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.