Design and development of polymeric transfection vectors for gene delivery in Recessive Dystrophic Epidermolysis Bullosa

Abstract

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is caused by mutations in the collagen VII gene (COL7A1) that lead to an alteration of function or a reduction in the amounts of collagen VII protein (C7). Any of these mutations will impair C7 assembly into anchoring fibrils that anchor the basement membrane zone (BMZ) to the underlying dermis. This in turn causes reduced skin resistance to mild trauma making the patients suffer from severe blistering and scarring in the skin and mucosa. Intensive efforts are being made to restore the anchoring protein at the BMZ as a means of providing a lasting cure for the disease. One of the methods examined as potential therapy utilizes viral vectors for genetic correction of the C7 expressing cells. However, toxicity and immunogenicity concerns have halted progress using viral vectors for gene therapy in many clinical trials. As a result, non-viral methods of delivery have attracted great interest as a replacement. The overall aim of this project was to develop a safe and efficient polymer based gene delivery method to encourage the production of functional C7 protein and restore the mechanical stability at the BMZ in RDEB mouse skin. We focused on one of the more versatile methods of non-viral based delivery that utilizes a dense polycation synthesized from deactivation-enhanced atom transfer radical polymerisation (multi-knot) or Michael addition (hyperbranched poly ([beta]-amino ester)). The polymers were created to deliver the therapeutic C7 plasmid DNA to RDEB keratinocytes and fibroblasts. The focus was mainly on keratinocytes and fibroblasts since they are the predominant cells found in the upper dermis and epidermis, and perhaps contribute more to C7 expression than any other group of cells. Using specially designed cationic polymers, we were able to restore some of the C7 expression in vitro and in RDEB skin equivalents (SEs) (3D organ cultures). SEs are being used to test for toxicity and effectiveness of many drugs, reducing the need for pre-clinical trials. Unfortunately, SEs do not fully replicate natural tissues, because they lack the complexity and array of cells and proteins found in natural tissue. In addition, the presence of immune response capability and blood circulation in pre-clinical models will give a more accurate account of the drug's safety. This encouraged us to test the effectiveness of polymer vectors for COL7A1 delivery in an in vivo pre-clinical model of RDEB. We used Col7[alpha] 1 null RDEB (Col7[alpha]1-/-) knockout mice (developed from immune-competent mice by targeted inactivation of Col7[alpha]1) to test our hypothesis. Clinically, these mice showed severe blistering and detachment of the epidermis from the dermis after birth similar to the human phenotype. We successfully observed expression of the therapeutic transgene product (C7) in the mice after intradermal injection of the HPAE/COL7A1 complex into the mouse paws and ventral region, although there was a noticeable inflammatory response around the injected area. This new approach has proved that it is possible to restore the expression of the missing protein C7 in RDEB skin using a polymer based gene delivery. Full restoration of the skin's mechanical stability requires further investigation into the delivery system, areas of injection and addressing the adverse effects of the delivery agents.