Epidermal proteins as drugs for topical application to the skin
: potential treatments for TGM1 and ALOX12B mutations in Autosomal Recessive Congenital Ichthyosis

  • Sari Thomforde

Student thesis: Doctoral Thesis


Autosomal Recessive Congenital Ichthyoses (singular ‘sis’) (ARCI) are a group of rare, non-syndromic skin diseases with varying degrees of severity characterised by scaling/cracking of the skin and erythema, often presenting with a collodion membrane at birth. There are many known mutations in different genes however approximately 50% of ARCI patients have mutations in TGM1, encoding for transglutaminase 1 (TG1) and ALOX12B, encoding for arachidonate 12-lipoxygenase, 12R type (12R-LOX). These two proteins are required in the assembly of the cornified lipid envelope, essential in skin barrier function and the regulation of transepidermal water loss. There is no cure for the condition; only symptomatic treatments exist to manage ARCI phenotype, through lengthy bathing sessions, creams, and oral retinoids. There is an ethical desire to move away from animal modelling where possible, and animal modelling systems for human skin therapies are often incompatible due to differences between orthologs and preferred substrates. TG1 protein replacement therapy (PRT) was tested as a treatment capable of mitigating or reversing the ARCI phenotype by using full-thickness human skin equivalents constructed with control and TGM1 -/- human keratinocytes (hK) and fibroblasts. A thermoresponsive nanogel (tNG) was used to facilitate TG1 delivery through the stratum corneum and into the viable epidermis. Using various assays to analyse barrier function of equivalents with and without TG1 treatment and with and without tNG coupling, it was shown barrier function was significantly improved only in TG1-deficient skin equivalents treated with TG1 plus tNG, comparable to that of the controls. As ALOX12B mutations make up a significant proportion of ARCI patients, it was hypothesised a similar approach could work for 12R-LOX PRT. The first consideration was to develop a stable knockdown (KD) hK line for ALOX12B for use in preliminary investigations. The second consideration was to develop a method to quantifiably measure 12R-LOX activity in hKs using high performance liquid chromatography (HPLC). A stable KD for ALOX12B was successfully achieved using lentiviral transduction for RNAi, and HPLC analysis of the metabolite +/-12-HETE in cell lysate incubated with arachidonic acid (AA) yielded inconsistent results. Chiral HPLC for 12R-HETE remains a possibility that warrants further investigation. Analysis of a commercially available human recombinant 12R-LOX enzyme appeared to demonstrate a loss of active protein when incubated with AA and analysed for +/-12-HETE using HPLC, therefore future 12R-LOX production, purification and storage of an active protein must also be achieved for further testing of PRT for ALOX12B mutations. Collaborations with labs with expertise in chiral HPLC and protein production and purification for therapeutics would be desired as a way to expand on limitations of these findings and further explore the use of topical PRT for other ARCI mutations.
Date of Award8 Jan 2024
Original languageEnglish
SupervisorBarbara Conway (Main Supervisor)

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