A. Dakiw Piaceski et al. Self-assembled skin and gene therapy to treat RDEB 73 www.ecmjournal.org Abstract The combination of gene therapy and tissue engineering is one of the most promising strategies for the treatment of recessive dystrophic epidermolysis bullosa (RDEB). RDEB is a rare genetic disease characterised by mutations in the COL7A1 gene, encoding type VII collagen (COLVII), which forms anchoring fbrils at the dermal-epidermal junction of the skin. This disease causes severe blistering and only palliative treatments are ofered. In this study, the base of a strategy combining gene therapy and a tissue-engineered skin substitute (TES), which would be suitable for the permanent closure of skin wounds, was set-up. As a high transduction efciency into fbroblasts and/or keratinocytes seems to be a prerequisite for a robust and sustained correction of RDEB, diferent envelope pseudotyped retroviral vectors and the transduction enhancer EF-C were tested. When green fuorescent protein (GFP) was used as a reporter gene to evaluate the retroviral-mediated gene transfer, the fbroblast infection efciency was 30 % higher with the Ampho pseudotyped vector as compared with the other pseudotypes. At least a 3.1-fold and a 1.3-fold increased transduction were obtained in fbroblasts and keratinocytes, respectively, with EF-C as compared with polybrene. A continuous and intense deposit of haemagglutinin (HA)-COLVII was observed at the dermal-epidermal junction of self-assembled TESs made of cells transduced with a HA-tagged COL7A1 vector. Furthermore, HA-tagged basal epidermal cells expressing keratin 19 were observed in TESs, suggesting stem cell transduction. This approach could be a valuable therapeutic option to further develop, in order to improve the long-term life quality of RDEB patients. Keywords: Acantholysis bullosa, epidermolysis bullosa dystrophica, cell- and tissue-based therapy, tissue engineering, cell culture techniques, genetic therapy, collagen type VII. *Address for correspondence: Manuel Caruso, Cancer Research Centre, Université Laval, Quebec City, Quebec, G1R 3S3, Canada. Telephone: +418 4185254444 Email: Manuel.Caruso@crchudequebec.ulaval.ca European Cells and Materials Vol. 35 2018 (pages 73-86) DOI: 10.22203/eCM.v035a06 ISSN 1473-2262 TRANSLATING THE COMBINATION OF GENE THERAPY AND TISSUE ENGINEERING FOR TREATING RECESSIVE DYSTROPHIC EPIDERMOLYSIS BULLOSA A. Dakiw Piaceski 1,2,4 , D. Larouche 1,2,4 , K. Ghani 3,4 , F. Bisson 1,2,4 , S. Cortez Ghio 1,2,4 , S. Larochelle 1,2,4 , V. J. Moulin 1,2,4 , M. Caruso 3,4,§, * and L. Germain 1,2,4,§ 1 Université Laval Research Center on Experimental Organogenesis/LOEX, Quebec City, Quebec, G1J 1Z4, Canada, Quebec City, Quebec, G1V 0A6, Canada 2 Department of Surgery, Faculty of Medicine, Université Laval, Quebec City, Quebec, G1V 0A6, Canada 3 Department of Molecular Biology, Medical Biochemistry and Pathology, Cancer Research Institute, Université Laval, Quebec City, Quebec, G1R 3S3, Canada 4 CHU de Québec - Université Laval Research Centre, Quebec City, Quebec, G1R 3S3, Canada § These authors contributed equally Introduction Epidermolysis bullosa (EB) is a genetic disease comprising a heterogeneous group of rare inherited skin disorders in which minor mechanical stress to the skin and mucous membranes causes the formation of blisters and erosions. There are four major types of EB, characterised by the location of the defective proteins and the level of blisters: EB simplex (EBS; epidermolytic), junctional EB (JEB; lucidolytic), dystrophic EB (DEB; dermolytic) and Kindler syndrome (mixed levels of blistering) (Aumailley et al., 2006; Fine et al., 2014). In the skin of patients with DEB, blister formation occurs beneath the lamina densa within the papillary dermis. Mutations in the COL7A1 gene are responsible for DEB, which can be autosomal dominant or recessive (RDEB), depending on the type of mutations (Kern et al., 2009). The COL7A1 gene encodes type VII collagen (COLVII), which is naturally secreted into the extracellular space by dermal fbroblasts and epidermal keratinocytes (Bruckner-Tuderman et al., 1999). These collagen molecules assemble into anchoring fbrils, a major component of the dermal-epidermal junction (DEJ),