Generation, characterization and genetic engineering of myogenic cells to restore dystrophin expression in a mouse model of Duchenne muscular dystrophyLenardič, Ajda (Avtor) Župunski, Vera (Mentor) Duchenne muscular dystrophycell therapyinduced myogenic progenitor cellsinduced pluripotent stem cellsgene editingDuchenne muscular dystrophy is the most severe form of muscular dystrophy. It is caused by frameshift mutations in dystrophin gene, which lead to the production of truncated, non-functional dystrophin protein. Treatment options for Duchenne muscular dystrophy are very limited and there is currently no cure. Cell therapies have great potential to treat the disease, but so far no efficient cell therapy has been developed. To contribute to overcoming the challenges that prevented previous attempts to develop such therapy from succeeding, we explored the possibilities of utilizing reprogramming and transdifferentiation strategies, combined with gene editing techniques, to prepare the cells that could restore dystrophin expression in two mouse models of Duchenne muscular dystrophy. The overall goal of our work was to generate high amount of genetically corrected myogenic, dystrophic mouse-derived cells that could be expanded extensively and would engraft well upon transplantation (back) to the dystrophic mouse. To this end, we utilized two different approaches for cell preparation. In the in vitro approach, we reprogrammed mouse embryonic fibroblasts, isolated from dystrophic mouse model, into induced myogenic progenitor cells. We then reversed the mutation in dystrophin gene utilizing CRISPR/Cas9 system and transplanted these genetically edited cells to the limb muscles of immunocompromised dystrophic mice. Despite successful gene editing and restoration of dystrophin expression in vitro, we were not able to detect any fibers with the restored dystrophin expression in vivo. In the in vivo approach, we reprogrammed dystrophic mouse-derived embryonic fibroblasts into induced pluripotent stem cells, corrected the mutation, and injected these cells into mouse blastocysts carrying two transgenes that allowed the ablation of blastocyst-derived satellite cells by administration of tamoxifen. Upon development of the injected blastocysts in the foster mother, newborn pups were injected with tamoxifen to ablate blastocyst-derived satellite cells and hence provide a niche for satellite cells derived from the injected induced pluripotent stem cells to proliferate. Based on the analysis of satellite cells, harvested from these mice, we concluded that contribution of induced pluripotent stem cells to the satellite cell niche in chimeric pups was too high to determine whether tamoxifen injections efficiently ablated blastocyst-derived satellite cells. However, high contribution of induced pluripotent stem cells to the satellite cell niche suggests that the amount of satellite cells obtained from the muscles of chimeric mice would likely be sufficient for efficient transplantation to dystrophic mouse regardless of tamoxifen administration. All in all, our work lays a good foundation for the potential development of cell therapy, based on the production of patient-derived, genetically edited myogenic cells in vitro or patient-derived, genetically edited satellite cells in animals such as pigs.20212022-02-18 14:18:33Magistrsko delo/naloga135055VisID: 18542COBISS_ID: 90914051 sl