Celebrating the impact of health research
Success story in muscle - Treating a devastating disease: the case of Duchenne Muscular Dystrophy
Dr. Jacques P. Tremblay PhD
Department of Molecular Medicine, Laval University
Duchenne Muscular Dystrophy (DMD) is caused by a mutation in the dystrophin gene. This devastating disease puts sufferers – who are always male – into a wheel chair by age 10 and on a respirator by 17, by which time many are dying. Few males with DMD reach the age of 30.
In 1987, dystrophin was identified as the culprit gene in the disease. Since that time, my lab has focused on finding a treatment for this untreatable disease. Our research has shown promising results.
DMD is caused by a mutation in the dystrophin gene that prevents the formation of dystrophin protein. In all people, intense muscle activity damages muscle fibers. In healthy people, though, these damaged fibers are repaired by the fusion of cells called myoblasts. In people with DMD, the absence of dystrophin leads to increased fragility of the muscle fibers and to their frequent damage, even following just normal activity. The increased frequency of damage, and therefore repairs, leads to a progressive inability of myoblasts to carry out their repair function, resulting in a reduction in the size and number of muscle fibers, which are gradually replaced by fat and connective tissues. This generally starts around four–to–five years of age, at which time affected boys will start to have trouble walking, running or climbing stairs – the first signs of a devastating disease that causes much suffering, both for the patients and their families, and ends in premature death.
In our efforts to find a treatment for DMD, we have focused on transplanting myoblasts with the normal dystrophin gene. These myoblasts can fuse with the host's damaged muscle fibers and not only repair the damage but also introduce in these fibers nuclei containing the normal dystrophin gene. Thus the transplantation of these normal myoblasts results in the expression of dystrophin in the patient's muscle fibers.
We first worked in vitro, showing that fusing one normal myoblast with several myoblasts containing a mutated dystrophin gene unable to produce dystrophin, led to the expression of dystrophin over whole small muscle fibers (myotubes) formed in culture (Huard, Labrecque et al. 1991). These results led to an initial clinical trial in 1990. The results were initially promising. Muscle fibers expressing dystrophin were observed in three of the four DMD patients who were transplanted (without immunosuppression) with myoblasts obtained from healthy, histocompatible brothers or sisters. One of these patients had 36% dystrophin–positive fibers (Huard, Bouchard et al. 1991; Huard, Bouchard et al. 1992; Tremblay, Malouin et al. 1993). We observed increases in strength in the patient who had the highest percentage of dystrophin–positive fibers and even more increases after a second transplantation from the same donor. However, subsequent transplantation did not lead to more improvements and the patient progressively lost the initial gains in strength.
When we looked more deeply into why the benefits did not last, we found that, while no antibodies were detected against the donor myoblasts themselves, antibodies were detected against the myotubes formed by the fusion of these myoblasts (Roy, Tremblay et al. 1993). To our surprise, we found that one of the proteins leading to this immune response was dystrophin itself! Because the patients had not previously produced dystrophin, the transplantation of normal myoblasts led to an immune reaction to this new protein.
We turned our attention to identifying the best immunosuppressive drug to prevent this immune response. We were conscious that four other research groups in Canada and the United States that had conducted similar clinical trials of myoblast transplantation, but not using histocompatible donors and using immunosuppression, had all obtained negative results. We later found out why that might have been – the drug that had been used by at least one of the research groups actually killed the myoblasts that were proliferating in the muscles after transplantation (Vilquin, Kinoshita et al. 1995).
Experiments to identify the best immunosuppressive drug for myoblast transplantation were made in a mouse model missing dystrophin, mimicking the DMD patients. After trying several drugs, we identified a new drug called FK506 (now commercialized under the names Tacrolimus or Prograf) that permitted successful transplantation of normal myoblasts in a mouse model of DMD, called mdx. Following myoblast transplantation using this drug, up to 90% of the muscle fibers expressed dystrophin in the mdx mice (Kinoshita, Vilquin et al. 1994; Kinoshita, Vilquin et al. 1994) . We were unsure about progressing straight to using the drug in humans, thus we first tried the drug in dogs also bred to be unable to express dystrophin. Unfortunately, the dogs did not respond well to FK506 immunosuppression and, even in combination with other immunosuppressive drugs, the results were modest (Ito, Vilquin et al. 1998) . However, FK506 showed more promise in monkeys, (Kinoshita, Vilquin et al. 1995; Kinoshita, Roy et al. 1996) , indicating that it might also work well in humans.
The monkey experiments also identified a second problem that had limited the success of the early trials of myoblast transplantation: limited migration of the transplanted myoblasts into muscle fibers. (Skuk, Goulet et al. 2000; Skuk and Tremblay 2001; Skuk and Tremblay 2001; Skuk, Goulet et al. 2002) . Finally, the monkey experiments also allowed us to determine that we had not injected enough myoblasts in the original clinical trial and that the ideal number of myoblasts to inject is 30 million myoblasts per cubic centimetre of muscle (Skuk, Caron et al. 2003; Skuk, Paradis et al. 2007).
With our knowledge advanced in this way, we received permission from Health Canada for a new phase I clinical trial, which successfully demonstrated that, with adequate immunosuppression, successful myoblast transplantation was possible. The trial, which began in 2002, involved nine DMD patients aged between five and 15 years old. We transplanted 30 million myoblasts in only one cubic centimetre, this time using donations from participants' father or mother, since using minor brothers or sisters as donors was no longer allowed by the Research Ethics Board. We used FK506 (Prograf), which had become the drug of choice for organ transplantation in children, to suppress their immune systems. A muscle biopsy done one month later confirmed that the normal dystrophin gene was present and expressed at both the messenger RNA and protein level. Indeed up to 26% dystrophin–positive fibers were detected (Skuk, Goulet et al. 2006; Skuk, Goulet et al. 2007).
We have now received approval from Health Canada for a phase I/II clinical trial to verify whether the transplantation of myoblasts throughout a complete muscle not only restores the expression of dystrophin but also increases strength of that muscle.
Outcomes: extending the research
Having shown that myoblast transplantation offers potential for treating DMD, we are now focusing on ways to improve the treatment, including the possibility of genetically correcting the patient's own myoblasts, thus removing the need for life–long immunosuppression therapy. We have already shown that such correction is possible using a lentivirus (a virus used to deliver DNA into a host cell) coding for micro–dystrophin (Moisset, Skuk et al. 1998). We are also working, with Dr. Mitsuo Oshimura of Japan on using a human artificial chromosome to introduce the full–length dystrophin gene. One problem is that the patient's own myoblasts may be difficult to grow because they are senescent after five years. We have, however, shown that it is possible to produce induced pluripotent stem cells (iPSCs) from the patient's own fibroblasts, which we were then able to differentiate into myoblasts. These myoblasts were then genetically corrected with a lentivirus coding for micro–dystrophin and were successfully transplanted in the muscles of immunodeficient mice, where they fused and led to muscle fibers expressing micro–dystrophin. This ex vivo gene therapy approach, if it is shown to be effective in humans, could be a means for avoiding the long–term immunosuppression currently required when using donor myoblasts.
Myoblast transplantation is an exciting development, not only for DMD, but also for other types of muscular dystrophies, in part because it can provide additional myogenic cells and restore strength to already–damaged muscles. We anticipate, following the completion of our new phase I/II clinical trial, further progress towards the development of a cellular therapy for DMD and other muscular dystrophies.
Dr. Daniel Skuk, Laval University; Dr. Jean–Pierre Bouchard, Laval University
Funding: Medical Research Council of Canada, Canadian Institutes of Health Research (CIHR), Association Française contre les Myopathies, Muscular Dystrophy Canada, Muscular Dystrophy Association, le Fonds de recherche du Québec – Santé, Stem Cell Network of Canada, National Institute of Health
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