Jeffrey Chamberlain

At the same time that plans for limb-girdle muscular dystrophy (LGMD) gene therapy move ahead, MDA researchers at the University of Michigan in Ann Arbor and elsewhere are working hard to solve the special set of technical problems that have slowed progress in gene therapy for Duchenne muscular dystrophy (DMD).

The immune system, designed to protect us from foreign invaders, has proved to be one of the biggest obstacles to developing gene therapy for DMD. The affected gene in DMD, the dystrophin gene, is enormous and requires a different viral delivery system than that being used in the LGMD trials. Unfortunately, the best available virus large enough to contain the dystrophin gene, the adenovirus, tends to attract a lot of attention from the immune system.

The adenovirus, unlike the virus being used for LGMD gene therapy, is one that normally causes a sickness in humans -- a cold-like illness. Many of the symptoms of a cold, such as runny nose and fever, actually result from the attempts by the immune system to fight off the invading cold virus. Although many of the original viral genes are removed from the adenovirus for use in gene therapy, animal tests have shown that the immune system still treats the initially developed therapeutic version of the virus somewhat as it would the cold-causing version. It tries to get rid of all cells in the body that contain the modified virus.

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STEALTH VIRUSES

To try to get around this problem, MDA researchers have been working on a new generation of "stealth" adenovirus carriers that contain none of the original viral genes and are designed to attract as little attention as possible from the body's immune system. These vectors are known as "gutted" vectors.

Last fall, at the American Society for Human Genetics meeting, MDA grantee Jeffrey Chamberlain of the University of Michigan reported that the new gutted adenovirus triggers little response from the immune system when tested in animals.

Some researchers have worried that these gutted viruses may be inherently unstable and may not last very long in the host muscle cells. However, Paula Clemens, an MDA grantee at the University of Pittsburgh, recently de-monstrated that a gutted adenovirus carrier is stable in the muscle cells of mice for at least several months while expressing nonviral genes.

Another problem that has worried researchers is that the immune system may react to the new dystrophin protein itself. This could happen because there is normally little or no dystrophin in the body of a person with DMD during fetal development, when the immune system is learning what proteins are considered "self" proteins. There has been less concern about this problem in the LGMD trials because most people make some of the sarcoglycan protein that's mutated in LGMD, even if the protein is nonfunctional.

New evidence from the lab of MDA grantee Andy Amalfitano of Duke University Medical Center in Durham, N.C., suggests that it may be possible to use the adenovirus to deliver a protein that the immune system considers foreign without triggering a full-blown immune response.

Initial data on the immune response to dystrophin carried by the new gutted adenovirus developed by Chamberlain also seems to suggest that dystrophin itself does not cause a significant response, but Chamberlain cautions that more sensitive tests may still detect some immune responses to the cells that contain dystrophin.

"The major challenges facing us now," Chamberlain says, "are perfecting methods to grow large amounts of the gutted adenovirus safely and efficiently, and completing the necessary preclinical studies on the gutted virus in animal systems."

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MULTIPLE STRATEGIES

MDA researchers are also working on alternative strategies to replace the defective dystrophin gene in people with DMD.

Some researchers, including MDA grantees Bernard Jasmin of the University of Ottawa in Canada and Kay Davies of Oxford University in England, have been looking for ways to cause muscle cells to make more of a protein related to dystrophin called utrophin. People with DMD normally make small amounts of utrophin, so this protein is less likely to trigger an immune response. Animals studies have shown that utrophin can substitute for dystrophin if enough utrophin is made in the muscle cells.

Other researchers are trying to find ways to use gene therapy not to replace the entire dystrophin gene, but to alter the way a cell reads a person's defective gene so that some dystrophin gets made. This approach, called exon-skipping, is being ex-plored by MDA grantees Steve Wilton of the Australian Neuromuscular Institute and Leslie Leinwand of the University of Colorado in Boulder. The advantage of this approach is that only small pieces of genetic material would need to be delivered to the cells, so the use of an immune-response-triggering virus could be avoided. The goal of this therapy would be to make the symptoms of DMD milder.

MDA's gene therapy research program continues to make the development of gene therapy techniques for DMD a priority.