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
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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

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Figure 5

Systemic delivery of AAV-mxABE rescues dystrophin expression and muscle function in multiple organs after 3 weeks.

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Systemic delivery of AAV-mxABE rescues dystrophin expression and muscle ...
(A) Schematic of systemic administration of AAV particles. AAV9-C3 and AAV9-M2 particles were injected intraperitoneally (i.p.) into postnatal day 3 (P3) DMDE30mut mice. Some DMDE30mut mice were injected with saline as mock-treated controls. Arrows indicate time points for tissue collection after i.p. injection. (B) Measurement by deep sequencing of dystrophin transcripts of the targeted A-to-I editing efficiency in TA, DI, and heart after systemic delivery (n = 4). (C) Western blot analysis shows restoration of dystrophin expression in the TA, DI, and heart of DMDE30mut mice 3 weeks after injection. Dilutions of protein extract from WT mice were used to standardize dystrophin expression (5%, 10%, and 50%). Vinculin (Cell Signaling Technology, 13901S) was used as the loading control. (D) Immunohistochemistry for dystrophin in TA, DI, and heart of DMDE30mut mice was performed 3 weeks after systemic injection. Dystrophin (Abcam, ab15277) is shown in green. Scale bar: 200 μm. (E) Quantification of Dys+ in cross sections of TA, DI, and heart muscles (n = 4). (F) CK levels were measured in WT, DMDE30mut mock-treated, and DMDE30mut AAV9-mxABE–treated mice 3 weeks after injection (n = 4). (G) Forelimb grip strength was measured in WT mice, DMDE30mut mice, and DMDE30mut mice treated with AAV9-C3/M2 particles (n = 6). (H) The remaining strength was also measured during 10 repetitions at 10-second intervals (n = 6). Dots and bars represent biological replicates and are mean ± SEM. Different asterisks represent statistical significance (P < 0.01) in multiple-comparison test using ANOVA.