Development of a novel biotherapy targeting inflammatory pathways in Netherton syndrome – TARGET-NS

Netherton Syndrome (NS) is an orphan genetic skin disease causing severe skin inflammation and scaling. We previously identified SPINK5 (serine protease inhibitor Kazal type 5) encoding LEKTI as being the defective NS gene. We recently identified by comparative mRNA sequencing, increased expression of a major pro-inflammatory pathway driven by a specific cytokine (which we will call A1) in murine models and in adult NS patient skin. We hypothesize that this pathway plays a central role in driving skin inflammation in NS and that its invalidation or pharmacological neutralization could rescue the disease phenotype.

This project aims to decipher the molecular and cellular basis of A1 signalling in NS pathogenesis using murine models and NS patients. The objectives are to uncover the contribution of A1 to skin inflammation, to determine the impact of KLKs activity on activation of A1 and to evaluate the blockade of the cytokine A pathway as a novel strategy capable to reverse skin inflammation in NS.This project brings together 2 complementary scientists (AH and ML) and 2 clinicians (EB and LW) who have a strong interest in NS and/or in murine models for inflammatory skin diseases.

WP1 will investigate the A1 signature in NS skin and the role of KLKs on biological activity of A1 in a 3D epidermal model for NS (AH, EB and LW). A cohort of adult and paediatric NS patients followed by EB and LW, respectively, will be characterized. Clinical features will be recorded during flares, extensive sera analysis with Luminex, mRNAseq from skin biopsies and PBMC phenotypic analysis will be performed. A 3D-epidermal model for NS (3D-NSKC) will be used to assess whether overexpression of cytokine A1 is an intrinsic characteristic of NS keratinocytes. The effects of cytokine A on a 3D-epidermal model made of normal keratinocytes will be compared to 3D-NSKC. Finally, the impact of KLKs on proteolytic activation of cytokine A1 and the effect of KLK inhibition or invalidation by CRISP/Cas9 will be studied.

WP2 will explore the role of the A1 pathway in NS murine models and will delineate its cellular and molecular mechanisms (AH and ML). The Spink5 inducible knock-out (ko) model (generated by AH in collaboration with ML) will be crossed with an existing ko murine model for the cytokine A1 receptor (provided by a Biopharma) to generate inducible double ko mice. These animals will be carefully characterized to evaluate the effect of ablation of cytokine A1 receptor on the NS phenotype. The existing TghKLK5 (generated by AH) will be crossed with the ko mouse for cytokine A1 receptor to assess the effects the receptor deletion onto the phenotype. We will also decipher the functional redundancy and specificity of cytokine A family in NS pathogenesis by generating mice which are defective for cytokine A1 and cytokine A2 (same cluster) (generated by ML) and for cytokine A1 only. Each of these mice will be crossed with the inducible Spink5 ko mice to analyse the effects on the NS phenotype.

In WP3, we will develop therapeutic strategies to block the cytokine A1 pathway in NS murine models. We will use intraperitoneal injections of an existing cytokine A receptor neutralizing antibody (provided by the same Biopharma) to treat inducible Spink5 ko mice for 21 days. Careful characterization of these mice will investigate whether the neutralizing antibody can attenuate the NS phenotype (AH). Other experiments will use topical calcipotriol (+/-betamethasone) to block the cytokine A pathway (as recently shown by ML) to attenuate the NS phenotype in inducible Spink5 mice (ML).

This project could represent a major step towards an effective treatment for this life threatening orphan skin disease with very high unmet medical needs. Rapid clinical translation towards a clinical trial would be highly feasible because of an existing highly efficient neutralizing antibody for cytokine A1 receptor developed for humans by a Biopharma which shows interest in NS.