Comparison of dystrophin expression following gene editing and gene replacement in an aged preclinical DMD animal model

• Published in: Molecular therapy Vol. 30; no. 6; pp. 2176 - 2185
• Main Authors: Bengtsson, Niclas E., Crudele, Julie M., Klaiman, Jordan M., Halbert, Christine L., Hauschka, Stephen D., Chamberlain, Jeffrey S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2022; American Society of Gene & Cell Therapy
• Subjects: AAV; Aging; Animals; Cas9; CRISPR; CRISPR-Cas Systems; CXMD; Disease Models, Animal; Disease Progression; DMD; Dogs; Duchenne; Dystrophin - genetics; gene editing; Gene Editing - methods; micro-dystrophin; muscle; Muscle, Skeletal - metabolism; Muscular Dystrophy, Duchenne - genetics; Muscular Dystrophy, Duchenne - therapy; Original; CRISPR; CXMD; micro-dystrophin; AAV; Duchenne; muscle; dogs; DMD; Cas9; gene editing
• ISSN: 1525-0016; 1525-0024; 1525-0024
• DOI: 10.1016/j.ymthe.2022.02.003

Gene editing has shown promise for correcting or bypassing dystrophin mutations in Duchenne muscular dystrophy (DMD). However, preclinical studies have focused on young animals with limited muscle fibrosis and wasting, thereby favoring muscle transduction, myonuclear editing, and prevention of disease progression. Here, we explore muscle-specific dystrophin gene editing following intramuscular delivery of AAV6:CK8e-CRISPR/SaCas9 in 3- and 8-year-old dystrophic CXMD dogs and provide a qualitative comparison to AAV6:CK8e-micro-dystrophin gene replacement at 6 weeks post-treatment. Gene editing restored the dystrophin reading frame in ∼1.3% of genomes and in up to 4.0% of dystrophin transcripts following excision of a 105-kb mutation containing region spanning exons 6–8. However, resulting dystrophin expression levels and effects on muscle pathology were greater with the use of micro-dystrophin gene transfer. This study demonstrates that our muscle-specific multi-exon deletion strategy can correct a frequently mutated region of the dystrophin gene in an aged large animal DMD model, but underscores that further enhancements are required to reach efficiencies comparable to AAV micro-dystrophin. Our observations also indicate that treatment efficacy and state of muscle pathology at the time of intervention are linked, suggesting the need for additional methodological optimizations related to age and disease progression to achieve relevant clinical translation of CRISPR-based therapies to all DMD patients. [Display omitted] Comparisons of CRISPR/Cas9 gene editing and micro-dystrophin gene replacement in old dystrophic dogs highlight the promise and limitations of AAV-mediated gene therapy in older subjects with advanced muscle pathology.