{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T18:34:22Z","timestamp":1790534062069,"version":"4.1.0"},"reference-count":35,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2020,12,23]],"date-time":"2020-12-23T00:00:00Z","timestamp":1608681600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008484","name":"BioMarin Pharmaceutical","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100008484","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the <jats:italic>DMD<\/jats:italic> gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52\/<jats:italic>mdx<\/jats:italic> mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human <jats:italic>DMD<\/jats:italic> genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human <jats:italic>DMD<\/jats:italic> exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the <jats:italic>hDMD<\/jats:italic> and <jats:italic>hDMDdel52<\/jats:italic> transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the <jats:italic>hDMD<\/jats:italic> and <jats:italic>hDMDdel52<\/jats:italic> transgene do not affect exon 51 and\/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52\/<jats:italic>mdx<\/jats:italic> mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52\/<jats:italic>mdx<\/jats:italic> mice and C57BL6\/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human <jats:italic>DMD<\/jats:italic> at both the functional and molecular level.<\/jats:p>","DOI":"10.1371\/journal.pone.0244215","type":"journal-article","created":{"date-parts":[[2020,12,23]],"date-time":"2020-12-23T19:04:41Z","timestamp":1608750281000},"page":"e0244215","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":33,"title":["Detailed genetic and functional analysis of the hDMDdel52\/mdx mouse model"],"prefix":"10.1371","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4787-7980","authenticated-orcid":true,"given":"Alper","family":"Yavas","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rudie","family":"Weij","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maaike","family":"van Putten","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eleni","family":"Kourkouta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chantal","family":"Beekman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jukka","family":"Puoliv\u00e4li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timo","family":"Bragge","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toni","family":"Ahtoniemi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeroen","family":"Knijnenburg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marlies Elisabeth","family":"Hoogenboom","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yavuz","family":"Ariyurek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Annemieke","family":"Aartsma-Rus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Judith","family":"van Deutekom","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicole","family":"Datson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2020,12,23]]},"reference":[{"issue":"10","key":"pone.0244215.ref001","doi-asserted-by":"crossref","first-page":"1049","DOI":"10.1038\/ejhg.2012.301","article-title":"Newborn bloodspot screening for Duchenne muscular dystrophy: 21 years experience in Wales (UK)","volume":"21","author":"SJ Moat","year":"2013","journal-title":"European journal of human genetics: EJHG"},{"issue":"3","key":"pone.0244215.ref002","first-page":"1223","article-title":"The Dystrophin Complex: Structure, Function, and Implications for Therapy","volume":"5","author":"QQ Gao","year":"2015","journal-title":"Compr Physiol"},{"issue":"39","key":"pone.0244215.ref003","first-page":"10992","volume":"113","author":"EP Rader","year":"2016","journal-title":"Role of dystroglycan in limiting contraction-induced injury to the sarcomeric cytoskeleton of mature skeletal muscle"},{"issue":"6","key":"pone.0244215.ref004","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1016\/j.nmd.2013.02.012","article-title":"Early neurodevelopmental assessment in Duchenne muscular dystrophy","volume":"23","author":"M Pane","year":"2013","journal-title":"Neuromuscular disorders: NMD"},{"issue":"6","key":"pone.0244215.ref005","first-page":"2471","article-title":"Recent advances in innovative therapeutic approaches for Duchenne muscular dystrophy: from discovery to clinical trials","volume":"8","author":"Y Shimizu-Motohashi","year":"2016","journal-title":"American journal of translational research"},{"issue":"8","key":"pone.0244215.ref006","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1038\/nrd3459","article-title":"Targeting RNA to treat neuromuscular disease","volume":"10","author":"F Muntoni","year":"2011","journal-title":"Nature reviews Drug discovery"},{"issue":"12","key":"pone.0244215.ref007","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1016\/S1474-4422(03)00585-4","article-title":"Dystrophin and mutations: one gene, several proteins, multiple phenotypes","volume":"2","author":"F Muntoni","year":"2003","journal-title":"The Lancet Neurology"},{"key":"pone.0244215.ref008","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1016\/j.neubiorev.2015.01.023","article-title":"A possible role of dystrophin in neuronal excitability: a review of the current literature","volume":"51","author":"RG Hendriksen","year":"2015","journal-title":"Neurosci Biobehav Rev"},{"issue":"2","key":"pone.0244215.ref009","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1002\/mus.20586","article-title":"Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule","volume":"34","author":"A Aartsma-Rus","year":"2006","journal-title":"Muscle & nerve"},{"issue":"4","key":"pone.0244215.ref010","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1002\/humu.22758","article-title":"The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations","volume":"36","author":"CL Bladen","year":"2015","journal-title":"Human mutation"},{"key":"pone.0244215.ref011","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1007\/978-1-4939-8651-4_10","article-title":"Creation of DMD Muscle Cell Model Using CRISPR-Cas9 Genome Editing to Test the Efficacy of Antisense-Mediated Exon Skipping","volume":"1828","author":"R Maruyama","year":"2018","journal-title":"Methods Mol Biol"},{"key":"pone.0244215.ref012","doi-asserted-by":"crossref","first-page":"534","DOI":"10.1016\/j.omtn.2018.10.002","article-title":"Shorter Phosphorodiamidate Morpholino Splice-Switching Oligonucleotides May Increase Exon-Skipping Efficacy in DMD","volume":"13","author":"U Akpulat","year":"2018","journal-title":"Mol Ther Nucleic Acids"},{"key":"pone.0244215.ref013","doi-asserted-by":"crossref","first-page":"53","DOI":"10.2147\/AHMT.S125739","article-title":"Duchenne and Becker muscular dystrophy in adolescents: current perspectives","volume":"9","author":"JG Andrews","year":"2018","journal-title":"Adolesc Health Med Ther"},{"issue":"7","key":"pone.0244215.ref014","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1038\/jhg.2016.28","article-title":"Deletion of exons 3\u20139 encompassing a mutational hot spot in the DMD gene presents an asymptomatic phenotype, indicating a target region for multiexon skipping therapy","volume":"61","author":"A Nakamura","year":"2016","journal-title":"Journal of human genetics"},{"issue":"4","key":"pone.0244215.ref015","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1212\/WNL.0000000000002891","article-title":"DMD genotypes and loss of ambulation in the CINRG Duchenne Natural History Study","volume":"87","author":"L Bello","year":"2016","journal-title":"Neurology"},{"issue":"3","key":"pone.0244215.ref016","first-page":"122","article-title":"Genetic diagnosis as a tool for personalized treatment of Duchenne muscular dystrophy","volume":"35","author":"L Bello","year":"2016","journal-title":"Acta myologica: myopathies and cardiomyopathies: official journal of the Mediterranean Society of Myology"},{"issue":"3","key":"pone.0244215.ref017","first-page":"293","volume":"30","author":"A Aartsma-Rus","year":"2009","journal-title":"Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations"},{"key":"pone.0244215.ref018","doi-asserted-by":"crossref","first-page":"533","DOI":"10.2147\/DDDT.S97635","article-title":"Eteplirsen in the treatment of Duchenne muscular dystrophy","volume":"11","author":"KR Lim","year":"2017","journal-title":"Drug design, development and therapy"},{"issue":"8","key":"pone.0244215.ref019","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1038\/s41582-018-0033-8","article-title":"Why dystrophin quantification is key in the eteplirsen saga","volume":"14","author":"A Aartsma-Rus","year":"2018","journal-title":"Nature reviews Neurology"},{"key":"pone.0244215.ref020","doi-asserted-by":"crossref","DOI":"10.1089\/nat.2020.0845","article-title":"The 10th Oligonucleotide Therapy Approved: Golodirsen for Duchenne Muscular Dystrophy","author":"A Aartsma-Rus","year":"2020","journal-title":"Nucleic Acid Ther"},{"issue":"1","key":"pone.0244215.ref021","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1089\/nat.2018.0756","article-title":"A Sequel to the Eteplirsen Saga: Eteplirsen Is Approved in the United States but Was Not Approved in Europe","volume":"29","author":"A Aartsma-Rus","year":"2019","journal-title":"Nucleic Acid Ther"},{"issue":"10","key":"pone.0244215.ref022","doi-asserted-by":"crossref","first-page":"1027","DOI":"10.1007\/s40265-020-01339-3","article-title":"Viltolarsen: First Approval","volume":"80","author":"S Dhillon","year":"2020","journal-title":"Drugs"},{"key":"pone.0244215.ref023","unstructured":"FDA NEWS RELEASE. FDA Approves Targeted Treatment for Rare Duchenne Muscular Dystrophy Mutation. 2020. https:\/\/www.fda.gov\/news-events\/press-announcements\/fda-approves-targeted-treatment-rare-duchenne-muscular-dystrophy-mutation."},{"issue":"3","key":"pone.0244215.ref024","first-page":"195","volume":"8","author":"JW McGreevy","year":"2015","journal-title":"Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy"},{"issue":"4912","key":"pone.0244215.ref025","first-page":"1578","volume":"244","author":"P Sicinski","year":"1989","journal-title":"The molecular basis of muscular dystrophy in the mdx mouse: a point mutation"},{"issue":"9","key":"pone.0244215.ref026","doi-asserted-by":"crossref","first-page":"5899","DOI":"10.1074\/jbc.M709410200","article-title":"Generation and characterization of transgenic mice with the full-length human DMD gene","volume":"283","author":"PA t Hoen","year":"2008","journal-title":"J Biol Chem"},{"issue":"2","key":"pone.0244215.ref027","doi-asserted-by":"crossref","first-page":"e0193289","DOI":"10.1371\/journal.pone.0193289","article-title":"A dystrophic Duchenne mouse model for testing human antisense oligonucleotides","volume":"13","author":"M Veltrop","year":"2018","journal-title":"PLoS One"},{"key":"pone.0244215.ref028","article-title":"Generation of embryonic stem cells and mice for duchenne research","volume":"5","author":"M Veltrop","year":"2013","journal-title":"PLoS Curr"},{"issue":"4","key":"pone.0244215.ref029","doi-asserted-by":"crossref","first-page":"e0195850","DOI":"10.1371\/journal.pone.0195850","article-title":"Use of capillary Western immunoassay (Wes) for quantification of dystrophin levels in skeletal muscle of healthy controls and individuals with Becker and Duchenne muscular dystrophy","volume":"13","author":"C Beekman","year":"2018","journal-title":"PLoS One"},{"issue":"4","key":"pone.0244215.ref030","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method","volume":"25","author":"KJ Livak","year":"2001","journal-title":"Methods"},{"issue":"9","key":"pone.0244215.ref031","doi-asserted-by":"crossref","first-page":"e0162467","DOI":"10.1371\/journal.pone.0162467","article-title":"Digital Droplet PCR for the Absolute Quantification of Exon Skipping Induced by Antisense Oligonucleotides in (Pre-)Clinical Development for Duchenne Muscular Dystrophy","volume":"11","author":"RC Verheul","year":"2016","journal-title":"PLoS One"},{"issue":"3","key":"pone.0244215.ref032","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1080\/15476286.2015.1125074","article-title":"Non-sequential and multi-step splicing of the dystrophin transcript","volume":"13","author":"I Gazzoli","year":"2016","journal-title":"RNA biology"},{"issue":"1","key":"pone.0244215.ref033","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1089\/nat.2019.0824","article-title":"Using a State-of-the-Art Toolbox to Evaluate Molecular and Functional Readouts of Antisense Oligonucleotide-Induced Exon Skipping in mdx Mice","volume":"30","author":"NA Datson","year":"2020","journal-title":"Nucleic Acid Ther"},{"issue":"1","key":"pone.0244215.ref034","first-page":"1","volume":"29","year":"2019","journal-title":"Cell-Penetrating Peptide Conjugates of Steric Blocking Oligonucleotides as Therapeutics for Neuromuscular Diseases from a Historical Perspective to Current Prospects of Treatment"},{"issue":"2","key":"pone.0244215.ref035","doi-asserted-by":"crossref","first-page":"139","DOI":"10.3233\/JND-170218","article-title":"Creation of a Novel Humanized Dystrophic Mouse Model of Duchenne Muscular Dystrophy and Application of a CRISPR\/Cas9 Gene Editing Therapy","volume":"4","author":"CS Young","year":"2017","journal-title":"J Neuromuscul Dis"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0244215","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2020,12,23]],"date-time":"2020-12-23T19:05:34Z","timestamp":1608750334000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0244215"}},"subtitle":[],"editor":[{"given":"Atsushi","family":"Asakura","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2020,12,23]]},"references-count":35,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2020,12,23]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0244215","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,12,23]]}}}