570. Efficient In Vitro Expansion and Gene Correction of Dystrophic Mouse Muscle Stem Cells

Abstract

Duchenne Muscular Dystrophy (DMD) is a progressive muscle wasting disease caused by mutations in the DYSTROPHIN gene. Gene correction and autologous transplantation of muscle stem cells (i.e., satellite cells) presents a promising therapeutic approach for restoring DYSTROPHIN expression to muscle fibers in these patients; however the low frequency of satellite cells in adult muscle is an obstacle for isolating sufficient numbers of cells for engraftment. To address this issue, we have sought culture conditions that would support the ex-vivo expansion of satellite cells and potentially provide more cells for transplantation. We found that forskolin, an adenylyl cyclase activator, dramatically expanded mouse satellite cells in culture. Forskolin-treated cultured cells retained the immunophenotypic characteristics of engraftable satellite cells, and transplantation of compound-treated cells into dystrophic muscle yielded a significantly higher level of engraftment compared to control cells. Forskolin also dramatically expands satellite cells from mdx mouse model of DMD in culture. As a proof of concept for a combined gene and cell therapy approach for treating DMD, we targeted the mutated Dystrophin locus in ex-vivo expanded forskolin treated dystrophic satellite cells using CRISPR/Cas9 gene editing technology and enriched for the targeted cells using an endogenous fluorescent reporter system. Expansion and gene correction of muscle satellite cells in culture as described here provides the possibility of improving combined gene and cell-based therapies for neuromuscular disorders.