By David N. Leff

Recently, a desperate father raised $140,000 in donations from his local community to enroll his son in an experimental trial for treating Duchenne muscular dystrophy (DMD). That price tag paid for a course of multiple-site injections of myoblasts ¿ primitive muscle cells with potential for becoming muscle fibers.

The ongoing, FDA-approved protocol calls for delivering 50 billion myoblasts, spread over 82 muscles, to the limbs of 22 dystrophic boys. But seeing no clinical benefit, the disillusioned parent soon withdrew his child from the study.

This highly controversial, and as yet unproven, treatment was developed by the Cell Therapy Research Foundation in Memphis, Tenn. Other efforts to implant myoblast cells, or propagate them by gene therapy, also remain experimental.

Two scientists at the Harvard-affiliated Boston Children¿s Hospital conceived that there must be a better way of replacing the dystrophin protein that is totally missing from the wasted muscles of DMD victims. Gene therapist Richard Mulligan is noted for his discovery of hematopoietic (blood-forming) stem cells. Geneticist Lou Kunkel, who discovered the dystrophin gene in 1986, specializes in muscle disorders. Both are co-senior authors of a paper in today¿s Nature, dated Sept. 23, 1999.

The article¿s title, ¿Dystrophin expression in the mdx mouse restored by stem cell transplantation,¿ sums up its authors¿ proof-of-principle advance toward practical therapy for DMD.

¿The mdx mouse,¿ the paper¿s lead author, Emanuela Gussoni, told BioWorld Today, ¿is an X-linked animal model of DMD. It has a mutation in the same gene seen in patients with the disease. They completely lack dystrophin, which is the protein product of the DMD gene.

¿That mdx mouse,¿ Gussoni went on, ¿was first discovered in the early 1980s, but until Kunkel identified the dystrophin gene, it wasn¿t possible to assess whether this animal model indeed corresponded to human DMD. Because the gene was not yet cloned, the lack of its protein, which causes muscular dystrophies, was not known. Then Kunkel cloned the gene and determined that its protein is the absent product in DMD. So that¿s how people went back and reanalyzed this animal model, which has now been used for at least a decade.

¿Mulligan is very famous for his gene therapy interest,¿ Gussoni continued, ¿and for the discovery and isolation of hematopoietic stem cells. So we figured that perhaps this technique could be used to isolate other types of stem cells in different tissues, particularly skeletal muscle.¿

This technique is the essence of bone marrow transplantation in which, say, a patient with lymphoma undergoes whole-body irradiation to wipe out his or her blood-forming stem cells, and thus eliminate their immune system cells. That permits engrafting donor cells to restore cancer-free marrow. (See BioWorld Today, Sept. 9, 1999, p. 1.)

Gussoni and her co-authors lethally irradiated female mdx mice, and separately injected into their tail veins two types of stem cells ¿ both from male donor animals ¿ to replenish their hematopoietic stem cells and generate healthy muscle cells.

Special Delivery ¿ Blood-Borne

¿We found,¿ she told BioWorld Today, ¿that both types of cells could not only protect the animals from the effects of lethal irradiation ¿ showing that muscle stem cells can differentiate into bone marrow ¿ but that they could also reconstitute the muscle. Which shows that highly purified hematopoietic stem cells are capable of differentiating into mature skeletal muscle. It means we can deliver these cells via the bloodstream.

¿But that¿s not all,¿ Gussoni continued. ¿The other part is that bone marrow stem cells, via the circulation, can also go into skeletal muscle and differentiate into adult muscle tissue.¿

What clinched these results was finding that the new stem-cell-generated marrow and muscle cells in the revitalized female mice now carried the Y chromosome in their nuclei ¿ the chromosomal mark of males-only donor cells. However, when it came to measuring the actual output of those newly minted muscle cells, the data proved conceptual rather than practical.

¿We counted the number of dystrophin-positive fibers in the muscles of these animals,¿ Gussoni recounted, ¿and we saw that if we inject bone marrow stem cells, and wait three months from the day of transplantation, we can achieve up to 10 percent of dystrophin-positive fibers in the muscle, which is a very encouraging result.

¿With skeletal muscle stem cells, we waited one month from the date of injection, and found that up to 9 percent was the best case. Not all the animals had that dystrophin-positive level. But it¿s still encouraging, though not yet a level we can consider therapeutic,¿ Gussoni allowed. ¿You¿d need at least 20 percent of dystrophin positivity in all myofibers. But the fact it was coming from the circulation into the muscles of the animals was also very encouraging, because it allows you to spread the cells everywhere in the body, as opposed to just giving local injections.¿

The co-authors, she said, ¿are now working very hard to try and increase that level of dystrophin positivity in the skeletal muscle, using these methods of delivery. There are several approaches that one can take. Certainly the easiest way of doing that would be to try and inject more cells, and see if we can get a better uptake. But I suspect that we may have to be more sophisticated than that.

¿I think we have to try to understand,¿ she added, ¿what signals are released from the target tissue to the cells in order to recruit the cells themselves, and we don¿t know very much about it. The other thing is basically understanding why the cells are recruited from the circulation into the muscle.

¿We are conducting experiments on new sets of mice right now,¿ Gussoni said, ¿but I can¿t tell you the new data yet because we¿re still in the process of analyzing their muscles.¿

Whole-Body Cancer Therapy?

Mulligan made the point that this technique has potential for treating diseases beyond muscle disorders ¿ ¿cancer, perhaps, being the most important one,¿ he said. ¿All of the gene-based strategies we have for treating cancer,¿ he told BioWorld Today, ¿suffer from the difficulty that we haven¿t yet figured out a way to disseminate good gene delivery systemically. Some of these viruses that are used to kill tumor cells are usually given in the context of local tumors. Obviously, we¿d like to treat metastatic disease.

¿We also have a hunch,¿ he concluded, ¿based on little or no facts, that these stem cells may target to sites of inflammation or other sites of injury.¿ n