Gene therapy for recessive dystrophic epidermolysis bullosa using genetically corrected autologous keratinocytes
AssigneesThe Board of Trustees of the Leland Stanford Junior University, The United States Government as represented by the Department of Veterans Affairs
InventorsZurab Siprashvili, Ngon T. Nguyen, M. Peter Marinkovich, Jean Tang, Alfred T. Lane, Paul A. Khavari
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Abstract
Methods are provided for the cell-based delivery of collagen VII for the treatment of Epidermolysis Bullosa and corneal erosion. The disclosure also provides a composition and a pharmaceutical composition comprises, comprise, or alternatively consist essentially of, or yet further consist of a keratinocyte sheet or a corneal cell sheet.
Claims17
A composition comprising ex vivo differentiated engineered autologous epidermal sheets,
- wherein the ex vivo differentiated engineered autologous epidermal sheets comprise a population of autologous keratinocytes derived from a human patient suffering from Recessive Dystrophic Epidermolysis Bullosa (RDEB) and having one or more mutations in both copies of a human collagen VII A1 (Col7A1) gene that are corrected ex vivo with a retroviral vector comprising a promoter operably linked to a genetic construct encoding a functional collagen VII (COL7A1) to generate a corrected population of autologous keratinocytes;
- wherein the corrected population of autologous keratinocytes is differentiated ex vivo into engineered autologous epidermal sheets with an epidermal sheet production medium, and
- wherein the epidermal sheet production medium comprises 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.
A method for differentiating keratinocytes into epidermal sheets ex vivo, the method comprising:
- (a) transducing ex vivo a population of isolated autologous keratinocytes isolated from a population of skin cells obtained from a human patient lacking expression of a functional collagen VII A1 (COL7A1) with a retroviral vector comprising a promoter operably linked to a genetic construct encoding the functional COL7A1 to generate a corrected population of autologous keratinocytes expressing the functional COL7A1,
- 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 in a keratinocyte culture medium without feeder layer cells prior to transduction;
- (b) culturing the genetically corrected population of autologous keratinocytes in a first culture medium to form an autologous corrected keratinocyte sheet;
- (c) differentiating the autologous corrected keratinocyte sheet into engineered autologous epidermal sheets by culturing the autologous corrected keratinocyte sheet in a second culture medium;
- wherein the second culture medium is 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;
- and
- (d) assembling the engineered autologous epidermal sheets.
A composition for differentiating a population of keratinocytes into epidermal sheets ex vivo 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.
Dependencies are inferred from the claim text.