Advanced Genome Editing Strategies for Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle wasting disease that causes loss of ambulation and premature death in affected boys. In the 1980s, the cause of the disease was attributed to mutations in the DMD gene, yet almost 40 years later there is still no cure. The large size of the gene, diversity of patient mutations, and delivery challenge of modifying skeletal muscle systemically have all limited the application of therapies to correct the disease-causing mutations. Gene editing with CRISPR-Cas technology is poised to revolutionize our ability to treat genetic diseases. For DMD, several landmark studies have demonstrated the strong therapeutic potential of using CRISPR to restore dystrophin protein in DMD models. Yet many challenges remain in converting proof-of-concept editing to a safe and effective therapy. Here, we focus on optimizing and developing methods to evaluate the two most promising strategies for using gene editing to treat DMD: exon deletion and exon skipping.In aim 1, we tackle the problem of searching for highly efficient gRNAs in the vast sequence space of DMD introns by applying high-throughput screening techniques to measure the relative deletion efficiency of gRNA pairs. We discover novel gRNA pairs for the deletion of DMD exon 51 and demonstrate an improvement over previous methods of gRNA pair discovery. In aim 2, we evaluate the potential of “CRISPR 2.0” editing tools that provide next-generation control over DNA editing to produce DMD exon skipping with reduced disruption to the genome. We discover a base editor and gRNA design that efficiently skips exon 45 and restores dystrophin expression and apply unbiased characterization methods to interrogate the impact of such editing.