STEM CELLS FOR DMD · Novel strategies for the cell therapy of muscular dystrophies

This project aims to develop an effective cell therapy for Duchenne Muscular Dystrophy (DMD). To reach this goal we plan to transfer a human artificial chromosome containing the whole dystrophin locus into dystrophic mesoangioblasts. As the transfer is inefficient and requires selection, it is impossible to use primary cells that would undergo senescence during selection. Therefore we need to reversibly immortalize DMD patient mesoangioblasts by the combined use of lentiviral vectors expressing either floxed human telomerase (h-Tert) or floxed Bmi1 (and also the Herpes Simplex Thymidine Kinase, HSTK). Immortal clones, selected for proper expression of h-Tert and Bmi1 and maintenance of a mesoangioblast phenotype, will be transfected with a human artificial chromosome expressing human dystrophin. Expressing clones will be selected and characterized. Before transplantation, selected clones will be infected with an adenoviral vector expressing the Cre recombinase and then treated with Gancyclovir. Transduced and selected cells will be transplanted into mdx/SCID (dystrophic and immune deficient) mice and the extent of dystrophin reconstitution and consequent functional benefit will be evaluated. In addition, the possible immune reaction to the transgenes or novel antigens generated by the experimental strategy will be monitored by an in vitro assay utilizing donor s dendritic cells and T lymphocytes. Finally this strategy will be repeated in Golden Retriever dystrophic dogs, the closest animal model to DMD. Dog mesoangioblasts will be reversibly immortalized, transduced with the dystrophin HAC and, after viral excision and GC selection, transplanted in the same dogs from which they had been directly isolated. Dystrophin expression, functional amelioration and possible toxic and immune reactions will be monitored. This project will therefore cover all pre-clinical experimentation and would set the stage for immediate clinical translation.