Exon Skipping Quantification by Quantitative Reverse-Transcription Polymerase Chain Reaction in Duchenne Muscular Dystrophy Patients Treated with the Antisense Oligomer Eteplirsen

Abstract Restoration of the open reading frame of the DMD gene and dystrophin protein production in Duchenne muscular dystrophy (DMD) can be achieved by exon skipping using antisense oligomers (AOs) targeted to splicing elements. Several such RNA-based gene therapy approaches are in clinical development in which all studies to date have assessed AO efficacy by semiquantitative nested reverse-transcription polymerase chain reaction (RT-PCR). Precise evaluation of dystrophin protein levels is complex and hindered by the large size and low abundance of dystrophin; thus an accurate and standardized measurement of DMD exon skipping at the RNA level remains important to assess and compare patient responses in DMD exon skipping clinical trials. Here we describe the development of a Taqman quantitative (q)RT-PCR assay to quantify exon skipping and highlight its use to determine the levels of exon skipping in DMD patients treated intramuscularly with a morpholino AO to skip exon 51, eteplirsen (AVI-4658). The musc...