mRNA-mediated delivery of gene editing tools to human primary muscle stem cells

Muscular dystrophies (MD) are a large group of monogenic muscle wasting disorders. Long-term regeneration of skeletal muscle is only attainable via its stem cell population, the satellite cells. These muscle stem cells (MuSC) can be isolated from human biopsy material and expanded ex vivo. Gene-corrected human primary MuSC are therefore an ideal candidate for autologous cell therapy to treat MD. We harnessed NCAM1 as an endogenous reporter locus to establish highly efficient mRNA-mediated gene editing in human primary MuSC from a wide range of donors. The NCAM1 gene encodes a membrane receptor expressed by all MuSC. We achieved gene editing efficiencies of >90% targeting NCAM1 with both SpCas9 and adenine base editor (ABE) mRNA and suitable sgRNAs. Furthermore, we validated our approach, by correcting the MD-causing mutation SGCA c.157 G>A with >80% efficiency in human carrier-derived MuSC via mRNA-mediated delivery of an ABE. We analysed on-target editing efficiencies of ABE-edited samples and controls using NGS amplicon sequencing.