Live-Imaging Of Revertant And Therapeutically Restored Dystrophin In Thedmd(Egfp-Mdx)Mouse Model For Duchenne Muscular Dystrophy

Background Dmd(mdx),harbouring the c.2983C>T nonsense mutation inDmdexon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinalin vivostudies. Methods We generated aDmd(EGFP-mdx)reporter allele carrying incisthemdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skipDmdexon 23, or after gene editing using AAV-encoded CRISPR/Cas9 forDmdexon 23 excision. Results Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persistedex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibresex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by themdx-23 mutation. Conclusion Intravital imaging ofDmd(EGFP-mdx)muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophinin vivoandex vivo.