Development of CRISPR base editing gene therapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy is a severe X-linked neuromuscular disorder caused by mutations in the DMD gene that disrupt the reading frame and abolish dystrophin expression. Many of these mutations can be corrected by exon skipping in order to restore the open reading frame of DMD. Current antisense oligonucleotide therapies can induce exon skipping and offer clinical benefit, but they require repeated administration, and they have only produced temporary effects. This limited temporality underscores the need for durable, genome-based strategies that can produce long-term corrections. In this context, base editing technologies provide a means to precisely modify single nucleotides in genomic DNA without introducing double-strand breaks. This precise nucleotide modification is achieved by coupling catalytically impaired Cas9 nucleases with nucleoside deaminases that convert specific bases through deamination and subsequent DNA repair. Among various designs, RNA-aptamer-mediated base editors offer a modular configuration that separates target recognition from base modification, allowing flexible combinations of Cas9 and deaminase components.My work expands this RNA-aptamer-mediated base editing system by incorporating Staphylococcus aureus Cas9 (SaCas9) and several cytidine deaminase orthologs from different vertebrate species, including novel variants from bats and lizards. Concomitantly, I also tested SaCas9-KKH variants with distinct PAM sequences to broaden targetable sites. These modular base editors achieved efficient editing at both reporter and genomic loci, with low indel formation and minimal off-target activity. To explore therapeutic applications, I applied these editors to promote exon skipping in the mouse Dmd and human DMD gene, focusing on mouse exons 23 and 44, and human exons 44, 45, and 51. By evaluating editing outcomes using episomal splicing reporters, genomic assays in K562 cells, and adeno-associated virus (AAV) delivery systems, I discovered an efficient and predictable base editing in the genome, as well as the successful induction of exon skipping at the mRNA level, with consistent outcomes across various experimental models.My study establishes RNA-aptamer-mediated base editing as a robust and versatile platform for inducing exon skipping at clinically relevant DMD loci. This modular base editor system not only expands the accessible genomic space but also provides a convenient framework for optimizing deaminase-Cas9 combinations tailored to specific therapeutic targets. This work demonstrates the potential of modular base editors to enable durable, genome-based exon-skipping therapies for Duchenne muscular dystrophy and other genetic disorders.