153 Characterization of interstitial collagenase MMP1 as a candidate gene for gene therapy in psoriasis

Abstract

Recessive dystrophic epidermolysis bullosa (RDEB), a rare skin blistering disorder, is caused by loss-of-function mutations in the gene encoding type VII collagen (C7), COL7A1. The main phenotype in RDEB is compromised skin architecture and fragility, resulting in constant wounding and excessive scarring. Nonfunctional C7 results in defective anchoring fibrils, which provide adhesion between the epidermis and dermis. In this study we are interested in premature termination codon (PTC) mutations in COL7A1. PTCs are associated with mRNA instability and nonsense mediated mRNA decay (NMD) as well as early termination of protein translation, thus leading to non-functional protein. We evaluate a read-through approach for overcoming PTC mutations in COL7A1 as potential therapy for RDEB. Reading-through PTCs allows for insertion of an amino acid at the mutation site and synthesis of full-length protein. Several drugs have been reported for their read-through ability but their efficacy is gene dependent. Here we evaluate an FDA approved drug-amlexanox-and demonstrate that it induces full-length C7 synthesis in patient derived RDEB keratinocytes and fibroblasts in a codon sequence specific manner. From six mutant alleles tested, four responded to amlexanox treatment. Treated RDEB cells showed 3-to 14-fold increase of C7 synthesis by western blot and 2-to 5-fold upregulation of COL7A1 mRNA levels, measured by qPCR. In our system read-through correlated with UPF1 phosphorylation state e the main factor in NMD. In conclusion, our study shows that amlexanox induces PTC read-through and synthesis of fulllength C7 in RDEB cells and upregulates COL7A1 transcript levels. Our results suggest amlexanox as potential therapy for RDEB patients harboring PTC mutations.