Keratin 14-null cells as a model to test the efficacy of gene therapy approaches in epithelial cells.

[epidermolysis bullosa simplex]

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 (K 5 ) or K 14 . 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 K 14 -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 K 14 -null cells by supplementation with wild-type K 14 complementary DNA (cDNA ). The K 14 -null cells show elevated levels of stress correlating with reduced normal keratin function . By transfecting wild-type K 14 into these cells , we demonstrate "proof of principle " that an add-back approach can significantly rescue the normal keratinocyte behavior profile . These K 14 -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 .