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Restoring Dystrophin Expression with Exon 44 and 53 Skipping in the DMD Gene in Immortalized Myotubes

Echigoya Y, Yokota T
Methods Mol Biol. 2023;2587:125-139. doi: 10.1007/978-1-0716-2772-3_7
Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is a therapeutic approach that applies to many Duchenne muscular dystrophy (DMD) patients harboring out-of-frame deletion mutations in the DMD gene. In particular, PMOs for skipping exon 44 have been developing in clinical trials, such as the drug NS-089/NCNP-02. Two exon 53 skipping PMOs, golodirsen and viltolarsen, have received conditional approval for treating patients due to their ability to restore dystrophin protein expression. Although promising, further development of exon-skipping technology is needed for patients to have more therapeutic benefit. This chapter describes evaluation methods of exon 44 and 53 skipping PMOs in immortalized DMD patient-derived skeletal muscle cells. We introduce how to quantify exon-skipping efficiencies and dystrophin rescue levels represented by RT-PCR and western blotting, respectively. The screening methods using immortalized patient myotubes can serve to find exon-skipping PMO drug candidates.
Not Epub
Organism or Cell Type: 
cell culture: myotubes