Base Editing of miRNA Binding Sites to Promote Utrophin Upregulation as a Compensatory Mechanism for Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked progressive degenerative disorder caused by the lack of dystrophin, a protein essential for maintaining membrane integrity in skeletal and cardiac muscles. Over 7.000 mutations have been identified in the dystrophin gene (DMD), with the majority involving exon deletions in the actin binding domain 1 (ABD1) or in the central rod domain (CRD). This significant genetic variability poses a challenge in the pursuit of a cure for DMD. Upregulation of utrophin, a paralogue of dystrophin, is considered a promising therapeutic strategy that could benefit all patients, regardless of their genetic mutation. Among the various regulatory mechanisms of utrophin, its expression is post-transcriptionally inhibited by several microRNAs (miRNAs) that bind to the 3’ untranslated region (UTR) of the utrophin transcript. Here, we present a strategy to induce permanent upregulation of utrophin by using the cutting-edge CRISPR-based technology of base editing to precisely destroy the miRNA binding sites in utrophin 3’ UTR. First, we identified the most effective combinations of single guide RNAs (sgRNAs) and base editors that target the miRNA binding sites. Then, we tested these combinations on a human induced pluripotent stem cell (hiPSC) line with exon deletion in the CRD. Finally, we generated a new DMD hiPSC line harbouring a single exon deletion in the ABD1. We intend to use this newly generated hiPSC line in combination with available DMD hiPSCs to demonstrate the universal applicability of the approach we are proposing. This work paves the way for developing a universally applicable gene-editing strategy that may provide a long-awaited therapeutic option for all DMD patients, regardless of their underlying mutation.