Patients with recessive dystrophic epidermolysis bullosa (RDEB) have incurable skin fragility, blistering, and scarring due to mutations in the gene that encodes for type VII collagen (C7) that mediates dermal-epidermal adherence in human skin. We showed previously that intradermal injection of recombinant C7 into transplanted human DEB skin equivalents stably restored C7 expression at the basement membrane zone (BMZ) and reversed the RDEB disease features. In this study, we evaluated the feasibility of protein therapy in a C7 null mouse (Col7a1(-/-)) which recapitulates the features of human RDEB. We intradermally injected purified human C7 into DEB mice and found that the injected human C7 stably incorporated into the mouse BMZ, formed anchoring fibrils, and corrected the DEB murine phenotype, as demonstrated by decreased skin fragility, reduced new blister formation, and markedly prolonged survival. After 4 weeks, treated DEB mice developed circulating anti-human C7 antibodies. Most surprisingly, these anti-C7 antibodies neither bound directly to the mouse's BMZ nor prevented the incorporation of newly injected human C7 into the BMZ. Anti-C7 antibody production was prevented by treating the mice with an anti-CD40L monoclonal antibody, MR1. We conclude that protein therapy may be feasible for the treatment of human patients with RDEB.

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.

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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.

Epidermolysis bullosa acquisita (EBA) is an acquired bullous disease of the skin characterized by IgG autoantibodies against type VII (anchoring fibril) collagen. We previously defined four immunodominant antigenic epitopes within the noncollagenous 1 (NC1) domain of type VII collagen. In this study, we produced an additional recombinant fusion protein from the NC1 domain corresponding to the N-terminal 227 amino acids (residues 1 to 227), which contains homology with cartilage matrix protein (CMP). Using enzyme-linked immunosorbent assay and immunoblot analysis, we tested sera from EBA patients (n ‫؍‬ 32), bullous systemic lupus erythematosus patients (n ‫؍‬ 3), bullous pemphigoid patients (n ‫؍‬ 15), and normal humans (n ‫؍‬ 12). Twenty-six of 32 EBA sera and two of three bullous systemic lupus erythematosus sera reacted with the CMP domain, whereas none of the control sera did. Affinity-purified anti-CMP EBA antibodies injected into hairless mice produced the clinical, histological, immunological, and ultrastructural features of EBA. F(ab) 2 fragments generated from anti-CMP EBA autoantibodies did not induce disease. Our studies provide the first evidence that EBA autoantibodies to the CMP subdomain of NC1 are pathogenic and induce blister formation. This is the first antigenic epitope on type VII collagen demonstrated to be a pathogenic target for EBA autoantibodies. Epidermolysis bullosa acquisita (EBA) is a severe, chronic, subepidermal bullous disease of the skin and mucosa characterized by skin fragility, blisters in traumaprone sites, scarring with milia formation, and nail dystrophy.1 It is a prototypic autoimmune disease in which EBA patients have in vivo tissue-bound and circulating IgG autoantibodies directed against type VII collagen, a major component of anchoring fibrils, structures that anchor the epidermis onto the dermis.2-8 EBA autoantibodies bind to type VII collagen within anchoring fibrils. EBA patients have a diminution of normal anchoring fibrils and subsequent epidermal-dermal disadherence. The clinical appearance of EBA patients and the histology of their cutaneous lesions are often very reminiscent of hereditary dystrophic epidermolysis bullosa. These two diseases are etiologically unrelated but share the common feature of decreased anchoring fibrils. In the case of inherited dystrophic epidermolysis bullosa, the cause of decreased or absent anchoring fibrils is a genetic defect in the gene that encodes for type VII collagen. 9,10Type VII collagen is composed of three identical ␣ chains, each consisting of a 145-kd central collagenous triple-helical segment characterized by repeating Gly-X-Y amino acid sequences, flanked by a large 145-kd amino-terminal noncollagenous domain (NC1), and a small 34-kd carboxyl-terminal noncollagenous domain (NC2). 6 -8,11,12 Within the extracellular space, type VII collagen molecules form anti-parallel, tail-to-tail dimers stabilized by disulfide bonding through a small carboxylterminal NC2 overlap between two type VII collagen molecules. The anti-para...

Epidermolysis bullosa acquisita (EBA) is an acquired, autoimmune, mechanobullous disease with clinical features reminiscent of genetic dystrophic epidermolysis bullosa (DEB). EBA patients have skin fragility, blisters, scars, and milia formation. DEB is due to a genetic defect in the gene-encoding type VII collagen, which makes anchoring fibrils, structures that attach the epidermis and its underlying basement membrane zone onto the papillary dermis. DEB patients have a decrease in normally functioning anchoring fibrils. EBA patients have the same problem, but their decrease in normally functioning anchoring fibrils is because of an abnormality in their immune system in which they produce anti-type VII collagen antibodies that attack their anchoring fibrils. These IgG anti-type VII collagen antibodies are "pathogenic" because when injected into a mouse, the mouse develops an EBA-like blistering disease. EBA has several distinct clinical presentations. It can present with features similar to DEB. It can also present with features reminiscent of bullous pemphigoid, cicatricial pemphigoid, Brunsting-Perry pemphigoid, or IgA bullous dermatosis. Treatment for EBA is unsatisfactory. Some therapeutic success has been reported with colchichine, dapsone, photopheresis, infliximab, and IVIG.

Epidermolysis bullosa acquisita (EBA) is an acquired, mechanobullous disease characterized by autoimmunity to type VII collagen. Type VII collagen makes anchoring fibrils, structures that connect the epidermis and its underlying basement membrane zone to the papillary dermis. EBA patients exhibit skin fragility, blisters, scars and milia formation reminiscent of genetic dystrophic epidermolysis bullosa (DEB). DEB patients have diminutive or absent anchoring fibrils due to a genetic defect in the gene encoding type VII collagen. EBA patients have a decrease in normally functioning anchoring fibrils secondary to an abnormality in their immune system in which they produce 'pathogenic' IgG anti-type VII collagen antibodies. The pathogenicity of these autoantibodies has been demonstrated by passive transfer animal models, in which anti-type VII collagen antibodies injected into a mouse produced an EBA-like blistering disease in the animal. EBA has several distinct clinical presentations. It can present with features similar to DEB, bullous pemphigoid, cicatricial pemphigoid, Brunsting-Perry pemphigoid or IgA bullous dermatosis. Treatment for EBA is unsatisfactory, however, some therapeutic success has been reported with colchicine, dapsone, photophoresis, infliximab and intravenous immunoglobulin.

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