title: A novel screening system improves genetic correction by internal exon replacement.
abstract: Trans-splicing is a powerful approach to reprogram the genome. It can be used to replace 5', 3' or internal exons. The latter approach has been characterized by low efficiency, as the requirements to promote internal trans-splicing are largely uncharacterized. The trans-splicing process is induced by engineered 'RNA trans-splicing molecules' (RTMs), which target a selected pre-mRNA to be reprogrammed via two complementary binding domains. To facilitate the development of more efficient RTMs for therapeutic applications we constructed a novel fluorescence based screening system. We incorporated exon 52 of the COL17A1 gene into a GFP-based cassette system as the target exon. This exon is mutated in many patients with the devastating skin blistering disease epidermolysis bullosa. In a double transfection assay we were able to rapidly identify optimal binding domains targeted to sequences in the surrounding introns 51 and 52. The ability to replace exon 52 was then evaluated in a more endogenous context using a target containing COL17A1 exon 51-intron 51-exon 52-intron 52-exon 53. Two selected RTMs produced significantly higher levels of GFP expression in up to 61% assayed cells. This novel approach allows for rapid identification of efficient RTMs for internal exon replacement.
authors.affiliation: Division of Molecular Dermatology and EB House Austria, Department of Dermatology, Paracelsus Medical University, 5020 Salzburg, Austria. u.koller@salk.at
authors.forename: Ulrich
authors.initials: U
authors.lastname: Koller
authors.forename: Verena
authors.initials: V
authors.lastname: Wally
authors.forename: Lloyd G
authors.initials: LG
authors.lastname: Mitchell
authors.forename: Alfred
authors.initials: A
authors.lastname: Klausegger
authors.forename: Eva M
authors.initials: EM
authors.lastname: Murauer
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mayr
authors.forename: Christina
authors.initials: C
authors.lastname: Gruber
authors.forename: Stefan
authors.initials: S
authors.lastname: Hainzl
authors.forename: Helmut
authors.initials: H
authors.lastname: Hintner
authors.forename: Johann W
authors.initials: JW
authors.lastname: Bauer
mesh_terms: D001324:Autoantigens / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D005091:Exons; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D024041:Non-Fibrillar Collagens / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D020040:Trans-Splicing; D000097805:Collagen Type XVII
chemical_list: D001324:Autoantigens; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D024041:Non-Fibrillar Collagens; D012333:RNA, Messenger; D000097805:Collagen Type XVII