Topical application of proteins as a new therapy option

The aim of our project is an efficient and targeted transport of functional proteins into the viable epidermis using innovative drug delivery systems. This approach aims to allow a local substitution of proteins that are endogenously missing or defective because of a genetic defect. The project investigates a novel therapeutic approach for the treatment of autosomal recessive congenital ichthyosis (ARCI) as an example for severe genetic skin diseases. As for today, the treatment of these diseases is exclusively symptomatic and the underlying cause is not addressed. In the first project period, funded by the DFG, the applicants have demonstrated successfully that a topical application of proteins is possible using microneedles, polyglycerol (PG)-based nanogels, or hyaluronic acid (HA) hydrogels. Through targeted inactivation of the gene coding for transglutaminase 1 (TGM1), we have generated disease skin models for ARCI in vitro, which are characterized by a disturbed skin barrier function. Most interestingly, the skin barrier function of TGM1-deficient disease models was successfully reconstituted following the topical application of the enzyme transglutaminase 1 (TGase-1) that was loaded onto nanogels. Based on these results we are confident that locally substituted proteins are functional in the viable epidermis and can compensate for the defect of disease-associated genes. In the present project we will focus on PG-based nanogels as protein transport system, which are characterized by high biocompatibility, high loading efficiencies, easy modifications, and switchability. We will use human reconstituted skin for these experiments since these models have several advantages, for instance, certain interspecies related problems relevant to the use of animal models are avoided. Moreover, we want to demonstrate that human skin models are suitable preclinical test systems and can be used for comprehensive drug testing in vitro. To this end, we will also generate less well characterized proteins and develop methods to determine their specific activity in cultured cells and tissues. Aside from cellular interactions, the cellular localization, biological availability, and fate of topically applied proteins in the skin are important for the therapeutic outcome and potential toxic effects: Do the proteins accumulate in the cells or does redistribution occur in the tissue? What does protein substitution mean for the skin homeostasis? To answer these questions, we will concentrate on further enzymes involved in sphingolipid metabolism of the skin, in particular 12R-LOX, eLOX-3, and CerS3. Mutations in their genes, ALOX12B, ALOXE3, and CERS3, can also be associated with ARCI, and they lead to disturbed epidermal barrier function and altered epidermal ceramide profiles. The effects of these proteins will be investigated in skin (disease) models, which are analysed for gene expression and lipid composition, and characterized regarding morphological changes and epidermal barrier function.

Autosomal recessive congenital ichthyoses (ARCI) are a group of severe genetic skin diseases that can be seen from birth. They are characterised by extensive scaling and variably severe redness of the whole skin. All types of congenital ichthyosis are associated with an impairment of the epidermal barrier function, which results in disturbance of the cutaneous water homeostasis and reduced protecting activities of the epidermis against adverse environmental effects. ARCI can be caused by mutations in various genes, among them TGM1, which codes for transglutaminase 1. Transglutaminase 1 is an enzyme of the outer skin layers and critical for the formation of the horny layer, which represents the protective barrier function of the skin. Therapies for congenital ichthyoses are currently insufficient, especially because causative therapies are not available although the role of transglutaminase 1 has been described more than 10 years ago. Transglutaminase 1 was synthesised in the laboratory in this project (recombinant transglutaminase 1) and used to establish a protein replacement therapy. The enzyme should be used topically, i.e. it should be applied directly onto the affected skin where it is supposed to be active. The transfer of active components through the horny layer of the skin is very limited even in the skin of people affected with congenital ichthyosis. Therefore, novel nanotransporters were used that are able to package the protein and transfer it into the metabolically active layers of the epidermis. Laboratory models of the skin were developed for these experiments that replicate the phenotype of congenital ichthyosis. For this purpose, either cells isolated from skin samples of affected persons were used or normal skin cells that were genetically manipulated to inactivate the gene TGM1 specifically. It was shown that the external, transferred protein is present in the viable layers of the epidermis and enzymatically active. The barrier function, which is characteristically disturbed in patients, was restored within two weeks in a cultured skin model. This was demonstrated with penetration experiments according to OECD guidelines using a range of substances. The permeability was strongly increased in disease models and was reduced to almost normal levels upon repeated treatment. These experiments showed that the external enzyme was effective in the cells as expected for the normal, intrinsic protein. Initial experiments concerning a potential toxicity of nanotransporters or the recombinant protein did not indicate any adverse effects. These results are promising for the development of a causative therapy for congenital ichthysis and, similarly, other genetic diseases of the epidermis. In next experiments the toxicity of the approach has to be assessed in more detail and the effective dose of the protein has to be determined. Clinical tests of the novel treatment will be initiated if these results are positive.