Rescue of human dystrophin after transplantation of exon-skipping-engineered DMD stem cells in a dystrophic animal model

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by genomic mutations that disrupt the dystrophin mRNA reading frame. This destabilizes the dystrophin and its associated complex proteins, provoking progressive and irreversible muscle degeneration. In some cases, forced exclusion (skipping) of a single or multiple exons can restore the reading frame, giving rise to a shorter, but still functional protein. Most of DMD mutations are localized into the central rod domain of the dystrophin gene; for this particular reason, this protein is well adapted for exon-skipping application since in frame removing of central spectrin-like repeats, was demonstrated to conserve its functionality. In a cell therapy perspective, exon skipping approach was used to treat a subpopulation of adult stem cells extracted from DMD patients. We previously shown that one population of human stem cells, harbouring the CD133 surface antigen, was able to efficiently participate in muscle regeneration in vivo. To extend this work, we evaluated the muscle regeneration potentiality of blood and muscle-derived CD133+ cells after in vitro exon skipping treatment. Lentiviral vectors were constructed to convey specific antisense oligonucleotides able to induce an efficient exon-skipping and to correct the initial frameshift caused by the DMD deletion. In our case, DMD cells yielding deletion of exons 49 and 50 were treated with vectors able to perform skipping of the exon 51, rendering in frame the dystrophin mRNA sequence. The skipped blood and muscle-derived stem cells were able to fuse in vivo with scid/mdx mice regenerative fibers and, not only perform expression of a functional human dystrophin, but also restructure the dystrophin-associated complex such as alpha and beta-sarcoglycans proteins. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, preliminary genetically modified to re-express a functional dystrophin, seems to represent a promising approach for DMD