Injection of Recombinant Human Type VII Collagen Corrects the Disease Phenotype in a Murine Model of Dystrophic Epidermolysis Bullosa

Abstract

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.

Immune reactivity to type VII collagen: implications for gene therapy of recessive dystrophic epidermolysis bullosa

“…More widely, these predictive and monitoring tests may be applied to other therapy approaches under consideration for RDEB, involving injection of allogenic or genetically engineered autologous fibroblasts or administration of recombinant type VII collagen protein. 16,[47][48][49] …”

“…12 The expression or injection of therapeutic proteins can also initiate humoral immune responses, 13,14 and several studies have shown a correlation between antibodies against the therapeutic protein and treatment failure, for example, antibodies against factor VIII in mouse and dog models of hemophilia A. 15 Recently, Remington et al 16 have shown that injection of recombinant type VII collagen in a type VII collagen knockout mouse model led to an immune response involving circulating type VII collagen antibodies, which was not located at the basement membrane zone. Although in these mice, the antibodies seemed to be no pathogenic, this study confirms the risk of developing an immune response against type VII collagen in patients who do not express this protein, as neutralizing antibodies may also well be raised.…”

Long-Term Type VII Collagen Restoration to Human Epidermolysis Bullosa Skin Tissue

“…Although the actual mechanism of autoantibody production in EBA is unknown, it is clear from clinical studies of EBA that type VII collagen antibodies are capable of inducing significant skin pathology consisting of sublamina densa separation of skin and mucosa and subsequent dystrophic scarring. Moreover, a protein therapy approach using recombinant type VII collagen injected into Col7a1 knockout mice induced elevated titers of anti-collagen VII antibodies, although these antibodies were absent at the BMZ and did not interfere with newly delivered protein incorporation into mouse skin (Remington et al, 2009). The 60% or more of patients with RDEB who retain expression of truncated NC1-containing type VII collagen protein, the most antigenic region of the protein (Ortiz-Urda et al, 2005), may thus represent the most attractive initial population for assessment of this approach.…”

“…Preclinical studies have demonstrated significant progress for a variety of therapeutic approaches, showing successful restoration of type VII collagen expression at the dermalepidermal junction. These studies have used direct gene (Woodley et al, 2004b) and protein (Woodley et al, 2004a;Remington et al, 2009) transfer as well as cell-based therapies using epidermal keratinocytes (Chen et al, 2002;Ortiz-Urda et al, 2002;Gache et al, 2004), dermal fibroblasts (Ortiz-Urda et al, 2003;Woodley et al, 2003;Kern et al, 2009), or even stem cells from bone marrow transplantation (Tolar et al, 2009). Although fibroblast-and protein-based therapies display attractive features, the short half-life of fibroblasts delivered to the dermis, as well as challenges in incorporating normal human type VII collagen into BMZ in clinical settings (Wong et al, 2008), supports the search for improved methods of administration in patients.…”

“…Subjects with RDEB develop painful blisters and wounds on the skin and mucous membranes, leading to a shortened life expectancy due to infection, organ failure, or squamous cell carcinoma (SCC). Preclinical RDEB studies have suggested various treatment strategies for the disease, such as direct virus-based (Woodley et al, 2004b), protein-based (Woodley et al, 2004a;Remington et al, 2009), and cell-based therapies including bone marrowderived cells (Tolar et al, 2009) and genetically modified keratinocytes (Chen et al, 2002;Ortiz-Urda et al, 2002;Gache et al, 2004) or fibroblasts (Ortiz-Urda et al, 2003;Woodley et al, 2003;Kern et al, 2009). The promise suggested by this body of preclinical research points to the emergence of new treatments for RDEB beyond currently available palliative wound care.…”