P03

Gene therapy is a promising approach to treat genetic diseases such as congenital myopathies. Gene replacement strategies have already been developed for other myopathies like Spinal Muscular Atrophy (SMA) and Duchene Muscular Dystrophy (DMD). For diseases associated with mutations in the RYR1 gene (RYR1-related myopathies), the size of the gene limits its insertion into a therapeutic vector. Therefore, gene editing seems to be the ideal strategy and especially with CRISPR/Cas9. Current methods to introduce gene editing tools into muscle cells use viral vectors, which are largely effective but are limited by prolonged Cas9 expression, increasing the risk of off-target cleavage. On the other hand, non-viral techniques are increasingly being developed to safely transduce target cells, but they have not been extensively studied in muscle cells. The aim of our study is to compare four methods of Cas9-gRNA delivery in human immortalized muscle cells : lentiviral vector-based transfer of Cas9 and gRNA, transduction with muscle-specific AAV vectors, electroporation of Cas9-gRNA ribonucleoproteins (RNPs) and nanoblades, a recently developed non-viral approach based on virus-like particles. The latter is a good candidate to combine the efficiency of viral transduction with the absence of DNA transfer into the host cell. This study will allow the development of therapeutic approaches for RYR1-related myopathies by using the most relevant and efficient strategy for Cas9 delivery.