An overview of MDA's progress toward treatments and cures

Around the time the first issue of Quest was published, in 1994, neurologists had just established that the steroid prednisone could slow the course of Duchenne muscular dystrophy (DMD). Molecular biologists were beginning to come up with realistic approaches to gene therapy — techniques for replacing or repairing the defective genes that cause DMD and other diseases in MDA's program. And, although MDA-funded investigators had begun a pioneering trial of cell transplantation for DMD in 1990, "stem cell therapy" was a treatment for blood disorders, seemingly irrelevant to neuromuscular disease.

Obviously, times have changed — so much so, in fact, that it's hard to keep pace. There isn't enough room in this magazine to recap MDA's research since the early 1990s, but to help bring you up to speed this article highlights some of the most recent and groundbreaking advances.

Prednisone

Treatment with prednisone — considered the only effective medication for DMD by many neurologists — could be undergoing a makeover. Standard protocols used since the early 1990s call for daily administration of prednisone, but given this way, the drug can cause weight gain, delayed growth, osteoporosis and behavioral problems.

Two pilot trials reported late last year show that a different dosing schedule may deliver prednisone's benefits without these unhealthy side effects. An MDA-funded trial in the United States tested high-dose prednisone given only on Friday and Saturday of each week, and a British trial tested the closely related drug prednisolone, given at a standard dose for the first 10 days of the month, or for 10 days on and 10 days off.

Both treatments produced significant strength gains with few side effects, and the "weekend protocol" is now slated for a larger trial. Meanwhile, the American Academy of Neurology is developing a set of guidelines for prednisone use in DMD.

Gentamicin

In mice with DMD, the antibiotic gentamicin stimulates the production of dystrophin — the protein missing in the disease — but the jury is still out on whether the drug will work in people. These mice fail to produce dystrophin because they have a mutation that inserts a "stop" signal in the dystrophin gene; 5 percent to 15 percent of boys with DMD have the same type of mutation. Gentamicin allows a cell's gene-reading machinery to bypass the stop signal and produce a full-length dystrophin protein.

In two small trials, the drug reduced the levels of creatine kinase, an indicator of muscle breakdown, but failed to increase dystrophin levels in boys with DMD. An expanded gentamicin trial, headed by MDA clinic director Jerry Mendell of Ohio State University in Columbus, is open for enrollment.

Utrophin

MDA-funded researcher Kay Davies continues to search for drugs that will stimulate muscle cells to produce utrophin — a protein that has similar functions to dystrophin, but is restricted to small patches of adult muscle.

In the late 1990s, Davies' research at the University of Oxford in England showed that mice with DMD are protected from muscle degeneration when they're genetically engineered to make more utrophin. Since then, she's been using a rapid search method called "high-throughput screening" to identify chemicals with utrophin-boosting activity.

A screen of over 160,000 chemicals, set up three years ago in collaboration with the Long Island, N.Y.-based company OSI Pharmaceuticals, was unsuccessful. More recently, Davies has begun collaborating with a fledgling company founded by Oxford chemists and with a large Swiss company, MyoContract Ltd. Testing a small custom-designed chemical library, both companies found a family of chemicals that increase the levels of utrophin in laboratory-grown cells. Those increases are probably too small to compensate for dystrophin, Davies says, but she's in discussions with MDA about setting up a larger screen to find more effective chemicals.

For nearly a decade, researchers were stymied by the search for the culprit gene behind facioscapulohumeral muscular dystrophy (FSHD), and thus, they had few clues to treatment.

In August 2002, MDA grantee Rossella Tupler at Massachusetts General Hospital in Boston discovered that the mutations underlying FSHD — mapped to chromosome 4 in 1992 — aren't in a single gene, but in a DNA region that controls genes. Deletions (missing pieces of DNA) in that region cause genes that are normally "off" to turn "on." Finding ways to block these overactive genes could lead to effective treatments.

Myotonic muscular dystrophy (MMD) turns out to arise from a similarly complicated mechanism.

In 1992, the disease was linked to repeating units of DNA in the DMPK gene on chromosome 19. Around the same time, John Day and Laura Ranum at the University of Minnesota in Minneapolis saw evidence for a chromosome 3 form of the disease (MMD2), and in August 2001, they reported that MMD2 is caused by repeats in a different gene, ZNF9. This was an important clue that the repeats — not the specific genes containing them — are central to MMD.

Shortly after that discovery, scientists began focusing on how the repeats might interfere with the functions of different cell types in the body, leading to MMD's diverse symptoms. It turns out that the repeats create abnormally long pieces of RNA (the intermediate between DNA and proteins), leading to a "traffic jam" that keeps cells from turning other RNAs into protein.

Targeting the repeats with small inhibitors derived from DNA or RNA might alleviate symptoms of the disease.

New Drugs

In late 2001, MDA-funded researchers began clinical trials to evaluate three potential drug treatments for ALS. This adult-onset disease destroys motor neurons (muscle-controlling nerve cells) in the brain and spinal cord. About 10 percent of cases are genetic, but in most, the cause is unknown.

All three of the drugs under evaluation have shown promising results in mice with genetic ALS, and are FDA-approved for treating other conditions.

Celecoxib (Celebrex), under testing in a multicenter trial coordinated from Johns Hopkins University in Baltimore and Massachusetts General Hospital in Boston, is used to reduce the inflammation associated with arthritis. Daniel Drachman, co-director of the MDA/ALS Center at Hopkins, became interested in the drug in light of recent evidence that inflammation contributes to ALS. The drug might also work against two other processes tied to ALS: oxidative stress, a buildup of oxygen-based free radicals, and glutamate excitotoxicity, a toxic accumulation of the brain chemical glutamate.

When a woman in Wisconsin developed breast cancer and ALS almost simultaneously, her oncologist prescribed the breast cancer drug tamoxifen (Nolvadex) — and surprisingly, her ALS appeared to stabilize. That happy accident led to an ALS trial of the drug at the University of Wisconsin in Madison. Benjamin Brooks, director of the university's MDA/ALS Center, believes that tamoxifen might slow ALS by blocking protein kinase C, an enzyme noted to be unusually active in people with ALS.

Minocycline (Minocin) is an antibiotic used to treat severe acne, but it has other properties that might make it effective against ALS. Recent studies show that the drug inhibits apoptosis, a process by which motor neurons appear to die in ALS. Pilot trials at California Pacific Medical Center in San Francisco and the University of New Mexico in Albuquerque showed that the drug is safe for ALS patients, and the two institutions are now conducting a large, multicenter trial.

Neurotrophic Factors

These proteins, found in brain and muscle, support the growth and survival of neurons, and were once viewed as a magic bullet for treating ALS — until trials of four types of neurotrophic factor turned out negative.

While researchers have all but given up on some of these factors, new studies suggest that inadequate delivery might have contributed to the failed trials of glial-derived neurotrophic factor (GDNF).Gyula Acsadi of Wayne State University in Detroit has found that delivering GDNF via gene therapy might prove more effective than the earlier method of delivering it into the brain. In an MDA-funded study published last year, Acsadi showed that intramuscular injections of the GDNF gene significantly increased the lifespan of mice with ALS. He's testing the same approach in mice with spinal muscular atrophy (SMA).

Vascular endothelial growth factor (VEGF) promotes blood vessel growth in response to hypoxia — a deficit in the body's oxygen supply — but recent studies suggest it's also a neurotrophic factor with a key role in ALS. In 2001, researchers at the Flanders Interuniver-sity Institute for Biotechnology in Belgium made the surprising discovery that mice with defects in the VEGF gene develop an ALS-like disease. With MDA support, the Belgian researchers are now investigating whether genetic enhancement of the VEGF response improves the survival of mice with ALS.

Meanwhile, a recent study at the University of Birmingham in England suggests that small changes, or polymorphisms, in the VEGF gene may be a risk factor for the sporadic (nongenetic) form of ALS in humans. Combined with a noted increase in the incidence of ALS among Air Force personnel and commercial airline pilots, these findings have some researchers speculating that people with VEGF polymorphisms might be predisposed to motor neuron damage from hypoxia experienced at high altitudes or in other low-oxygen conditions.

Better Immunosuppressants

In clinical trials, several new drugs have shown promise against autoimmune diseases, which occur when the immune system attacks other tissues in the body. Prednisone is a mainstay of treatment for these diseases, which include myasthenia gravis (MG) and the inflammatory myopathies, but in some cases it fails to produce improvement, and in others it has intolerable side effects.

In 2001, two trials conducted at MDA clinics — at Johns Hopkins University in Baltimore and Duke University in Durham, N.C. — showed that MG sometimes responds to mycophenylate mofetil (CellCept), a drug originally developed to prevent immune rejection of transplanted organs. In both trials, about 65 percent of MG patients experienced improved strength or a reduced need for prednisone after receiving CellCept for several months.

More recent trials at Hopkins and in Argentina suggest similar benefits from intravenous cyclophosphamide, a drug traditionally used to treat cancers of the immune system.

In a multicenter trial funded by NIH, researchers are testing the multiple sclerosis drug beta-interferon-1a (Avonex) against inclusion-body myositis (IBM). In an MDA-funded pilot trial completed in 2001, the drug was found to be safe but not effective for IBM patients. The new trial doubles the dose and is expected to yield results later this year.

Vaccines

J. Edwin Blalock, an MDA grantee at the University of Alabama in Birmingham, is making progress in his efforts to develop "vaccines" for MG.

Traditionally, a vaccine is an inactivated virus that stimulates the immune system to boost its defenses against future viral infections. The MG vaccines are designed to resemble proteins on the errant immune cells that cause MG; they're meant to stimulate the immune system to destroy those cells.

Blalock has shown that the vaccines increase strength in mice primed to develop MG. In preparation for a human trial, he's begun treating pet dogs that naturally developed MG, and with help from a Belgian entrepreneur who has MG, he's established a biotech company called CuraVac.

Blalock's vaccine strategy also holds promise for treating other autoimmune diseases.

In 1999, MDA clinic director Jerry Mendell of Ohio State University began a trial of gene therapy for limb-girdle muscular dystrophy (LGMD) — one of the first efforts to test this fledgling science against a human disease. That same year, a young man died in an unrelated gene therapy trial, and the government suspended all gene therapy research involving human subjects.

Although Mendell's trial was never completed, he was able to salvage some data from it. Last year, he announced that his experimental protocol — intramuscular injection of the alpha-sarcoglycan gene — was safe, but not beneficial for people with alpha-sarcoglycan deficiency (a form of LGMD).

Meanwhile, Mendell and other MDA scientists have redoubled their efforts to bring gene therapy back to the clinic.

Vector Questions

In Mendell's trial, the alpha-sarcoglycan gene was packaged into an adeno-associated virus (AAV), considered among the safest and most effective vectors (gene-delivery vehicles) partly because of its small size. Since then, vector technology has improved remarkably, with new versions of AAV that home to muscle with higher efficiency.

Barry Byrne, director of the Powell Gene Therapy Center at the University of Florida in Gainesville, was recently awarded an MDA grant to fine-tune these viruses in preparation for a new LGMD gene therapy trial.

Meanwhile, other researchers have laid the groundwork for a Duchenne MD gene therapy trial by testing viral delivery of the dystrophin gene in mice with the disease. Two MDA-funded research groups, one led by Jeffrey Chamberlain at the University of Washington in Seattle and the other headed by Xiao Xiao at the University of Pittsburgh, have developed small versions of the very large gene — called mini- or microdystrophins — that are easily accommodated by AAVs. When given to mice with DMD by intramuscular injection, this type of gene transfer slows degeneration and improves contractile force in the injected muscles.

Immune System Obstacles

Still, researchers face several obstacles before gene therapy can live up to its promise, including the body's immune system — which has the potential to destroy gene therapy vectors and the therapeutic genes inside.

In fact, immune reactions could lead to harmful side effects — even death. Scientists and FDA officials now believe that the 1999 gene therapy-related death of Jesse Gelsinger, a teen-ager being treated for a liver disease, was caused by an immune reaction to the vector used in the trial.

Mendell, Chamberlain and Xiao believe AAV provokes almost no response from the immune system. Unfortunately, recent studies suggest that even dystrophin itself might cause an immune response in someone whose body has never made the protein.

And in other ways, the AAV-microdystrophin system isn't a perfect solution to DMD. While microdystrophins retain the most essential parts of the full-length protein, Chamberlain predicts that, at best, they may bring DMD closer to Becker MD (a less severe version of dystrophin deficiency).

For these reasons, Chamberlain has been testing a larger vector, a "gutted" adenovirus, for its ability to deliver full-length dystrophin to the muscles of DMD mice. Other researchers have made vectors from retroviruses (like HIV) and from plasmids, or condensed circles of DNA.

Reaching Enough Muscles

But the biggest obstacle to gene therapy for muscle diseases, Chamberlain says, is that so far, no one has come up with a way to deliver a gene to all of the muscles in the body.

"There's a lot of talk about scaling up intramuscular injection, for example, just targeting some of the most critical muscles, like those that control posture and hand function, to improve the quality of life for boys [with DMD]," he says. "But clearly, we have to get beyond that and find ways to deliver dystrophin to the heart and the diaphragm [a chest muscle that controls breathing]."

Two MDA-funded researchers, Hansell Stedman at the University of Pennsylvania in Philadelphia and Leaf Huang at the University of Pittsburgh, are at the forefront of efforts to develop systemic gene-delivery methods. In a rodent model, Stedman has shown that he can deliver the delta-sarcoglycan gene to the muscles of an entire limb by using clamps and tourniquets to increase local blood pressure and medications to increase local blood flow. Huang has used a similar procedure to deliver dystrophin to the diaphragm in mice with DMD.

For other neuromuscular diseases, would-be gene therapists have learned much from research on DMD. Gyula Acsadi, who's developing a gene therapy approach to treat ALS, spent his early scientific career studying ways to deliver dystrophin to muscle cells.

Byrne and Andrea Amalfitano (at Duke University) are both working on gene therapy for Pompe's disease, a fatal infant disease caused by altered muscle metabolism. Amalfitano spent his early career working with Chamberlain.

Chamberlain predicts that DMD gene therapy trials will begin in two years, and probably will involve intramuscular injections of AAV-microdystrophin. In the meantime, he says, researchers need to validate systemic gene delivery methods in large animals with DMD.

Scientists once thought that adult stem cells could be found only in tissues with a high rate of cell turnover, such as bone marrow and skin. Now, it's clear that they're found in tissues once thought incapable of regeneration, like the brain, and that they have some capacity to cross tissue boundaries.

With new techniques for isolating and growing embryonic stem cells and adult-derived stem cells, scientists could one day have a tool kit for counteracting neuromuscular diseases. Stem cell transplants to repair damaged muscles and nerves could become as commonplace as organ transplants.

But for the present, stem cell therapy poses a set of challenges much like those of gene therapy (see "Gene Therapy"). In fact, "stem cell therapy basically is gene therapy — you're just using the cell to deliver the gene," says Louis Kunkel of Children's Hospital in Boston, who hopes to use stem cells to treat muscular dystrophy.

Like any gene therapy vector, transplanted stem cells could trigger an immune response. Also, scientists don't entirely understand how stem cells choose their fate; and the signals that control their mobilization to different tissues in the body are poorly understood.

MDA-funded researchers have addressed these problems by experimenting with both embryonic and adult-derived stem cells, each of which has distinct potential advantages. (In accord with federal policy set by President Bush, MDA's support of human embryonic stem cell research is limited to some 75 stem cell "lines" created before August 2001.) In principle, adult-derived stem cells could be harvested from the person in need of treatment, corrected for any genetic defects, and transplanted where they're needed, circumventing the problem of immune rejection. Embryonic stem cells, on the other hand, are believed capable of generating more cell progeny and a greater variety of cell types.

Making New Muscle

Kunkel and his colleague Emmanuela Gussoni have isolated muscle-forming stem cells from the muscle tissue and bone marrow of healthy adult mice. In a 1999 study funded by MDA, they used a bone marrow transplant procedure to deliver the cells to mice with DMD. Some of the injected cells migrated through the bloodstream to form new muscle fibers, but not in sufficient numbers to improve muscle function.

More recently, nature performed a similar experiment on a boy with DMD. At 1 year of age, the boy received a bone marrow transplant for an immune disorder, and 11 years later, he was discovered to have a slowly progressive form of DMD. When Kunkel and Gussoni were asked to perform a muscle biopsy on the boy, they found that a small number of transplanted marrow cells had made muscle cells — not enough to account for the boy's slow course of DMD.

"The fact that cells from a bone marrow transplant can be found in muscle is a big finding," Kunkel says. "But the levels are not high enough to be therapeutic. In mice, dogs and humans [with DMD], we've found that after a transplant, less than 1 percent of the fibers in a given muscle produce dystrophin."

Other stem cells, delivered by other methods, have produced better repair. Johnny Huard, an MDA grantee at the University of Pittsburgh, has found stem cells in adult mouse muscle that can form muscle fibers, nerves and blood vessels. When given to mice with DMD by intramuscular injection, these cells can restore dystrophin in up to 25 percent of the fibers in the injected muscle.

Recently, he's also shown that a chemical in the body called TGF-beta can stimulate the cells to form scar tissue, an important clue as to why muscle-derived stem cells sometimes fail to produce muscle.

"During an injection of stem cells, we may have to block [TGF-beta activity] to keep the cells from making scar tissue," he says. Other chemicals, he and Kunkel note, might be used to attract stem cells to muscle and push them toward a muscle cell fate.

Renewing Nerves

Several research groups have shown that embryonic and marrow-derived stem cells can be induced to become motor neurons, the muscle-controlling nerve cells destroyed by ALS and SMA. But given the complex connections neurons must make with each other and with muscle cells, many scientists believe stem cells might be more efficient at rescuing sick neurons than replacing dead ones.

This thinking recently gained support from a study by Jeffrey Rothstein, who co-directs the MDA/ALS Center at Johns Hopkins. Rothstein found that intraspinal injections of human embryonic stem cells significantly improved the motor function of rats with an ALS-like disease.

But when he examined the rats' spinal cords, he found that very few of the stem cells had produced motor neurons. Instead, most of them had formed astrocytes — "support" cells in the nervous system. The cells appear to release neurotrophic factors that nurture dying neurons back to health.

Stanley Appel, director of the MDA/ALS Center at Baylor College of Medicine in Houston, is hopeful that bone marrow stem cells will be similarly beneficial to people with ALS. But he doesn't expect the cells to form neurons or astrocytes; Appel and others believe that autoimmunity might contribute to ALS.

In an MDA-funded clinical trial, he's giving bone marrow transplants to 10 ALS patients, hoping that the procedure will "reboot" their immune systems. Scientists at the University of Turin in Italy recently announced they're testing direct intraspinal injection of bone marrow stem cells in ALS patients.