Duchenne’s Muscular Dystrophy and Myoblast Transfer

Duchenne’s Muscular Dystrophy is a genetic disorder linked to the X chromosome that is caused by a deficiency in the protein dystrophin (Mendell et al., 1995). This disease weakens skeletal and cardiac muscles, and may pose obstacles when sitting, standing, walking, and speaking. Treatment by myoblast transfer showed promising results in animal trials. This method strives to replace dystrophin through the injection of donor myoblast cells into skeletal muscles. The donor cells are able to replace dystrophin by fusing with muscle fibers and supplying the absent gene that causes the production of dystrophin. Clinical trials of this method were used to treat murine dystrophies, and they showed favorable results. One account showed dystrophin produced from the donor cells one month after the myoblast administration. However, other reports were not able to find any donor produced dystrophin or messenger RNA after the injection. From all of the controlled trials, there was no report of increased strength after the myoblast application. On the contrary, uncontrolled trials showed increases in strength. To avoid rejection of the myoblast cells, immunosuppressors were used in some of the trials. Furthermore, incorporation of anti-inflammatory factors affects the analysis of the myoblast administration; prednisone and cyclosporine have demonstrated the ability to increase strength in Duchenne’s muscular dystrophy patients. As a result of the promising conclusions of these studies, the researchers in this study hypothesized that myoblast transfer will be a viable treatment for replacing the lacking gene that causes the production of dystrophin in patients with Duchenne’s muscular dystrophy. Also, the researchers hypothesized that administering cyclosporine, in conjunction with the myoblast transfer, will help to increase muscle strength for these patients.

This study differs from previous studies in a few ways. In contrast to previous studies, this study conducted myoblast injections once a month for a six-month period (Mendell et al., 1995). Previous studies only administered myoblasts on a single occasion. Also, the measurement of dystrophin expression in this study was done using peptide antibodies that are distinct to the missing exons in the dystrophin gene. This allows examiners to differentiate between revertant fibers, or fibers from the host that return to normal, and donor cell produced dystrophin-positive fibers. Lastly, the administration of cyclosporine or a placebo was randomly assigned. This allowed for the evaluation of the effect of cyclosporine on increasing muscle strength, and the determination of the need for immunosuppressors.

Results

Twelve boys with Duchenne’s muscular dystrophy took part in a double-blind trial of myoblast transfer. Normal human myoblasts were isolated and mixed with muscle samples from the biceps brachii of all donors. The samples were maintained until being dissociated in 0.01 percent trypsin, collagenases I and II, and 0.02 percent EDTA. The patient's arms were then randomly assigned to receive a myoblast injection or a sham injection, both patients and researchers were unaware of which arm received myoblasts and which received sham injections. The maximal voluntary isometric strength of elbow flexion was the primary variable tested, the average of three consecutive trials was recorded. One year after the trial started (6 months after the final myoblast transfer) biopsies of biceps brachii were performed.

The results showed that myoblast transfer had no effect on muscle strength. Six myoblast transfers were completed, at month 0 and months 1, 2, 3, 4, and 5. Maximal voluntary isometric strength was tested the day before each myoblast transfer and at twelve months. The difference in the degree of change from the base-line test (month 0) between arms injected with myoblasts and sham injected arms. Additionally there was no difference in the mean number of dystrophin positive fibers between the myoblast injected and sham injected arms. Dystrophin mutations are what causes Duchenne muscular dystrophy. Peptide specific antibodies raised against deleted exons was used to determine whether the dystrophin positive fibers in the arms that received myoblast infusions were expressing donor-derived dystrophin or native mutated fibers. Only four of the twelve patients muscle fibers expressed donor-derived dystrophin. Of those patients, only one fiber was in the entire cross section of Patients 3 and 6 and two positive fibers were found in the entire cross section of Patient 12. These positive cross sections made up less than 1 percent of the revertant fibers that express dystrophin in patients with Duchenne’s muscular dystrophy. However, patient 5 expressed that 10.3 percent of dystrophin fibers were donor derived, which was 420 dystrophin positive fibers from a total of 4080 fibers. Six boys were treated with 5 mg of cyclosporine per kilogram each day and the other sic were given a placebo. There was no significant difference in the average muscle scores of the boys treated with cyclosporine and those given a placebo. The loss of muscle strength was similar to the natural rate of decline. There was no significant difference between boys treated with cyclosporine and those given a placebo. There was no significant difference in muscular strength after six rounds of myoblast transfer.

Discussion

The study assessed the effects of myoblast transfer as a way to treat Duchenne’s muscular dystrophy. The patients each received a total of 660 million myoblasts to the biceps muscle over the course of 6 months, which differs from a previous study in which the patients received half the amount. None of the patients had any muscular strength increase following the conclusion of the treatment. It is suspected that treatment for muscular dystrophy depends on the expression of dystrophin gene. Of the twelve patients that participated in this study, four had donor-produced dystrophin. One patient in particular had significant amounts of dystrophin-positive fibers, with a total of 10.3%. The positive fibers were found to be equally distributed throughout the muscle, in both the superficial and deep muscles. In a previous study, it was concluded that 1% of host muscle fibers expressed donor-produced dystrophin following a method using reverse transcriptase polymerase chain reaction. The researchers also found no difference in muscular strength and dystrophin-positive fibers between myoblast-injected and sham-injected patients. It is suspected that the dystrophin fibers found in the sham-injected sites were originally mutant fibers that have reverted back to their original forms having emerged from a second mutation. In another study, a patient was observed to have 5% of dystrophin-positive fibers with no donor-produced DNA, further showing that detecting the source of dystrophin is very difficult.

Future studies would be beneficial as to understanding the origin of dystrophin-positive fibers and discovering other ways to detect the source of them. It would also be helpful to use a bigger sample size than this study used, which was only 12 patients. By doing so, researchers would have a higher confidence level and would be able to differentiate between the source of dystrophin-positive fibers. Since this study had secondary outcome regarding the use of cyclosporine and its effects, it would be interesting to note how that treatment coincides with myoblast transfer. A previous study observing the effects of cyclosporine found positive results in increased muscle strength after 12 weeks of treatment. This would be interesting to follow up using myoblast transfer to see if there would be further positive results or if the myoblast transfer has no effects at all.

In conclusion, this study showed no improvements of muscle strength in patients with Duchenne’s muscular dystrophy using myoblast transfer. This is despite the fact that the patients received high numbers of myoblasts to the muscles. It was demonstrated that myoblast transfer can result in cells with the capability of fusing with host fibers and expressing dystrophin-positive fibers. Future studies would help to further this one and assessing the efficiency of myoblast transfer to attain positive results.