Abstract
Abstract
Background
Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD.
Methods
In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors.
Results
Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan.
Conclusions
We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.
Funder
National Natural Science Foundation of China
Strategic Priority Research Program of the Chinese Academy of Sciences
National Key Research and Development Program of China Stem Cell and Translational Research
Guangdong Provincial Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases
Science and Technology Planning Project of Guangdong Province, China
Southern China International Cooperation Base for Early Intervention and Functional Rehabilitation of Neurological Diseases
Bureau of Science and Technology of Guangzhou Municipality
Science and Technology Planning Project of Guangdong Province
Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences
Key Research & Development Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory
Publisher
Springer Science and Business Media LLC
Subject
Genetics(clinical),Genetics,Molecular Biology,Molecular Medicine
Cited by
11 articles.
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