Proof of principle that it is possible to deliver new genes body-wide to all the muscles of an adult animal

MDA-supported researchers at the University of Washington-Seattle have delivered the gene for the dystrophin protein to all voluntary muscles with a single intravenous injection in mice with Duchenne muscular dystrophy (DMD).

Such widespread gene delivery to muscle tissue has until now eluded researchers. DMD is the most common childhood form of MD, and it leads to progressive muscle weakness and usually to death in the patient’s 20s.

Principal investigator Jeffrey Chamberlain, an MDA grantee in the Departments of Neurology, Biochemistry and Medicine, used several innovative techniques to deliver the gene for dystrophin, which is missing in DMD and deficient in Becker muscular dystrophy, a less severe disease.

Chamberlain and colleagues are part of the Sen. Paul D. Wellstone Cooperative Muscular Dystrophy Research Center at the university, one of three MD “centers of excellence” co-funded by the National Institutes of Health and MDA. They published their results online today in the journal Nature Medicine. (Results will be in the August print issue.)

MDA Medical Director Valerie Cwik noted, “The ultimate goal of gene therapy for muscle diseases is improvement of strength and function, which will require treatment in multiple muscles simultaneously. These exciting results achieve that in mice, and bring us one step closer to an effective treatment for humans.”

“A major limitation to gene therapy until now has been that no one had found a method by which a new gene could be delivered to all the muscles of an adult animal, including one that had already developed muscular dystrophy,” Chamberlain said.

“Our new work identifies, for the first time, a method by which a new dystrophin gene can be delivered, using a safe and simple method, to all the affected muscles of a mouse with muscular dystrophy.”

The research differs from other strategies in gene transfer to muscle in several ways.

First, the researchers packaged the gene inside a new type of viral “vector,” or delivery system, known as a type 6 adeno-associated virus. The virus has all its own genes removed and can’t replicate in the body, but it appears to be highly efficient and safe for delivering genes to muscle cells.

Second, the researchers augmented the ability of the vector with its dystrophin gene cargo by giving the mice a compound called VEGF, or vascular endothelial growth factor. VEGF increases the permeability of blood vessel walls so that the vector can “leak out” into surrounding muscles after being injected into the bloodstream. The researchers say the compound has been used in humans (for other reasons) and appears to be safe.

A third hurdle the investigators overcame is the unwanted immune response that has sometimes occurred with other gene therapy techniques.

The researchers tried two different molecular promoters, or “on switches,” that tell cells to begin making protein molecules from a gene’s instructions.

One promoter, called CK6, turns on genes only in muscle tissue, and mice that got this one didn’t show any immune response, even when they were given a bacterial protein gene that makes a protein foreign to them. Unfortunately, CK6 doesn’t turn on genes very well in the diaphragm or heart.

With another promoter, called CMV, mice showed some evidence of an immune response to a bacterial protein when it was activated in their heart muscle cells. But experiments conducted later showed that the mice don’t reject the dystrophin protein when it’s delivered with the CMV promoter, Chamberlain says.

The duration of gene activity -- dystrophin protein production from the gene -- was at least eight weeks after treatment, and the research group has seen activity for even longer in the time since these reported experiments were conducted.

Mouse muscles tested after treatment with dystrophin genes encased in a viral shell with the CK6 promoter were more resistant to injury than were muscles from untreated mice. And blood levels of creatine kinase, an enzyme whose presence in the circulation indicates damaged muscle tissue, were cut by half.

“We now have obtained a proof of principle that it is possible to deliver new genes body-wide to all the muscles of an adult animal,” Chamberlain said. “The immediate goal is to find out if we can extend this work to people.” He’s now planning safety tests of the AAV6 vector to see if it can be used in patients.

MDA is a voluntary health agency working to defeat more than 40 neuromuscular diseases, including the MDs, through programs of worldwide research, comprehensive services, and far-reaching professional and public health education.

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