{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,8]],"date-time":"2026-07-08T07:27:34Z","timestamp":1783495654887,"version":"3.55.0"},"reference-count":43,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2020,10,18]],"date-time":"2020-10-18T00:00:00Z","timestamp":1602979200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000925","name":"National Health and Medical Research Council","doi-asserted-by":"publisher","award":["APP1144791"],"award-info":[{"award-number":["APP1144791"]}],"id":[{"id":"10.13039\/501100000925","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The COL7A1 gene encodes homotrimer fibrils essential for anchoring dermal and epidermal layers, and pathogenic mutations in COL7A1 can cause recessive or dominant dystrophic epidermolysis bullosa. As a monogenic disease gene, COL7A1 constitutes a potential target for antisense oligomer-mediated exon skipping, a therapy applicable to a growing number of other genetic disorders. However, certain characteristics of COL7A1: many exons, low average intron size, and repetitive and guanine-cytosine rich coding sequence, present challenges to the design of specific and effective antisense oligomers. While targeting COL7A1 exons 10 and 73 for excision from the mature mRNA, we discovered that antisense oligomers comprised of 2\u2032-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers produced similar, but distinctive, splicing patterns including excision of adjacent nontargeted exons and\/or retention of nearby introns in some transcripts. We found that the nonsequential splicing of certain introns may alter pre-mRNA processing during antisense oligomer-mediated exon skipping and, therefore, additional studies are required to determine if the order of intron removal influences multiexon skipping and\/or intron retention in processing of the COL7A1 pre-mRNA.<\/jats:p>","DOI":"10.3390\/ijms21207705","type":"journal-article","created":{"date-parts":[[2020,10,18]],"date-time":"2020-10-18T21:26:06Z","timestamp":1603056366000},"page":"7705","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Nonsequential Splicing Events Alter Antisense-Mediated Exon Skipping Outcome in COL7A1"],"prefix":"10.3390","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7036-5334","authenticated-orcid":false,"given":"Kristin A.","family":"Ham","sequence":"first","affiliation":[{"name":"Centre for Molecular Medicine and Innovative Therapeutics, Health Futures Institute, Murdoch University, Murdoch 6150, Australia"},{"name":"Perron Institute for Neurological and Translational Science, Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Nedlands 6009, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6379-2303","authenticated-orcid":false,"given":"May Thandar","family":"Aung-Htut","sequence":"additional","affiliation":[{"name":"Centre for Molecular Medicine and Innovative Therapeutics, Health Futures Institute, Murdoch University, Murdoch 6150, Australia"},{"name":"Perron Institute for Neurological and Translational Science, Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Nedlands 6009, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8632-641X","authenticated-orcid":false,"given":"Sue","family":"Fletcher","sequence":"additional","affiliation":[{"name":"Centre for Molecular Medicine and Innovative Therapeutics, Health Futures Institute, Murdoch University, Murdoch 6150, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steve D.","family":"Wilton","sequence":"additional","affiliation":[{"name":"Centre for Molecular Medicine and Innovative Therapeutics, Health Futures Institute, Murdoch University, Murdoch 6150, Australia"},{"name":"Perron Institute for Neurological and Translational Science, Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Nedlands 6009, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,10,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"553","DOI":"10.1111\/j.1600-0625.2008.00723.x","article-title":"Mutation analysis and characterization of COL7A1 mutations in dystrophic epidermolysis bullosa","volume":"17","author":"Dang","year":"2008","journal-title":"Exp. Dermatol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/j.det.2009.10.020","article-title":"Dystrophic epidermolysis bullosa: Pathogenesis and clinical features","volume":"28","year":"2010","journal-title":"Dermatol. Clin."},{"key":"ref_3","first-page":"611","article-title":"Adeno-associated virus (AAV) dual vector strategies for gene therapy encoding large transgenes","volume":"90","author":"McClements","year":"2017","journal-title":"Yale J. Biol. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1006\/geno.1994.1239","article-title":"Structural organization of the human type VII collagen gene (COL7A1), composed of more exons than any previously characterized gene","volume":"21","author":"Christiano","year":"1994","journal-title":"Genomics"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1302","DOI":"10.1038\/mt.2016.92","article-title":"Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy","volume":"24","author":"Bornert","year":"2016","journal-title":"Mol. Ther. J. Am. Soc. Gene Ther."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"551","DOI":"10.1083\/jcb.131.2.551","article-title":"Immunohistochemical and mutation analyses demonstrate that procollagen VII is processed to collagen VII through removal of the NC-2 domain","volume":"131","year":"1995","journal-title":"J. Cell Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1016\/S0923-1811(99)00018-3","article-title":"Hereditary skin diseases of anchoring fibrils","volume":"20","year":"1999","journal-title":"J. Dermatol. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1577","DOI":"10.1083\/jcb.103.4.1577","article-title":"Type VII collagen is a major structural component of anchoring fibrils","volume":"103","author":"Sakai","year":"1986","journal-title":"J. Cell Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"398","DOI":"10.1046\/j.1523-1747.1999.00518.x","article-title":"Clustering of COL7A1 mutations in exon 73: Implications for mutation analysis in dystrophic epidermolysis bullosa","volume":"112","author":"Mecklenbeck","year":"1999","journal-title":"J. Investig. Dermatol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1136\/jmg.2006.045302","article-title":"Epidermolysis bullosa. II. Type VII collagen mutations and phenotype\u2013genotype correlations in the dystrophic subtypes","volume":"44","author":"Varki","year":"2007","journal-title":"J. Med. Genet."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2387","DOI":"10.1016\/j.jid.2016.07.029","article-title":"Targeted Exon Skipping Restores Type VII Collagen Expression and Anchoring Fibril Formation in an In Vivo RDEB Model","volume":"136","author":"Turczynski","year":"2016","journal-title":"J. Investig. Dermatol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3568","DOI":"10.1093\/nar\/gkg616","article-title":"ESEfinder: A web resource to identify exonic splicing enhancers","volume":"31","author":"Cartegni","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_13","first-page":"6211","article-title":"Order of intron removal during splicing of endogenous adenine phosphoribosyltransferase and dihydrofolate reductase pre-mRNA","volume":"13","author":"Kessler","year":"1993","journal-title":"Mol. Cell Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3017","DOI":"10.1016\/S0140-6736(16)31408-8","article-title":"Treatment of infantile-onset spinal muscular atrophy with nusinersen: A phase 2, open-label, dose-escalation study","volume":"388","author":"Finkel","year":"2016","journal-title":"Lancet"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Frank, D.E., Schnell, F.J., Akana, C., El-Husayni, S.H., Desjardins, C.A., Morgan, J., Charleston, J.S., Sardone, V., Domingos, J., and Dickson, G. (2020). Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy. Neurology.","DOI":"10.1212\/WNL.0000000000009233"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1002\/ana.24555","article-title":"Longitudinal effect of eteplirsen versus historical control on ambulation in Duchenne muscular dystrophy","volume":"79","author":"Mendell","year":"2016","journal-title":"Ann. Neurol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"baw153","DOI":"10.1093\/database\/baw153","article-title":"GeneBase 1.1: A tool to summarize data from NCBI gene datasets and its application to an update of human gene statistics","volume":"2016","author":"Piovesan","year":"2016","journal-title":"Database"},{"key":"ref_18","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":"Gazzoli","year":"2016","journal-title":"RNA Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"10376","DOI":"10.3390\/ijms160510376","article-title":"Identification and validation of evolutionarily conserved unusually short pre-mRNA introns in the human genome","volume":"16","author":"Shimada","year":"2015","journal-title":"Int. J. Mol. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1038\/ng0295-184","article-title":"The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced","volume":"9","author":"Tennyson","year":"1995","journal-title":"Nat. Genet."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Jonkers, I., Kwak, H., and Lis, J.T. (2014). Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons. eLife, 3.","DOI":"10.7554\/eLife.02407"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1312","DOI":"10.1038\/nprot.2008.112","article-title":"PCR-amplification of GC-rich regions: \u2018slowdown PCR\u2019","volume":"3","author":"Frey","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3340","DOI":"10.1093\/nar\/26.14.3340","article-title":"Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR","volume":"26","author":"McDowell","year":"1998","journal-title":"Nucleic Acids Res."},{"key":"ref_24","first-page":"403","article-title":"Basic local alignment search tool","volume":"215","author":"Altschul","year":"1990","journal-title":"J. Med. Biochem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"S12","DOI":"10.1186\/1471-2105-11-S12-S12","article-title":"The MOLGENIS toolkit: Rapid prototyping of biosoftware at the push of a button","volume":"11","author":"Swertz","year":"2010","journal-title":"BMC Bioinform."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1100","DOI":"10.1002\/humu.21551","article-title":"The international dystrophic epidermolysis bullosa patient registry: An online database of dystrophic epidermolysis bullosa patients and their COL7A1 mutations","volume":"32","author":"Jonkman","year":"2011","journal-title":"Human Mutat."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1002\/humu.21651","article-title":"The COL7A1 mutation database","volume":"33","author":"Kowalewski","year":"2012","journal-title":"Hum. Mutat."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Bornert, O., Hogervorst, M., Nauroy, P., Bischof, J., Swildens, J., Athanasiou, I., Tufa, S.F., Keene, D.R., Kiritsi, D., and Hainzl, S. (2020). QR-313, an antisense oligonucleotide, shows therapeutic efficacy for treatment ofdominant and recessive dystrophic epidermolysis bullosa: A preclinical study. J. Investig. Dermatol.","DOI":"10.1016\/j.jid.2020.08.018"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Flynn, L.L., Ruohan, L., Aung-Htut, M.T., Pitout, I.L., Cooper, J., Hubbard, A., Griffiths, L., Bond, C., Wilton, S.D., and Fox, A.H. (2018). Interaction of modified oligonucleotides with nuclear proteins, formation of novel nuclear structures and sequence-independent effects on RNA processing. bioRxiv, 446773.","DOI":"10.1101\/446773"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Adams, A.M., Harding, P.L., Iversen, P.L., Coleman, C., Fletcher, S., and Wilton, S.D. (2007). Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: Cocktails and chemistries. BMC Mol. Biol., 8.","DOI":"10.1186\/1471-2199-8-57"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Aung-Htut, M.T., McIntosh, C.S., West, K.A., Fletcher, S., and Wilton, S.D. (2019). In vitro validation of phosphorodiamidate morpholino oligomers. Molecules, 24.","DOI":"10.3390\/molecules24162922"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2853","DOI":"10.1093\/nar\/gkaa126","article-title":"Comparison of the efficacy of MOE and PMO modifications of systemic antisense oligonucleotides in a severe SMA mouse model","volume":"48","author":"Sheng","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e379","DOI":"10.1038\/mtna.2016.87","article-title":"Antisense Oligonucleotide-Mediated Exon Skipping as a Systemic Therapeutic Approach for Recessive Dystrophic Epidermolysis Bullosa","volume":"5","author":"Bremer","year":"2016","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/j.csbj.2014.09.008","article-title":"The supraspliceosome\u2014A multi-task machine for regulated pre-mRNA processing in the cell nucleus","volume":"11","author":"Shefer","year":"2014","journal-title":"Comput. Struct. Biotechnol. J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1016\/j.omtn.2019.09.009","article-title":"Natural exon skipping sets the stage for exon skipping as therapy for dystrophic epidermolysis bullosa","volume":"18","author":"Bremer","year":"2019","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1111\/j.1346-8138.2010.01008.x","article-title":"Exon 87 skipping of the COL7A1 gene in dominant dystrophic epidermolysis bullosa","volume":"38","author":"Koga","year":"2011","journal-title":"J. Dermatol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1046\/j.1523-1747.1999.00709.x","article-title":"Moderation of phenotypic severity in dystrophic and junctional forms of epidermolysis bullosa through in-frame skipping of exons containing non-sense or frameshift mutations","volume":"113","author":"McGrath","year":"1999","journal-title":"J. Investig. Dermatol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"e934","DOI":"10.1111\/j.1365-2230.2009.03254.x","article-title":"A novel de novo splice-site mutation in the COL7A1 gene in dominant dystrophic epidermolysis bullosa (DDEB): Specific exon skipping could be a prognostic factor for DDEB pruriginosa","volume":"34","author":"Saito","year":"2009","journal-title":"Clin. Exp. Dermatol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1308","DOI":"10.1111\/bjd.13945","article-title":"A COL7A1 variant leading to in-frame skipping of exon 15 attenuates disease severity in recessive dystrophic epidermolysis bullosa","volume":"173","author":"Weibel","year":"2015","journal-title":"Br. J. Dermatol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1288","DOI":"10.1038\/sj.mt.6300095","article-title":"Antisense oligonucleotide-induced exon skipping across the human dystrophin gene transcript","volume":"15","author":"Wilton","year":"2007","journal-title":"Mol. Ther. J. Am. Soc. Gene Ther."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Aung-Htut, M., McIntosh, C., Ham, K., Pitout, I., Flynn, L., Greer, K., Fletcher, S., and Wilton, S. (2019). Systematic Approach to Developing Splice Modulating Antisense Oligonucleotides. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20205030"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"642","DOI":"10.2144\/97224bm14","article-title":"Bandstab: A PCR-based alternative to cloning PCR products","volume":"22","author":"Wilton","year":"1997","journal-title":"Biotechniques"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"W43","DOI":"10.1093\/nar\/gkm234","article-title":"OligoCalc: An online oligonucleotide properties calculator","volume":"35","author":"Kibbe","year":"2007","journal-title":"Nucleic Acids Res."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/20\/7705\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:23:29Z","timestamp":1760178209000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/20\/7705"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,10,18]]},"references-count":43,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2020,10]]}},"alternative-id":["ijms21207705"],"URL":"https:\/\/doi.org\/10.3390\/ijms21207705","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,10,18]]}}}