Mini-dCas13X-mediated RNA editing restores dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy

• Published in: The Journal of clinical investigation Vol. 133; no. 3; pp. 1 - 14
• Main Authors: Li, Guoling, Jin, Ming, Li, Zhifang, Xiao, Qingquan, Lin, Jiajia, Yang, Dong, Liu, Yuanhua, Wang, Xing, Xie, Long, Ying, Wenqin, Wang, Haoqiang, Zuo, Erwei, Shi, Linyu, Wang, Ning, Chen, Wanjin, Xu, Chunlong, Yang, Hui
• Format: Journal Article
• Language: English
• Published: United States American Society for Clinical Investigation 01-02-2023
• Subjects: Analysis; Animals; Biomedical research; Cardiac muscle; Care and treatment; Codon; Codons; CRISPR-Cas Systems; Diaphragm; Disease Models, Animal; Duchenne muscular dystrophy; Duchenne's muscular dystrophy; Dystrophin; Dystrophin - genetics; Editing; Editors; Gene Editing - methods; Gene mutations; Gene therapy; Genetic aspects; Genetic research; Genetic Therapy - methods; Heart; Humans; Kinases; Mice; Muscle function; Muscle, Skeletal - metabolism; Muscular dystrophy; Muscular Dystrophy, Duchenne - genetics; Muscular Dystrophy, Duchenne - therapy; Mutation; Nonsense mutation; Pathogenicity; Patients; Point mutation; Proteins; RNA Editing; RNA processing; Skeletal muscle; Therapeutics; Utrophin; China; Muscle; Monogenic diseases; Gene therapy; Therapeutics
• ISSN: 1558-8238; 0021-9738; 1558-8238
• DOI: 10.1172/JCI162809

Approximately 10% of monogenic diseases are caused by nonsense point mutations that generate premature termination codons (PTCs), resulting in a truncated protein and nonsense-mediated decay of the mutant mRNAs. Here, we demonstrate a mini-dCas13X-mediated RNA adenine base editing (mxABE) strategy to treat nonsense mutation-related monogenic diseases via A-to-G editing in a genetically humanized mouse model of Duchenne muscular dystrophy (DMD). Initially, we identified a nonsense point mutation (c.4174C>T, p.Gln1392*) in the DMD gene of a patient and validated its pathogenicity in humanized mice. In this model, mxABE packaged in a single adeno-associated virus (AAV) reached A-to-G editing rates up to 84% in vivo, at least 20-fold greater than rates reported in previous studies using other RNA editing modalities. Furthermore, mxABE restored robust expression of dystrophin protein to over 50% of WT levels by enabling PTC read-through in multiple muscle tissues. Importantly, systemic delivery of mxABE by AAV also rescued dystrophin expression to averages of 37%, 6%, and 54% of WT levels in the diaphragm, tibialis anterior, and heart muscle, respectively, as well as rescued muscle function. Our data strongly suggest that mxABE-based strategies may be a viable new treatment modality for DMD and other monogenic diseases.