Junctional epidermolysis bullosa (JEB) is an inherited mechanobullous disease characterized by reduced adherence of the epidermal keratinocytes to the underlying dermis, and is often caused by the absence of functional
laminin 332 due to the lack or dysfunction of its beta3 chain. As there are no specific
therapies for JEB, we tested whether a
protein replacement strategy could be applicable for the restoration of the
laminin 332 assembly and reversion of the JEB phenotype in human keratinocytes that lack beta3 subunit. Here, we developed the protocol for production and purification of the biologically active recombinant beta3 chain. Next, we demonstrated that delivery of recombinant beta3
polypeptide into the endoplasmic reticulum of the immortalized beta3-null keratinocytes led to the restoration of the
laminin 332 assembly, secretion, and deposition into the basement membrane zone, as confirmed by Western blot analysis, confocal immunofluorescent microscopy in vitro, and on cultured organotypic human JEB skin reconstructs. Although the amount of
laminin 332 produced by
protein-treated beta3-null keratinocytes is lower than that in normal human keratinocytes, our results demonstrate the applicability of the
recombinant proteins for JEB treatment and open new perspectives for the development of novel
therapeutics for this inherited, currently intractable, skin disorder.