204 Epidermodysplasia verruciformis: Clinical, viral, and histopathological phenotype in patients with EVER3 mutation

Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disorder caused by mutations in COL7A1 that encodes for the anchoring fibrils of type VII collagen. Our lab has demonstrated that non-viral gene delivery by polymer is able to restore the expression of type VII collagen (C7) to human RDEB skin and could overcome the possibility of generating an immune response when compared to viral vectors. Silencing caused by viral promoters is a major problem for an episomal non-viral gene therapy. To increase the safety profile of COL7A1 therapy more focus needs to be in the DNA cassette itself as an important tool for resisting transgene silencing and extending C7 expression. The feasibility of this approach was demonstrated using a novel gene delivery polymer as a non-viral vector combined with minicircle (MC) DNA encoding full-length COL7A1 under the control of three different promoters, including human COL7A1 promoter (C7p) as a tissue-specific promoter, human elongation factor one alpha (EF1a) and cytomegalovirus (CMV). The three MC plasmid (MC-C7p-C7, MC-EF1a-C7 and MC-CMV-C7) were used to transfect human RDEB Keratinocytes (K) and Fibroblasts (F). Type VII collagen expression was detected and compared among the three promoters and confirmed in in vitro at the gene level using RT-qPCR as well as at the protein level using immuno-blotting. All the three MC constructs are capable of achieving expression levels comparable to native protein from healthy human primary cells, there is an indication of a 20 fold C7 expression change detected in transfected RDEBF cells compared to un-transfected cells. A multifaceted analysis of the promoters expression is being performed. In conclusion, our results suggest that an optimal promoter selection is an important enhancement that can be added to the minicircle vector in our non-viral system and therefore will enhance transgene expression and help to reduce immuneimmunogenicity.