Exploring Therapies for Duchenne Muscular Dystrophy Using Transdifferentiated Patient Fibroblasts

Duchenne muscular dystrophy (DMD) is caused by a wide variety of mutations that disrupt the reading frame of the DMD gene, leading to the absence of dystrophin. Several therapies have been explored, but, to date, there is no curative treatment for this disease. One reason for this is the lack of good models to test personalized therapy, as DMD is caused by more than 8558 different mutations. Although several DMD mouse models have been created, with some carrying the human dystrophin gene, it is practically impossible to generate a mouse model for each unique mutation identified in humans. Thus, patient-derived cell lines are the best option to study the impact of specific mutations and to screen potential therapies. While primary myoblasts derived from muscle biopsies are the most relevant model, they have limited proliferative capacity. To overcome this limitation, the immortalization of human primary myoblasts has been explored as an alternative. However, acquisition of the cells remains dependent on invasive muscle biopsies. In contrast, skin biopsies offer a less invasive and more accessible option. By immortalizing and transdifferentiating fibroblasts derived from skin biopsies into myoblasts, it is possible to establish a cell source with robust myogenic potential. This protocol describes a rapid and direct method for transdifferentiating fibroblasts into a myogenic lineage. The process involves transduction with two lentiviruses: one carrying hTERT for primary culture immortalization and another with a tetracycline-inducible MyoD. Upon the addition of doxycycline, MyoD expression induces the conversion of fibroblasts into myoblasts and subsequently mature myotubes expressing DMD mRNA and late differentiation markers, including dystrophin. This efficient transdifferentiation protocol serves as a valuable tool for investigating the effect of mutations in the DMD gene and exploring innovative gene-based or pharmacological biotherapies for DMD and other neuromuscular disorders.