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.