Penn Researchers Develop Gene Therapy Technique That Reverses Muscle Membrane Weakness In Muscular Dystrophy Variant

It is hoped that the Penn-developed technique would be ultimately transferable to human clinical trials involving gene therapy treatments of many forms of muscular dystrophy, as well as other types of debilitating or fatal disorders - such as metabolic myopathy and cardiomyopathy (heart failure). "We've demonstrated proof of concept that the methodology can work for both skeletal and cardiac muscle," says principal author Hansell Stedman, MD, assistant professor of surgery at Penn's Institute for Human Gene Therapy, "so the next step will be to establish its safety and effectiveness in patients with this form of muscular dystrophy." Stedman cautions that clinical trials will have to address safety issues one step at a time before significant therapeutic benefit can be approached.

The research is being published as the cover article in the April 1999 issue of Nature Medicine.

Induced Vessel Leakage Nets Massive Cell Saturation

In limb girdle muscular dystrophy, the instability of muscle tissue is linked directly to the level of genetic disruption that occurs within the sarcoglycan complex - a critical muscle structure composed of four membrane-spanning proteins. Depending on the nature of the mutations that can affect any one of these proteins, the integrity of the muscle membrane is compromised - eventually resulting in muscle weakness that can range from a very mild form to a more severe, rapidly-progressing type.

Building on prior work (much of which has been done at Penn's Institute for Human Gene Therapy) that has shown the adeno-associated virus as an effective gene-transfer vehicle, the Penn team sought to overcome the existing problem of how best to gain access to the millions and millions of muscle cells that require genetic re-engineering. Dismissing intramuscular injection as an impractical technique (since literally thousands of injections might be needed), they proposed an intravascular route that would require some means of allowing infused genetic material (being carried in adeno-associated viruses) to seep out of the blood vessels into the surrounding muscle tissue.

"We thought long and hard about this problem because you can't just take virus from the test-tube, put it into an intravenous line, inject it into the bloodstream and have it go to all the muscle cells of the body," explains Stedman. "It's not like oxygen, which gets to every muscle cell with every breath you take. The viruses are literally millions of times bigger than oxygen molecules and they're too big to leave the blood vessels under normal circumstances." The solution, they realized, was to make the blood vessels become leaky, temporarily, so that the viruses could squeeze out of the vessels and make contact with nearby muscle tissue. Thus, histamine - a natural vessel destabilizer -- was added to the liquid solution carrying the adeno-associated viruses. Infusing just a single limb - a leg - with the histamine-enhanced solution, the scientists were able to achieve widespread gene transfer to all the muscles in that particular area of the body. (Following infusion, the histamine was flushed out of the vascular area with a simple saline rinse, which then encouraged the walls of the vessels to automatically snap back into position.)

The infusions were well tolerated, so that normal activity was resumed within hours. Restoration of the sarcoglycan protein complex was noted at all time-points tested, suggesting the potential for permanent correction.

"Fatal Contraction" Analyzed

Another important contribution of the Penn study relates to a clearer understanding of the nature of the disease itself. Indeed, by developing a sophisticated in vitro assay process to accurately gauge gene expression, the researchers were able to describe new aspects of the step-by-step process that occurs as part of the biochemical deterioration of muscle cells in limb girdle muscular dystrophy and, perhaps, all other forms of the disorder.

By removing a muscle in its entirety and then inducing rapid injury, the investigators learned that within minutes of a forced lengthening of individual muscle fibers, there is a loss in the mechanical integrity of part of the muscle membrane. This loss results in an in-rush of calcium - which, in turn, launches a rapid destruction process that adversely effects several other proteins in the muscle fiber. "Once a muscle fiber sustains this sort of lethal blow, or 'fatal contraction,' it's like a breach in the hull of the Titanic - with calcium rushing in faster than it can be pumped out," explains Stedman. "In a matter of hours, the muscle fibers reach a point of no return and become irreversibly damaged. Progression of the disease is temporarily offset, however, by the remarkable capacity of muscle fibers to regenerate for a time."

By better understanding this sequence of cellular events, researchers can develop better ways to monitor and hopefully prevent the progression of this process.

This work was supported, in part, by grants from the National Institutes of Health (NIAMS and NINDS) and the Muscular Dystrophy Association (MDA). Additional support was provided by the Department of Surgery and the Institute of Human Gene Therapy at the University of Pennsylvania Health System, and the Association Francaise contre les Myopathies.