tacrolimus and Muscular-Dystrophy--Duchenne

A clinical trial was conducted to test a new protocol of normal muscle precursor cell (MPC) allotransplantation in skeletal muscles of patients with Duchenne muscular dystrophy (DMD). Cultured MPCs obtained from one of the patient's parents were implanted in 0.25 or 1 cm of a Tibialis anterior in 9 patients with DMD. MPC injections were placed 1 to 2 mm from each other, and a similar pattern of saline injections was done in the contralateral muscle. The patients were immunosuppressed with tacrolimus. Muscle biopsies were performed at the injected sites 4 weeks later. In the biopsies of the cell-grafted sites, there were myofibers expressing donor's dystrophin in 8 patients. The percentage of myofibers expressing donor's dystrophin varied from 3.5% to 26%. Evidence of small myofiber neoformation was observed in some patients. Donor-derived dystrophin transcripts were detected by reverse transcriptase-polymerase chain reaction in the cell-grafted sites in all patients. The protocol of immunosuppression was sufficient to obtain these results, although it is not certain whether acute rejection was efficiently controlled in all the cases. In conclusion, intramuscular allotransplantation of normal MPCs can induce the expression of donor-derived dystrophin in skeletal muscles of patients with DMD, although this expression is restricted to the sites of MPC injection.

Topics: Adolescent; Animals; Child; Dystrophin; Fluorescent Antibody Technique; Graft Rejection; Histocompatibility Antigens; Humans; Image Processing, Computer-Assisted; Immunosuppressive Agents; In Situ Hybridization, Fluorescence; Mice; Mice, SCID; Muscle Cells; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Reverse Transcriptase Polymerase Chain Reaction; Stem Cell Transplantation; Tacrolimus

Topics: Animals; Apoptosis; Cell Differentiation; Cell Transplantation; Clinical Trials as Topic; Genetic Therapy; Graft Rejection; Humans; Muscular Dystrophy, Duchenne; Myoblasts; Tacrolimus; Treatment Outcome

Three Duchenne muscular dystrophy (DMD) patients received injections of myogenic cells obtained from skeletal muscle biopsies of normal donors. The cells (30 x 10 (6)) were injected in 1 cm3 of the tibialis anterior by 25 parallel injections. We performed similar patterns of saline injections in the contralateral muscles as controls. The patients received tacrolimus for immunosuppression. Muscle biopsies were performed at the injected sites 4 weeks later. We observed dystrophin-positive myofibers in the cell-grafted sites amounting to 9 (patient 1), 6.8 (patient 2), and 11% (patient 3). Since patients 1 and 2 had identified dystrophin-gene deletions these results were obtained using monoclonal antibodies specific to epitopes coded by the deleted exons. Donor dystrophin was absent in the control sites. Patient 3 had exon duplication and thus specific donor-dystrophin detection was not possible. However, there were fourfold more dystrophin-positive myofibers in the cell-grafted than in the control site. Donor-dystrophin transcripts were detected by RT-PCR (using primers reacting with a sequence int eh deleted exons) only in the cell-grafted sites in patients 1 and 2. Dystrophin transcripts were more abundant in the cell-grafted than in the control site in patient 3. Therefore, significant dystrophin expression can be obtained in teh skeletal muscles of DMD patients following specific conditions of cell delivery and immunosuppression.

Topics: Adolescent; Antibodies, Monoclonal; Biopsy; Cell Transplantation; Child; Cytoskeletal Proteins; DNA Primers; Dystrophin; Epitopes; Genetic Therapy; Haplotypes; Histocompatibility Testing; Humans; Immunohistochemistry; Immunosuppressive Agents; Membrane Glycoproteins; Microscopy, Fluorescence; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Mutation; Reverse Transcriptase Polymerase Chain Reaction; Sarcoglycans; Tacrolimus

The goal of the present study was to determine the feasibility, success, and toxicity of myoblast transplantation (MT) in the whole muscle of primates. Allogenic myoblasts transduced with the beta-galactosidase (beta-Gal) gene were transplanted in the whole Biceps brachii of 5 monkeys immunosuppressed with FK506. Myoblast injections were spaced at every 1 to 1.5 mm in 7 muscles, as well as at every 5 mm in 2 muscles. Myoblasts were resuspended in HBSS, notexin 1 microg/ml or notexin 5 microg/ml. Depending on the number of beta-Gal labeled myoblasts and the injection protocol, biopsies of transplanted muscles exhibited 7% to 74% beta-Gal+ fibers 1 month after MT. Beta-Gal+ fibers were present in muscle biopsies made 3, 8, and 12 months after MT. Myoglobinuria and hyperkalemia, the risk factors after extensive muscle damage and notexin toxicity, were not observed. The withdrawal of immunosuppression led to histological evidences of cellular rejection of the graft. We concluded that MT can be successfully performed in large primate muscles without toxicity due to muscle damage. An effective immunosuppression allowed the maintenance of beta-Gal+ fibers up to 1 year after MT. These results suggest parameters that may allow effective MT in humans.

Topics: Animals; beta-Galactosidase; Biopsy; Cell Transplantation; Cells, Cultured; Elapid Venoms; Genes, Reporter; Graft Rejection; Immunosuppressive Agents; Macaca mulatta; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Tacrolimus