RAAV2-mediated restoration of LEKTI in LEKTI-deficient cells from Netherton patients

Background: Netherton syndrome (NS, MIM 256500) is a potential live threatening autosomal-recessive skin disorder clinically characterized by the trias of congenital erythroderma, hair shaft anomalies and atopic diathesis. It is caused by mutations in the gene SPINK5 resulting in a deficiency of its processed protein named lympho-epithelial Kazal-type related inhibitor (LEKTI). LEKTI controls the activity of several serine proteases in the skin that are involved in terminal differentiation. Loss of LEKTI results in protease hyperactivity, increased degradation of intercellular junctions, reduced stratum corneum adhesion and impaired skin barrier function. Today NS can only be treated symptomatically. Objective: Does gene transfer offer a therapeutic option for NS in the future? Methods: A recombinant adeno-associated virus type 2 vector was constructed containing the full length cDNA (rAAV2/C-SPINK5) of functional human LEKTI. Infectious virus particles were used for transfection of LEKTI-deficient-keratinocytes of NS patients in vitro. Results: Gene transfer of SPINK5 in NS-keratinocytes led to a five-fold increase in mRNA expression of SPINK5 reaching almost 75% of normal value. The functionality of the expressed LEKTI was proven in a hydrolytic activity assay demonstrating that the activity of LEKTI after gene transfer increased closely to the level seen in keratinocytes of healthy individuals. Conclusion: The results provide first evidence that gene transfer of SPINK5 results in increased LEKTI activity in NS-keratinocytes, thus offering a rational to further pursue such a gene therapy approach for NS.