Tubulyzine, a novel tri-substituted triazine, prevents the early cell death of transplanted myogenic cells and improves transplantation success.

Myoblast transplantation (MT) is a potential therapeutic approach for several muscular dystrophies. A major limiting factor is that only a low percentage of the transplanted myoblasts survives the procedure. Recent advances regarding how and when the myoblasts die indicate that events preceding actual tissue implantation and during the first days after the transplantation are crucial. Myoseverin, a recently identified tri-substituted purine, was shown to induce in vitro the fission of multinucleated myotubes and affect the expression of a variety of growth factors, and immunomodulation, extracellular matrix-remodeling, and stress response genes. Since the effects of myoseverin are consistent with the activation of pathways involved in wound healing and tissue regeneration, we have investigated whether pretreatment and co-injection of myoblasts with Tubulyzine (microtubule lysing triazine), an optimized myoseverin-like molecule recently identified from a triazine library, could reduce myoblast cell death following their transplantation and consequently improves the success of myoblast transplantation. In vitro, using annexin-V labeling, we showed that Tubulyzine (5 microM) prevents normal myoblasts from apoptosis induced by staurosporine (1 microM). In vivo, the pretreatment and co-injection of immortal and normal myoblasts with Tubulyzine reduced significantly cell death (assessed by the radio-labeled thymidine of donor DNA) and increased survival of myoblasts transplanted in Tibialis anterior (TA) muscles of mdx mice, thus giving rise to more hybrid myofibers compared to transplanted untreated cells. Our results suggest that Tubulyzine can be used as an in vivo survival factor to improve the myoblast-mediated gene transfer approach.

Systemic production of human granulocyte colony-stimulating factor in nonhuman primates by transplantation of genetically modified myoblasts.

Clinical use of human granulocyte-colony stimulating factor (hG-CSF) to treat various diseases involving neutropenia has been previously shown to (1) successfully increase circulating neutrophils, (2) reduce condition-related infections, and (3) cause few side effects in patients. To alleviate the symptoms of neutropenia, the patient must receive frequent injections of recombinant hG-CSF. Permanent ways to deliver stable levels of the molecule to the patient are being investigated. Among them, the transplantation of hG-CSF-secreting cells has been proposed and performed successfully in rodents, using fibroblast cell lines and primary muscle cells. We thus investigated whether similar results could be obtained by intramuscular myoblast transplantation in a large animal model. When 1-3 x 10(8) myoblasts were injected into three Macaca mulatta, hG-CSF was detected at high levels (300-900 pg/ml), which in turn led to a four- to fivefold increase in circulating neutrophils. However, both the concentrations of hG-CSF and neutrophil levels were found to decrease over time. Nonetheless, neutrophils were found at higher levels from the fourth week until the end the experiment (up to 29 weeks) in G-CSF monkeys compared with control animals. These results show that transplantation of hG-CSF-secreting myoblasts may indeed be a therapeutic option for the treatment of neutropenic patients.

Myoblast transplantation in whole muscle of nonhuman primates.

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.

Progress in myoblast transplantation: a potential treatment of dystrophies.

Myoblast transplantation (MT) consists of injecting normal or genetically modified myogenic cells into muscles, where they are expected to fuse and form mature fibers. As an experimental approach to treat severe genetic muscle diseases, MT was tested in dystrophic patients at the beginning of the 1990s. Although these early clinical trials were unsuccessful, MT has progressed through the research on animal models. Many factors that may condition the success of MT were identified in the last years. The present review updates our knowledge on MT and describes the different problems that have limited its success. Factors that were first underestimated, like the specific immune response after MT, are presently well characterized. Destruction of the hybrid fibers by activated T-lymphocytes and production of antibodies against the transplanted myoblasts take place after MT and are responsible for the graft rejection. The choice of the immunosuppression seems to be very important, and FK506 is the best agent known to allow the best results after MT. Under FK506 immunosuppression, very efficient MT were obtained both in mice and monkeys. Moreover, in dystrophic mice it was demonstrated that MT ameliorates some phenotypical characteristics of the disease. The improvement of the survival of the transplanted cells and the increase of their migration into the injected tissue are presently under investigation. Some of the present research is directed also to bypass the immunosuppression by using the patient's own cells for MT. In this sense, efforts are conducted to introduce the normal gene into the patient's myoblasts before MT and to improve the ability of these cells to proliferate in vitro. Micros. Res. Tech. 48:213-222, 2000.

Transplantation of human myoblasts in SCID mice as a potential muscular model for myotonic dystrophy.

Myotonic dystrophy (DM), the most frequent hereditary myopathy in adults, is characterized clinically by muscle weakness, myotonia, and systemic symptoms. Although the specific genetic basis for DM has been established, less is known about the cellular defects responsible for its pleiotropic manifestations. DM pathogenesis studies are presently limited due to the absence of animal models. In the present study, we transplanted myoblasts of DM patients into the Tibialis anterior of Severe Combined Immunodeficient (SCID) mice to determine whether this approach could reproduce the muscular characteristics of DM. One to 4 months after transplantation, a variable number of innervated human muscle fibers, recognized by an antibody specific for the human dystrophin, were found in the transplanted muscles. The CTG expansion was retained in human muscle fibers as determined by Southern blot analysis. Although the histological characteristics of DM were absent in these fibers, electromyographic recording showed typical myotonic discharges in muscles transplanted with DM myoblasts. The specificity of the myotonic runs was demonstrated by its inhibition by apamin, a drug that specifically blocks DM myotonia. We conclude that transplantation of myoblasts from DM patients into SCID mice represents a potential in vivo model for basic studies of this disease.

Successful myoblast transplantation in primates depends on appropriate cell delivery and induction of regeneration in the host muscle.

Myoblast transplantation (MT) may be a potential treatment for severe recessive hereditary myopathies. The limited results of MT in clinical trials led us to improve this technique in monkeys, an animal model phylogenetically similar to humans. Three Macaca mulata monkeys were used as donors and six as receivers for MT. Myoblasts were grown in culture from muscle biopsies of adult monkeys and infected with a retroviral vector encoding the LacZ gene. Different numbers of cells (i.e., 4 x 10(6), 8 x 10(6), and 24 x 10(6) cells) were transplanted into different muscles and 8 x 10(6) cells (resuspended in a notexin solution) were injected in one muscle of four monkeys. For these transplantations, the cell suspension (in a volume of about 100 microl) was injected at 35 sites less than 1 mm apart. Two other monkeys received 100 x 10(6) myoblasts resuspended in 1 ml of HBSS or 1 ml of notexin. For these two monkeys, the myoblasts were injected at 200-250 sites within a small portion of the muscle. All monkeys were immunosuppressed with daily injections of FK506. Four weeks after MT, the transplanted muscle portions were biopsied and the presence of beta-galactosidase-positive (beta-Gal+) muscle fibers was investigated. The number of beta-Gal+ fibers was 822 +/- 150 (site grafted with 4 x 10(6) cells), 1253 +/- 515 (8 x 10(6) cells), 1084 +/- 278 (24 x 10(6)), and 2852 +/- 1211 (notexin). In the monkeys grafted with 100 x 10(6) myoblasts, the number of beta-Gal+ fibers was 4850 (site without notexin) and 9600 (site with notexin). We demonstrated that a precise mechanical distribution of myoblasts into the tissue improves substantially MT in primates. The presence of notexin with the transplanted cells further increased the success of their transplantation. These are the best results obtained either with MT or gene therapy in primates and they encourage the possibility to human MT trials.

Complement deposition and cell death after myoblast transplantation.

One of the problems limiting myoblast transplantation (MT) is the early death of the transplanted cells. Because complement can be fixed by myoblasts in vitro, and because it has the capacity to induce cell lysis, its possible role in the early death of transplanted myoblasts was investigated. CD1 mice and Macaca mulata monkeys were used as recipients for MT. In some mice, C3 was depleted before MT using Cobra Venom Factor. Mice were sacrificed during the first hour and up to 3 days after MT. Monkeys were biopsied 1 to 4 h after MT. Myoblast necrosis was assessed by the presence of intracellular calcium. Complement deposition was demonstrated by immunohistochemistry with anti-C3 and anti-C5b-9 neoantigen antibodies. In mice, C3 deposition was observed in damaged muscle fibers and in regions containing necrosed myoblasts. Complement depletion did not diminish the proportion of necrosed cells. In monkeys, only a small percentage of transplanted myoblasts showed C3 or C5b-9 deposition, mostly intracellular. Complement activation seems not to be implicated in directly damaging the transplanted cells, but seems secondary to cellular death. Taking into account its chemotactic functions, complement could be implicated in the migration of neutrophils and macrophages into the clusters of transplanted cells.

Myoblast transplantation in non-dystrophic dog.

Dog myoblasts obtained from muscle biopsies were infected in vitro with a defective retroviral vector containing a cytoplasmic beta-galactosidase (beta-Gal) gene. These myoblasts were initially transplanted in the irradiated muscles of SCID mice and beta-Gal positive muscle fibers were observed. beta-Gal myoblasts were also transplanted back either in the donor dogs (autotransplantation model) or in unrelated recipient dogs (allotransplantation model). Following these myoblast injections, a rapid inflammatory reaction developed within the muscle as indicated by an expression of P-selectin and of pro-inflammatory cytokine mRNAs (interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta), and by a neutrophil infiltration. Following either auto- or allotransplantation in inadequately or non-immunosuppressed dogs, a specific immune reaction also developed within 2 weeks as indicated by the infiltration of CD4+ and of CD8+ lymphocytes, the increased expression of IL-10 and granzyme B mRNAs and the presence of antibodies reacting with the injected cells. Some dogs were immunosuppressed with several combinations of FK506, cyclosporine (CsA) and RS-61443. In dogs immunosuppressed with CsA combined with RS-61443, only a few myoblasts and myotubes expressing beta-Gal were observed 1-2 weeks after the transplantation, but no muscle fibers expressing beta-Gal were observed after 4 weeks, and antibodies against the injected cells were formed. In dogs immunosuppressed with FK506 alone, although no antibodies against the injected cells were produced, there were no small cells and no muscle fibers expressing beta-Gal 1 month after the transplantation. However, FK506 triggered diarrhea and vomiting in dogs. When the dogs were immunosuppressed with FK506 combined with CsA and RS-61443, muscle fibers expressing beta-Gal were present 4 weeks after the transplantation and no antibodies reacting with donor myoblasts were detected. These results indicate that the combination of three immunosuppressive agents (i.e., FK506, CsA and RS-61443) is effective in controlling the specific immune reactions following myoblast transplantation in dogs and they underline that the outcome of myoblast transplantation is dependent in part on an adequate immunosuppression. These results obtained here in normal dogs may justify myoblast transplantation in dystrophic dogs despite the side effects of FK506.

Successful transplantation of genetically corrected DMD myoblasts following ex vivo transduction with the dystrophin minigene.

Myoblast transplantation and gene therapy are two promising therapeutical approaches for the treatment of Duchenne Muscular Dystrophy (DMD). So far, both strategies have met many hurdles, mainly because of immune reactions. In this study, we investigated a third and novel strategy based on the combination of these two basic ones, i.e., transplantation of genetically modified myoblasts. We first derived a primary culture from a muscle biopsy of a young DMD patient (3 years old). Adenoviral-mediated dystrophin gene transfer into these DMD cultures and expression of the dystrophin transgene were achieved in vitro. The transduced cultures were then transplanted the same day in immunodeficient SCID mouse muscles. Three weeks following the graft, many human dystrophin-positive fibers were observed throughout sections of the injected muscles. However, many fibers expressed human MHC antigens without expressing human dystrophin due to the low percentage of infected primary muscle cells in vitro (even when a high MOI [400] was used) and to a reduction and even to a complete loss of transgene copy number during myoblast replication. From our results, we conclude that, although not at a high proportion, (1) DMD primary myoblast cultures are infectable by adenoviruses; (2) they can be efficiently transplanted back in a muscle, leading to normal fusion of infected myoblasts with the host fibers; and (3) they can correct the dystrophin deficiency in the host fibers by the expression of a mini-dystrophin transgene.

Oculopharyngeal muscular dystrophy in Uruguay.

Within the last 30 years, sixty-five patients exhibiting the clinical symptoms of oculopharyngeal muscular dystrophy (OPMD) were studied at the Neuromuscular Diseases Unit of the Neurological Institute of Montevideo. They are members of five unrelated families which came from the Canary Islands to Uruguay between 1850 and 1900. In the three families examined, the typical inclusions characteristic of OPMD were found in the nuclei of muscle fibers. Treatment for ptosis and dysphagia was discussed. The particular migratory pattern of this group of patients could be of considerable interest in the study of molecular genetics.

Prevention by anti-LFA-1 of acute myoblast death following transplantation.

Myoblast transplantation is a potential treatment for Duchenne muscular dystrophy. One of the problems possibly responsible for the limited success of clinical trials is the rapid death of the myoblasts after transplantation. To investigate this problem, myoblasts expressing beta-galactosidase were injected in the tibialis anterior muscles of mice. Beta-galactosidase activity was reduced by 74.7% after 3 days. Myoblast death observed at 3 days was reduced to 57.2% when the hosts were irradiated. This result suggested that host cells were contributing to this phenomenon. Transplantation in SCID and FK506-treated mice did not reduce cell death, indicating that mortality was not due to an acute specific reaction. In contrast, administration of the anti-LFA-1 (TIB-213) mAb markedly reduced myoblast death at 3 days without altering leukocyte tissue infiltration. We postulated that neutrophils were mediating myoblast mortality by an LFA-1-dependent mechanism. To test this hypothesis, IL-1beta-activated myoblasts were loaded with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethylester) (DCFH), a marker for oxidative stress. Addition of neutrophils and zymosan-activated serum resulted in a time-dependent DCFH fluorescence; this neutrophil-induced oxidation was considerably inhibited by TIB-213. These results indicate that an effective control of the inflammatory reaction will be necessary for any new clinical trials of myoblast transplantation and suggest that neutrophil-mediated myoblast injury occurs by an LFA-1-dependent pathway.

Control of inflammatory damage by anti-LFA-1: increase success of myoblast transplantation.

Myoblast transplantation is a potential treatment for Duchenne Muscular Dystrophy. This article confirms by experiments in mice that one problem that has limited the success of clinical trials of this procedure is a rapid (within 3 days) inflammatory reaction which kills most of the injected myoblasts. The death of the transplanted myoblasts can be prevented by treating the host with a mAb against LFA-1. This led to a 27-fold increase in the number of muscle fibers expressing a reporter gene present in the donor myoblasts when the host is also adequately immunosuppressed with FK506. Therefore, both the nonspecific inflammatory reaction and the specific immune response should be adequately controlled following myoblast transplantation.