title: Trans-splicing improvement by the combined application of antisense strategies.
abstract: Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.
authors.affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. u.koller@salk.at.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Koller
authors.affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. s.hainzl@salk.at.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hainzl
authors.affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. t.kocher@salk.at.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kocher
authors.affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. c.huettner@salk.at.
authors.forename: Clemens
authors.initials: C
authors.lastname: Hüttner
authors.affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. a.klausegger@salk.at.
authors.forename: Alfred
authors.initials: A
authors.lastname: Klausegger
authors.affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. c.gruber@salk.at.
authors.forename: Christina
authors.initials: C
authors.lastname: Gruber
authors.affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. el.mayr@salk.at.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mayr
authors.affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. v.wally@salk.at.
authors.forename: Verena
authors.initials: V
authors.lastname: Wally
authors.affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. jo.bauer@salk.at.
authors.forename: Johann W
authors.initials: JW
authors.lastname: Bauer
authors.affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. e.murauer@salk.at.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Murauer
mesh_terms: D024143:Collagen Type VII / Q000235:genetics / Q000378:metabolism; D004820:Epidermolysis Bullosa / Q000235:genetics / Q000473:pathology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007438:Introns; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D020040:Trans-Splicing; D014162:Transfection
chemical_list: D024143:Collagen Type VII; D016376:Oligonucleotides, Antisense; D049452:Green Fluorescent Proteins