US 12,173,314 B2
Gene therapy for recessive dystrophic epidermolysis bullosa using genetically corrected autologous keratinocytes
Zurab Siprashvili, San Mateo, CA (US); Ngon T. Nguyen, Union City, CA (US); M. Peter Marinkovich, Redwood City, CA (US); Jean Tang, Stanford, CA (US); Alfred T. Lane, Los Altos, CA (US); and Paul A. Khavari, Palo Alto, CA (US)
Assigned to The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US); and The United States Government as represented by the Department of Veterans Affairs, Washington, DC (US)
Appl. No. 16/066,253
Filed by The Board of Trustees of the Leland Stanford Junior University, Stanford, CA (US); and The United States Government as represented by the Department of Veterans Affairs, Washington, DC (US)
PCT Filed Jan. 3, 2017, PCT No. PCT/US2017/012061
§ 371(c)(1), (2) Date Jun. 26, 2018,
PCT Pub. No. WO2017/120147, PCT Pub. Date Jul. 13, 2017.
Claims priority of provisional application 62/274,700, filed on Jan. 4, 2016.
Claims priority of provisional application 62/414,533, filed on Oct. 28, 2016.
Prior Publication US 2019/0382724 A1, Dec. 19, 2019
Int. Cl. A61K 38/39 (2006.01); A61K 48/00 (2006.01); A61L 27/00 (2006.01); A61L 27/36 (2006.01); C12N 5/00 (2006.01); C12N 5/071 (2010.01)
CPC C12N 5/0629 (2013.01) [A61K 48/0058 (2013.01); A61K 48/0075 (2013.01); A61K 48/0091 (2013.01); C12N 2510/00 (2013.01); C12N 2760/16043 (2013.01)] 16 Claims
OG exemplary drawing
 
1. 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.