Efficient Cas9 nuclease-based editing in skeletal muscle via lipid nanoparticle delivery

Gene editing holds great promise for muscular dystrophy treatment, but the rapid evaluation of different editing modalities in skeletal musclein vivoremains challenging due to lack of simple, effective delivery tools. Here we demonstrate that selective organ targeting (SORT) lipid nanoparticles (LNP) encapsulating optimized Cas9 cargo can facilitate efficient, local delivery to skeletal muscles achieving editing rates ≥35% and restore protein expression for a proof-of-concept muscular dystrophy target. Interestingly, efficient editing in skeletal muscle was observed despite a strong adaptive immune response to repeat dosing of the Cas9 LNPs. High efficiency editing mediated by LNP-based delivery of Cas9 to skeletal muscle permitted detailed analysis of insertion and deletion (InDel) outcomesin vivofor a set of potential therapeutic target sites, which differed substantially from InDel outcomes observed in proliferating cells in one specific instance. Overall, our findings on enhanced LNP delivery of Cas9, platform-specific immune responses, and differential editing patterns observed betweenin vitroandin vivomodels provide valuable insights that should inform the development of gene editing therapeutics for neuromuscular diseases.