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 keratinocGene-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.[+][-]
