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

A method for treating Recessive Dystrophic Epidermolysis Bullosa (RDEB) in a human patient having one or more mutations in both copies of a human collagen VII A1 (Col7A1) gene and suffering from RDEB, the method comprising:

• (a) transducing ex vivo a population of isolated autologous keratinocytes isolated from a population of skin cells obtained from the human patient with a retroviral vector comprising a promoter operably linked to a genetic construct encoding a functional collagen VII (COL7A1) protein to generate corrected autologous keratinocytes expressing a COL7A1 protein,
• wherein the corrected autologous keratinocytes meet pre-release criteria for viral transduction efficiency (VTE) of >50% and a proviral genome copy number (PGCN) of less than or equal to 1.5, and
• wherein the population of isolated autologous keratinocytes are cultured on collagen I peptide in a keratinocyte culture medium without feeder layer cells prior to transduction;
• (b) culturing the genetically corrected autologous keratinocytes to form an autologous COL7A1 corrected keratinocyte sheet;
• (c) maturing the autologous COL7A1 corrected keratinocyte sheet to form engineered autologous epidermal sheets,
• wherein maturing comprises culturing the autologous COL7A1 corrected keratinocyte sheet in a second culture medium;
• and wherein the second culture medium comprises DFF31;
• (d) assembling the engineered autologous epidermal sheets;
• and
• (e) transplanting a graft of the assembled engineered autologous epidermal sheets to a RDEB-induced wound of the human patient;
• wherein the assembled engineered autologous epidermal sheets are transplanted to the human patient within about 9 to 20 days from the time the population of autologous keratinocytes were transduced.