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 journalArticleResearchpeer-review

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

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Duchenne Muscular Dystrophy
Antisense Oligonucleotides
Exons
Cell Culture Techniques
Polymerase Chain Reaction
Dystrophin
Frameshift Mutation
Sepharose
Muscle Cells
Real-Time Polymerase Chain Reaction
Software
Gels
RNA

Cite this

Hiller, Monika ; Falzarano, Maria Sofia ; Garcia-Jimenez, Iker ; Sardone, Valentina ; Verheul, Ruurd C ; Popplewell, Linda ; Anthony, Karen ; Ruiz-Del-Yerro, Estibaliz ; Osman, Hana ; Goeman, Jelle J ; Mamchaoui, Kamel ; Dickson, George ; Ferlini, Alessandra ; Muntoni, Francesco ; Aartsma-Rus, Annemieke ; Arechavala-Gomeza, Virginia ; Datson, Nicole A ; Spitali, Pietro. / A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures. In: PLoS ONE. 2018 ; Vol. 13, No. 10. pp. 1-15.
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title = "A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures",
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.",
author = "Monika Hiller and Falzarano, {Maria Sofia} and Iker Garcia-Jimenez and Valentina Sardone and Verheul, {Ruurd C} and Linda Popplewell and Karen Anthony and Estibaliz Ruiz-Del-Yerro and Hana Osman and Goeman, {Jelle J} and Kamel Mamchaoui and George Dickson and Alessandra Ferlini and Francesco Muntoni and Annemieke Aartsma-Rus and Virginia Arechavala-Gomeza and Datson, {Nicole A} and Pietro Spitali",
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Hiller, M, Falzarano, MS, Garcia-Jimenez, I, Sardone, V, Verheul, RC, Popplewell, L, Anthony, K, Ruiz-Del-Yerro, E, Osman, H, Goeman, JJ, Mamchaoui, K, Dickson, G, Ferlini, A, Muntoni, F, Aartsma-Rus, A, Arechavala-Gomeza, V, Datson, NA & 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, vol. 13, no. 10, pp. 1-15. https://doi.org/10.1371/journal.pone.0204485

A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures. / Hiller, Monika; Falzarano, Maria Sofia; Garcia-Jimenez, Iker; Sardone, Valentina; Verheul, Ruurd C; Popplewell, Linda; Anthony, Karen; Ruiz-Del-Yerro, Estibaliz; Osman, Hana; Goeman, Jelle J; Mamchaoui, Kamel; Dickson, George; Ferlini, Alessandra; Muntoni, Francesco; Aartsma-Rus, Annemieke; Arechavala-Gomeza, Virginia; Datson, Nicole A; Spitali, Pietro.

In: PLoS ONE, Vol. 13, No. 10, 02.10.2018, p. 1-15.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures

AU - Hiller, Monika

AU - Falzarano, Maria Sofia

AU - Garcia-Jimenez, Iker

AU - Sardone, Valentina

AU - Verheul, Ruurd C

AU - Popplewell, Linda

AU - Anthony, Karen

AU - Ruiz-Del-Yerro, Estibaliz

AU - Osman, Hana

AU - Goeman, Jelle J

AU - Mamchaoui, Kamel

AU - Dickson, George

AU - Ferlini, Alessandra

AU - Muntoni, Francesco

AU - Aartsma-Rus, Annemieke

AU - Arechavala-Gomeza, Virginia

AU - Datson, Nicole A

AU - Spitali, Pietro

PY - 2018/10/2

Y1 - 2018/10/2

N2 - 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.

AB - 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.

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