397. Correction of the Dystrophin Gene By the CRISPR/Cas9 System in a Mouse Model of Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a monogenic and fatal genetic disorder characterized by muscle wasting, loss of ambulation, and death by the age of 20 due to the loss of functional dystrophin – an essential musculoskeletal protein. Gene therapy has held promise for treating monogenetic diseases, however, delivery barriers and the requirements for costly repetitive treatments are a few of the challenges of technologies in the therapeutic pipeline. Recently, the CRISPR/Cas9 system has been adapted for genome editing to make specific modifications in the host genome. This technology would enable permanent genome corrections and a potentially curative approach to DMD.Previously we adapted CRISPR/Cas9 for targeted deletions of regions of the human dystrophin gene that restore expression of a functional protein (Ousterout et al., Nature Communications (in press)). The delivery of Cas9 with single guide RNAs (sgRNAs) targeting the introns surrounding exons 51 and 45-55 created double strand breaks which are repaired through non-homologous end joining to create genomic deletions of these regions. These deletions restored the reading frame and recovered dystrophin expression in cultured patient-derived myoblasts. Therapeutically, this approach could be used to convert the fatal DMD phenotype to the milder phenotype associated with partial dystrophin function characteristic of Becker muscular dystrophy.Here we utilize adeno-associated virus (AAV) to deliver the CRISPR/Cas9 system to the mdx mouse model of DMD. AAV vectors were designed to package and deliver genes encoding the Staphylococcus aureus Cas9 (SaCas9), due to its smaller size (3.2 kb) compared to the widely used Cas9 from Streptococcus pyogenes. Two single guide RNAs (sgRNAs) were designed to target sites flanking exon 23 of the dystrophin gene and validated to direct SaCas9-mediated deletion of this exon, which contains a premature stop codon in this model. Mice were anesthetized and AAV1 containing the SaCas9 and sgRNA expression cassettes was injected into the gastrocnemius muscle of 8 week old mice. At 4 and 8 weeks post-injection, the gastrocnemius was harvested and genomic DNA and mRNA were extracted. PCR of the genomic DNA demonstrated the expected ~1200 bp deletion of exon 23. Further, RT-PCR of the extracted mRNA showed removal of the 216 bp encoding exon 23. Furthermore, sgRNAs compatible with SaCas9 have been developed and validated for the deletion of human exon 51.This work demonstrates proof-of-principle of CRISPR/Cas9-mediated genome editing in skeletal muscle in an adult mammal, opening up new possibilities for gene therapy and the study of gene function. Optimization of delivery and activity is underway to improve the therapeutic potential of this approach. Additional work to characterize functional improvement in the mouse hind limb is also ongoing. This work establishes CRISPR/Cas9-mediated genome editing as a potential therapeutic approach to DMD.