Live‐imaging of revertant and therapeutically restored dystrophin in the <i>Dmd<sup>EGFP‐mdx</sup></i> mouse model for Duchenne muscular dystrophy
Mina Petkova, Amalia Stantzou, A. Morin, Olga N. Petrova, Susanne Morales-Gonzalez, Franziska Seifert, J. Bellec-Dyèvre, Tudor Manoliu, Aurélie Goyenvalle, Laurynn Garcia, Isabelle Richard, Corinne Laplace‐Builhé, Markus Schuelke, Helge Amthor
Abstract
BACKGROUND: , harbouring the c.2983C>T nonsense mutation in Dmd exon 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 longitudinal in vivo studies. METHODS: reporter allele carrying in cis the mdx-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 skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 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 persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex 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 the mdx-23 mutation. CONCLUSION: muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.