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- Title
811. Correction of Laminin-5 β3 Chain Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors.
- Authors
Di Nunzio, Francesca; Maruggi, Giulietta; Ferrari, Stefano; del Rio, Marcela; Larcher, Fernando; De Luca, Michele; Mavilio, Fulvio
- Abstract
Mutations in any of the genes encoding the laminin 5 heterotrimer (α3, β3 and γ2) cause junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. We and others have shown that expression of a retrovirally transferred β3-chain cDNA in keratinocytes from affected patients reconstitutes normal synthesis, assembly and secretion of laminin 5, and corrects the adhesion defect in vitro and in vivo. We have recently started a phase-I clinical trial of gene therapy of JEB based on transplantation of cultured skin derived from autologous epidermal stem cells transduced with a MLV-derived retroviral vector. Since gamma- retroviral vectors have raised safety concerns for the genotoxic risk associated with the insertion of LTR elements into the human genome, we developed an alternative gene transfer strategy based on LTR- modified, HIV-derived lentiviral vectors. Two self-inactivating (SIN) lentiviral vectors were built, in which expression of either GFP or a LAMB3 cDNA is under the control of either a constitutive promoter (PGK) or the keratinocyte-specific, 2.2-kb promoter-enhancer of keratin 14 (K14). In a third construct, expression of the transgene is under the control of the viral LTR, modified by replacing the U3 region with two K14 enhancer elements. Analysis in human keratinocyte cultures and in full-thickness human skin equivalents reconstituted onto immunodeficient mice showed that GFP expression directed by the K14 elements is tissue-specific and restricted to the basal layer of the epidermis. Expression of laminin5 from the three alternative vectors was evaluated in keratinocyte cultures derived from skin biopsies of JEB patients. Biochemical and cell kinetics assays demonstrated transduction of epidermal clonogenic stem/progenitor cells and full phenotypic correction of JEB keratinocytes with all vectors. Southern blot analysis of individual cell clones showed that LTR-modified lentiviral vectors are genetically stable and integrate in multiple copies in the human genome. This study shows that the use of lentiviral vectors transcriptionally targeted to the basal keratinocytes by the insertion of restricted enhancer elements is an effective, and potentially safer, alternative for gene therapy of JEB.Molecular Therapy (2006) 13, S314–S315; doi: 10.1016/j.ymthe.2006.08.896
- Subjects
STEM cells; KERATINOCYTES; HUMAN gene mapping; GENE therapy; MICROBIAL genetics
- Publication
Molecular Therapy, 2006, Vol 13, pS314
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.896