University of Florida researchers have used a common gel to successfully deliver gene therapy to the diaphragm

“The heart and diaphragm are two critical muscles for sustaining life,” said study researcher Dr. Barry Byrne, director of the UF Powell Gene Therapy Center and associate chairman of UF’s department of Pediatrics. “This approach essentially makes the genetic background of the muscle normal again and could significantly improve the quality of life for people on ventilators and those who care for them.”

People with muscular dystrophy inherit a mutated gene unable to produce a critical enzyme, which causes their muscles to become increasingly weak as the disease progresses. In Pompe’s disease, the weakness leads to respiratory failure and is fatal.

Cathryn Mah, the study’s principal investigator and a UF research assistant professor of pediatrics, collaborated on the research with several UF scientists, including Byrne, a professor of pediatrics and of molecular genetics and microbiology, and Tom Fraites, a former doctoral student. The study was funded by grants from the National Institutes of Health, and the Florida and Puerto Rico affiliate of the American Heart Association.

In the current study, UF researchers applied a glycerin-based polymer gel they modified to contain corrective copies of the gene to the diaphragms of mice sick with a disorder that mimics Pompe’s disease, thereby strengthening the muscle. This approach was the first time transferring a corrective gene to mouse diaphragm muscle cells was efficient and had a sustained effect, said Byrne, a member of the UF Genetics Institute.

Until now, scientists have been stymied by the mouse diaphragm’s extreme thinness, which prevents direct injection of genes into the muscle. Infusing or injecting genes into veins or arteries also was problematic. In the two-part study, UF researchers first compared the gel-delivery method with a saline rinse used to deliver copies of a gene that stained the cells blue when the cell accepted them.

Next, they tested the gel’s ability to deliver the gene therapy to the weakened muscle cells using the apparently harmless recombinant adeno-associated virus, or rAAV.

Byrne said the polymer gel was crucial to the study’s success because it clung to muscle cells longer than the saline. The gel acted as a time-release mechanism, increasing the muscle’s exposure to the rAAV, which was modified to deliver copies of the gene that produces the missing enzyme, acid alpha-glucosidase. The enzyme also is known as GAA, or acid maltase.

Scientists elsewhere have studied the effectiveness of a water-based gel in certain tissues but found it interfered with the stability of the virus commonly used to carry copies of therapeutic genes into cells. Other gels dissipate too quickly, before enough corrective genes can be transferred to yield beneficial results, researchers said.

“We tested this gel for ease of handling and for its ability to retain its consistency at certain temperatures,” Mah said. “It had the right properties to make it useful for this study.”

Cells normally must use GAA to break down the carbohydrate glycogen to create energy. The 24 mice in the study were missing the GAA enzyme, causing massive amounts of glycogen to accumulate in their muscle cells. This accumulation disrupts the cells’ architecture, disabling their ability to power efficient breathing, Mah said. Researchers painted the gene therapy gel onto the underside of the thin diaphragm muscle in the live study mice, then took tissue samples six weeks later.

Because the treated cells eliminated the excess stored glycogen, the diaphragm could work normally and breathing was repaired, the researchers said.

Other studies to address the adult and infantile form of Pompe’s disease is in various stages of development, Mah said. Byrne, who has a long-standing interest in the ailment, is currently participating in second-stage clinical trials to develop an enzyme replacement therapy to administer by infusion. Other researchers are in the early stages of working to modify the outer protein shell of the delivery virus so that it could be injected into a vein and target a specific area or organ.

Jeffrey Chamberlain, a professor of neurology and director of the Muscular Dystrophy Cooperative Research Center at the University of Washington School of Medicine, said the findings could have important implications for a variety of neuromuscular disorders as well as for Pompe’s disease.

“The methods described by the Byrne lab are a new and exciting approach that have led to a significant level of gene delivery to this important muscle,” Chamberlain said. “Unfortunately, it is not a very accessible muscle, and it has proved quite difficult to deliver new genes to this muscle for the purpose of gene therapy interventions. In the short term, this work will help advance research into studying these diseases, and in the long term should help accelerate the ability to treat a variety of very serious diseases.”