Intravenously Administered Recombinant Human Type VII Collagen Derived from Chinese Hamster Ovary Cells Reverses the Disease Phenotype in Recessive Dystrophic Epidermolysis Bullosa Mice

Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited disorder characterized by skin fragility, blistering, and multiple skin wounds with no currently approved or consistently effective treatment. It is due to mutations in the gene encoding type VII collagen (C7). Using recombinant human C7 (rhC7) purified from human dermal fibroblasts (FB-rhC7), we showed previously that intravenously injected rhC7 distributed to engrafted RDEB skin, incorporated into its dermal-epidermal junction (DEJ), and reversed the RDEB disease phenotype. Human dermal fibroblasts, however, are not used for commercial production of therapeutic proteins. Therefore, we generated rhC7 from Chinese hamster ovary (CHO) cells. The CHO-derived recombinant type VII collagen (CHO-rhC7), similar to FB-rhC7, was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. CHO-rhC7 bound to fibronectin and promoted human keratinocyte migration in vitro. A single dose of CHO-rhC7, administered intravenously into new-born C7-null RDEB mice, incorporated into the DEJ of multiple skin sites, tongue and esophagus, restored anchoring fibrils, improved dermal-epidermal adherence, and increased the animals' life span. Furthermore, no circulating or tissue-bound anti-C7 antibodies were observed in the mice. These data demonstrate the efficacy of CHO-rhC7 in a preclinical murine model of RDEB.

Gentamicin induces functional type VII collagen in recessive dystrophic epidermolysis bullosa patients

“…Collagens are long-lived, stable macromolecules with slow turnover times (41,42). We have shown previously that injected human recombinant type VII collagen stably incorporated into the DEJ of RDEB-like mice and persisted there for at least 3 months (7,10). In this study, 3 of the treated patients maintained type VII collagen levels in excess of 50% of that in NHS for at least 3 months.…”

“…These have included intradermal injection of allogeneic dermal fibroblasts or gene-corrected RDEB fibroblasts (11,14,16), intradermal injection of lentiviral vectors expressing type VII collagen (48), intradermal injection or topical application of recombinant type VII collagen protein (6)(7)(8), intravenous injection of type VII collagen protein itself or fibroblasts that synthesize and secrete type VII collagen (9,10,12), transplantation of bone marrow stem cells, and gene-corrected keratinocyte autografts (13,15,17,49). None of the above are consistently efficacious, and some of them involve substantial risk and considerable invasiveness.…”

“…Prior to the onset of the study, baseline levels of type VII collagen and AFs in the DEJ of each patient were assessed at day 0. At sites of intact skin, 8-mm punch biopsies were obtained, divided into 2 parts -one part for quantitative IF staining for the expression of type VII collagen at the DEJ and for standard histology and a second part for IEM to assess AF structures at the DEJ, as previously described (6)(7)(8)(9)(10)(11)(12)(13). At 1-and 3-month follow-up visits, biopsies from the test sites were obtained and processed in an identical fashion.…”

“…At 1-and 3-month follow-up visits, biopsies from the test sites were obtained and processed in an identical fashion. For the IF staining, a minimum of 60 vertical frozen sections through the entire specimen were prepared and labeled with a polyclonal anti-type VII collagen antibody as described previously (6)(7)(8)(9)(10)(11)(12)(13). Additionally, IF staining was performed on at least 20 sections with 2 different monoclonal anti-type VII collagen antibodies (clone LH7.2; Sigma-Aldrich; mAb 185, clone 32; Millipore) as well as a mouse monoclonal antibody to NC2 of type VII collagen (49).…”

“…Unfortunately, there are no cures or even effective treatments for RDEB. Various therapeutic strategies have been envisioned for RDEB, including recombinant type VII collagen-based protein therapy (6)(7)(8)(9)(10), bone marrow stem cells, or allogeneic dermal fibroblast-based cell therapy and transplantation of gene-corrected keratinocyte autografts (11)(12)(13)(14)(15)(16)(17). However, none of these therapies are consistently effective, and some have associated morbidity and mortality.…”