By Sean Henahan, Access Excellence

PHILADELPHIA, Pa. (Sept. 17, 1996) The successful delivery of a missing gene into the muscle cells of mice representa major step forward in the development of a gene-therapy strategy to treat Duchenne and other muscular dystrophies, report University of Pennsylvania researchers.

Patients with muscular dystrophy are born with a flawed, nonfunctional version of the unusually large gene that codes for a crucial muscle protein called dystrophin. The gene has presented a challenge for gene therapists because its size limited its use in available viral vectors.

Researchers have now managed to use a stripped-down version of an ordinary cold virus (adenovirus) to ferry working constructs of the full-length gene into the muscle cells of dystrophin-deficient mice. Moreover, the treatments have enabled the mice to produce varying amounts of the needed protein.

Scientists reported similar results earlier, but those experiments used a truncated and only partially functional form of the dystrophin gene. Researchers disclosed in June that they had achieved effective dystrophin gene transfer and subsequent protein production in laboratory cell cultures. The current research confirms and substantially extends that work by showing that the new technology can also succeed in animals.

"What this study suggests is that by a simple injection into the muscle, one might be able to begin to restore the critically needed dystrophin in patients," says Penn's Hansell H. Stedman, MD, an assistant professor of surgery and member of the Institute for Human Gene Therapy.

To create the new viral vector, the researchers began with a type of adenovirus that easily infects many kinds of cells. They then deleted all of the genes responsible for producing normal viral proteins, including those used by the virus to reproduce, retaining only enough of the virus to ensure that it would be able to enter the muscle cells after injection to deliver its genetic load. That load, a rebuilt version of the working dystrophin gene with nonessential sections of the naturally occurring gene removed to save space, was then incorporated into the adenovirus.

To allow laboratory propagation of the completed vector, a so-called helper virus containing genes needed for replication is temporarily added and then later chemically cut away. The researchers also hope that an important advantage of the genetically engineered virus will be that, because it does not include viral genes nor produce viral proteins, it will not spark as vigorous an immune-system response as the wild virus does.

While adenoviruses have proven themselves highly capable of introducing therapeutic genes into cells, their presence in the body also triggers the immune system to begin their removal, often limiting their effectiveness in gene therapy protocols. When the new vector with its dystrophin gene construct was injected into the muscles of a strain of mice that naturally lack an analogous gene, between 30 and 40 percent of the muscle fibers in one experimental group of mice produced the essential dystrophin at two weeks postinjection, after which the level of protein expression appeared to gradually diminish.

Several hurdles remain before the gene therapy might move to clinical trials for muscular dystrophy. One is the need to develop methods to make amounts of the vector sufficient to treat human patients, another is to further decrease the immune system's response to the vector, and a third is to find ways to deliver the vector to muscles throughout the body. Still, a treatment based on the new strategy for delivering the dystrophin gene may have the potential to benefit many patients, according to Stedman.

The study will be published in the October 1 issue of Human Gene Therapy.