title: Correction of laminin-5 deficiency in human epidermal stem cells by transcriptionally targeted lentiviral vectors.
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.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204.
authors.affiliation: Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Di Nunzio
authors.forename: Giulietta
authors.initials: G
authors.lastname: Maruggi
authors.forename: Stefano
authors.initials: S
authors.lastname: Ferrari
authors.forename: Enzo
authors.initials: E
authors.lastname: Di Iorio
authors.forename: Valentina
authors.initials: V
authors.lastname: Poletti
authors.forename: Marta
authors.initials: M
authors.lastname: Garcia
authors.forename: Marcela
authors.initials: M
authors.lastname: Del Rio
authors.forename: Michele
authors.initials: M
authors.lastname: De Luca
authors.forename: Fernando
authors.initials: F
authors.lastname: Larcher
authors.forename: Graziella
authors.initials: G
authors.lastname: Pellegrini
authors.forename: Fulvio
authors.initials: F
authors.lastname: Mavilio
mesh_terms: D000818:Animals; D015815:Cell Adhesion Molecules / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D006262:Health; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D013234:Stem Cells / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*; D000097794:Kalinin
chemical_list: D015815:Cell Adhesion Molecules; D000097794:Kalinin