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

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 muscle biopsies of these patients were previously thoroughly characterized, providing a valuable benchmark for the evaluation of novel methodology. We demonstrate that levels of dystrophin protein restoration, and thus patient response, correlate accurately with the RNA level. Furthermore, this sensitive assay detects revertant exon 51 skipped fibers in untreated biopsies, providing an important baseline to precisely quantify treatment success. This study represents the first quantitative assessment of exon skipping in a clinical trial setting. We present a standardized and reproducible method to assess patient response that will complement protein studies in future preclinical and clinical exon skipping-based gene therapy studies for DMD.