1. ¹Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy
2. ²Veneto Eye Bank Foundation, Venice, Italy
3. ³Epithelial Biomedicine Division, CIEMAT-CIBERER (Centre for Biomedical Research on Rare Diseases), Madrid, Spain

Correspondence: Fulvio Mavilio, Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy. E-mail: fulvio.mavilio@unimore.it

The first two authors contributed equally to this work.

Received 11 August 2007; Accepted 27 August 2008; Published online 23 September 2008.

Abstract

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer–restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.