Integration Into Chromosome Enhances
Dystrophin Gene Therapy in Mice

MDA grantees at Stanford (Calif.) University have announced that, in experiments with mice, they have achieved excellent production and distribution of a muscle protein needed by patients with Duchenne muscular dystrophy (DMD). The scientists used a new technique in which genes are injected into the muscles without the use of viruses but with a second gene that coaxes the new genes to integrate into an existing chromosome.

Tom Rando, an MDA-supported associate professor in Stanford’s Department of Neurology and Neurological Sciences, and Carmen Bertoni, an MDA-supported instructor in that department, injected the leg muscles of DMD-affected mice with genes for dystrophin, a muscle protein missing in DMD, and integrase, a protein that causes genetic material to integrate into a chromosome instead of remaining separate from the chromosomes in the cell nucleus.

The research team, whose results are scheduled for publication online next week in Proceedings of the National Academy of Sciences, also included investigators from Stanford’s Genetics Department and the Veterans Affairs Palo Alto Health Care System.

The team found that the mouse muscles receiving both dystrophin and integrase genes had more than eight times the amount of dystrophin six months after injection than did mouse muscles that received dystrophin genes alone.

Moreover, while the muscle fibers that received dystrophin genes alone showed dystrophin production centered around the injection site, fibers that got the dystrophin and integrase combination produced dystrophin along the whole length of the fiber, which is thought to give the fiber much better protection against damage.

The superior protection afforded by the increased level and distribution of dystrophin with the combined gene transfer strategy was demonstrated when the investigators tested the permeability of the muscle fibers to a blue dye six months after the gene transfer injections.

Healthy muscle fibers don’t take up the dye, but damaged fibers, such as those missing dystrophin, do. In these experiments, relatively few fibers that got the integrase plus dystrophin gene combination were permeable to the dye. They remained their usual color, while many of those injected with dystrophin genes alone turned blue.

“The issue of the distribution of dystrophin from end to end of the fiber is an important factor that really pertains to any form of muscle gene delivery. You need to consider not just the amount of the protein produced, but where it is,” Rando said.

“The idea of using an integrating gene is a step forward for virus-free gene therapy, but with the caveat that it carries an inherent risk, because gene integration in other situations has led to genetic mutations. The development of this technology will involve producing more specific integrases with the hope of targeting integration to one place. Knowing where the gene is, knowing it’s safe, and getting high levels of protein production are the ultimate goals.”