Functional improvement of dystrophic muscle by utrophin gene editing

Utrophin upregulation is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). Utrophin expression is post-transcriptionally repressed via microRNAs binding the 3' UTR of utrophin, particularly at the inhibitory microRNA target region (IMTR). Guide RNA pairs for editing the IMTR and a larger IMTR⁺ region were designed and ranked by their ability to edit and increase utrophin expression in normal and dystrophic muscle cells in vitro. Optimized guide RNA pairs and SpCas9 were used to generate MyoAAV vectors and tested for the ability to ameliorate the dystrophic phenotype of the mdx mouse model of DMD, in vivo. Systemic delivery of MyoAAV targeting the IMTR and IMTR⁺ using intraperitoneal injections in neonatal mdx mice resulted in efficient gene editing and increased utrophin expression in muscle. Increased utrophin was appropriately localized and accompanied by the appearance α-sarcoglycan at the sarcolemma, indicating dystrophin-associated glycoprotein complex recovery. Functional analyses revealed increased specific force generation and improved resistance to eccentric contractions. Histological analyses showed a reduction in the proportion of centrally nucleated fibers that are indicative of dystrophic pathology in the mdx mice. Together, this provides proof-of-concept for a novel therapeutic strategy that, in principle, would be applicable to all patients with DMD, independent of mutation status.