Evaluation of Exon Skipping and Dystrophin Restoration in In Vitro Models of Duchenne Muscular Dystrophy

Abstract Several exon skipping antisense oligonucleotides (eteplirsen, golodirsen, viltolarsen, and casimersen) have been approved for the treatment of Duchenne muscular dystrophy, but many more are in development targeting an array of different DMD exons. Preclinical screening of the new oligonucleotide sequences is routinely performed using patient-derived cell cultures, and evaluation of their efficacy may be performed at RNA and/or protein level. While several methods to assess exon skipping and dystrophin expression in cell culture have been developed, the choice of methodology often depends on the availability of specific research equipment. In this chapter, we describe and indicate the relevant bibliography of all the methods that may be used in this evaluation and describe in detail the protocols routinely followed at our institution, one to evaluate the efficacy of skipping at RNA level (nested PCR) and the other the restoration of protein expression (myoblot), which provide good results using equipment largely available to most research laboratories.