Gene therapy for recessive dystrophic epidermolysis bullosa using genetically corrected autologous keratinocytes

A pharmaceutical composition for treating Recessive Dystrophic Epidermolysis Bullosa (RDEB) in a human patient, the pharmaceutical composition comprising an engineered autologous epidermal sheet, and an acellular matrix, a collagen matrix, or a biocompatible mesh;

• wherein the engineered autologous epidermal sheet comprises a population of corrected and differentiated autologous keratinocytes derived from the human patient suffering from RDEB and having one or more mutations in both copies of a human collagen VII A1 (Col7A1) gene;
• wherein the corrected and differentiated autologous keratinocytes in the population are transduced with a retroviral vector comprising a promoter operably linked to a genetic construct encoding a functional collagen VII (COL7A1) protein, express a COL7A1 protein, and have a viral transduction efficiency (VTE)>50% and an average proviral genome copy number (PGCN) of no more than 3;
• and
• wherein the population of corrected and differentiated autologous keratinocytes is differentiated ex vivo with an epidermal sheet production medium comprising Dulbecco's Modified Eagle Medium, F12 medium, 10% fetal bovine serum, 36 ng/ml hydrocortisone, 25 μg/ml adenine, 5 μg/ml insulin, 5 μg/ml transferrin, 10 ng/ml epidermal growth factor, 30 μg/mL amikacin, 20 μg/mL vancomycin, and 2 ng/ml liothyronine.