Design and Development of Minicircle Vectors with Human Promoters for Non-viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa

Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare, inherited, skin blistering disorder caused by mutations in COL7A1 that encodes for type VII collagen (C7). Gene replacement therapy offers an attractive therapeutic approach to treating RDEB. The use of non-viral delivery systems avoids the safety concerns related with the use of virus’ in gene therapy. Non-viral gene therapy requires a suitable safe and efficient DNA cassette for delivery of COL7A1 into a target skin tissue, resisting transgene silencing and extending C7 expression. Using human promoters and especially tissue-specific promoters in minicircle DNA (MC) (containing no accessory bacterial sequences), will control the expression of C7 in RDEB skin cells and increase the safety profile and potential clinical application of non-viral gene delivery systems. In this thesis, MC encoding full-length human COL7A1 with two different human promoters, a human COL7A1 tissue-specific promoter (C7p) and a human elongation factor one alpha (EF1α) ubiquitous promoter, were developed and compared with a control viral cytomegalovirus (CMV) ubiquitous promoter for their efficiency in driving COL7A1 gene expression in RDEB cells. A biocompatible cationic polymer (HPAE) was used to successfully transfect RDEB cells with MCC7C7p, MCC7EF1α and MCC7CMV. Promoter efficiency was evaluated at various stages at COL7A1 mRNA level (qRT-PCR) and protein level (flow cytometry, immunoblotting and immunofluorescence) after transfection in 2D cultures and in 3D human RDEB skin equivalent (SE). At the COL7A1 mRNA level, transfected RDEB keratinocytes (RDEBK) showed that the human EF1α promoter outperformed the control viral CMV promoter by 1.8-fold, while both ubiquitous EF1α and CMV promoters outperformed the tissue-specific C7p by 2.7-fold and 1.5-fold respectively. At the protein level, the mean percentage of C7-positive transfected RDEBK was significantly higher when compared with un-transfected cells (59.7% by human EF1α, 51.6% by CMV and 40.5% by human C7p). The C7 expression by EF1α promoter was higher than that by CMV and C7p in RDEBK cell lysate. In addition, the full-length C7 expression levels from the supernatant by human promoters exceeded the level of endogenous protein observed in healthy keratinocytes. Moreover, C7 expression was still detected after 23 days of treatment with the developed systems MCC7C7p and MCC7EF1α in the 3D human RDEB skin equivalents. Non-viral gene therapy using minicircle DNA carrying the full-length COL7A1 under the control of human promoters was capable of restoring type VII collagen expression in immortalized human RDEBK cells and in 3D RDEB human skin equivalents, showing a longer duration of C7 expression when using human promoters. This developed system has a high potential to be used as a topical treatment to restore C7 in RDEB patients, and to be adapted to other genetic diseases involving non-functional or truncated proteins.