No abstract

Five billion normal myoblasts were injected into each of 21 Duchenne muscular dystrophy (DMD) boys aged 6-14 yr to assess the feasibility, safety, and efficacy of the Phase II myoblast transfer therapy (MTT). The Phase II study was designed to strengthen muscles of both lower limbs. Forty-eight intramuscular injections transferred the myoblasts into 22 major muscles at 55.6 x 10(6)/mL in 10 min under general anesthesia. Eleven boys had received 8 million myoblasts each 1 yr ago in the Phase I MTT. In the Phase II study, eight of them had their myoblasts subcultured from reserves frozen 1 yr ago. The donor myoblasts for each of the remaining boys were cultured from satellite cells derived from a 1-g muscle biopsy of a normal male who might or might not be histocompatible with the recipient. The immunosuppressant cyclosporine (Cy) is being administered to recipients for 6 mo after MTT to facilitate donor cell survival. There was no evidence of an adverse reaction to MTT or Cy as determined by serial laboratory evaluations including electrolytes, creatinine, and urea. Early objective functional tests using the KinCom Robotic Dynamometer were conducted on 13 subjects aged 6 to 13 before MTT and at 3 mo after MTT. Of the 69 muscle groups (knee extensors, knee flexors, plantar flexors) tested for isometric force generation in these subjects, 43% showed mean increase of 41.3% +/- 5.9 SEM, 38% showed no change, and 19% showed continuous force reduction of 23.4% +/- 3.1 SEM. The remaining subjects await the 3-mo post-MTT evaluation. The results indicate that 1) MTT is safe; 2) MTT increases muscle strength in DMD: 81% of the muscles tested showed either increase in strength or did not show continuous loss of strength; 3) more than 5 billion myoblasts can be cultured from 1 g normal muscle biopsy, providing unprecedented numbers of cells for MTT; 4) myoblasts, frozen over 1 yr, retain the ability to proliferate from 10 million to 5 billion, and to form normal myofibers; 5) injections of 5 billion myoblasts have not provoked any immunological rejection symptoms in the Phase II subjects, 11 of whom received 8 million myoblasts in the Phase I MTT a year ago; 6) it is safe to perform multiple injections of myoblasts into lower limb muscles without formation of emboli; and 7) donor cell rejection by the recipient can be prevented with Cy when properly managed.

A treatment has been developed to alleviate muscle weakness in murine dystrophy. Cultured myoblasts from genetically normal mouse embryos were injected into the right soleus of 20-day-old normal or dystrophic mice. Hosts and donors were immunocompatible but exhibited different genotype markers. Donor cells produced GPl-1CC. Host cells produced GPl-1BB. When compared with contralateral controls 6 months postoperatively, test dystrophic solei exhibited greater cross-sectional area, total fiber number, wet weight, and twitch and tetanus tensions. They contained more normal-appearing and less abnormal-appearing fibers. Their mean fiber resting potential was similar to that of normal controls. Presence of GPl-1CC with or without the hybrid isozyme GPl-lBC in these muscles implied the survival and development of donor myoblasts into normal myofibers, and fusion of normal myoblasts with dystrophic satellite cells to form genetically mosaic myofibers. Injection of fibroblasts instead of myoblasts caused detrimental effects.

The feasibility, safety, and efficacy of myoblast transfer therapy (MIT) were assessed in an experimental lower body treatment (LBT) involving 32 Duchenne muscular dystrophy (DMD) boys aged 6-14 yr, half of whom were nonambulatory. Through 48 injections, five billion (55.6 x 10(6)/mL) normal myoblasts were transferred into 22 major muscles in both lower limbs, in 10 min with the subject under general anesthesia. Ten subjects received myoblasts cultured from satellite cells derived from 1-g fresh muscle biopsies of normal males aged 9-21 yr. Donor myoblasts for the remaining 22 boys were subcultured from reserves frozen 1 mo-1.5 yr ago. Only four donors were known to have identical histocompatibility with their recipients. All subjects took oral doses of the immunosuppressant cyclosporine (Cy), beginning at 2 days before MTT and lasting for 6 mo after MTT to facilitate donor cell survival. There was no evidence of an adverse reaction to MTT or Cy as determined by serial laboratory evaluations including electrolytes, creatinine, and urea. Objective functional tests using the KinCom Robotic Dynamometer measured the maximum isometric contractile forces of the ankle plantar flexors (AF), knee flexors (KF), and knee extensors (KE) before MTT and at 3, 6, and 9 mo after MTT. The AF, being distal muscles and less degenerative than the KE and the KF, showed no decrease in mean contractile force 3 mo after MTT, and progressive increases in force at 6 and 9 mo after MTT. At 9 mo after MTT, 60% of the 60 AF examined showed a mean increase of 50% in force; 28% showed no change; and only 12% showed a mean decrease in force of 29% when compared to the function of the same muscles before MTT. The KF, being proximal muscles and more degenerative, showed no change in function at 9 mo after MTT. The KE, being proximal and anti-gravitational, were most degenerative before MTT. They showed no statistically significant change in force at 3 mo after MTT but showed decreases at 6 and 9 mo after MTT. At 9 mo after MTT, 23% of the 60 KE examined showed a mean increase of 65% in force; 22% showed no change; and 55% showed a mean decrease of 24% in force. When results of all muscle groups (AF, KF, KE) were pooled, there was no change in force at 3, 6, or 9 mo after MTT vs. before MTT according to the Wilcoxon signed rank test.(ABSTRACT TRUNCATED AT 400 WORDS)

A randomly selected extensor digitorum brevis (EDB) muscle in each of three Duchenne muscular dystrophy (DMD) boys aged 9 to 10 was injected with approximately 8 x 106 myoblasts. The contralateral EDBs were sham-injected with carrier solution. Donor myoblasts were derived from cell culture of muscle biopsies from the normal ward or normal brothers of the recipients. Cyclosporine (CsA) treatment began two days before myoblast injection and continued for three months. Three days prior to myoblast injection and three months after, the isometric twitch and maximum voluntary contraction of the left and the right EDBs were measured. Myoblast-injected EDBs showed increases in tensions whereas sham-injected EDBs showed reductions. Both immunocytochemical staining and immunoblot revealed dystrophin in the myoblast-injected EDBs. Dystrophic characteristics such as fiber splitting, central nucleation, phagocytic necrosis, variation in fiber shape and size, and infiltration of fat and connective tissues were less frequently observed in these muscles. Sham-injected EDBs exhibited significant structural and functional degeneration and no dystrophin. Throughout the study, there was no sign of erythema, swelling or tenderness at the injection sites. Serial laboratory evaluation including electrolytes, creatinine, and urea did not reveal any significant changes before or after myoblast transfer. We conclude that myoblast transfer therapy is a safe and efficacious procedure to improve the biochemistry, structure, and function of degenerative EDB muscles in DMD.