Netherton Syndrome (NS) is a rare autosomal recessive skin disorder caused by mutations in the gene encoding Serine Protease Inhibitor Kazal-type 5 (SPINK5), a protease inhibitor expressed in the stratum corneum of the epidermis. In NS patients, the inhibitory activities of SPINK5 are abolished, triggering hyperactivity of epidermal proteases which leads to skin dryness, hair shaft defects, elevated IgE levels, chronic skin inflammation and scaling that predisposes the patient to life-threatening infections. Currently, there are no curative treatments for NS, and novel targeted therapeutics are necessitated. To this end, we have developed KB104, a replication-defective HSV-1 gene therapy vector encoding human SPINK5 for direct topical application to the skin. Here we show that KB104 efficiently transduces target skin cells in vitro and in vivo and produces functional SPINK5. Human keratinocytes infected with KB104 at varying multiplicities of infection produced and secreted full-length SPINK5 in a dose-dependent manner, as determined by real time PCR, immunocytochemistry, ELISA, and western blotting. Importantly, the KB104-expressed SPINK5 was functional, being capable of inhibiting a native target of SPINK5, the serine protease Kallikrein 5, in an enzymatic inhibition assay. KB104 also efficiently expressed human SPINK5 after either topical or intradermal administration in immunocompetent mice, as assessed by real time PCR and immunohistochemistry. Human SPINK5 co-localized with mouse filaggrin, a protein expressed in the stratum corneum, indicating that KB104 successfully transduced the targeted epidermal layer. Moreover, no inflammatory infiltration or structural changes were observed at the KB104-treated site, confirming that KB104 is well-tolerated. Taken together, these preclinical studies support the potential for KB104 to be a convenient, safe, and efficacious gene therapy vector for direct molecular correction of SPINK5 deficiency in Netherton Syndrome.(33%) by all investigators. The concordance rate was 60% (free-marginal k¼0.59, moderate agreement). Non-blaschkoid lesions primarily coursed perpendicular to or lacked the curvilinear nature of Blaschko's lines. These results suggest that factors beyond segmental mosaicism must underlie linear morphea patterning. Further analysis is planned to gain consensus and seek "morpheatomes" that better characterize disease patterning.