Full-length dystrophin restoration via targeted exon integration by AAV-CRISPR in a humanized mouse model of Duchenne muscular dystrophy

• Published in: Molecular therapy Vol. 29; no. 11; pp. 3243 - 3257
• Main Authors: Pickar-Oliver, Adrian, Gough, Veronica, Bohning, Joel D., Liu, Siyan, Robinson-Hamm, Jacqueline N., Daniels, Heather, Majoros, William H., Devlin, Garth, Asokan, Aravind, Gersbach, Charles A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.11.2021; American Society of Gene & Cell Therapy
• Subjects: Animals; CRISPR; CRISPR-Cas Systems; Dependovirus - genetics; Disease Models, Animal; Dystrophin - genetics; Exons; Gene Editing; Gene Expression; Gene Order; Gene Transfer Techniques; Genetic Engineering; Genetic Therapy - methods; Genetic Vectors - genetics; genome editing; Humans; Mice; Mice, Transgenic; Muscle, Skeletal - metabolism; muscular dystrophy; Muscular Dystrophy, Duchenne - genetics; Muscular Dystrophy, Duchenne - therapy; Mutation; Myocardium - metabolism; Original; targeted integration; viral vectors; Virus Integration; targeted integration; CRISPR; genome editing; muscular dystrophy; viral vectors
• ISSN: 1525-0016; 1525-0024; 1525-0024
• DOI: 10.1016/j.ymthe.2021.09.003

Targeted gene-editing strategies have emerged as promising therapeutic approaches for the permanent treatment of inherited genetic diseases. However, precise gene correction and insertion approaches using homology-directed repair are still limited by low efficiencies. Consequently, many gene-editing strategies have focused on removal or disruption, rather than repair, of genomic DNA. In contrast, homology-independent targeted integration (HITI) has been reported to effectively insert DNA sequences at targeted genomic loci. This approach could be particularly useful for restoring full-length sequences of genes affected by a spectrum of mutations that are also too large to deliver by conventional adeno-associated virus (AAV) vectors. Here, we utilize an AAV-based, HITI-mediated approach for correction of full-length dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy (DMD). We co-deliver CRISPR-Cas9 and a donor DNA sequence to insert the missing human exon 52 into its corresponding position within the DMD gene and achieve full-length dystrophin correction in skeletal and cardiac muscle. Additionally, as a proof-of-concept strategy to correct genetic mutations characterized by diverse patient mutations, we deliver a superexon donor encoding the last 28 exons of the DMD gene as a therapeutic strategy to restore full-length dystrophin in >20% of the DMD patient population. This work highlights the potential of HITI-mediated gene correction for diverse DMD mutations and advances genome editing toward realizing the promise of full-length gene restoration to treat genetic disease. [Display omitted] This study applies targeted Cas9-based gene insertion strategies for the correction of full-length dystrophin in a pre-clinical humanized mouse model of Duchenne muscular dystrophy. Following intramuscular or intravenous delivery, full-length dystrophin is restored in skeletal and cardiac muscle.