title: Human involucrin promoter mediates repression-resistant and compartment-specific LEKTI expression.
abstract: Gene-modified skin grafts, produced through gene transfer to human keratinocyte stem cells, offer the possibility of therapeutic benefit for inherited skin diseases. We have previously described efficient lentiviral vector-mediated gene transfer to keratinocyte stem cells and the generation of human skin grafts for the inherited skin disease, Netherton syndrome, which arises due to mutations in serine protease inhibitor Kazal-type 5 (SPINK5). Vectors incorporating an internal murine retroviral-derived promoter [spleen focus-forming virus (SFFV)] in combination with a codon-optimized SPINK5 transgene supported high levels of reconstitution and robust correction of skin architecture. Subsequent longer-term experiments have uncovered unanticipated silencing phenomena, with loss of SPINK5 gene expression over time. The inadvertent introduction of CpG sites during codon optimization appears to have rendered vectors susceptible to silencing due to methylation across the promoter-transgene boundary. Substitution of the methylation-susceptible SFFV promoter with a 572-bp minimal human involucrin promoter (INVOp), which encodes very few CpG sites, prevented repression of the SPINK5 transgene and resulted in durable and highly compartment-specific reconstitution of lympho-epithelial Kazal-type-related inhibitor (LEKTI) in human skin grafted onto immunodeficient mice. We conclude that skin grafts modified with lentiviral vectors encoding INVOp offer a suitable platform for therapeutic gene therapy in Netherton syndrome, and our experience highlights unanticipated effects of transgene codon optimization.
authors: Immunobiology, UCL Institute of Child Health , London, WC1N 1EH, UK. w.di@ich.ucl.ac.uk
authors: Wei-Li
authors: WL
authors: Di
authors: Ekaterina
authors: E
authors: Semenova
authors: Fernando
authors: F
authors: Larcher
authors: Marcela
authors: M
authors: Del Rio
authors: John I
authors: JI
authors: Harper
authors: Adrian J
authors: AJ
authors: Thrasher
authors: Waseem
authors: W
authors: Qasim
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D053491:Proteinase Inhibitory Proteins, Secretory / Q000235:genetics / Q000378:metabolism*; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D012867:Skin / Q000166:cytology / Q000378:metabolism; D016038:Skin Transplantation / Q000379:methods*; D019076:Transgenes
chemical_list: D003062:Codon; D011498:Protein Precursors; D053491:Proteinase Inhibitory Proteins, Secretory; C120650:SPINK5 protein, human; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; C032253:involucrin