Gene therapy for recessive dystrophic epidermolysis bullosa using genetically corrected autologous keratinocytes
Inventors
Siprashvili, Zurab • Nguyen, Ngon T. • Marinkovich, M. Peter • Tang, Jean • Lane, Alfred T. • Khavari, Paul A.
Assignees
US Department of Veterans Affairs • Leland Stanford Junior University
Publication Number
US-12173314-B2
Publication Date
2024-12-24
Expiration Date
2037-01-03
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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.
Core Innovation
The invention provides methods and compositions for treating Epidermolysis Bullosa (EB), specifically Recessive Dystrophic Epidermolysis Bullosa (RDEB), by engineering a population of human keratinocytes to express functional type VII collagen (C7) through integration of a genetic construct encoding wild-type human COL7A1. The genetically corrected keratinocytes can be cultured ex vivo into autologous keratinocyte sheets, which are then grafted onto wound beds of affected individuals to restore C7 expression and improve skin integrity.
RDEB is a genetic blistering skin disorder caused by mutations in the COL7A1 gene leading to the loss of functional type VII collagen, resulting in fragile skin characterized by blistering, erosions, and disabling scarring. Current therapies are limited to palliative care, and alternative treatments such as topical or intradermal application of recombinant C7 have limitations including poor penetration and diffusion. The invention addresses the need for a localized delivery method for functional C7 to the skin and mucosal tissues that can prevent blistering and scarring, while avoiding systemic toxicity associated with systemic administration.
Claims Coverage
The patent contains one independent claim that defines a method for treating RDEB using genetically corrected autologous keratinocytes. The main inventive features extracted focus on the ex vivo genetic correction, keratinocyte culture conditions, sheet formation, and transplantation timing.
Ex vivo transduction of autologous keratinocytes with retroviral vector encoding functional COL7A1
The method comprises transducing isolated autologous keratinocytes from the patient using a retroviral vector carrying a promoter linked to a genetic construct for a functional collagen VII protein. The corrected cells meet pre-release criteria of viral transduction efficiency greater than 50% and proviral genome copy number of no more than 1.5.
Keratinocyte culture on collagen I peptide without feeder layer
Prior to transduction, the autologous keratinocytes are cultured on collagen I peptide in a keratinocyte culture medium that is free of feeder layer cells, enabling efficient genetic correction and subsequent sheet formation.
Formation and maturation of autologous COL7A1 corrected keratinocyte sheets
Genetically corrected keratinocytes are cultured to form keratinocyte sheets, which are matured into engineered autologous epidermal sheets by use of a second culture medium comprising DFF31.
Transplantation schedule and wound preparation
The engineered autologous epidermal sheets are assembled and transplanted to RDEB-induced wounds on the patient within about 9 to 20 days post-transduction. Optionally, the target wound is cauterized prior to transplantation to remove non-corrected keratinocytes, enhancing graft incorporation.
The independent claim defines a comprehensive method of ex vivo genetic correction of autologous keratinocytes with a retroviral vector expressing functional COL7A1, formation and maturation of keratinocyte sheets in defined culture conditions, and transplantation onto prepared wounds within a specified timeframe. This method addresses key challenges in treating RDEB by restoring functional collagen VII in patient skin.
Stated Advantages
Genetically corrected autologous epidermal skin grafts increase type VII collagen deposition and reduce blistering in patients with severe RDEB.
The method provides encouraging efficacy and acceptable safety in human subjects with durable correction of skin integrity for several months post-grafting.
The localized cell-based delivery circumvents limitations of topical or intradermal protein therapies and avoids potential systemic toxicity.
Documented Applications
Treatment of Recessive Dystrophic Epidermolysis Bullosa (RDEB) by grafting genetically corrected autologous keratinocyte sheets expressing functional type VII collagen onto skin wounds.
Treatment of corneal erosion in RDEB patients by transplantation of genetically corrected corneal cell sheets expressing functional collagen VII.
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