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.
Claims23
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) subjecting the human patient to one or more tests selected from replication competent retrovirus (RCR), COL7A1-sensitive cytotoxic T-cells, or a combination thereof;
- (b) obtaining from the human patient a population of skin cells comprising keratinocytes;
- (c) isolating a population of keratinocytes comprising the one or more mutations in the col7A1 gene from the population of skin cells, and culturing the isolated population of keratinocytes on a collagen 1 peptide in a first keratinocyte culture medium;
- (d) transducing the isolated population of keratinocytes ex vivo with a retroviral vector comprising a promoter operably linked to a genetic construct encoding a functional human collagen VII (COL7A1) protein to generate genetically corrected keratinocytes that meet pre-release criteria for viral transduction efficiency (VTE) >50% and proviral genome copy number (PGCN) of less than or equal to 1.5;
- (e) culturing the genetically corrected keratinocytes in the first keratinocyte culture medium to form an autologous COL7A1 corrected keratinocyte sheet;
- (f) subjecting the autologous COL7A1 corrected keratinocyte sheet to one or more pre-release tests selected from a group consisting of VTE test, PGCN test, and replication competent retrovirus (RCR) test;
- (g) 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 medium;
- (h) assembling the engineered autologous epidermal sheets;
- and
- (i) transplanting a graft of the assembled engineered autologous epidermal sheets to an RDEB-induced wound of the human patient.
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) subjecting the human patient to one or more tests selected from replication competent retrovirus (RCR), COL7A1-sensitive cytotoxic T-cells, or a combination thereof;
- (b) obtaining from the human patient a population of skin cells comprising keratinocytes;
- (c) isolating a population of keratinocytes comprising the one or more mutations in the col7A1 gene from the population of skin cells, and culturing the isolated population of keratinocytes in a first serum-free human keratinocyte culture medium;
- (d) transducing the isolated population of keratinocytes ex vivo with a retroviral vector comprising a promoter operably linked to a genetic construct encoding a functional human collagen VII (COL7A1) protein to generate genetically corrected keratinocytes that meet pre-release criteria for viral transduction efficiency (VTE) >50% and proviral genome copy number (PGCN) of less than or equal to 1.5;
- (e) culturing the genetically corrected keratinocytes in the first serum-free human keratinocyte culture medium to form an autologous COL7A1 corrected keratinocyte sheet;
- (f) subjecting the autologous COL7A1 corrected keratinocyte sheet to one or more pre-release tests selected from a group consisting of VTE test, PGCN test, and replication competent retrovirus (RCR) test;
- (g) 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 medium;
- (h) assembling the engineered autologous epidermal sheets;
- and
- (i) transplanting a graft of the assembled engineered autologous epidermal sheets to an RDEB-induced wound of the human patient.
Dependencies are inferred from the claim text.