title: Keratin 14-null cells as a model to test the efficacy of gene therapy approaches in epithelial cells.
abstract: Skin fragility disorders caused by keratin mutations are incurable, and a better understanding of their etiology is needed to find new ways to improve and treat these conditions. The best-studied skin fragility disorder is epidermolysis bullosa simplex (EBS), an autosomal dominant condition caused by mutations in keratin 5 (K5) or K14. To analyze disease mechanisms and develop gene therapy strategies, we have used keratinocyte cell lines derived from EBS patients as model systems. Here, we describe two cell lines established from EBS patients with K14-null mutations. We analyze the responses of these cells to stress assays previously shown to discriminate between wild-type and keratin-mutant keratinocytes, to directly evaluate the efficacy of rescuing K14-null cells by supplementation with wild-type K14 complementary DNA (cDNA). The K14-null cells show elevated levels of stress correlating with reduced normal keratin function. By transfecting wild-type K14 into these cells, we demonstrate "proof of principle" that an add-back approach can significantly rescue the normal keratinocyte behavior profile. These K14-null cell lines provide a disease model for studying the effects of keratin ablation in EBS patients and to test the efficacy of gene add-back and other therapy approaches in keratinocytes.
authors.affiliation: CR UK Cell Structure Research Group, Division of Molecular Medicine, College of Life Sciences, University of Dundee, Dundee, UK. m.dalessandro@dundee.ac.uk
authors.forename: Mariella
authors.initials: M
authors.lastname: D'Alessandro
authors.forename: Stephanie E
authors.initials: SE
authors.lastname: Coats
authors.forename: Marcel F
authors.initials: MF
authors.lastname: Jonkman
authors.forename: Marcel F
authors.initials: MF
authors.lastname: Jonkmann
authors.forename: Irene M
authors.initials: IM
authors.lastname: Leigh
authors.forename: E Birgitte
authors.initials: EB
authors.lastname: Lane
mesh_terms: D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D016110:Epidermolysis Bullosa Simplex / Q000235:genetics / Q000628:therapy*; D015316:Genetic Therapy; D018869:Heat-Shock Response; D006801:Humans; D053547:Keratin-14 / Q000235:genetics* / Q000502:physiology; D015603:Keratinocytes / Q000502:physiology*; D009154:Mutation; D013312:Stress, Physiological; D014162:Transfection
chemical_list: C507540:KRT14 protein, human; D053547:Keratin-14