Correction of laminin-5 deficiency in human epidermal stem cells by transcriptionally targeted retroviral vectors

Deficiency of the basement membrane component lami- nin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autol- ogous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and cor- rects 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 expres- sion 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 immunodefi- cient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentivi- ral vectors efficiently transduced clonogenic stem/pro- genitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratino- cytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodefi- cient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.