BioWorld International Correspondent

LONDON - It may one day be possible to treat Duchenne’s muscular dystrophy with injections every few years, a study using a mouse model of the disease suggested.

A team of Italian researchers said they were able to correct the abnormal gene throughout the bodies of mice using a novel gene therapy approach. Importantly, the muscle groups that responded to the treatment included the animals’ hearts and diaphragms.

Irene Bozzoni, professor of molecular biology at the University of Rome, told BioWorld International: "We used a new gene therapy strategy, which involves correcting the mutation present in the endogenous gene at the level of the RNA, rather than by providing the cells with a new wild-type gene."

Bozzoni, together with collaborators at other Italian centers, has reported the study in a paper in the Feb. 24, 2006, issue Proceedings of the National Academy of Sciences. Its title is "Body-wide gene therapy of Duchenne’s muscular dystrophy in the mdx mouse model."

The group hopes to embark on studies in humans soon. Those probably would involve injections into individual muscles in the first instance, to see whether the treatment helps the muscles to recover function, and whether there are any adverse immunological responses.

Duchenne’s muscular dystrophy affects about one in 3,500 males. The disease is caused by mutations in the gene that encodes the protein dystrophin.

The dystrophin gene is the largest in the human genome - too large to make it possible to deliver "good" copies of it to cells using any of the gene therapy techniques that have been tried so far. To circumvent that problem, Bozzoni and her colleagues decided several years ago to devise a way of working with the RNA produced by the defective gene instead.

They knew that dystrophin often could function normally even if the gene had some mutations leading to deletions in its middle. That happens, for example, in people with Becker muscular dystrophy, whose symptoms are mild or absent altogether.

Many of the mutations that cause Duchenne’s result in a premature stop codon. Bozzoni said, "We thought that if we could somehow prevent the inclusion of the exon that carries the mutation, it might be possible for the cell to carry on making a shorter but still functional version of dystrophin."

When the cell makes dystrophin, it first transcribes the DNA into messenger RNA before splicing sections of RNA together to provide the code for making the protein. By delivering a short piece of antisense RNA that would complement and thus cover up the signal to the cell to "splice here," the researchers were able to induce the cell to skip the unwanted exon.

To deliver the antisense RNA to faulty cells, Bozzoni’s team coupled DNA encoding the RNA onto DNA encoding green fluorescent protein, so that they could track which cells were expressing the additional genetic material. They then inserted that "cassette" into an adeno-associated viral vector (AAV).

Initial tests on cultures of cells from patients with Duchenne’s proved promising. The PNAS paper reported the next stage of the research, using an animal model of Duchenne’s, the mdx mouse.

The researchers injected the AAV incorporating the antisense-expressing cassettes into the tail vein of the animals. At one, three and six months, they then looked to see how green the various muscle groups of the animals were. "We found we had colonization of all muscle groups, including heart and diaphragm," Bozzoni said. "This is important because these organs are strongly affected" in Duchenne’s.

Additional tests showed that the antisense molecules were active in all muscle groups, and that "exon skipping" was obtained, together with rescue of dystrophin synthesis. The muscles of treated mdx mice were stronger than those of untreated mdx mice, and had not degenerated in the same way. Treated mdx mice were able to run for 15 to 20 percent longer than untreated mdx mice.

"The results show that even a small percentage recovery of dystrophin is enough to confer improvement on the animal," Bozzoni said.

The group now is working on how to apply the technique to large animals, such as humans. Work by other groups has shown that AAV can remain stable in primate cells for up to five years. After that time, further injections would have to be given, but these could cause an immunological reaction.

"This could be a problem on the second injection," Bozzoni said, "but we could maybe get around this by using a different serotype of AAV for the second injection."

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