Duchenne muscular dystrophy (DMD) is a severe, neuromuscular disorder caused by mutations in the DMD gene, precluding synthesis of functional dystrophin protein. Antisense oligonucleotide (AON)-mediated exon skipping has been developed as a method to restore the reading frame, which allows the synthesis of internally truncated, but partially functional dystrophin proteins, as found in the less severe Becker muscular dystrophy (BMD). This approach is species specific, since AONs targeting human exons often will not have full homology to mouse exons. As such, mouse models with mutations in the murine Dmd gene are of limited use to study human specific AONs in vivo. However, our del52hDMD/mdx mouse model contains mutated copies of both the mouse (nonsense mutation in exon 23) and human (deletion of exon 52) dystrophin-encoding genes. This model allows for testing effects of treatment with human specific exon 51 or 53 targeting AONs on RNA, protein, histological, and functional levels. Therefore, the model can be used to optimize human specific AONs, e.g., by comparing dystrophin protein and exon skipping levels.
Absolute quantification of exon skipping levels can be obtained by digital droplet PCR (ddPCR). This method compartmentalizes samples into thousands of droplets that represent individual micro PCR reactions, and can be either positive or negative after amplification depending on whether there was a template molecule present or not. This allows for precise determination of the copy numbers of template molecules. The protocol described here uses probes binding to exon–exon junctions (EEJs) of human DMD transcripts with and without skipping of exon 51. We report that this method is specific for human transcripts so that exon skipping levels can be quantified accurately by ddPCR in del52hDMD/mdx mice.
Duchenne muscular dystrophy (DMD) Exon skipping Antisense oligonucleotides (AONs) Humanized mouse model Digital droplet PCR (ddPCR)
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Ruurd Verheul from BioMarin B.V. (Leiden, the Netherlands) is acknowledged for sharing the ddPCR exon skipping quantification protocol and designing the TaqMan assays for the del52hDMD/mdx mouse model. The authors are thankful for obtaining RNA samples to perform the experiment from Laura van Vliet and Maaike van Putten of the Leiden University Medical Center (Leiden, the Netherlands). Rolf Vossen from the Leiden Genome Technology Center (Leiden, the Netherlands) is acknowledged for technical assistance with the ddPCR.
Monika Hiller’s salary is covered by a grant from the Prinses Beatrix Spierfonds (grant W.OR14-13) to Pietro Spitali.
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