Abstract
Intramuscular injection and electroporation of naked plasmid DNA (IMEP) has emerged as a potential alternative to viral vector injection for transgene expression into skeletal muscles. In this study, IMEP was used to express the DUX4 gene into mouse tibialis anterior muscle. DUX4 is normally expressed in germ cells and early embryo, and silenced in adult muscle cells where its pathological reactivation leads to Facioscapulohumeral muscular dystrophy. DUX4 encodes a potent transcription factor causing a large deregulation cascade. Its high toxicity but sporadic expression constitutes major issues for testing emerging therapeutics. The IMEP method appeared as a convenient technique to locally express DUX4 in mouse muscles. Histological analyses revealed well delineated muscle lesions 1-week after DUX4 IMEP. We have therefore developed a convenient outcome measure by quantification of the damaged muscle area using color thresholding. This method was used to characterize lesion distribution and to assess plasmid recirculation and dose–response. DUX4 expression and activity were confirmed at the mRNA and protein levels and through a quantification of target gene expression. Finally, this study gives a proof of concept of IMEP model usefulness for the rapid screening of therapeutic strategies, as demonstrated using antisense oligonucleotides against DUX4 mRNA.
Highlights
Intramuscular injection and electroporation of naked plasmid DNA (IMEP) has emerged as a potential alternative to viral vector injection for transgene expression into skeletal muscles
Contrary to Duchenne Muscular Dystrophy (DMD), resulting from the loss of dystrophin, Facioscapulohumeral muscular dystrophy (FSHD)[43] is a gain of function disease caused by the inappropriate expression in skeletal muscle of DUX4, a gene normally only expressed in germline and early e mbryogenesis[44,45,46,47,48,49,50,51,52,53,54,55]
Non-viral methods for transgene delivery have received increased attentions thanks to their relative safety, simplicity and cost-effectiveness compared to the administration of viral vectors which were found associated with cytotoxicity, inflammation and immune r esponse[1,2]
Summary
Intramuscular injection and electroporation of naked plasmid DNA (IMEP) has emerged as a potential alternative to viral vector injection for transgene expression into skeletal muscles. Development of in vivo proof-of-concept studies for emerging therapies are required as a step towards clinical trials Several hurdles such as DUX4 toxicity and its stochastic low expression have made the generation of an animal model recapitulating all the pathophysiological aspects of FSHD very challenging. Some of these mouse models allow inducible conditional DUX4 expression, bypassing DUX4 high toxicity during embryonic development and enabling mice to grow up and develop muscular dystrophy[78,79,80,83] These models open new ways to investigate molecular mechanisms leading to FSHD symptoms. We describe a convenient in vivo model of DUX4 local muscle expression using an EP procedure This model is simple, unexpansive, reproducible and associated with an easy read out that facilitates quantitative analysis. This model can be useful at the forefront for high throughput therapeutic screening
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