Targeting Duchenne Muscular Dystrophy by Skipping DMD Exon 45 with Base Editors
Duchenne muscular dystrophy is an X-linked monogenic disease caused by mutations in the dystrophin gene (DMD) and characterized by progressive muscle weakness leading to loss of ambulation and significantly decreased life expectancy. Since the current standard of care for Duchenne muscular dystrophy is to merely treat symptoms, there is a dire need for novel treatment modalities that can correct the underlying genetic mutations. While several gene replacement therapies are being explored in clinical trials, one emerging approach that can directly correct mutations in genomic DNA is base editing. We have recently developed CRISPR-SKIP, a base editing strategy to induce permanent exon skipping by introducing C>T or A>G mutations at splice acceptors in genomic DNA, which can be utilized therapeutically to recover dystrophin expression when a genomic deletion leads to an out-of-frame DMD transcript. We now demonstrate that CRISPR-SKIP can be adapted to correct some forms of Duchenne muscular dystrophy by disrupting the splice acceptor in human DMD exon 45 with high efficiency, which enables open reading frame recovery and restoration of dystrophin expression. We also demonstrate that AAV-delivered split-intein base editors edit the splice acceptor of DMD exon 45 in cultured human cells and in vivo, highlighting the therapeutic potential of this strategy. Overall design: Determining the ability of CRISPR base editors to edit the splice acceptor of DMD exon 45, and the effect that that editing has on exon skipping. Sequencing data from targeted amplicon sequencing of edited genomic DNA from cell lines and mouse tissues, as well sequencing of RNA from engineered tissue