BOSTON—Stem cells may prove to be an effective shuttle for delivering corrective genes to tissues throughout the body, according to recent muscular dystrophy research. Louis M. Kunkel, PhD, Chief of the Division of Genetics at Children's Hospital, Boston, Massachusetts, described a novel population of stem cells that, when transplanted into the bloodstream of a mouse model for Duchenne's muscular dystrophy, can restore a small percentage of the dystrophin-deficient muscle tissue. Speaking at the 125th Annual Meeting of the American Neurological Association, Dr. Kunkel reviewed dystrophin replacement therapies and their potential uses in the management of muscular dystrophy.

DELIVERING DYSTROPHIN

For years, researchers have been looking for ways to introduce normal copies of missing proteins into the muscle of patients with muscular dystrophy. The genetic defects associated with muscular dystrophy include mutations in the dystrophin gene and in the genes of its associated sarcoglycan proteins. The proteins form a complex responsible for maintaining muscle-cell integrity, Dr. Kunkel said.

Gene therapy trials aimed at introducing g-sarcoglycan into patients with limb-girdle muscular dystrophy are currently on hold. However, one would expect only a local benefit since the viral vectors carrying the gene products are injected directly into muscle tissue, Dr. Kunkel noted.

Injection of whole muscle cells has also been successful in the local restoration of dystrophin, Dr. Kunkel said. In trials that involved six centers across the United States and Canada, the muscles of boys with Duchenne's muscular dystrophy were injected with normal myoblasts from their fathers or one of their unaffected siblings. Despite multiple injections of myoblast cells into a single muscle, however, most of the patients showed no appreciable dystrophin expression one month and six months after the injection.

FISHING IN THE SEA OF DNA

In an attempt to detect the donor cells in the patients who underwent muscle cell injections, Dr. Kunkel and his colleagues refined a technique called fluorescence in situ hybridization (FISH) analysis that labeled the missing exons in the dystrophin gene. Donor nuclei have a complete dystrophin gene, he explained, whereas patient nuclei have deletions in the gene. In addition to locating donor cells, the researchers used immunohistochemistry to detect dystrophin expression. Taking serial sections from a muscle biopsy of a patient injected with his father's myoblasts, they were able to visualize donor cells producing dystrophin. "That's quite amazing when you think about it," Dr. Kunkel said.

Unfortunately, the vast majority of donor nuclei (about 90%) were not found in the patient's muscle six months after the transplantation. And, among the donor cells that did fuse into host myofibrils, only half produced dystrophin. Nonetheless, "you walk away from this experiment with a conclusion that basically the cells were capable of doing what we asked them to do," Dr. Kunkel said.

A SPECIAL POPULATION OF STEM CELLS

The researchers took a step back and wondered whether stem cells could also be a source of mature dystrophin-producing muscle cells. Bone marrow–derived stem cells, Dr. Kunkel said, circulate throughout the body and have been shown to give rise to both hematopoietic and muscle cells. In fact, muscle tissue itself appears to support a unique stem cell population that can give rise to both hematopoietic and muscle cells, they found.

Stem cells isolated from either the bone marrow or muscle tissue of normal male mice gave rise to healthy muscle cells expressing dystrophin in irradiated female mouse models for Duchenne's muscular dystrophy, according to a study Dr. Kunkel and his colleagues published in the September 23, 1999 Nature. Although no clinical benefit was seen, as many as 10% of the muscle fibers were expressing dystrophin 12 weeks after the intravenous injection of bone marrow cells. Dr. Kunkel noted that relatively few transplanted muscle stem cells made it back to the muscle; however, dystrophin expression was seen as early as five weeks post-transplant. These muscle stem cells, he said, appear to have a "memory of where they came from and they home back to that tissue much more readily than bone marrow transplant."

EARLY INTERVENTION OF STEROIDS

Dr. Kunkel described a 14-year-old boy who was born with severe combined immunodeficiency and had (at age 1 year) received bone marrow cells from his father. He began having difficulty walking and climbing stairs at age 13 and was recently confirmed to have a deletion in exon 45 of the dystrophin gene. His mother is a carrier for the deletion, confirming that the boy should have a severe course of disease.

However, the boy is still ambulatory; and it may be that the bone marrow transplant ameliorated his symptoms, Dr. Kunkel said. The researchers are planning to study a biopsy of the boy's muscle to determine whether his father's cells are contributing to dystrophin expression. Dr. Kunkel noted that the patient received steroid therapy because of severe graft-versus-host disease after the bone marrow transplant. This early intervention of steroids, he said, may also have had an influence on the boy's disease course. Dr. Kunkel hopes to learn a great deal from this patient and believes that stem cell transplantation can be optimized to provide clinically useful levels of engraftment in muscle.

NR