Mini-dCas13X–mediated RNA editing restores dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy
Guoling Li, Ming Jin, Zhifang Li, Qingquan Xiao, Jiajia Lin, Dong Yang, Yuanhua Liu, Xing Wang, Long Xie, Wenqin Ying, Haoqiang Wang, Erwei Zuo, Linyu Shi, Ning Wang, Wanjin Chen, Chunlong Xu, Hui Yang
Guoling Li, Ming Jin, Zhifang Li, Qingquan Xiao, Jiajia Lin, Dong Yang, Yuanhua Liu, Xing Wang, Long Xie, Wenqin Ying, Haoqiang Wang, Erwei Zuo, Linyu Shi, Ning Wang, Wanjin Chen, Chunlong Xu, Hui Yang
View: Text | PDF
Research Article

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

Abstract

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.

Authors

Guoling Li, Ming Jin, Zhifang Li, Qingquan Xiao, Jiajia Lin, Dong Yang, Yuanhua Liu, Xing Wang, Long Xie, Wenqin Ying, Haoqiang Wang, Erwei Zuo, Linyu Shi, Ning Wang, Wanjin Chen, Chunlong Xu, Hui Yang

×

Figure 1

Establishment and characterization of a humanized DMD mouse model.

Options: View larger image (or click on image) Download as PowerPoint
Establishment and characterization of a humanized DMD mouse model. (A) P...
(A) Pedigree of patient with DMD (proband) with a nonsense mutation p.Gln1392*. Squares represent males; circles represent females. (B) Histological analysis of left biceps muscle from normal and proband. Dystrophin (MilliporeSigma, D8168) is shown in brown. (C) Strategy for generating humanized DMD mouse model. CRISPR/Cas9 editing using 2 sgRNAs flanking an exon was used to delete mouse Dmd exon 30 and replace it with human DMD exon 30 carrying the nonsense mutation. (D) RT-PCR products from muscle of DMDE30mut mice were sequenced to validate the exon 30 mutation. (E) Sirius red staining and H&E staining of TA, DI, and heart muscle of WT and DMDE30mut mice. (F) Dystrophin immunohistochemistry from indicated muscles of WT and DMDE30mut mice. Dystrophin (Abcam, ab15277) and spectrin (Millipore, MAB1622) are shown in green and magenta, respectively. (G) Western blot confirming the absence of dystrophin in indicated muscle tissues. (H) WT and DMDE30mut mice were subjected to forelimb grip strength testing to measure muscle performance (n = 6). (I) Serum CK, a marker of muscle damage and membrane leakage, was measured in WT and DMDE30mut mice (n = 8). All mice were 8 weeks old at the time of the experiment. Data are represented as mean ± SEM. Each dot represents an individual mouse. **P < 0.01 using unpaired 2-tailed Student’s t test. Scale bars: 200 μm.