Generation of a de novo intronic junction in the DMD gene through CRISPR/Cas genome editing as a potential therapy for a high proportion of Duchenne Muscular Dystrophy patients.

Duchenne Muscular Dystrophy is caused by mutations across the DMD gene, leading to absence of dystrophin protein and giving rise to progressive muscle wasting. Different gene therapies are being investigated, such as AAV microdystrophin delivery, premature termination codon read-through and exon-skipping. Nevertheless, these therapies require repeated administration, could carry an adverse immunological risk and some are restricted by mutation specificity. Such problems may be circumvented with genome editing. We aim to express an internally truncated functional dystrophin from the endogenous DMD locus using a SaCas9 CRISPR system. Our gRNAs would delete exons 19 to 55 by creating a de novo junction between introns 18-55 and producing a ~800 kbp deletion. This strategy would eliminate ~81% of total DMD mutations. As a positive control, a cDNA construct expressing D19-55 dystrophin was generated and tested in mdx mice. This construct significantly increased dystrophin positive fibres by plasmid intramuscular injection, indicating that D19-55 dystrophin is expressible and has potential for beneficial effects when expressed in sufficient levels. To produce the deletion by genome editing, optimal gRNAs for each intronic site in murine Dmd were multiplexed into an SaCas9 construct and tested in Neuro2A and C2C12 cells. Deletion of exons 19-55 was confirmed by PCR and Sanger Sequencing. Multiplexed constructs were packaged into AAV9 and assessed in-vivo by local injection into 2-months old mdx mice. Functional efficacy was assessed by muscle electrophysiology. However, no beneficial effects were seen likely due to inability to detect a deletion in treated muscle samples with the employed techniques.