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ABSTRACT: Patients with dystrophic epidermolysis bullosa (DEB) have incurable skin fragility, blistering, and multiple skin wounds because of mutations in the gene that encodes for type VII collagen (C7), which holds together the epidermal and dermal layers of human skin. The intradermal injection of gene-corrected DEB fibroblasts, recombinant C7 protein, or lentiviral vectors expressing C7 is a potential therapy for DEB. Nevertheless, severe DEB causes widespread wounds and treatment would require multiple injections. An alternative strategy might be to inject genetically engineered cells into the patient's circulation that home to the skin wounds and deposit the transgene product. In this study, we demonstrated that intravenously (IV) injected, molecularly engineered DEB fibroblasts (overexpressing human C7) homed to murine skin wounds and continuously delivered C7 at the wound site, where it incorporated into the skin's basement membrane zone and formed anchoring fibril structures. Wounds made on murine or grafted human skin demonstrated accelerated healing when the animals were IV injected with gene-corrected DEB fibroblasts. Our data demonstrate that abundant C7 promotes wound healing. This is also the first evidence that IV injected, molecularly engineered skin fibroblasts can deliver C7 to skin wounds. This strategy could be useful for treating DEB patients.
Molecular Therapy 04/2007; 15(3):628-35. · 6.87 Impact Factor
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ABSTRACT: Epidermolysis bullosa acquisita (EBA) is an autoimmune sub-epidermal blistering disease characterized by autoantibodies to type VII (anchoring fibril) collagen. To date, however, direct evidence for a pathogenic role of human EBA autoantibodies has not been demonstrated. In this study, we affinity-purified anti-type VII collagen antibodies from EBA patients' sera and then injected them into adult hairless immunocompetent mice. Mice injected with EBA autoantibodies developed skin fragility, blisters, erosions, and nail loss on their paws - all features of EBA patients. By clinical, histological, immunological, and ultrastructural parameters, the induced lesions were reminiscent of human EBA. Histology showed bullous lesions with an epidermal-dermal separation. IgG and C3 deposits were observed at the epidermal-dermal junction. All mice had serum antibodies that labeled the dermal side of salt-split human skin like EBA sera. Direct immunogold electron microscopy specifically localized deposits of human IgG to anchoring fibrils. (Fab')(2) fragments generated from EBA autoantibodies did not induce disease. We conclude that EBA human patient autoantibodies cause sub-epidermal blisters and induce EBA skin lesions in mice. These passive transfer studies demonstrate that human EBA autoantibodies are pathogenic. This novel EBA mouse model can be used to further investigate EBA autoimmunity and to develop possible therapies.
Journal of Investigative Dermatology 07/2006; 126(6):1323-30. · 6.31 Impact Factor
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ABSTRACT: Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechanobullous disorders caused by mutations in the gene, COL7A1, that codes for type VII, (anchoring fibril), collagen, which is critical for epidermal-dermal adherence. Most gene therapy approaches have been ex vivo, involving cell culture and culture graft transplantation, which is logistically difficult. To develop a more simplified approach, we engineered a self-inactivating lentiviral vector expressing human type VII collagen and injected this vector intradermally into hairless, immunodeficient mice and into a human DEB composite skin equivalent grafted onto immunodeficient mice. In both situations, the vector transduced dermal cells, which in turn synthesized and exported type VII collagen into the extracellular space. Remarkably, the type VII collagen selectively adhered to and incorporated into the basement membrane zone (BMZ) between the dermis and the epidermis, where it formed anchoring fibril structures. In the case of the DEB skin equivalent, the newly expressed type VII collagen reversed the DEB phenotype characterized by poor epidermal-dermal adherence and anchoring fibril defects. A single lentiviral vector injection provided stable type VII collagen at the BMZ for at least 3 months. These data demonstrate efficient and long-term type VII collagen gene transfer in vivo using direct intradermal injection of an engineered lentiviral vector.
Molecular Therapy 09/2004; 10(2):318-26. · 6.87 Impact Factor
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ABSTRACT: Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders that are caused by mutations in the type VII collagen gene and for which ex vivo gene therapy has been considered. To develop a simpler approach for treating DEB, we evaluated the feasibility of protein-based therapy by intradermally injecting human recombinant type VII collagen into mouse skin and a DEB human skin equivalent transplanted onto mice. The injected collagen localized to the basement membrane zone of both types of tissues, was organized into human anchoring fibril structures and reversed the features of DEB disease in the DEB skin equivalent.
Nature Medicine 08/2004; 10(7):693-5. · 22.46 Impact Factor
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ABSTRACT: Type VII collagen is synthesized and secreted by both human keratinocytes and fibroblasts. Although both cell types can secrete type VII collagen, it is thought that keratinocytes account for type VII collagen at the dermal-epidermal junction (DEJ). In this study, we examined if type VII collagen secreted solely by dermal fibroblasts could be transported to the DEJ. We established organotypic, skin-equivalent cultures composed of keratinocytes from patients with recessive dystrophic epidermolysis bullosa (RDEB) and normal dermal fibroblasts. Immuno-labeling of skin equivalent sections with the anti-type VII collagen antibody revealed tight linear staining at the DEJ. RDEB fibroblasts, were gene-corrected to make type VII collagen and used to regenerate human skin on immune-deficient mice. The human skin generated by gene-corrected RDEB fibroblasts or normal human fibroblasts combined with RDEB keratinocytes restored type VII collagen expression at the DEJ in vivo. Further, intradermal injection of normal human or gene-corrected RDEB fibroblasts into mouse skin resulted in the stable expression of human type VII collagen at the mouse DEJ. These data demonstrate that human dermal fibroblasts alone are capable of producing type VII collagen at the DEJ, and it is possible to restore type VII collagen gene expression in RDEB skin in vivo by direct intradermal injection of fibroblasts.
Journal of Investigative Dermatology 12/2003; 121(5):1021-8. · 6.31 Impact Factor
