735. Functional Restoration of Dystrophin Protein in HiPSC-Derived Skeletal Myotubes and Cardiomyocytes After CRISPR/Cas9-Mediated Deletion of 530-725kb of DMD

Abstract

Duchenne muscular dystrophy (DMD) is typically due to frameshifting mutations in the DMD gene encoding dystrophin. Loss of dystrophin protein results in progressive muscle degeneration and premature death. Approximately 60% of DMD patients have frameshifting mutations in a hotspot region within exons 45-55 in the rod domain of dystrophin. Genotype/phenotype assessments have revealed that in-frame deletion of exons 45-55 leads to the milder, allelic disease, Becker muscular dystrophy. This finding suggests that restoration of the reading frame by targeting exons 45-55 could treat ~60% of DMD patients to greatly reduce disease severity. We have developed a platform using clustered regularly interspaced short palindromic repeats (CRISPR) and- associated protein (Cas9) gene editing to achieve this purpose. We have utilized CRISPR/Cas9-mediated deletion and rejoining of up to 725kb to restore the reading frame in DMD human induced pluripotent stem cells (hiPSCs). This is the largest deletion shown to date in DMD. Clonal hiPSC lines containing the exon 45-55 deletion were differentiated to disease relevant types, such as cardiomyocytes and skeletal muscle myotubes, which had restored dystrophin protein. We demonstrated, for the first time, that the internally deleted dystrophin generated by CRISPR/Cas9 was functional and improved membrane integrity, reduced miR31 expression, and restored the dystrophin glycoprotein complex in vitro and after engraftment of skeletal muscle cells in vivo. This gene editing platform restores the reading frame for the majority of DMD patients and offers potential as an ex vivo correction for stem cell therapy or for use in vivo.