title: A microinjected COL7A1-PAC vector restores synthesis of intact procollagen VII in a dystrophic epidermolysis bullosa keratinocyte cell line.
abstract: Dystrophic epidermolysis bullosa (DEB) comprises a family of inherited blistering skin disorders for which no corrective therapy currently exists. In the most severe form, the Hallopeau-Siemens subtype (RDEB-HS), the epidermal adhesion protein collagen VII is absent from the skin as a consequence of null mutations in the COL7A1 gene. In order to develop an ex vivo gene therapy approach for DEB, we aimed to restore expression of intact procollagen VII in RDEB-HS keratinocytes. The entire human COL7A1 locus in a P1-derived artificial chromosome (PAC) was transferred to RDEB-HS keratinocytes by microinjection, after which sustained biosynthesis and secretion of procollagen VII was detected for 1 year in vitro. Protein chemical analysis demonstrated that the chain composition, domain structure, N-glycosylation and protein folding of the newly produced procollagen VII were similar, if not identical, to its authentic counterpart, indicating that transgenic procollagen VII was structurally normal. These data demonstrate a "proof of principle" for genomic DNA vectors as a means of restoring collagen VII production in RDEB-HS skin and help develop future gene therapy protocols.
authors.affiliation: Department of Dermatology, University of Münster, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Mecklenbeck
authors.forename: Sarah H
authors.initials: SH
authors.lastname: Compton
authors.forename: Jose E
authors.initials: JE
authors.lastname: Mejía
authors.forename: Riccardo
authors.initials: R
authors.lastname: Cervini
authors.forename: Alain
authors.initials: A
authors.lastname: Hovnanian
authors.forename: Leena
authors.initials: L
authors.lastname: Bruckner-Tuderman
authors.forename: Yann
authors.initials: Y
authors.lastname: Barrandon
mesh_terms: D027042:Chromosomes, Artificial, P1 Bacteriophage / Q000235:genetics*; D024143:Collagen Type VII / Q000096:biosynthesis / Q000235:genetics*; D016108:Epidermolysis Bullosa Dystrophica / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D005822:Genetic Vectors / Q000008:administration & dosage; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D008845:Microinjections; D011347:Procollagen / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D024143:Collagen Type VII; D011347:Procollagen