Disease exacerbation by fibroblast inclusion in Duchenne Muscular Dystrophy MYOrganoids reveals limitations of microdystrophin therapeutic efficacy

Current gene therapy approaches for Duchenne muscular dystrophy (DMD) using AAV-mediated delivery of microdystrophin ({micro}Dys) have shown limited efficacy in patients, contrasting with the favorable outcomes observed in animal models. This discrepancy is partly due to the lack of models that replicate key pathogenic features associated with the severity of the human disease, such as fibrosis and muscle dysfunction. To tackle the translational gap, we develop a human disease model that recapitulates these critical hallmarks of DMD for a more predictive therapeutic investigation. Using a muscle engineering approach, we generate MYOrganoids from iPSC-derived muscle cells co-cultured with fibroblasts that enable functional maturation for muscle force analysis upon contractions. Incorporation of DMD fibroblasts within DMD iPSC-derived muscle cells allows phenotypic exacerbation by unraveling of fibrotic signature and fatiguability through cell-contact-dependent communication. Although {micro}Dys gene transfer partially restores muscle resistance, it fails to fully restore membrane stability and reduce profibrotic signaling. These findings highlight the persistence of fibrotic activity post-gene therapy in our human DMD system, an unparalleled aspect in existing DMD models, and provide the opportunity to explore the underlying mechanisms of dysregulated cellular communication to identify anti-fibrotic strategies empowering gene therapy efficacy.