title: Construction and validation of an RNA trans-splicing molecule suitable to repair a large number of COL7A1 mutations.
abstract: RNA trans-splicing has become a versatile tool in the gene therapy of monogenetic diseases. This technique is especially valuable for the correction of mutations in large genes such as COL7A1, which underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa. Over 800 mutations spanning the entire length of the COL7A1 gene have been associated with defects in type VII collagen, leading to excessive fragility of epithelial tissues, the hallmark of dystrophic epidermolysis bullosa (DEB). In the present study, we designed an RNA trans-splicing molecule (RTM) that is capable of repairing any given mutation within a 4200 nucleotide region spanning the 3' half of COL7A1. The selected RTM, RTM28, was able to induce accurate trans-splicing into endogenous COL7A1 pre-mRNA transcripts in a type VII collagen-deficient DEB patient-derived cell line. Correct trans-splicing was detected at the RNA level by semiquantitative RT-PCR and correction of full-length type VII collagen was confirmed at the protein level by immunofluorescence and western blot analyses. Our results demonstrate that RTM28, which covers >60% of all mutations reported in DEB and is thus the longest RTM described so far for the repair of COL7A1, represents a promising candidate for therapeutic applications.
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: B
authors.initials: B
authors.lastname: Tockner
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: T
authors.initials: T
authors.lastname: Kocher
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Hainzl
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: J
authors.initials: J
authors.lastname: Reichelt
authors.affiliation: Department of Dermatology, University Hospital, Paracelsus Medical University, Salzburg, Austria.
authors.forename: J W
authors.initials: JW
authors.lastname: Bauer
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: U
authors.initials: U
authors.lastname: Koller
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: E M
authors.initials: EM
authors.lastname: Murauer
mesh_terms: D002478:Cells, Cultured; D024143:Collagen Type VII / Q000235:genetics* / Q000378:metabolism; D004820:Epidermolysis Bullosa / Q000628:therapy*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020040:Trans-Splicing
chemical_list: C569053:COL7A1 protein, human; D024143:Collagen Type VII; D012333:RNA, Messenger