rAAV2-mediated restoration of LEKTI in LEKTI-deficient cells from Netherton patients

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Abstract

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.

Introduction

Netherton syndrome (NS, MIM 256500) is an autosomal-recessive skin disorder clinically characterized by congenital erythroderma, trichorrhexis invaginata (bamboo hair), and atopic diathesis. It accounts for up to 18% of congenital erythrodermas [1], which can be life-threatening in the neonatal period due to severe complications such as hypernatremic dehydration, hypothermia, extreme weight loss, bronchopneumonia, recurrent bacterial infections and sepsis [2].

NS is caused by genetic defects in the gene SPINK5 (serine protease inhibitor kazal-type 5) [3], [4], [5], [6]. SPINK5 is located on chromosome 5q32 and encodes serine protease inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor). The protein LEKTI is a 15-domain Kazal-type related serine protease inhibitor [7], [8]. In the epidermis, LEKTI is confined to the granular layer and stratum corneum [6], [8] where it co-localizes with kallikreins (KLK) 5 and 7 [9]. Several domains of LEKTI have shown to inhibit KLK 5 and 7 [10] that are involved in the degradation of corneodesmosomes such as desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1) [11]. Normally, the interplay between the inhibitory pressure of LEKTI and the proteolytic activity of KLK 5 und 7 is well-balanced leading to a controlled desquamation process and an intact skin barrier.

In NS, mutations result in a complete or severe loss of LEKTI and subsequently a reduced inhibition of serine proteases. The enhanced activity of KLK 5 and 7 induce premature degradation of corneodesmosomal cadherins as demonstrated by a dramatically reduction of Dsg1 and Dsc1 [11], [12]. This mechanism has been suggested to result in loss of stratum corneum integrity and severe skin barrier defect.

Various therapeutic options have been used in NS to reduce clinical symptoms, among them topical steroids, calcipotriene, calcineurin-inhibitors, topical and systemic retinoids, PUVA, and intravenous immunoglobulin all with variable and only temporary success [13], [14]. Today, no causal and permanent treatment for NS does exist, although the severity and continuity of the diseases are demanding new developments. Herein, we describe the first developmental steps towards a genetic treatment approach for NS.

Section snippets

Cell culture

Human primary NS-keratinocytes were isolated from punch biopsies derived from 4 NS patients after informed consent had been given; normal human keratinocytes (NHK) came from circumcised foreskin (provided by Dr. Thomas A. Angerpointer, Pediatric Surgery Practice, Munich, Germany). The specimens were processed according to a standard protocol described previously [15], [16], [17]. Briefly, skin was cleaned of fat, cut into small piece (if necessary) and incubated overnight at 4 °C in thermolysin

Netherton patients

Keratinocytes of four patients with typical clinical presentation of NS were available for the study. The underlying genetic defects are summarized in Table 1. Mutation c.316delAG and c.1432-13 G > A had been published previously [6], [9]. All mutations resulted in premature termination of SPINK5. In addition all demonstrated reduced mRNA expression of SPINK5, and increase hydrolytic activity consistent with a deficiency of LEKTI.

Increased expression of SPINK5 in Netherton keratinocytes following gene transfer of SPINK5

In the skin, SPINK5 expression is known to be restricted to the

Discussion

Netherton syndrome is a genetic disorder with severe and chronic skin manifestations that can currently only be treated in a symptomatic way. There are two promising molecular concepts at the horizon that might offer causal therapeutic benefit – protein replacement therapy and gene therapy. In theory, protein replacement by means of topical application of recombinant LEKTI might allow LEKTI to permeate the impaired skin barrier of NS patients and become active at the granular layer and thus

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by grants of the Wilhelm-Sander Stiftung (2003.117.1), Munich, Germany. Work presented in this publication was performed as part of the doctoral thesis of D.R.

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