Researchers from the University of Pittsburgh report 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, their results, published in this week's online edition of the Proceedings of the National Academy of Sciences (PNAS), indicate that a single treatment can have expansive reach to 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, an organ that accounts for 40 percent of body weight, gene therapy can only have real therapeutic benefit if it is able to reverse genetic defects in every cell of the body's 600 muscle groups.
By using a miniature gene, similar in function to the one defective in CMD, and applying a newly developed method for "systemic" gene delivery, the 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, the team, led by Xiao Xiao, Ph.D., associate professor of orthopaedic surgery and molecular genetics and biochemistry at the University of Pittsburgh School of Medicine, report that treated mice had physiological improvements in the muscles of the heart, diaphragm, abdomen and legs; and they grew faster, were physically more active and lived four times as long as untreated animals.
"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 Dr. Xiao.
The most common form of CMD, and also one of the most severe, is due 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 extra-cellular matrix, disintegrates, and the muscle cells become vulnerable to damage.
Simply replacing the defective gene with a good laminin alpha-2 gene is not possible because its size makes it impossible for researchers to get it to squeeze inside viral vectors – disarmed viruses that are used to shuttle genes into cells. But the 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. Dr. Xiao's laboratory is known for its work developing this vector, which they have previously shown is the most efficient means for delivering genes to muscle cells.
In the current study, the authors show 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 locally, given by intramuscular injection to different muscles of the leg. With both approaches, muscle cells were able to 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.
Clearly, the authors are most excited about the impressive results achieved in their experiments using systemic gene delivery, which proved there could be significant therapeutic improvements and even be life-saving. Yet they say their results are far from ideal and 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. Unless we address the underlying cause of congenital muscular dystrophy we're not likely to be able to completely arrest or cure CMD," added Chungping Qiao, M.D., Ph.D., the study's first author and a research associate fellow in Dr. Xiao's lab.
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. The 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.
In addition to Drs. Xiao and Qiao, other authors are Jianbin Li; Tong Zhu, M.D., Ph.D.; Xiaojung Ye, M.D., Ph.D.; Chunlian Chen; and Juan Li, M.D., all from the department of orthopaedic surgery; and from the department of cell biology and physiology, Romesh Draviam and Simon Watkins, Ph.D.