title: A reporter-based screen to identify potent 3' trans-splicing molecules for endogenous RNA repair.
abstract: In the treatment of genetic disorders, repairing defective pre-mRNAs by RNA trans-splicing has become an emerging alternative to conventional gene therapy. Previous studies have made clear that the design of the binding domains of the corrective RNA trans-splicing molecules (RTMs) is crucial for their optimal functionality. We established a reporter-based screening method that allows for selection of highly functional RTMs from a large pool of variants. The efficiency and functionality of the screen were validated in the COL7A1 gene, in which mutations are the cause of the skin disease dystrophic epidermolysis bullosa. Comparison of RTMs containing different binding domains hybridizing to COL7A1 intron 64/exon 65 revealed highly different trans-splicing efficiencies. Isolated RTMs were then adapted for endogenous trans-splicing in a recessive dystrophic epidermolysis bullosa (RDEB) keratinocyte cell line expressing reduced levels of COL7A1 mRNA. Our results confirm the applicability and relevance of prescreening reporter RTMs, as significant levels of endogenous COL7A1 mRNA repair were seen with RTMs identified as being highly efficient in our screening system.
authors: Division of Molecular Dermatology and EB House Austria, Department of Dermatology, Paracelsus Medical University, Salzburg 5020, Austria.
authors: Eva M
authors: EM
authors: Murauer
authors: Ulrich
authors: U
authors: Koller
authors: Stefan
authors: S
authors: Hainzl
authors: Verena
authors: V
authors: Wally
authors: Johann W
authors: JW
authors: Bauer
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D024143:Collagen Type VII / Q000235:genetics / Q000378:metabolism; D016108:Epidermolysis Bullosa Dystrophica / Q000235:genetics / Q000503:physiopathology; D005091:Exons; D005434:Flow Cytometry; D005808:Genes, Recessive; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D020040:Trans-Splicing / Q000235:genetics*; D014162:Transfection
chemical_list: C569053:COL7A1 protein, human; D024143:Collagen Type VII; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins