Abstract
Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects in type-VII collagen (C7), a protein encoded by the COL7A1 gene and essential for anchoring fibril formation at the dermal-epidermal junction. Gene therapy of RDEB is based on transplantation of autologous epidermal grafts generated from gene-corrected keratinocytes sustaining C7 deposition at the dermal-epidermal junction. Transfer of the COL7A1 gene is complicated by its very large size and repetitive sequence. This article reports a gene delivery approach based on the Sleeping beauty transposon, which allows integration of a full-length COL7A1 cDNA and secretion of C7 at physiological levels in RDEB keratinocytes without rearrangements or detrimental effects on their clonogenic potential. Skin equivalents derived from gene-corrected RDEB keratinocytes were tested in a validated preclinical model of xenotransplantation on immunodeficient mice, where they showed normal deposition of C7 at the dermal-epidermal junction and restoration of skin adhesion properties. These results indicate the feasibility and efficacy of a transposon-based gene therapy approach to RDEB.
Highlights
Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by a functional deficit of type VII collagen (C7) protein due to loss of function mutations in the C7 gene (COL7A1)
To transpose the COL7A1 cassette in primary clonogenic RDEB keratinocytes, which are resistant to plasmid transfection, we developed an adenoviral (AdV) vector platform consisting in a helper-dependent (HD) AdV vector carrying the transposon cassette flanked by flippase recognition target (FRT) sites (AdVHDC7.from adenovirus serotype 50 (F50)), a second-generation vector (i.e., E1- and E2A-deleted) coding for enhanced flippase (FLPe) recombinase (AdVD2FLPe.F50), and a first-generation AdV vector (i.e., E1-deleted) coding for the SB100X transposase (AdVD1SB100X.F50)
Sleeping beauty (SB)-mediated transposition of a full-length human COL7A1 cDNA in iRDEB keratinocytes The SB100X and the T2-based transposon were exploited to investigate the restoration of C7 expression in patientderived, iRDEBs carrying null mutations in the COL7A1 gene
Summary
Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by a functional deficit of type VII collagen (C7) protein due to loss of function mutations in the C7 gene (COL7A1). Current ex vivo gene therapy approaches, based on autologous transplantation of genetically corrected epidermal grafts, aim at transferring the correct, full-length COL7A1 cDNA (w9 kb) in keratinocyte stem cells by oncoretroviral or lentiviral vectors (Droz-Georget Lathion et al, 2015; Jackow et al, 2016; Sebastiano et al, 2014; Siprashvili et al, 2010; Titeux et al, 2010). These vectors are associated with a certain risk of insertional mutagenesis and have limited cargo capacity, resulting in genomic instability and rearrangements when used to transfer large C7 expression cassettes (Droz-Georget Lathion et al, 2015; Titeux et al, 2010). This finding represents a promising patientspecific therapy, it has to cope with the low efficiency of homologous recombination in primary cells (Coluccio et al, 2013; Duarte et al, 2014), which requires selection/enrichment of the genome-edited cells before graft generation and transplantation
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