title: Self-inactivating MLV vectors have a reduced genotoxic profile in human epidermal keratinocytes.
abstract: Transplantation of epithelia derived from keratinocyte stem cells transduced by retroviral vectors is a potential therapy for epidermolysis bullosa (EB), a family of inherited skin adhesion defects. The biosafety characteristics of retroviral vectors in keratinocytes are, however, poorly defined. We developed self-inactivating (SIN) vectors derived from the Moloney murine leukemia (MLV) and the human immunodeficiency (HIV) viruses expressing therapeutic levels of LAMB3, a transgene defective in junctional EB, and tested their integration profile in human primary keratinocytes. The SIN-HIV vector showed the expected preference for transcribed genes while the SIN-MLV vector integrated preferentially in regulatory elements, but showed a significantly lower tendency to target cell growth-related genes, transcription start sites and epigenetically defined promoters compared with a wild-type MLV vector in an epithelial cell context. A quantitative gene expression assay in individual keratinocyte clones showed that MLV-derived vectors deregulate expression of targeted genes at a lower frequency than in hematopoietic cells, and that the SIN-MLV design has the lowest activity compared to both MLV and SIN-HIV vectors. This study indicates that SIN-MLV vectors may have a better safety profile in keratinocyte than in hematopoietic cells, and be a reasonable alternative to lentiviral vectors for gene therapy of inherited skin disorders.
authors.affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Cavazza
authors.forename: F
authors.initials: F
authors.lastname: Cocchiarella
authors.forename: C
authors.initials: C
authors.lastname: Bartholomae
authors.forename: M
authors.initials: M
authors.lastname: Schmidt
authors.forename: C
authors.initials: C
authors.lastname: Pincelli
authors.forename: F
authors.initials: F
authors.lastname: Larcher
authors.forename: F
authors.initials: F
authors.lastname: Mavilio
mesh_terms: D000818:Animals; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D004820:Epidermolysis Bullosa / Q000235:genetics* / Q000378:metabolism / Q000628:therapy*; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000502:physiology; D011401:Promoter Regions, Genetic; D041701:Swiss 3T3 Cells; D014161:Transduction, Genetic; D019076:Transgenes; D038301:Virus Inactivation; D016662:Virus Integration; D000097794:Kalinin
chemical_list: D015815:Cell Adhesion Molecules; D000097794:Kalinin