Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy.
| Autor: | Verheul RC; BioMarin Nederland BV, Leiden, The Netherlands., van Deutekom JC; BioMarin Nederland BV, Leiden, The Netherlands., Datson NA; BioMarin Nederland BV, Leiden, The Netherlands. |
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| Jazyk: | angličtina |
| Zdroj: | PloS one [PLoS One] 2016 Sep 09; Vol. 11 (9), pp. e0162467. Date of Electronic Publication: 2016 Sep 09 (Print Publication: 2016). |
| DOI: | 10.1371/journal.pone.0162467 |
| Abstrakt: | Antisense oligonucleotides (AONs) in clinical development for Duchenne muscular dystrophy (DMD) aim to induce skipping of a specific exon of the dystrophin transcript during pre-mRNA splicing. This results in restoration of the open reading frame and consequently synthesis of a dystrophin protein with a shorter yet functional central rod domain. To monitor the molecular therapeutic effect of exon skip-inducing AONs in clinical studies, accurate quantification of pre- and post-treatment exon skip levels is required. With the recent introduction of 3rd generation digital droplet PCR (ddPCR), a state-of-the-art technology became available which allows absolute quantification of transcript copy numbers with and without specific exon skip with high precision, sensitivity and reproducibility. Using Taqman assays with probes targeting specific exon-exon junctions, we here demonstrate that ddPCR reproducibly quantified cDNA fragments with and without exon 51 of the DMD gene over a 4-log dynamic range. In a comparison of conventional nested PCR, qPCR and ddPCR using cDNA constructs with and without exon 51 mixed in different molar ratios using, ddPCR quantification came closest to the expected outcome over the full range of ratios (0-100%), while qPCR and in particular nested PCR overestimated the relative percentage of the construct lacking exon 51. Highest accuracy was similarly obtained with ddPCR in DMD patient-derived muscle cells treated with an AON inducing exon 51 skipping. We therefore recommend implementation of ddPCR for quantification of exon skip efficiencies of AONs in (pre)clinical development for DMD. Competing Interests: All authors are employees (which includes contribution to patent [applications] and participation in stock-option plans) of BioMarin Nederland BV, a company that develops RNA therapeutics for Duchenne muscular dystrophy. JCTvD is inventor on exon skipping patents (WO2002/024906 Induction of exon skipping in eukaryotic cells, WO2004/083446 Modulation of exon recognition in pre-mRNA by interfering with the secondary RNA structure, WO2006/112705 Modulation of exon recognition in premRNA by interfering with the binding of SR proteins and by interfering with secondary RNA structure, WO2009/054725 Means and methods for counteracting muscle disorders, WO2009/139630 Method for efficient exon 44 skipping in Duchenne muscular dystrophy and associated means, WO2010/050801 Methods and means for efficient skipping of exon 45 in Duchenne muscular dystrophy pre-mRNA, WO2010/050802 Methods and means for efficient skipping of at least one of the following exons of the human Duchenne muscular dystrophy gene: 43, 46, 50-53, WO2013/112053 RNA modulating oligonucleotides with improved characteristics for the treatment of Duchenne en Becker muscular dystrophy, WO2014/007620 Oligonucleotide for the treatment of muscular dystrophy patients). This did not alter the authors' adherence to PLOS ONE policies on sharing data and materials. |
| Databáze: | MEDLINE |
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