A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures

Monika Hiller, Maria Sofia Falzarano, Iker Garcia-Jimenez, Valentina Sardone, Ruurd C Verheul, Linda Popplewell, Karen Anthony, Estibaliz Ruiz-Del-Yerro, Hana Osman, Jelle J Goeman, Kamel Mamchaoui, George Dickson, Alessandra Ferlini, Francesco Muntoni, Annemieke Aartsma-Rus, Virginia Arechavala-Gomeza, Nicole A Datson, Pietro Spitali

Research output: Contribution to JournalArticle

Abstract

Background: Duchenne muscular dystrophy is a lethal disease caused by lack of dystrophin. Skipping of exons adjacent to out-of-frame deletions has proven to restore dystrophin expression in Duchenne patients. Exon 51 has been the most studied target in both preclinical and clinical settings and the availability of standardized procedures to quantify exon skipping would be advantageous for the evaluation of preclinical and clinical data. Objective: To compare methods currently used to quantify antisense oligonucleotide–induced exon 51 skipping in the DMD transcript and to provide guidance about the method to use. Methods: Six laboratories shared blinded RNA samples from Duchenne patient-derived muscle cells treated with different amounts of exon 51 targeting antisense oligonucleotide. Exon 51 skipping levels were quantified using five different techniques: digital droplet PCR, single PCR assessed with Agilent bioanalyzer, nested PCR with agarose gel image analysis by either ImageJ or GeneTools software and quantitative real-time PCR. Results: Differences in mean exon skipping levels and dispersion around the mean were observed across the different techniques. Results obtained by digital droplet PCR were reproducible and showed the smallest dispersion. Exon skipping quantification with the other methods showed overestimation of exon skipping or high data variation. Conclusions: Our results suggest that digital droplet PCR was the most precise and quantitative method. The quantification of exon 51 skipping by Agilent bioanalyzer after a single round of PCR was the second-best choice with a 2.3-fold overestimation of exon 51 skipping levels compared to digital droplet PCR.
Original languageEnglish
Pages (from-to)1-15
Number of pages15
JournalPLoS ONE
Volume13
Issue number10
DOIs
Publication statusPublished - 2 Oct 2018

Fingerprint Dive into the research topics of 'A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures'. Together they form a unique fingerprint.

  • Impacts

    FDA approves first drug to treat Duchenne muscular dystrophy (DMD)

    Karen Anthony (Co-Investigator)

    Impact: Public policy impacts, Health and Well-Being impacts, Quality of life impacts, 03: Good Health and Well-Being (UN SDG)

    Activities

    • 2 Participating in a conference or workshop

    Final COST Action meeting

    Karen Anthony (Participant)

    22 Mar 2017

    Activity: Participating in or organising a conference or workshopParticipating in a conference or workshopResearch

    11th Annual Meeting of the Oligonucleotide Therapeutics Society

    Karen Anthony (Participant)

    11 Oct 201514 Oct 2015

    Activity: Participating in or organising a conference or workshopParticipating in a conference or workshopResearch

    Cite this

    Hiller, M., Falzarano, M. S., Garcia-Jimenez, I., Sardone, V., Verheul, R. C., Popplewell, L., Anthony, K., Ruiz-Del-Yerro, E., Osman, H., Goeman, J. J., Mamchaoui, K., Dickson, G., Ferlini, A., Muntoni, F., Aartsma-Rus, A., Arechavala-Gomeza, V., Datson, N. A., & Spitali, P. (2018). A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures. PLoS ONE, 13(10), 1-15. https://doi.org/10.1371/journal.pone.0204485