Novel Therapeutic Approaches for Recessive Dystrophic Epidermolysis Bullosa
The heritable blistering disorder recessive dystrophic epidermolysis bullosa (RDEB) is a devastating disease caused by loss of function mutations in the COL7A1 gene, encoding type VII collagen (C7). C7 is the main component of anchoring fibrils which tether the epidermis and dermis together. The main phenotypic features of these patients are skin fragility, and trauma induced blisters which heal with excessive scarring and fibrosis. The most severe complication in RDEB is the development of aggressive, rapidly metastasizing squamous cell carcinoma (SCC), which is fatal in most cases. Significant progress has been made in the past decade in testing various therapeutic approaches, but currently, there is no cure available and the standard of care is limited to managing and relieving the symptoms and prevention of blister erosions. This work focuses on two novel therapeutic approaches for RDEB, which address the main burdens of the disease: a) lack of functional C7, and b) mechanisms contributing to the severe fibrotic microenvironment in RDEB skin. We tested an FDA approved drug, amlexanox, for its efficacy to read-through premature termination codon mutations (PTCs) and recover full length C7 protein in patient derived keratinocytes and fibroblasts. We further showed functionality of the full length protein by assessment of its localization at the dermal-epidermal junction (DEJ) in 3D skin equivalents and the stability of its triple helix after secretion from the cell. The second part of this project investigated the mechanisms of thrombospondin 1 (TSP1) driven TGFβ signaling in RDEB. We identified TSP1 as a novel binding partner of C7 by three independent methods: (a) proximity ligation assay, (b) immunoprecipitation from cell lysates, and (c) real-time assay of binding interactions. We propose that lack of binding of these two proteins in RDEB due to loss of C7 promotes increased TGFβ signaling in RDEB cells. Transient knockdown of TSP1 in RDEB SCC and non-SCC fibroblasts downregulated phospho-Smad3 levels, used as read-out of TGFβ signaling, showing that TSP1 levels directly affect this pathway in RDEB. Inhibition of TSP1 activation of TGFβ in a novel cell derived matrix assay prevented fibrosis as evaluated by picrosirius red stain and collagen fiber thickness. These results suggest a novel mechanism of TGFβ activation in RDEB and we propose TSP1 as a potential pharmacologic target for fibrosis reduction in the tumor promoting microenvironment in patients with RDEB, a currently intractable disorder.
Molecular biology|Cellular biology
Atanasova, Velina S, "Novel Therapeutic Approaches for Recessive Dystrophic Epidermolysis Bullosa" (2018). ETD Collection for Thomas Jefferson University. AAI10812907.