G.P.86: Digital droplet RT-PCR for the absolute quantification of exon skipping induced by antisense oligonucleotides in (pre-) clinical development for Duchenne muscular dystrophy

Abstract

Antisense oligonucleotide (AON) therapies for Duchenne muscular dystrophy (DMD) are aimed at inducing skipping of specific exons during pre-mRNA splicing. This results in restoration of the reading frame and consequently synthesis of a shorter yet functional version of the dystrophin protein, as has been demonstrated in healthy control and DMD patient-derived muscle cell cultures, various DMD mouse and dog models, and recently in DMD patients treated with eteplirsen or drisapersen. As in many DMD patients a low level of spontaneous, rescuing background exon skipping occurs, accurate quantification of exon skip levels before and after AON treatment is required to monitor molecular AON drug effect in clinical studies. So far exon skip levels have generally been assessed using conventional 1st generation RT-PCR, in which non-skipped and skipped transcripts are co-amplified followed by visual comparison or densitometry of exon skip levels by gel electrophoresis or lab-on-chip. As dystrophin is not a highly abundant protein, underlying DMD transcript levels are relatively low and two rounds of PCR amplification (primary and nested) are required, often resulting in more efficient amplification of the shorter ‘skipped’ fragment compared to the longer ‘non-skipped’ fragment and end-stage saturation of the amplified products. With the recent introduction of 3rd generation digital droplet RT-PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of copy numbers of transcripts with and without exon skip in a single PCR reaction, with high precision, sensitivity and reproducibility. We have developed ddPCR TaqMan probes targeting specific exon–exon junctions in control, BMD, DMD and mdx samples and will present data demonstrating its suitability for assessing DMD transcript copy numbers and exon skip levels. We recommend ddPCR for quantifying exon skip efficiencies in the various stages of (pre-) clinical development of AONs.