MDA Working to Restart Stalled LGMD Gene Therapy Trial

Last year, MDA announced the beginning of the first gene therapy trial for a muscular dystrophy - a phase 1 trial designed to test the safety of replacing missing genes for sarcoglycan proteins in patients with sarcoglycan-deficient forms of limb-girdle muscular dystrophy (LGMD).

Preparations for the trial took many months and involved investigators at several institutions. Shortly after MDA's trial began, an unrelated situation involving a separate gene therapy trial at the University of Pennsylvania in Philadelphia caused the Food and Drug Administration to put on hold all trials with which this institution was involved, including the LGMD gene therapy trial taking place at Ohio State University in Columbus.

The MDA trial was using a different viral vector (gene carrier) than the one believed to have caused problems at the University of Pennsylvania, and there has been no evidence of inflammation or toxicity in the two people injected with a therapeutic sarcoglycan gene before the trial was put on hold. Tissue samples have been taken from both trial participants to judge whether their muscle cells took up the sarcoglycan genes and began to make sarcoglycan proteins.

To date, however, the researchers doing the analysis haven't been told which tissue samples came from the saline-injected muscles and which came from the gene-injected muscles.

MDA continues to fund the portion of the trial that involves the identification of appropriate trial participants.

Also, since it now appears that the Institute of Human Gene Therapy at the University of Pennsylvania will be unable to manufacture the viral vectors used to carry genes into cells, MDA has brought together a number of investigators from medical institutions across the country to see which ones can produce the required vector for our trial.

MDA is also working with other institutions to develop ways to deliver healthy genes to disabled cells.

Conference Spotlights Progress in SMA, MMD, DMD

Arthur Burghes

In May, 450 muscle researchers from all over the world gathered in Pacific Grove, Calif., for the MDA-supported Molecular Biology of Muscle Development and Disease meeting.

Topics included muscle development, methods of regulating genes that affect muscles, the biology of muscle stem cells, and the latest research on specific muscle disorders. Progress in three disorders, in particular, was noteworthy.

New mice shed light on SMA. Mice developed by Judith Melki at the INSERM research institute in Strasbourg, France, show that the problem in spinal muscular atrophy (SMA), a disorder of muscle-controlling nerve cells, may lie with muscle cells themselves as well as with the nerve cells that control them (motor neurons).

Melki's team developed a line of mice that lacked only in their muscle cells the motor neuron survival protein known in humans as SMN-T. She found that they developed an SMA-like disease, just as did mice that lacked the SMN-T-like protein in their motor neurons.

The implications of this finding and its relevance to the treatment of the human disorder aren't fully clear.

The findings add to those of MDA grantee Arthur Burghes of the Department of Medical Biochemistry at Ohio State University (see Quest, vol. 7, no. 2), who recently reported that mice lacking an SMN-T-like protein and showing an SMA-like disorder can be cured with extra copies of the gene for a related protein, human SMN-C. (SMN-T is also called SMN1, and SMN-C is also called SMN2.)

Genetic defect underlying MMD explored. Charles Thornton, an MDA grantee at the University of Rochester (N.Y.) Department of Neurology and an MDA clinic co-director at that university's medical center, reported on a line of mice developed by his research group that may shed light on the underlying genetic defect in myotonic muscular dystrophy (MMD). This disorder affects not only muscle but many other organs.

It's been known since 1992 that some extra DNA on chromosome 19, known as an "expanded triplet repeat," is found in virtually everyone with MMD and not in people who don't have the disease, but its exact significance and mechanism haven't been well understood. The triplet repeat is thought to affect at least two nearby genes.

Thornton's group developed mice with an expanded triplet repeat section in a gene entirely different from the genes associated with MMD in humans and found that the mice developed myotonia - an inability to relax muscles - which is one of the symptoms of MMD.

The research suggests that the presence of a triplet repeat itself may be significant in MMD, regardless of its exact location.

Engineering the dystrophin gene for DMD, BMD gene therapy. Jeffrey Chamberlain, MDA grantee in the Department of Human Genetics at the University of Michigan in Ann Arbor, reported on his team's efforts to develop gene therapy for Duchenne and Becker muscular dystrophies (DMD and BMD), disorders involving a lack of functional dystrophin, a muscle protein.

Chamberlain's team has developed a new, high-capacity adenoviral vector (gene delivery vehicle) with special modifications that direct it to enter only muscle cells, not cells of the immune system or other nonmuscle tissues. It lacks all genes normally present in adenoviruses.

This new vector, carrying a dystrophin gene and tested in mice, not only held the line against ongoing muscle loss from lack of dystrophin, but also appeared to reverse exercise-induced muscle damage in older mice that lacked dystrophin. It also attracted less unwanted attention from the immune system.

Chamberlain has at the same time been working to develop a dystrophin "minigene" that will fit into the smaller and potentially safer adeno-associated viral vector.

New Hope for Gene Therapy of DMD, BMD Using AAV Vectors

Xiao Xiao

Gene therapy for Duchenne and Becker muscular dystrophy (DMD and BMD), long stalled because of technical hurdles, may again surge ahead if strategies being developed by MDA grantee Xiao Xiao at the University of Pittsburgh continue to look as promising as early experiments suggest.

Xiao, of the university's Department of Molecular Genetics and Biochemistry, has been working on a plan that would allow the dystrophin gene, flawed in DMD and BMD, to be delivered with a type of virus that he says has been very safe and effective at delivering genes to muscle tissue. Until now, that virus has been considered too small to carry the very large dystrophin gene.

Building on the work of other MDA grantees, Xiao has developed new approaches that would allow researchers to use the adeno-associated virus (AAV) to deliver the large dystrophin gene to muscle cells.

"The adeno-associated virus is thus far the best vector [delivery vehicle] for muscle," Xiao says. He notes that the AAV virus, even in its natural state, doesn't seem to cause any human disease, provokes minimal unwanted responses from the immune system, is small enough to cross barriers into muscle cells that larger viruses can't cross, and inserts itself into the cell's DNA, thus making itself a permanent part of the cell.

AAV's disadvantage for gene therapy has been its small carrying capacity. Even when all of the virus's own genes are removed, leaving extra room for a therapeutic gene to be inserted, it can only carry very small genes.

Now, Xiao says, his group has developed two possible ways to overcome this problem. In one plan, they've developed a way to break the large dystrophin gene into two parts, put one of those parts into each of two AAV viral shells (with the AAV's original viral genes removed), insert the AAV viral particles into cells, and have the dystrophin DNA rejoin and become one whole dystrophin gene later on, in the cell.

For these experiments, the investigators used a slightly miniaturized version of the dystrophin gene that was originally developed by MDA grantee Kay Davies in 1991. This gene is based on one from a man with mild BMD and works almost as well as a full-length dystrophin gene.

Xiao says this type of DNA joining, called dimerization, is something AAV viruses normally do with their own DNA and that exploiting this aspect of natural AAV biology may give scientists the break they've needed to get very large genes like dystrophin into cells. These new findings are published in the May issue of Nature Medicine.

In another set of experiments, presented at the recent meeting of the American Society of Gene Therapy in Denver, Xiao's group inserted an even more miniaturized version of a dystrophin gene (one developed by his own lab) into a leg muscle in dystrophin-deficient mice and got a "very encouraging therapeutic effect."

The appearance of the muscle fibers that received these genes was "indistinguishable from healthy muscle," Xiao says.

Enzyme Therapy Trials to Start Soon in Acid Maltase Deficiency

Yuan-Tsong Chen

Three biotechnology companies - Genzyme General of Cambridge, Mass.; Synpac NC of Durham, N.C.; and Pharming of Leiden, the Netherlands - have joined forces for clinical trials of a new enzyme replacement therapy for children with acid maltase deficiency (Pompe's disease). The work of MDA grantee Yuan-Tsong Chen of the Department of Pediatrics at Duke University in Durham, N.C., underlies this research (see Research Updates in Quest, vol. 5, no. 4). Chen's group modified the natural enzyme in such a way that it could be taken up by muscle cells from the bloodstream.

In Pompe's disease, the lack of the enzyme acid maltase, which normally breaks down the complex carbohydrate known as glycogen so that the body can use it as the fuel glucose, leads to weakness of the skeletal, heart and respiratory muscles. A complete lack of acid maltase can cause profound heart and respiratory impairment and death in infancy. A partial deficiency can cause less severe effects.

Chen oversaw a small (phase 1) trial of the replacement enzyme, now called by the trade name Pompase, last year at Duke. Three infants who received the enzyme are alive, despite an initial poor prognosis. Results of that study have been submitted for publication.

For more information about upcoming trials, call Genzyme's information line at (800) 745-4447. The company's Web site is www.genzyme.com.

Gene Repair Strategy Looks Promising in Dog, Mice With DMD

Two scientific teams have shown that the gene that's flawed in Duchenne and Becker muscular dystrophies (DMD and BMD) can sometimes be coaxed into repairing itself when muscle cells are injected with a synthetic, paper-clip-shaped molecule known as a "chimeric oligo-nucleotide" (a short string of DNA and its close chemical relative, RNA).

The benefits for muscle tissue at this stage are small, but significant, say investigators on both projects.

A paper-clip-shaped molecule known as a chimeric oligoncleotide can apparently supply a DNA correction patch (blue) to correct a point mutation in a gene (red). The patch is surrounded by RNA (orange) and DNA (green) and leads to a normal DNA sequence (green bar).

MDA grantee Thomas Rando of the Department of Neurology and Neurological Sciences at Stanford University School of Medicine in Palo Alto, Calif., was on a team that used chimeric oligonucleotides to repair genes for dystrophin, the muscle protein affected in DMD and BMD, in a leg muscle in mice. These mice have a DMD-like disease because of a mutation in the dystrophin gene.

The results, published in the May 9 issue of Proceedings of the National Academy of Sciences, were encouraging, although not directly applicable to patients at this point. "This is a technology in its infancy," Rando said. "We're not on the verge of a therapeutic transition, but it's a first step, perhaps."

He emphasized that delivery to large muscle groups remains a problem and that the correction strategy only applies at this time to very small genetic defects known as point mutations. These affect a minority of people with DMD and haven't been seen in BMD.

It remains to be seen whether the technique has any application to deletions, the usual type of genetic mutation in DMD and BMD.

MDA grantee Joe Kornegay of the Department of Veterinary Medicine and Surgery at the University of Missouri in Columbia was part of a study that used a very similar approach to repair dystrophin genes in a leg muscle of a dystrophin-deficient dog. His group, under the direction of Richard Bartlett of the National Institutes of Health in Bethesda, Md., published its results in the June issue of Nature Biotechnology.

"This technique relies upon an innate system that the body has to correct genetic lesions [abnormalities]," Kornegay says. "By introducing specially prepared oligonucleotides, you can induce this innate function to repair a particular defect."

He notes that the technique "avoids some of the problems inherent with certain gene therapy techniques." In particular, it doesn't require the use of viruses, which can have undesirable effects, such as alerting the body's immune system to attack the cells carrying the new genes.

Also, Kornegay says, the correction would "theoretically be permanent," in contrast to some current gene therapy strategies, in which the desired effects are sometimes temporary.

Albuterol Study in FSHD Negative,
but 'Provocative'

A one-year, MDA-supported clinical trial of long-acting albuterol in adults with facioscapulohumeral muscular dystrophy (FSHD) showed that the drug didn't improve muscle strength in most muscle groups. It did, however, improve grip strength and skeletal muscle mass (bulk), suggesting that it may preserve or develop muscle to a limited extent in this disorder.

For this study, 90 adult participants were divided into high-dose, low-dose and placebo (inert substance) groups.

The investigators reported results in May at an MDA-supported meeting on FSHD at the National Institutes of Health in Bethesda, Md.

Albuterol is a beta-2 adrenergic agonist, a class of drugs that has shown muscle-building effects in animals and humans.

The results, although basically negative, are "provocative enough to lead to more studies," said neurologist John Kissel, who co-directs the MDA clinic at Ohio State University in Columbus and received MDA funding for the albuterol study. "It's hard to know what to make of it."

Kissel says his group now plans to study why the drug appears to improve muscle mass but not strength and to see whether certain doses of the drug or certain groups of people (such as men versus women) responded differently to the drug - a technique called subgroup analysis.

Kissel says the team will look at whether gender, age, initial muscle strength scores or other factors affected how people did on albuterol.

Depending on the conclusions from these analyses, the researchers may try to give the drug in a different way (such as in pulses instead of continuously) or make other alterations in the treatment approach or targeted group.

Albuterol is available by prescription because it's used in asthma therapy. For people with muscular dystrophy who are taking it or considering taking it, Kissel says: "I tell people that if you think it's helping you, I think there's enough data, some encouraging findings, to suggest staying on it is reasonable; but there isn't enough data to suggest that people who are not on it should go on it." He said cardiac complications (a side effect) are a theoretical concern with this drug, although they didn't see any in the study.

As for albuterol's future in FSHD and possibly other forms of muscular dystrophy, he said: "I don't want to throw out the baby with the bath water."

Study Supports Prednisone Use in Duchenne MD

Research at the MDA clinic at the University of Rochester (N.Y.) Medical Center adds to data showing that the steroid drug prednisone slows the decline in skeletal muscle function and prolongs walking in boys with Duchenne muscular dystrophy (DMD) by showing that it also slows the decline in pulmonary function and delays or prevents the need for assisted ventilation. The findings were presented at the spring meeting of the American Academy of Neurology in San Diego.

The researchers studied 25 boys with DMD who had been on prednisone since 1991 and compared them with a "natural history" group of boys seen in the clinic in earlier years who had the same characteristics but were not on prednisone. There were about 200 boys in the natural history database.

"In this [prednisone-treated group] of patients, only three of the 25 have had scoliosis that required surgery, whereas in the natural history group, you will find that almost 75 percent to 80 percent developed scoliosis," says Shree Pandya, a physical therapist and coordinator for the trials. Scoliosis is a curvature of the spine often associated with decline in muscle function in DMD.

"Hardly anyone has required pulmonary support [ventilation] as of now," Pandya says. "That's not to say that they will not in the next five to 10 years, but for sure prednisone has changed the natural history of Duchenne dystrophy."

Pandya said the respiratory data compare favorably with the data from the natural history group. She says, however, that it's hard to directly compare the two groups because "the [older] database only goes to age 20; these kids [prednisone-treated] are reaching age 29, so there is no data to compare them to."

"We also feel - and this gets complicated - that there is a whole interrelationship between how long someone continues to walk, when they develop scoliosis and the effect of scoliosis on pulmonary function. What we're finding is that not only have we had our kids walk up to age 14 or 15 without braces, but they have not developed scoliosis."

Pandya says this difference from the natural history of DMD may be responsible for the improved pulmonary function as much as any change in respiratory muscles themselves.

As for side effects of prednisone, Pandya says: "There was a phase five years ago where people were really concerned about side effects. Really, we have not seen the side effects that we were so concerned about."

She says they haven't seen diabetes, brittle bones, increased infections or other serious effects known to be associated with prednisone. Three patients did, however, drop out of the study because of unacceptable weight gain (a common side effect of prednisone), and three patients died during the study.

Europeans Cautious, Positive on Gene Therapy

The mood was cautiously optimistic at a June meeting of Euregenethy, a European gene therapy society seeking to standardize gene therapy regulations among the 15 member states of the European Union.

Sobered by the death of an American teen-ager at the University of Pennsylvania in Philadelphia in a gene therapy clinical trial last year, investigators and regulators put their heads together for three days in Paris to try to figure out what kinds of safety issues are most important in this relatively young field.

Noting that gene therapy has matured in the last 10 years from a gleam in researchers' eyes to a reality that has shown at least some therapeutic benefit in certain cases, one researcher said that gene therapy has reached "the end of the beginning."

The European researchers reported that two early concerns weren't as worrisome as they had once feared they might be.

The chance that a virus used to deliver a therapeutic gene (a common gene therapy strategy) could be passed between patients and others is minimal within a few days after the gene transfer, investigators reported. Also, the chance that a newly inserted gene could cause cancer or a genetic disorder is likewise minimal, studies showed.

However, participants agreed that vigilance on these and other safety issues would continue to be necessary as regulations are developed and that procedures to ensure fully informed consent for patients in clinical trials are of the utmost importance.

A French study in which babies with a severe immune-system disorder were restored to apparent health using gene therapy was hailed as a sign that gene transfer strategies might be turning a corner.

In the bleeding disorder hemophilia, a gene transfer study at Stanford University in Palo Alto, Calif., has also shown promise and was also widely acclaimed as a sign of successes to come.

In fact, the Stanford team used an adeno-associated virus (AAV) injected into muscle as a way of delivering the blood-clotting gene to patients. AAV is likewise a promising approach for muscular dystrophy gene therapy.

European research on AAV vectors also suggests the virus looks good for gene therapy but that high standards of quality control during its production and careful monitoring during studies are needed to ensure safety.