Pitt researchers have reported the first study to achieve success with gene therapy for the treatment of congenital muscular dystrophy (CMD) in mice, demonstrating that the formidable scientific challenges that have cast doubt on gene therapy ever being feasible for children with muscular dystrophy can be overcome.

Moreover, the results, published last month in the online edition of the Proceedings of the National Academy of Sciences (PNAS), indicate that a single treatment can reach muscles throughout the body and significantly increase survival.

CMD is a group of some 20 inherited muscular dystrophies characterized by progressive and severe muscle wasting and weakness first noticed soon after birth. No effective treatments exist and children usually die quite young.

Despite gene therapy being among the most vigorously studied approaches for muscular dystrophy, it has been beset with uniquely difficult hurdles. The genes to replace those that are defective in CMD are larger than most, so it has not been possible to apply the same methods successfully used for delivering other types of genes. And because CMD affects all muscles, which account for 40 percent of body weight, gene therapy can only have real therapeutic benefit if it can reverse genetic defects in every cell of the body’s 600 muscle groups.

By using a miniature gene, similar in function to the defective one in CMD, and applying a new method for “systemic” gene delivery, Pitt researchers have shown that gene therapy for muscular dystrophy is both feasible and effective in a mouse model of especially profound disease.

Using this approach, a Pitt team led by Xiao Xiao, associate professor of orthopaedic surgery and of molecular genetics and biochemistry in Pitt’s School of Medicine, reported physiological improvements in the muscles of the heart, diaphragm, abdomen, and legs in treated mice. Those mice also grew faster, were physically more active than untreated mice, and lived four times as long.

“While we have much farther to go until we can say gene therapy will work in children, we have shown here a glimmer of hope by presenting the first evidence of a successful gene therapy approach that improved both the general health and longevity in mice with congenital muscular dystrophy,” said Xiao.

The most common form of CMD, and also one of the most severe, is owing to a genetic mutation of laminin alpha-2, a protein that is essential for maintaining the structures that surround muscle cells and is an integral link in the chain of proteins that regulate the cell’s normal contraction and relaxation. If the protein is defective, or is lacking, this outside scaffold, called the extracellular matrix, disintegrates, and the muscle cells become vulnerable to damage.

Scientists can’t simply replace the defective gene with a good laminin alpha-2 gene, because its size makes it impossible to be squeezed inside viral vectors—disarmed viruses that are used to shuttle genes into cells. But the Pitt team found a good stand-in in a similar protein called agrin that when miniaturized could be inserted inside an adeno-associated virus (AAV) vector. Xiao’s laboratory is known for its work in developing this vector, which, they have shown, is the most efficient means for delivering genes to muscle cells.

In the recent study, Xiao’s team showed that two strains of AAV (AAV-1 and AAV-2) were effective in transferring the mini-agrin gene to cells in two mouse models. The AAV-1 vector was given by systemic delivery—a single infusion into the abdominal cavity—a method the authors only recently described and which they used for the first time in this study to transfer a therapeutic gene. The AAV-2 vector was delivered by intramuscular injection to different leg muscles. With both approaches, muscle cells could assimilate and copy the genetic instructions for making mini-agrin. Once produced, the mini-agrin protein functionally took the place of the laminin alpha-2 protein by binding to the key proteins on either end, thus restoring the cell’s outside scaffolding and reestablishing the missing link to key structures inside the cell.

Pitt scientists cautioned that their research results, while impressive, are far from ideal and that more work lies ahead.

“It’s probably not realistic to expect that we can achieve complete success using the mini-agrin gene, which, while somewhat similar, is structurally unrelated to laminin alpha-2,” said Chungping Qiao, the study’s first author and a research associate fellow in Xiao’s lab. “Unless we address the underlying cause of congenital muscular dystrophy, we’re not likely to be able to completely arrest or cure CMD.”

Future directions for research include finding a way to engineer the laminin alpha-2 gene. For this study, the authors chose to use the mini-agrin gene because researchers from the University of Basel, Switzerland, had already demonstrated it could improve the symptoms of muscular dystrophy in a transgenic mouse model, which has little clinical relevance. Pitt researchers might also explore approaches that combine genes that promote both muscle and nerve growth, as well as focus on improving the AAV vectors.

The research was supported by the National Institutes of Health (NIH) Paul Wellstone Muscular Dystrophy Cooperative Research Center and the NIH’s National Institute of Arthritis and Musculoskeletal and Skin Diseases, as well as through a fellowship awarded to Qiao by the Muscular Dystrophy Association.