Muscle-derived stem cells (MDSCs) can differentiate into multiple lineages, including haematopoietic lineages. However, it is unknown whether MDSCs preserve their myogenic potential after differentiation into other lineages. To address this issue, we isolated from dystrophic muscle a population of MDSCs that express stem-cell markers and can differentiate into various lineages. After systemic delivery of three MDSC clones into lethally irradiated mice, we found that differentiation of the donor cells into various lineages of the haematopoietic system resulted in repopulation of the recipients' bone marrow. Donor-derived bone-marrow cells, isolated from these recipients by fluorescence-activated cell sorting (FACS), also repopulated the bone marrow of secondary, lethally irradiated, recipients and differentiated into myogenic cells both in vitro and in vivo in normal mdx mice. These findings demonstrate that MDSC clones retain their myogenic potential after haematopoietic differentiation.
Control of cancer by irradiation therapy alone or in conjunction with combination chemotherapy is often limited by organ specific toxicity. Ionizing irradiation toxicity is initiated by damage to normal tissue near the tumor target and within the transit volume of radiotherapy beams. Irradiation-induced cellular, tissue, and organ damage is mediated by acute effects, which can be dose limiting. A latent period follows recovery from the acute reaction, then chronic irradiation fibrosis (late effects) pose a second cause of organ failure. We have developed the technology for radioprotective gene therapy using the transgene for the antioxidant manganese superoxide dismutase, delivered to specific target organs (lung, esophagus, oral cavity, oropharynx, and bladder) using gene transfer vectors including plasmid/liposomes (PL) and adenovirus. Irradiation protection by MnSOD transgene overexpression at the cellular level has been demonstrated to be localized to the mitochondrial membrane. Using MnSOD transgene constructs lacking the mitochondrial localization leader sequence, and in other experiments attaching this localization signal to otherwise non-radioprotective cytoplasmic Cu/ZnSOD, mitochondrial localization has been demonstrated to be critical to protection. Organ specific injection of MnSOD-PL prior to irradiation demonstrates transgene expression for 48-72 hours, and an associated decrease in ionizing irradiation-induced expression of inflammatory cytokine mRNA and protein. Significant reduction of organ specific tissue injury has been demonstrated in several organ systems in rodent models. Application of MnSOD-PL gene therapy in the setting of fractionated chemo-radiotherapy is being tested in clinical trials for prevention of esophagitis during treatment of non-small cell carcinoma of the lung, and in prevention of mucositis during combination therapy of carcinomas of the head and neck. Encouraging results in pre-clinical models suggest that radioprotective gene therapy may facilitate dose escalation protocols to allow increases in the therapeutic ratio of cancer radiotherapy.
Muscle-based gene therapy using adenovirus, retrovirus, and herpes simplex virus has been hindered by viral cytotoxicity, host immune response, and the maturation-dependent viral transduction of muscle fibers. The development of new mutant vectors has greatly reduced the toxicity and the immune rejection problems, but the inability of viral vectors to penetrate and transduce mature myofibers remains an important issue. Research has been focused on the characterization of barriers to viral transduction in mature myofibers to develop strategies to circumvent the maturation-dependent viral transduction of myofibers. Here, we report that adeno-associated virus (AAV) can be used to overcome the maturation-dependent viral transduction of myofibers. We have investigated by which mechanism AAV can penetrate and efficiently transduce mature muscle fibers, and have shown that this viral vector is not blocked by the basal lamina and that AAV transduction of myofibers is independent of myoblast mediation. Although AAV can efficiently transduce mature myofibers, a differential transduction is still observed among the different types of myofibers that correlates with the expression of the heparan sulfate proteoglycan receptors, the muscle maturity, the number of viral particles used, and the time postinjection. The identification of the mechanisms by which AAV transduces mature myofibers will help in the development of strategies to achieve an efficient muscle-based gene therapy for inherited and acquired diseases.
The mechanisms causing age-dependent loss of muscle fiber infectivity observed in vivo for both adenoviral (Ad) and herpes simplex virus type 1 (HSV-1) gene delivery vectors remain poorly understood. Here we investigate the possible bases for this phenomenon using the novel application of enzymatically isolated, viable, single muscle fibers. We show that maturation-dependent loss of fiber infectivity is recapitulated in single fibers, and, thus, is not solely due to host immune response. Using localized irradiation of muscle in vivo, we show data suggesting that Ad infectivity of differentiated myofibers depends, at least in part, on myoblasts to mediate fiber transduction. On the other hand, infection of single fibers by HSV-1 is not affected by irradiation. Using confocal microscopy, we show that the basal lamina of myogenic cells efficiently infected by HSV-1 is structurally less organized than that of fibers resistant to infection by HSV-1. As well, we show that single myofibers isolated from adult, basal lamina-defective mice (merosin-deficient, dy/dy) are at least 10-fold more susceptible to infection by HSV-1 than are myofibers isolated from control mice. Together, these observations support the hypothesis that the basal lamina acts as a physical barrier to HSV-1 infection of mature muscle.
Duchenne muscular dystrophy (DMD) is an X-linked associated proteins. A greater amount of dystrophin recessive muscle disease characterized by a lack of dysreplacement occurred in mdx muscle following transplantrophin expression. Myoblast transplantation and gene tation of mdx myoblasts isolated from a transgenic mouse therapy have the potential of restoring dystrophin, thus overexpressing dystrophin suggesting that engineering decreasing the muscle weakness associated with this disautologous myoblasts to express high amounts of dystroease. In this study we present data on the myoblast phin might be beneficial. The ex vivo approach possesses mediated ex vivo gene transfer of full-length dystrophin to attributes that make it useful for gene transfer to skeletal mdx (dystrophin deficient) mouse muscle as a model for muscle including: (1) creating a reservoir of myoblasts capautologous myoblast transfer. Both isogenic primary mdx able of regenerating and restoring dystrophin to dystrophic myoblasts and an immortalized mdx cell line were transmuscle; and (2) achieving a higher level of gene transfer duced with an adenoviral vector that has all viral coding to dystrophic muscle compared with adenovirus-mediated sequences deleted and encodes -galactosidase and fulldirect gene delivery. However, as observed in direct gene length dystrophin. Subsequently, these transduced myotransfer studies, the ex vivo approach also triggers a cellublasts were injected into dystrophic mdx muscle, where the lar immune response which limits the duration of transinjected cells restored dystrophin, as well as dystrophingene expression.
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