Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD

Duchenne muscular dystrophy (DMD) is the most prevalent inherited myopathy affecting children, caused by genetic loss of the gene encoding the dystrophin protein. There are currently four FDA-approved drugs for DMD that aim to restore expression of dystrophin by exon skipping using splice switching oligonucleotides. While these therapies require lifelong repeat administration, recent advancements in gene editing technologies have raised the possibility of achieving permanent exon skipping, and thereby curing the disease with a single treatment. Here we have investigated the use of the Staphylococcus aureus CRISPR/Cas9 system and a double-cut strategy, delivered using a pair of AAV9 vectors, for dystrophin restoration in the severely-affected dystrophin/utrophin double knock-out (dKO) mouse. Single guide RNAs were designed to induce double-strand DNA breaks on either side of Dmd exon 23, such that the intervening exon 23 sequence is excised when the flanking intronic regions are joined via the non-homologous end joining repair pathway. Exon 23 deletion was confirmed at the DNA level by PCR and Sanger sequencing, and at the RNA level by RT-qPCR. Restoration of dystrophin protein expression was demonstrated by western blot and immunofluorescence staining in mice treated via either intraperitoneal or intravenous routes of delivery. Dystrophin restoration was most effective in the diaphragm, where a maximum of 5.7% of wild-type dystrophin expression was observed. CRISPR treatment was insufficient to extend lifespan in the dKO mouse, and dystrophin was expressed in a within-fiber patchy manner in skeletal muscle tissues. Further analysis revealed a plethora of non-productive DNA repair events, including AAV genome integration at the CRISPR cut sites. This study highlights potential challenges for the successful development of CRISPR therapies in the context of DMD.