570 Skipping to Improve Scar Management: Modulation of TGF-β Signalling in Hypertrophic Scars Via Exon Skipping

In burn patients wound healing is often accompanied by hypertrophic scar (HTS) development, resulting in both functional and aesthetic problems. HTS’s are characterized by abundant presence of myofibroblasts, which contribute to excessive production of extracellular matrix (ECM) components. The TGF-β signalling pathway plays a key role in the differentiation and activity of the myofibroblasts. Previous studies have shown that inhibition of TGF-β receptors in fibrotic diseases, such as Dupuytren’s disease, results in a significant reduction of the fibrotic load. In this study we have investigated the effects of exon skipping using antisense oligonucleotides (AON’s) to inactivate ALK5 (TGF-beta receptor I) in HTS’s. HTS biopsies were used to set up fibroblast monocultures and an ex vivo model. In order to induce exon skipping, AONs targeting ALK5 were supplemented to fibroblast monocultures or injected in HTS tissue. Chemical inhibition was performed with the ALK5 inhibitor SB431542. Analysis of TGF-β signalling downstream targets and collagens was performed by qPCR, touchdown PCR, immunohistochemistry and western blot. Our data demonstrate that 1) exon skipping of ALK5 was successful in HTS fibroblast monocultures, 2) exon skipping affects the expression of ECM-related genes in monolayers of HTS derived fibroblasts and, 3) chemical inhibition of ALK5 in HTS ex vivo tissue reduces the expression of ECM- related components. Moreover, preliminary data on the effect of exon skipping on fibroblast differentiation suggests that this technique could lead to a less severe phenotype in HTS tissue. In conclusion, exon skipping is a promising tool in order to modulate the TGF-β signalling pathway and, thereby, the expression of ECM components in HTS.