561. CRISPR-Induced Deletion (CinDel) Method Allows Permanent and Efficient Restoration of the DMD Gene Reading Frame in Duchenne Patient Myoblasts and Preserves Truncated Dystrophin Structure

The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne Muscular Dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. Here we present CRISPR-induced deletion (CinDel), a new gene therapy approach to correct the DMD gene in Duchenne patients with one or more exons deletions. By using adequate pair of gRNAs targeting specifically the exons precede and follow the patient deletion in DMD gene, CinDel induces precise DSB in targeted sequences and allows an additional deletion. The remaining parts of the exons were fusioned by NHEJ to form a hybrid exon and restored the DMD reading frame in 62 % of hybrid exons in vitro in patient myoblasts and in vivo in electroporated muscle hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted dystrophin protein was detected following the formation of myotubes by the unselected treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.