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-12110504-B2
Publication Date
2024-10-08
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 the treatment of Epidermolysis Bullosa (EB), specifically Recessive Dystrophic Epidermolysis Bullosa (RDEB), by genetically engineering human keratinocytes to express functional wild-type human collagen VII (COL7A1, C7). These genetically corrected keratinocytes are cultured to form keratinocyte sheets which are then grafted onto wound beds of affected individuals. The approach includes ex vivo retroviral transduction of keratinocytes with a genetic construct encoding full-length wild-type COL7A1 protein, yielding a therapeutic composition in the form of engineered autologous epidermal sheets (LEAES). This allows localized delivery of functional collagen VII directly to wounded or eroded skin sites, restoring the structural integrity compromised in RDEB.
Recessive Dystrophic Epidermolysis Bullosa is a severe genetic blistering disorder caused by mutations in the COL7A1 gene, resulting in absent or dysfunctional type VII collagen. This protein is essential for anchoring fibrils that connect the dermal-epidermal basement membrane, maintaining skin stability. The absence of functional collagen VII leads to widespread blistering, erosions, painful wounds, and debilitating scarring, including mitten deformities and increased risk of invasive squamous cell carcinoma, the main cause of death in these patients. Current therapies are limited to palliative care, with no effective method to provide sustained, systemic correction of the mutated collagen VII.
The invention addresses the limitations of current treatments by enabling ex vivo genetic correction of autologous keratinocytes, efficient viral transduction achieving high expression levels of functional collagen VII, and robust grafting methods that promote long-term epidermal regeneration. It provides methods for preparing keratinocyte sheets with proviral genome copy numbers controlled to ensure efficacy and safety. Further, it includes detailed protocols for the isolation, genetic modification, culturing, quality testing, and transplantation of these sheets, demonstrating clinical improvements in wound healing, reduced blistering, and anchoring fibril formation in treated subjects during clinical studies.
Claims Coverage
The patent contains one main independent claim directed to a method for treating RDEB by ex vivo gene correction of patient keratinocytes followed by transplantation as engineered autologous epidermal sheets.
Ex vivo viral transduction of keratinocytes with functional COL7A1 construct
Isolating keratinocytes from a human RDEB patient and transducing them ex vivo with a retroviral vector encoding a functional human collagen VII protein under control of a promoter, ensuring viral transduction efficiency >50% and proviral genome copy number ≤1.5.
Formation and cultivation of autologous collagen VII corrected keratinocyte sheets
Culturing the genetically corrected keratinocytes in a first keratinocyte culture medium to form a keratinocyte sheet, then maturing this sheet in a second culture medium (DFF31) to produce engineered autologous epidermal sheets (LEAES).
Pre-release and quality testing of engineered sheets
Subjecting the keratinocyte sheets to pre-release tests including viral transduction efficiency, proviral genome copy number, and replication competent retrovirus tests, plus optional sterility, endotoxin, mycoplasma, viability, cytotoxic T cell assay, immunofluorescence, and electron microscopy.
Preparation and transplantation of engineered sheets onto prepared wounds
Assembling the mature engineered autologous epidermal sheets and grafting them onto RDEB-induced wounds of the patient, wherein wounds can be ablative-treated (e.g., cauterized) to remove non-corrected keratinocytes prior to transplantation.
Use of specific retroviral vectors and promoter combinations
Utilizing retroviral vectors such as MoMLV or GalV-pseudotyped LZRSE carrying the COL7A1 gene under control of a MoMLV LTR promoter for effective gene transfer into keratinocytes.
The claims focus on a comprehensive therapeutic method involving ex vivo retroviral gene correction of patient-derived keratinocytes with functional COL7A1, culture into keratinocyte sheets under defined media, thorough quality testing, and transplantation onto RDEB wounds. The inventive features ensure effective collagen VII expression, safety, and clinical applicability in human subjects with RDEB.
Stated Advantages
Genetic correction of autologous keratinocytes enables sustained, localized delivery of functional collagen VII to skin wounds, improving wound healing and skin durability.
The method overcomes limitations of current treatments which are primarily palliative, offering a potential disease-modifying therapy for a severe genetic blistering disorder.
Use of autologous cells and controlled viral integration reduces immunogenicity and risks associated with systemic treatments and protein injections.
The engineered grafts show a favorable safety profile with no serious adverse events such as systemic viral infection, autoimmunity, or skin cancer observed in clinical trials.
The approach allows targeting of stem cells within the epidermis, providing long-term correction spanning multiple epidermal renewal cycles.
Documented Applications
Treatment of Recessive Dystrophic Epidermolysis Bullosa (RDEB) by transplantation of genetically corrected autologous keratinocyte sheets to open skin wounds.
Use in treatment of corneal erosion associated with RDEB by transplantation of genetically corrected corneal cell sheets.
Ex vivo genetic correction of autologous skin or corneal cells using retroviral vectors encoding functional COL7A1 for personalized gene therapy.
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