Cystatin M/E knockdown by lentiviral delivery of shRNA impairs epidermal morphogenesis of human skin equivalents

The protease inhibitor cystatin M/E (CST6) regulates a biochemical pathway involved in stratum corneum homeostasis, and its deficiency in mice causes ichthyosis and neonatallethality. Cystatin M/E deficiency has not been described in humans so far, and we did not detect disease-causing mutations in the CST6 gene in a large number of patients with autosomal recessive congenital ichthyosis, who were negative for mutations in known ichthyosisassociated genes. To investigate the phenotype of CST6 deficiency in human epidermis, we used lentiviral delivery of short hairpin RNAs that target CST6 in a 3D reconstructed skin model. Surprisingly, CST6 deficiency did not cause an ichthyosis-like phenotype, butprevented the development of a multilayered epidermis. From this study, we conclude that CST6 deficiency may be incompatible with normal human foetal development. Abbreviations: ARCI, autosomal recessive congenital ichthyosis; CST6, cystatin M/E; CTSL, cathepsin L; CTSV, cathepsin V; IFAP, ichthyosis follicularis with atrichia and photophobia; KP, keratosis pilaris; LGMN, legumain; LI, lamellar ichthyosis; qPCR, real-time quantitative PCR; shRNA, short hairpin RNA