J Clin Invest. 2023 Feb 1
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, and Hui Yang
Products used in the paper Details Operation
AAV vector packaging AAV9 production and delivery to DMDE30mut mice. AAVs were produced using PackGene Biotech (Guangzhou, China) and purified by iodixanol density gradient centrifugation. Request Quote

Research Field: Duchenne muscular dystrophy

Keywords: Therapeutics

AAV Serotype: AAV9

Dose: For intramuscular injection, DMDE30mut mice were anesthetized, and TA muscle was injected with 50 μL of AAV9 (3 × 10^11 vg) preparations or with the same volume saline solution. For intraperitoneal injection, the P3 DMDE30mut mice were injected using an ultrafine needle (31 gauge) with 50 μL of AAV9 (1 × 10^12 vg) preparations or with saline solution.

Routes of Administration: For intramuscular injection, DMDE30mut mice were anesthetized, and TA muscle was injected with 50 μL of AAV9 (3 × 10^11 vg) preparations or with the same volume saline solution. For intraperitoneal injection, the P3 DMDE30mut mice were injected using an ultrafine needle (31 gauge) with 50 μL of AAV9 (1 × 10^12 vg) preparations or with saline solution.

Targeted organ: muscle

Animal or cell line strain: DMDE30mut mice were generated in the C57BL/6J background using the CRISPR/Cas9 system

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.

Popular Services

AAV Packaging Service

AAV Analytical Service

Vector Design