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Netherton syndrome (NS) is a rare autosomal recessive skin disorder caused by mutations in SPINK5 . It is a debilitating condition with notable mortality in the early years of life. There is no curative treatment. We undertook a nonrandomized, open-label, feasibility, and safety study using autologous keratinocytes transduced with a lentiviral vector encoding SPINK5 under the control of the human involucrin promoter. Six NS subjects were recruited, and gene-modified epithelial sheets were successfully generated in three of five subjects. The sheets exhibited expression of correctly sized lympho-epithelial Kazal-type-related inhibitor (LEKTI) protein after modification. One subject was grafted with a 20 cm 2 gene-modified graft on the left anterior thigh without any adverse complications and was monitored by serial sampling for 12 months. Recovery within the graft area was compared against an area outside by morphology, proviral copy number and expression of the SPINK5 encoded protein, LEKTI, and its downstream target kallikrein 5, which exhibited transient functional correction. The study confirmed the feasibility of generating lentiviral gene-modified epidermal sheets for inherited skin diseases such as NS, but sustained LEKTI expression is likely to require the identification, targeting, and engraftment of long-lived keratinocyte stem cell populations for durable therapeutic effects. Important learning points for the application of gene-modified epidermal sheets are discussed.

Netherton syndrome (NS) is a serious inherited skin disorder caused by mutations in the serine protease inhibitor Kazal type 5 gene (SPINK5), which encodes for a serine protease inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI). Patients with NS have defective keratinization, hair shaft defects, recurrent infections, atopy, and a predisposition to skin malignancies. Historically, 1 in 10 infants has died before their first birthday. Currently, there are no proven treatments to cure this condition. A SIN-lentiviral vector encoding the codon-optimized SPINK5 gene under the control of a 572 bp element derived from the human involucrin promoter can confer compartment-specific LEKTI expression in NS keratinocytes with restoration of normal skin architecture. Here we detail a study protocol for a phase I trial for feasibility and safety evaluations of autologous epidermal sheets generated from ex vivo gene-corrected keratinocyte stem cells, which will be grafted onto patients with mutation-proven NS.