Journal of Investigative Dermatology - Keratin 14-Null Cells as a Model to Test the Efficacy of Gene Therapy Approaches in Epithelial Cells

full-text ► Journal of Investigative Dermatology - Keratin 14-Null Cells as a Model to Test the Efficacy of Gene Therapy Approaches in Epithelial Cells: "Original Article

Subject Category: Cell Biology

Journal of Investigative Dermatology (2011) 131, 1412–1419; doi:10.1038/jid.2011.19; published online 17 February 2011
Keratin 14-Null Cells as a Model to Test the Efficacy of Gene Therapy Approaches in Epithelial Cells

Mariella D'Alessandro1, Stephanie E Coats1, Marcel F Jonkmann2, Irene M Leigh3 and E Birgitte Lane1,4

1. 1CR UK Cell Structure Research Group, Division of Molecular Medicine, College of Life Sciences, University of Dundee, Dundee, UK
2. 2Center for Blistering Diseases, Department of Dermatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
3. 3College of Medicine, Dentistry and Nursing, University of Dundee, Dundee, UK
4. 4Institute of Medical Biology, Singapore, Singapore

Correspondence: Mariella D'Alessandro, Division of Molecular Medicine, College of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK. E-mail: m.dalessandro@dundee.ac.uk

Received 28 June 2010; Revised 23 November 2010; Accepted 12 January 2011; Published online 17 February 2011.

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.

Abbreviations:
cDNA, complementary DNA; DM-EBS, Dowling–Meara EBS; EBS, epidermolysis bullosa simplex; JNK, c-Jun N-terminal kinase; K, keratin; SAPK, stress-activated protein kinase

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