title: Functional correction of type VII collagen expression in dystrophic epidermolysis bullosa.
abstract: Functional defects in type VII collagen, caused by premature termination codons on both alleles of the COL7A1 gene, are responsible for the severe autosomal recessive types of the skin blistering disease, recessive dystrophic epidermolysis bullosa (RDEB). The full-length COL7A1 complementary DNA (cDNA) is about 9 kb, a size that is hardly accommodated by therapeutically used retroviral vectors. Although there have been successful attempts to produce functional type VII collagen protein in model systems of RDEB, the risk of genetic rearrangements of the large repetitive cDNA sequence may hamper the clinical application of full-length COL7A1 cDNA in the human system. Therefore, we used trans-splicing to reduce the size of the COL7A1 transcript. Retroviral transduction of RDEB keratinocytes with a 3' pre-trans-splicing molecule resulted in correction of full-length type VII collagen expression. Unlike parental RDEB keratinocytes, transduced cells displayed normal morphology and reduced invasive capacity. Moreover, transduced cells showed normal localization of type VII collagen at the basement membrane zone in skin equivalents, where it assembled into anchoring fibril-like structures. Thus, using trans-splicing we achieved correction of an RDEB phenotype in vitro, which marks an important step toward its application in gene therapy in vivo.
authors: Division of Molecular Dermatology and EB House Austria, Department of Dermatology, Paracelsus Medical University, Salzburg, Austria. e.murauer@salk.at
authors: Eva M
authors: EM
authors: Murauer
authors: Yannick
authors: Y
authors: Gache
authors: Iris K
authors: IK
authors: Gratz
authors: Alfred
authors: A
authors: Klausegger
authors: Wolfgang
authors: W
authors: Muss
authors: Christina
authors: C
authors: Gruber
authors: Guerrino
authors: G
authors: Meneguzzi
authors: Helmut
authors: H
authors: Hintner
authors: Johann W
authors: JW
authors: Bauer
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D001706:Biopsy; D002478:Cells, Cultured; D018389:Codon, Nonsense / Q000235:genetics*; D024143:Collagen Type VII / Q000235:genetics*; D016108:Epidermolysis Bullosa Dystrophica / Q000235:genetics / Q000473:pathology / Q000628:therapy; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D015603:Keratinocytes / Q000473:pathology / Q000502:physiology; D009924:Organ Culture Techniques; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D018389:Codon, Nonsense; D024143:Collagen Type VII; D012333:RNA, Messenger