice has shown that the same gene can largely rescue mice from the effects of Duchenne muscular dystrophy (DMD).

Insulin-like growth factor (Igf1) is a natural protein that stimulates the growth of cardiac and skeletal (voluntary) muscles. Last year, MDA grantees H. Lee Sweeney and Nadia Rosenthal created mice with an mIgf1 gene, which causes enhanced production of the Igf1 protein in skeletal muscle. The mice grew large muscles and resisted the normal muscle wasting that occurs with old age (see "Research Updates," Quest, vol. 8, no. 2).

In a new study, Sweeney and Rosenthal introduced the mIgf1 gene into mice with DMD, and found that it counteracted the accelerated muscle wasting, the buildup of scar tissue and the decline in strength associated with the disease.

Sweeney, a molecular biologist at the University of Pennsylvania in Philadelphia, said he envisions treating DMD by using a combination of gene therapy and stem cells to administer mIgf1. A virus could deliver the gene to stem cells grown in the lab, and those cells could be injected into the body, he explained.

Treatment with mIgf1 wouldn't correct the cause of DMD — the loss of dystrophin, a structural component in muscle cells. Instead, it works by stimulating muscle stem cells to divide and build new muscle tissue.

In mice, that increased muscle mass largely compensated for the lack of dystrophin and the resulting frailty of individual muscle fibers. Diseased mice that were given the mIgf1 gene showed a 40 percent to 60 percent increase in the size and strength of their leg muscles compared to diseased mice not given mIgf1. Treated mice also had larger, healthier-looking diaphragms (a chest muscle that controls breathing), and a 40 percent drop in blood levels of creatine kinase (a protein that leaks out of damaged muscles).

Those results were published in the April 1 issue of the Journal of Cell Biology.

Gene therapy with mIgf1 could have some advantages over gene therapy with dystrophin, Sweeney said. Unlike dystrophin, mIgf1 can spread from cell to cell, so that a small amount of it could have "a profound impact" on someone with DMD, he said. Also, mIgf1 might be useful for treating muscle diseases caused by other protein deficiencies, including limb-girdle muscular dystrophy.

Sweeney has entered discussions with biotechnology companies capable of producing the viruses and stem cells that will be needed to test mIgf1 in people.

MDA's Gene Therapy Program Back on Track

With a $2.2 million grant to the University of Florida at Gainesville, MDA has boosted its efforts toward clinical trials of gene therapy for two forms of muscular dystrophy.

Gene for Microdystrophin or Sarcoglycan In gene therapy trials for LGMD and DMD, MDA-supported investigators plan to use adeno-associated viruses (far right) to deliver genes to muscle cells.

After the death of a participant in a non-MDA-related trial in 1999, gene therapy trials across the country were set back while regulatory reforms and basic science questions were investigated. Now, many researchers are ready to resume gene therapy preclinical and clinical testing.

The new MDA grant is for the developmentof "vectors" — gene therapy delivery vehicles — to carry corrective genes for at least one type of limb-girdle muscular dystrophy (LGMD) to muscle cells. Some forms of LGMD are related to deficiencies of genes for proteins known as sarcoglycans, and these genes, which are relatively small, will be packaged inside viral shells so they can penetrate muscle tissue.

The virus the team will use, known as the adeno-associated virus (AAV), is thought to be both safe and effective for delivering genes to muscle. Newer versions of the AAV vectors, which will be thoroughly tested in animal models, are thought to be superior to those used even as recently as 1999.

Gene therapy expert Barry Byrne of the Powell Gene Therapy Center at the University of Florida and molecular biologist Kevin Campbell of the University of Iowa in Iowa City will develop the vectors, while neuromuscular disease specialist Jerry Mendell of Ohio State University in Columbus plans to use that technology to launch a new LGMD clinical trial at his institution.

Meanwhile, MDA is also laying the groundwork for a clinical trial of AAV-based gene therapy for Duchenne muscular dystrophy (DMD). DMD results from any of a number of mutations in a large gene for the muscle protein known as dystrophin. The full-size gene can't be packaged inside an AAV vector.

To overcome this problem, MDA grantee Jeffrey Chamberlain, a molecular biologist at the University of Washington in Seattle, has developed highly miniaturized dystrophin genes that lead to the production of small proteins. Studies using these microdystrophin genes, which were published in the March issue of Nature Medicine, showed extremely promising results in correcting muscle abnormalities in dystrophin-deficient mice.

The researchers hope to have clinical trials under way for both LGMD and DMD in about two years.

Transplant Study Reveals Heart's Regenerative Powers

A study of heart transplant recipients suggests the heart has a surprising capacity for self-repair. If scientists can tap into that capacity, they might be able to counteract the cardiac damage that occurs in some neuromuscular diseases.

The body appears to contain stem cells that form new heart muscle cells when needed.

It's known that shortly after a heart transplant, cells in the recipient's bloodstream invade the donated heart, sometimes leading to rejection or other complications that can cause the transplant to fail. Piero Anversa of New York Medical College in Valhalla wondered if cells from the recipient could migrate into the donated heart and form new cardiac muscle cells that might improve the transplant's outcome.

To test the idea, he and his colleagues performed autopsies on eight men who had received heart transplants from female donors. Examining the female hearts, they detected a Y chromosome in 7 percent to 10 percent of cardiac muscle cells, indicating that those cells had come from the male recipients. (Females have no Y chromosomes.)

Anversa and his group also found evidence that the new cardiac muscle cells were derived from stem cells — master cells, perhaps reserved from early development, that can produce a variety of cell types.

In hearts of people who had never undergone heart transplants, the group detected a lower number of cells with stem cell "markers" (proteins found almost exclusively in stem cells).

Anversa and his group speculate that the stem cells found in the transplanted hearts could have come from the recipients' bone marrow or from remnants of the removed heart.

"These cells are not confined to restricted regions of the heart; they migrate where they are needed," the group writes in the Jan. 3 issue of the New England Journal of Medicine. With further study, it might be possible to transplant the cells into damaged cardiac tissue or use drugs to enhance their natural repair capacity.

Myotonic Dystrophy Clinical Studies Explore Heart, Brain, Proteins

As new studies shed further light on the toxic RNA theory of what causes myotonic muscular dystrophy (MMD), (see "Family Histories Help Solve Medical Mysteries," Quest, February 2002), clinical research is proceeding in this complex disorder.

Interference With Muscle Proteins

MDA-sponsored investigators recently examined the fate of a group of proteins in the muscles of people with type 1 and type 2 MMD and found that these important proteins accumulated in clumps in the nuclei of muscle cells.

MDA grantees Charles Thornton and Ami Mankodi of the Department of Neurology of the University of Rochester (N.Y.) School of Medicine and Dentistry, and Maurice Swanson of the Department of Molecular Genetics and Microbiology at the University of Florida College of Medicine in Gainesville, were on the team, which studied muscle samples from people with either type of MMD.

Their findings, published in the Sept. 15 issue of Human Molecular Genetics, suggest that the clumping up of these molecules, known as muscleblind proteins, would likely interfere with muscle cell maturation. In flies, cells without muscleblind proteins don't go through the final steps needed to become mature muscle tissue. A similar interference with this process could be occurring in the human cells, the research suggests.

Brain Protein Abnormalities

European investigators have linked abnormalities in a brain protein known as tau with the enlarged DNA on chromosome 19 in type 1 MMD. Researchers at several institutions in France studied tau in samples of brain tissue from deceased MMD patients and found alterations in tau that could lead to brain abnormalities.

They later found that mice carrying the type 1 MMD defect also had abnormal forms of tau in their brains.

Nicolas Sergeant and colleagues published their results in the Sept. 15 issue of Human Molecular Genetics, and Hervé Seznec and colleagues published theirs in the Nov. 1 issue.

Abnormal tau has been implicated in several brain disorders, including Alzheimer's disease, and may play a role in the neurological dysfunction sometimes seen in myotonic dystrophy.

Heart-Monitoring Device

Type 1 MMD is known to cause cardiac arrhythmia, or irregular heartbeat.

People with MMD can experience fainting, a sensation of fluttering in the chest or even sudden death because of an arrhythmia. A study at Indiana University's Krannert Institute of Cardiology in Indianapolis is seeking to understand which MMD patients need careful cardiac monitoring and which ones may benefit from implantation of a cardiac device, such as a pacemaker or defibrillator.

As of January, some 400 patients were enrolled in the study. In a subsection of the study, six people had a device called a loop recorder implanted just underneath the skin of the chest to constantly record heart rate and rhythm. A serious problem was detected in three people and two received an electronic device to correct it.

Early funding from MDA to cardiologist William Groh at the institute has been followed by funding from Medtronic (makers of the loop recorder and other implantable electronic devices) and the National Institutes of Health.

The study isn't open at this time, but further information can be obtained from Miriam Lowe, research coordinator, at (800) 843-2786.

Muscle Contraction Drug Trial

Researchers at the University of Rochester (N.Y.) Medical Center are studying the drug mexiletine (Mexitil) to see if it helps with the myotonia (the inability to relax muscles after use) that occurs in MMD. The drug is now marketed for the control of irregular heartbeats.

Some openings in the trial may remain. For information, contact Cheryl Barbieri at (585) 275-5409 or cheryl_barbieri@urmc.rochester.edu.

Children Over 12 With SMA Excel at Applying Intelligence

Investigators at the University of Cologne and the University of Aachen in Germany recently published the results of a study comparing 96 children ages 6 to 18 with types 1, 2 and 3 spinal muscular atrophy (SMA) with 45 unaffected siblings in the same age range and 59 unrelated, unaffected children and adolescents.

After employing several kinds of intelligence tests, including those that measure "general" and "environmentally mediated" (applied) intelligence, the researchers concluded that the general intelligence of children with SMA is in the average range, but that the use of general intelligence by those with SMA was superior by age 12.

By later childhood and adolescence, skills required for the verbal components of certain intelligence tests were more developed in those with SMA than in their unaffected peers. Intelligence wasn't influenced by the type of SMA.

"By adolescence," the authors say, "environmentally mediated aspects of intelligence are, indeed, higher in patients with SMA. It could be speculated that children need the time until adolescence to develop effective and useful strategies to 'compensate' [for] their physical handicap by the acquisition of cognitive skills and knowledge. In view of the many restrictions in their lives, with few possibilities to express themselves motorically, the domain of thinking and learning becomes their main area of creativity."

SMA Tests Refined, Precautions Noted

Arthur Burghes | Thomas Prior

MDA grantees Thomas Prior and Arthur Burghes at Ohio State University in Columbus are among investigators who recently made some refinements to the understanding of genetic testing for spinal muscular atrophy (SMA), publishing their results in the January/February issue of Genetics in Medicine.

The researchers confirmed what's been strongly suspected for several years among SMA experts: that the presence of multiple copies of the SMN2 gene (also called SMN-C) can to some extent compensate for flawed or missing SMN1 (SMN-T) genes.

The Ohio State investigators studied 142 people with SMA. Of those with type 1 SMA, the most severe form, most of the patients had two copies of the compensating SMN2 gene. Of those with type 3 SMA, the least severe form, most people had three copies of the SMN2 gene.

Similar results were found by a German research team, which published its results in the February issue of the American Journal of Human Genetics.

The findings indicate that, in general, the more SMN2 genes a person has, the better able he or she is to withstand the effects of mutations in the SMN1 gene. The correlation, however, isn't perfect and can't be used to predict with certainty the course of someone's disorder.

Most labs in the United States don't at this time count the copies of the SMN2 gene in a diagnostic test. Nor can most laboratories detect small, but significant, flaws in SMN1 or SMN2 genes that can influence the course of SMA or the potential to be a carrier. For these and other reasons, caution is needed in interpreting genetic test results in SMA.

Prednisone Alternatives Debated in Treatment of Myasthenia Gravis

The corticosteroid drug prednisone (Deltasone) has long been a mainstay of treatment for myasthenia gravis (MG), a disorder in which the immune system attacks specialized areas on muscle cells so they can't receive signals from nerve cells. MG is one of many autoimmune disorders, in which the immune system mistakenly attacks the body's own tissues.

Donald Sanders

Although prednisone, an immunosuppressant drug, can keep MG under control for many patients, it often has to be taken for several years in this disorder, which allows serious side effects to occur and worsen with time. Among the common effects of long prednisone use are high blood sugar, weight gain, depression, changes in appearance, mood swings, low blood potassium and high blood pressure. After months of prednisone treatment, osteoporosis (loss of bone density), skin damage, redistribution of fat to the face and center of the body, and sometimes destruction of the hip joint are common.

In the January issue of Muscle & Nerve, neurologist Michael Rivner of the Medical College of Georgia in Augusta argues that the use of prednisone as first-line therapy in MG should be questioned in light of data about newer immunosuppressants. In a debate format, neurologists Richard Bedlack and Donald Sanders of Duke University Medical Center in Durham, N.C., argue in favor of using prednisone for MG patients with more than minimal weakness, then adding other medications if prednisone can't be reduced to an acceptable level. At Duke, Bedlack directs the MDA/ALS Center, and Sanders co-directs the MDA clinic.

Better Diagnosis,Treatment in the Works for MNGIE & MDS

Scientists have discovered that two forms of mitochondrial myopathy (MM) are caused by altered processing of thymidine, one of four chemicals that serve as building blocks for DNA. For one form of the disease, those findings have already led to a quick and easy diagnostic procedure and to experimental treatments aimed at restoring the body's normal balance of thymidine.

Mutations in nuclear DNA may lead to errors in mitochondrial DNA.

Mitochondrial myopathies are genetic diseases associated with defects in mitochondria — the tiny powerhouses that provide energy to each cell in the body.

MDA grantee Michio Hirano of Columbia University in New York studies MMs that result from a breakdown of communication between mitochondria and the cell's control center, the nucleus. In these diseases, mutations in nuclear DNA (genetic material housed within the nucleus) lead to errors in the manufacture of mitochondrial DNA (genetic material housed within the mitochondria), which is essential for making mitochondrial proteins.

One such disease, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), strikes young people and causes degeneration of the nerves, muscles and digestive system. In a 1999 MDA-funded study, Hirano showed that MNGIE is caused by mutations in the gene for thymidine phosphorylase, an enzyme that breaks down thymidine. Hirano speculated that those defects might increase thymidine levels in mitochondria, upsetting the delicate balance of chemicals needed to make mitochondrial DNA.

In a new MDA-funded study published in December in the Journal of Biological Chemistry, Hirano found that people with MNGIE have blood levels of thymidine more than 60-fold above normal. "Elevated thymidine in plasma is a reliable way to confirm the diagnosis of MNGIE" and can be checked using a simple test, Hirano said.

Hirano also has evidence that clearing excess thymidine from the blood might be an effective treatment for MNGIE. He's used a procedure called hemodialysis to filter thymidine from the blood of several MNGIE patients. The treatment causes a sharp drop in thymidine and temporary relief from symptoms, but within 24 hours, the thymidine levels spring back up again.

"Hemodialysis is not a long-term solution for this disease. We have tried some medications, but none have increased thymidine clearance. We're considering other drugs," Hirano said.

Diagnostic tools and treatments for MNGIE might also work for mitochondrial DNA depletion syndrome (MDS), which can cause life-threatening muscle weakness during infancy.

Late last year, a group based at Hebrew University in Jerusalem showed that one form of MDS is caused by mutations in the gene for thymidine kinase-2 (TK2), which converts thymidine into deoxythymidine triphosphate (dTTP). Although studies haven't yet been done, Hirano said people with this disease may have low plasma levels of dTTP and might benefit from supplemental dTTP.

Gene Therapy Explored for mtDNA Diseases

For certain mitochondrial myopathies (MM), the road to gene therapy is filled with obstacles, but a new MDA-funded study shows it's possible to overcome one of the most challenging.

MMs involve a breakdown of the mitochondria — tiny factories inside our cells that produce the energy molecule ATP. Some MMs arise from mutations in DNA from the cell's nucleus (nDNA) while others are caused by mutations in mitochondrial DNA (mtDNA). Both types of DNA encode an assembly line of mitochondrial proteins required for making ATP.

Diseases caused by nDNA mutations are potentially treatable with a host of gene therapy tools, including vectors (gene delivery vehicles) that can send corrective genes to the nucleus. But most vectors aren't very good at ferrying genes to mitochondria, so scientists have been exploring new gene therapy methods for treating mtDNA diseases (see "Research Updates," Quest, vol. 8, no. 1).

Now, MDA grantees Eric Schon of Columbia University and Giovanni Manfredi of Cornell University (both in New York) have shown that it's possible to compensate for mtDNA mutations by engineering a mitochondrial gene so that it's delivered to the nucleus, made into a protein and then sent to the mitochondria.

An identical mutation in the mitochondrial gene MTATP6 can cause two mtDNA diseases — neuropathy, ataxia and retinitis pigmentosa (NARP) and maternally inherited Leigh's syndrome (MILS). The MTATP6 protein works at the end of the ATP assembly line, and people with NARP and MILS have a deficiency of ATP in multiple tissues, including muscle and brain. Schon, Manfredi and their colleagues sought to restore MTATP6 in the mitochondria of laboratory-grown cells partially derived from an MILS patient.

Since mtDNA follows a different genetic code than nDNA, they first had to create a version of the MTATP6 gene that matched the nDNA code. Then, they fitted the gene with a "mitochondrial targeting signal," a kind of routing slip that's required for sending nuclear-encoded proteins to the mitochondria. When they used conventional gene therapy vectors — a virus or a circle of bacterial DNA — to deliver the modified gene to the cells' nuclei, they found that the MTATP6 protein worked its way into the cells' mitochondria, leading to an increase in ATP production.

"A number of other issues need to be addressed if gene therapy is to become practical" for treating mtDNA diseases, the researchers say in the April issue of Nature Genetics.

Exercise Study Opens in CPT2 Deficiency

Ronald Haller

Ronald Haller, an MDA research grantee at the University of Texas Southwestern Medical Center in Dallas, is conducting a study to see whether 14 weeks of supervised exercise training can improve the ability of muscles to resist injury in people with carnitine palmityl transferase 2 (CPT2) deficiency. The researchers plan to study 10 people in this pilot trial.

CPT1 and CPT2 are involved in transporting fat-derived molecules from the main part of the cell (cytoplasm) into the energy-producing parts, the mitochondria. Without these enzymes, cells fail to adequately use fats and don't make enough energy for prolonged exercise.

If the researchers' exercise training regimen proves to help cells make more mitochondria and alleviates the exercise intolerance and muscle injury seen in CPT2 deficiency, it could also have implications for related disorders of energy metabolism.

To find out more about this study, contact Haller or Tanja Taivassalo at (214) 345-4611 or rhaller2@earthlink.net.

Blood Test Devised for Dysferlin-Related Disorders

MDA-supported researcher Robert Brown at Massachusetts General Hos-pital in Boston was part of a team that recently found that the muscle protein known as dysferlin can be detected in white blood cells as a convenient way of estimating its quantity in muscle.

Current testing involves either analysis of the dysferlin gene, or examination of a muscle sample for a deficiency of the dysferlin protein, both expensive procedures.

Dysferlin gene mutations underlie two muscular dystrophies — limb-girdle MD type 2B and a form of distal MD known as Miyoshi myopathy. Recently, another form of distal MD was also found to be related to a dysferlin abnormality.

The researchers, who published their work in the January issue of Annals of Neurology, found that the level of dysferlin in white blood cells known as monocytes correlates well with the level found in muscle biopsy samples, allowing them to substitute the blood test for the muscle examination.

Cloning Tested in Mice With Genetic Disease

For the first time, scientists have used a controversial procedure called therapeutic cloning to partially correct a genetic disease in a lab animal.

In therapeutic cloning, an individual's genetic material is injected into an egg cell that's had its own genetic material removed, and the egg cell is stimulated to develop into an embryo. The embryo is used to derive stem cells, the master cells that assemble the body's tissues.

A team led by Rudolf Jaenisch of the Whitehead Institute for Biomedical Research in Cambridge, Mass., put the procedure to the test on mice with a genetic deficiency of the immune system. The researchers cloned embryonic stem cells from the mice and corrected the cells' genetic deficiency before reimplanting them.

The implanted cells partially restored the immune system of the mice, but strangely, also provoked an immune reaction.

The work, published in the March 8 issue of Cell, raises some obvious doubts about the immune compatibility of cloned stem cells.

But it also suggests that therapeutic cloning combined with gene therapy might hold promise for a variety of genetic diseases, conclude Jaenisch and his team.