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• W1428940282 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. 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. basement membrane zone Chinese hamster ovary CHO-derived recombinant C7 dystrophic epidermolysis bullosa dermal–epidermal junction fibroblast-derived recombinant C7 N-terminal noncollagenous domain of type VII collagen recessive dystrophic epidermolysis bullosa. recombinant human type VII collagen Type VII collagen (C7) is the major component of anchoring fibrils (AFs), attachment structures in the dermal–epidermal junction (DEJ) of the skin that serve to adhere the epidermal layer of the skin onto the dermis (Sakai et al., 1986Sakai L.Y. Keene D.R. Morris N.P. et al.Type VII collagen is a major structural component of anchoring fibrils.J Cell Biol. 1986; 103: 1577-1586Crossref PubMed Scopus (435) Google Scholar; Lin and Carter, 1992Lin A.N. Carter D.M. Epidermolysis Bullosa: Basic and Clinical Aspects. New York, NY, Springer1992Crossref Google Scholar; Burgeson, 1993Burgeson R.E. Type VII collagen, anchoring fibrils, and epidermolysis bullosa.J Invest Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Scopus (236) Google Scholar). It is composed of three identical α-chains, each consisting of a 145-kDa central collagenous triple-helical segment, flanked by a large 145-kDa amino-terminal, noncollagenous domain (NC1), and a small 34-kDa carboxyl-terminal non-collagenous domain (NC2) (Lunstrum et al., 1986Lunstrum G.P. Sakai L.Y. Keene D.R. et al.Large complex globular domains of type VII procollagen contribute to the structure of anchoring fibrils.J Biol Chem. 1986; 261: 9042-9048Abstract Full Text PDF PubMed Google Scholar; Burgeson et al., 1990Burgeson R.E. Lunstrum G.P. Rokosova B. et al.The structure and function of type VII collagen.Ann NY Acad Sci. 1990; 580: 32-43Crossref PubMed Scopus (82) Google Scholar). Within the extracellular space, C7 molecules form antiparallel dimers that aggregate laterally to form AFs. Our study using recombinant NC1 demonstrated that NC1 interacts with various extracellular matrix components including fibronectin, laminin 332, type I collagen, and type IV collagen (Chen et al., 1997Chen M. Marinkovich M.P. Veis A. et al.Interactions of the amino-terminus noncollagenous domain (NC1) of type VII collagen with extracellular matrix components: a potential role in epidermal-dermal adherence in human skin.J Biol Chem. 1997; 272: 14516-14522Crossref PubMed Scopus (160) Google Scholar, Chen et al., 1999Chen M. Marinkovich M.P. Jones J.J. et al.NC1 domain of type VII collagen binds to the beta 3 chain of laminin 5 via a unique subdomain within the fibronectin-like repeats.J Invest Dermatol. 1999; 112: 177-183Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Further, we demonstrated that C7 is the primary extracellular matrix protein that most potently promotes human keratinocyte migration (Woodley et al., 2008Woodley D.T. Hou Y.P. Martin S. et al.Characterization of molecular mechanisms underlying mutations in dystrophic epidermolysis bullosa using site-directed mutagenesis.J Biol Chem. 2008; 283: 17838-17845Crossref PubMed Scopus (29) Google Scholar). Defects in the C7 gene, designated COL7A1, result in hereditary dystrophic epidermolysis bullosa (DEB) (Uitto and Christiano, 1992Uitto J. Christiano A.M. Molecular genetics of the cutaneous basement membrane zone. Perspectives on epidermolysis bullosa and other blistering skin diseases.J Clin Invest. 1992; 90: 687-692Crossref PubMed Scopus (138) Google Scholar, Uitto and Christiano, 1994Uitto J. Christiano A.M. Molecular basis for the dystrophic forms of epidermolysis bullosa: mutations in the type VII collagen gene.Arch Dermatol Res. 1994; 287: 16-22Crossref PubMed Scopus (96) Google Scholar). DEB is a group of heritable mechanobullous skin diseases characterized by skin fragility, separation of the epidermis from the dermis (blister formation), milia, and scarring. The DEJ of patients with DEB is characterized by a paucity or a diminutive size of AFs (Lin and Carter, 1992Lin A.N. Carter D.M. Epidermolysis Bullosa: Basic and Clinical Aspects. New York, NY, Springer1992Crossref Google Scholar). RDEB skin wounds heal with marked fibrosis, contraction, and scarring (Fine et al., 2008Fine J.D. Eady R.A. Bauer E.A. et al.The classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB.J Am Acad Dermatol. 2008; 58: 931-950Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar). RDEB is often lethal because of an aggressive squamous cell carcinoma that develops within a chronically open wound or scar area (Fine et al., 2009Fine J.D. Johnson L.B. Weiner M. et al.Epidermolysis bullosa and the risk of life-threatening cancers: The National EB Registry experience, 1986–2006.J Am Acad Dermatol. 2009; 60: 203-211Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). The current mainstays of treatment for RDEB are palliative interventions, which primarily consist of bandaging of the skin, and nutritional sustenance (Fine et al., 2014Fine J.D. Bruckner-Tuderman L. Eady R.A. et al.Inherited epidermolysis bullosa: Updated recommendation on diagnosis and classification.J Am Acad Dermatol. 2014; 70: 1103-1126Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar). Unfortunately, to date, there are no approved treatments for RDEB, thus generating numerous investigations toward developing successful treatments. Although there are no currently approved treatments for RDEB, several investigative groups are developing various therapies for RDEB including ex vivo gene therapy, cell therapy, and protein replacement therapy (Ortiz-Urda et al., 2002Ortiz-Urda S. Thyagarajan B. Keene D.R. et al.Stable nonviral genetic correction of inherited human skin disease.Nat Med. 2002; 8: 1166-1170Crossref PubMed Scopus (225) Google Scholar; Chen et al., 2002Chen M. Kasahara N. Keene D.R. et al.Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa.Nat Genet. 2002; 32: 670-675Crossref PubMed Scopus (163) Google Scholar; Woodley et al., 2003Woodley D.T. Krueger G.G. Jorgensen C.M. et al.Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone.J Invest Dermatol. 2003; 121: 1021-1028Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, Woodley et al., 2004Woodley D.T. Keene D.R. Atha T. et al.Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa.Nat Med. 2004; 10: 693-695Crossref PubMed Scopus (111) Google Scholar, Woodley et al., 2004Woodley D.T. Atha T. Huang Y. et al.Intradermal injection of lentiviral vectors corrects regenerated human dystrophic epidermolysis bullosa skin tissue in vivo.Mol Ther. 2004; 11: 318-326Abstract Full Text Full Text PDF Scopus (74) Google Scholar; Wong et al., 2008Wong T. Gammon L. Liu L. et al.Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa.J Invest Dermatol. 2008; 128: 2179-2189Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar; Remington et al., 2009Remington J. Wang X. Hou Y. et al.Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.Mol Ther. 2009; 17: 26-33Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar; Wagner et al., 2010Wagner J.E. Ishida-Yamamoto A. McGrath J.A. et al.Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.N Engl J Med. 2010; 363: 629-639Crossref PubMed Scopus (265) Google Scholar). C7 is an unusual collagen that, unlike other collagens, can be soluble in neutral buffer and blood and does not initiate the clotting cascade or platelet aggregation (Saelman et al., 1994Saelman E.U. Nieuwenhuis H.K. Hese K.M. et al.Platelet adhesion to collagen types I through VIII under conditions of stasis and flow is mediated by GPIa/IIa (alpha 2 beta 1-integrin).Blood. 1994; 83: 1244-1250Crossref PubMed Google Scholar). We have recently been exploring the possibility of delivering C7 intravenously. This would have the advantage of delivering C7 simultaneously to multiple skin wounds and wounds in the oral mucosa and esophagus. We showed that intravenously injected, molecularly engineered RDEB fibroblasts (overexpressing human C7) and fibroblast-derived recombinant C7 (FB-rhC7) itself distributed to murine skin wounds, incorporated into the skin’s DEJ, formed AF structures, and accelerated wound closure (Woodley et al., 2007Woodley D.T. Remington J. Huang Y. et al.Intravenously injected human fibroblasts home to skin wounds, deliver type VII collagen and promote wound healing.Mol Ther. 2007; 15: 628-635Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, Woodley et al., 2013Woodley D.T. Wang X. Amir M. et al.Intravenously injected recombinant human type VII collagen homes to skin wounds and restores skin integrity of dystrophic epidermolysis bullosa.J Invest Dermatol. 2013; 133: 1910-1913Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Although our genetically engineered human dermal fibroblast cell line is an excellent source for producing large quantities of purified rhC7, human dermal fibroblasts are not currently used for large-scale commercial production of therapeutic proteins. Chinese hamster ovary (CHO) cells, in contrast, are commonly used for large-scale production of therapeutic proteins (Jayapal et al., 2007Jayapal K.P. Wlaschin K.F. Hu W.S. et al.Recombinant protein therapeutics from CHO cells—20 years and counting.Chem Eng Prog. 2007; 103: 40-47Google Scholar; Kim et al., 2012Kim J.Y. Kim Y.G. Lee G.M. CHO cells in biotechnology for production of recombinant proteins: current state and further potential.Appl Microbiol Biotechnol. 2012; 93: 917-930Crossref PubMed Scopus (495) Google Scholar). We have expressed recombinant human C7 in a CHO cell line using a eukaryotic expression vector and generated rhC7 molecules in sufficient amounts for biological characterization and further functional analysis. Our structural and functional characterization of rhC7 purified from CHO cells (CHO-rhC7) shows that CHO-derived rhC7 is similar to FB-rhC7 based on a number of physicochemical assessments. We also evaluated the efficacy of intravenously administration of CHO-rhC7 in the neonatal C7-null (Col7a1−/−) mouse model. We showed that the intravenously injected CHO-rhC7 distributed and incorporated into the DEJ of RDEB mice at multiple skin sites as well as the tongue and esophagus and formed AFs. As a consequence, intravenous administration of CHO-rhC7 significantly improved the RDEB murine phenotype as demonstrated by reduced dermal–epidermal separation and markedly prolonged survival. Furthermore, we did not detect any tissue deposited or circulating anti-human C7 antibodies in any of the neonatal RDEB mice administered a single intravenous injection of CHO-rhC7. These data provide evidence of intravenous CHO-rhC7 injection in RDEB-like C7 knockout mice with improvement of key features of the disease. Finally, these data show that molecularly engineered CHO cells are capable of synthesizing and secreting a large human extracellular matrix macromolecule that retains the physical and functional attributes of the authentic human protein. As shown in Figure 1a, CHO-rhC7 migrates at the same position as FB-rhC7 (Figure 1a). In addition, similar to FB-rhC7, CHO-rhC7 was also glycosylated (Figure 1b). When the purified CHO-rhC7 was subjected to 4–12% SDS-PAGE under non-reducing conditions (Figure 1c), it migrated at the same position as FB-rhC7 with an apparent molecular weight of 870 kDa, in agreement with the reported molecular weight of an rhC7 trimer (Chen et al., 2002Chen M. Costa F.K. Lindvay C.R. et al.The recombinant expression of full-length type VII collagen and characterization of molecular mechanisms underlying dystrophic epidermolysis bullosa.J Biol Chem. 2002; 277: 2118-2124Crossref PubMed Scopus (69) Google Scholar). To determine whether the rhC7 trimers formed in vitro are stable and have a proper triple-helical conformation in their collagenous domain, the purified rhC7 was treated with chymotrypsin and then subjected to immunoblot analysis using a polyclonal antibody against the triple-helical domain. Under these digestion conditions, the noncollagenous NC1 and NC2 domains were removed. The triple-helical domain is resistant to digestion, with the exception of cleavage into the P1 and P2 fragments (Figure 1e), as previously described by Burgeson, 1993Burgeson R.E. Type VII collagen, anchoring fibrils, and epidermolysis bullosa.J Invest Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Scopus (236) Google Scholar. As shown in Figure 1d, digestion of both the 290-kDa CHO-rhC7 and FB-rhC7 with chymotrypsin yielded 200 and 119 kDa fragments that were resistant to further protease degradation, corresponding to the intact triple-helical domain (TH) and its carboxyl-terminal half, the P1 fragment. These data suggest that similar to FB-rhC7, CHO-rhC7 forms a triple helix with a collagenous domain resistant to protease digestion. We previously demonstrated that the NC1 domain of FB-rhC7 interacts with fibronectin (Chen et al., 1997Chen M. Marinkovich M.P. Veis A. et al.Interactions of the amino-terminus noncollagenous domain (NC1) of type VII collagen with extracellular matrix components: a potential role in epidermal-dermal adherence in human skin.J Biol Chem. 1997; 272: 14516-14522Crossref PubMed Scopus (160) Google Scholar). To evaluate the ability of purified CHO-rhC7 to bind to fibronectin, we subjected both CHO-rhC7 and FB-rhC7 to our solid-phase ligand-binding assays. As shown in Figure 2a, CHO-rhC7 binds to fibronectin similar to FB-rhC7 in a dose-dependent manner. We previously demonstrated that rhC7 isolated from fibroblasts promotes skin cell migration. Therefore, we assessed the ability of rhC7 isolated from CHO cells to promote the migration of human keratinocytes (Woodley et al., 2008Woodley D.T. Hou Y.P. Martin S. et al.Characterization of molecular mechanisms underlying mutations in dystrophic epidermolysis bullosa using site-directed mutagenesis.J Biol Chem. 2008; 283: 17838-17845Crossref PubMed Scopus (29) Google Scholar). Figure 2b shows representative microscopic fields. CHO-rhC7 strongly promoted human keratinocyte migration at levels similar to those generated by FB-rhC7. In contrast, laminin 1 failed to promote keratinocyte migration. These data indicate that, similar to FB-rhC7, CHO-rhC7 is a potent promotility matrix for human keratinocytes. To evaluate the feasibility of intravenous protein replacement therapy for RDEB, we tested the ability of intravenously injected CHO-rhC7 to correct the RDEB phenotype in murine C7-knockout mice (Col7a1−/−) that recapitulate the clinical, genetic, immunohistochemical, and ultrastructural characteristics of severe human RDEB. Neonatal Col7a1−/− mice were administered a single intravenous injection of 16 μg of CHO-rhC7 (n=53) or a vehicle control (n=23) via the superficial temporal vein. Tissue sections from various skin and internal sites were then obtained 1 week after initial injection and subjected to immunostaining with a polyclonal rabbit antibody that recognized the amino-terminal noncollagenous domain (NC1) of human C7. As shown in Figure 3a (top two rows), the injected CHO-rhC7 distributed and incorporated into the DEJ of multiple skin sites including abdomen, back, front leg, rear leg, chest, head, front and back paw, and tongue and esophagus. No C7 was found at any site when C7-knockout mice were injected with vehicle (Figure 3a, bottom row). Moreover, the injected CHO-rhC7 was detected at the DEJ of tongue and esophagus up to 5 months after a single injection (Supplementary Figure S1 online). Our data provide evidence that the intravenously administered CHO-rhC7 distributes to the tongue and esophagus that would not be accessible by topical or intradermal therapies. Download .pdf (4.55 MB) Help with pdf files Supplementary Information After demonstrating that intravenously administered CHO-rhC7 distributed to the DEJ of the RDEB mouse’s skin wounds, we next determined whether there is a dose-dependent distribution to the DEJ. We intravenously injected neonatal RDEB mice with 0 μg (n=12), 5 μg (n=16), 16 μg (n=25), and 28 μg (n=11) of CHO-rhC7. We then killed the mice 4 days after injection and examined a single organ, the tongue, for the distribution of CHO-rhC7. As shown in Figure 3b, there was a dose-dependent increase in the incorporation of CHO-rhC7 into the mouse’s DEJ. Next, we wanted to know whether intravenously injected CHO-rhC7 protein also distributed to non-target tissues. We examined tissue sections from stomach, tongue, small intestine, brain, kidney, liver, lung, spleen, and heart 2 to 3 weeks following a single intravenous injection and subjected them to immunostaining with an antibody specific for C7. The rhC7 was only readily observed in the tongue but not in any organs described above (Supplementary Figure S2 online). We next determined whether intravenously administered CHO-rhC7 could reverse the RDEB phenotype of the RDEB mice. Histological analysis of multiple skin sites from intravenously injected RDEB mice revealed markedly improved dermal–epidermal adherence as shown in Figure 4a. In contrast, the skin from the vehicle control-injected RDEB mice continued to exhibit poor dermal–epidermal adherence. As RDEB mice typically die within the first week of life (Heinonen et al., 1999Heinonen S. Mannikko M. Klement J.F. et al.Targeted inactivation of the type VII collagen gene (Col7a1) in mice results in severe blistering phenotype: a model for recessive dystrophic epidermolysis bullosa.J Cell Sci. 1999; 112: 3641-3648Crossref PubMed Google Scholar), we also determined their survival over time following a single intravenous administration of CHO-rhC7. The Kaplan–Meier curve revealed that a single intravenous administration of CHO-rhC7 significantly increased the survival of RDEB mice (Figure 4b) compared with vehicle-treated mice (log-rank P-value <0.001). The median overall survival was 12 days for CHO-rhC7-injected mice compared with 3 days for vehicle-injected RDEB mice, implying a 4-fold prolongation of survival time. Of note, 14 of the 53 (26%) CHO-rhC7-injected mice survived beyond 8 weeks after receiving a single intravenous infusion with 16 μg of CHO-rhC7. Therefore, intravenous CHO-rhC7 administered to RDEB mice not only corrected their skin RDEB phenotype but also improved their survival. We next sought to determine whether the CHO-rhC7 could form AFs at the DEJ in vivo. Transmission immunoelectron microscopy was performed on the tongue of RDEB mice after the intravenous administration of CHO-rhC7. As shown in Figure 5, intravenous administered CHO-rhC7 incorporated into the mouse’s DEJ. As expected, there were no detectable AFs in mice treated with vehicle control. Taken together, these data indicate that protein-based therapy by intravenous injection of CHO-rhC7 can correct the abnormal RDEB dermal–epidermal separation and restore C7 expression and AF formation at the DEJ in RDEB mice. We next examined whether intravenously injected CHO-rhC7 would induce an immune response in neonatal RDEB mice. Specifically, we looked for the induction of anti-C7 antibodies in the blood and skin of these mice after a single intravenous CHO-rhC7 administration. Sera from CHO-rhC7-injected mice (n=12) were taken at 30 and 60 days following a single intravenous administration of CHO-rhC7 and then subjected to a commercially available ELISA for anti-C7 antibodies. Interestingly, there were no detectable anti-C7 antibodies in all 12 mice examined at both time points (Supplementary Figure S3 online). In addition, direct immunofluorescence staining performed on the skin of RDEB mice treated with intravenous CHO-rhC7 was negative for IgG deposits at the DEJ, despite the presence of human C7 at their DEJ (Supplementary Figure S4 online). Protein replacement therapy has been successful in a number of hereditary diseases other than RDEB, including hereditary angioedema, Gaucher’s disease, Fabry’s disease, and others (Schiffmann et al., 2001Schiffmann R. Kopp J.B. Austin III, H.A. et al.Enzyme replacement therapy in Fabry disease: a randomized controlled trial.JAMA. 2001; 285: 2743-2749Crossref PubMed Scopus (1126) Google Scholar; Beutler, 2004Beutler E. Enzyme replacement in Gaucher disease.PLoS Med. 2004; e21: 118-121Google Scholar; Desnick, 2004Desnick R.J. Enzyme replacement and enhancement therapies for lysosomal diseases.J Inherit Metab Dis. 2004; 27: 385-410Crossref PubMed Scopus (180) Google Scholar; Leader et al., 2008Leader B. Baca Q.J. Golan D.E. Protein therapeutics: a summary and pharmacological classification.Nat Rev Drug Dis. 2008; 7: 21-39Crossref PubMed Scopus (1422) Google Scholar; Cicardi and Zanichelli, 2010Cicardi M. Zanichelli A. Replacement therapy with C1 esterase inhibitors for hereditary angioedema.Drugs Today. 2010; 46: 867-874Crossref PubMed Scopus (14) Google Scholar). CHO cells are widely used for large-scale production of therapeutic recombinant proteins. In this paper, we demonstrated that CHO cells generate high-quality rhC7 that behaves similar to FB-rhC7, both structurally and functionally. In the study herein, we demonstrated that intravenously injected CHO-rhC7 distributed and incorporated into multiple skin sites (legs, paws, back, abdomen, and chest), and the tongue and esophagus of RDEB-like C7-knockout mice. We noticed that strong C7 staining was often detected in the tongue, front and back paws, and the front leg. It is possible that these sites are more trauma-prone areas with consistent wounds and immature vessels that allow more intravenous C7 to extravasate from the blood into the skin and incorporate into the DEJ. In addition, we detected CHO-rhC7 in the esophagus. These data provide direct evidence that an intravenous administration in RDEB mice is capable of delivering CHO-rhC7 to affected areas including the posterior oral cavity and esophagus. RDEB mice typically die within the first week of life (Heinonen et al., 1999Heinonen S. Mannikko M. Klement J.F. et al.Targeted inactivation of the type VII collagen gene (Col7a1) in mice results in severe blistering phenotype: a model for recessive dystrophic epidermolysis bullosa.J Cell Sci. 1999; 112: 3641-3648Crossref PubMed Google Scholar). In this study, we found that intravenously administered CHO-rhC7 significantly increases the survival of RDEB mice. This is most likely because the intravenously injected C7 incorporates into multiple widespread skin and mucosal wounds improving the dermal–epidermal adherence, which allows the mice to live beyond this critical first week of life. Once past this critical period, we speculate that hair growth in the animals may provide additional protection and lessen new blister formation. It is conceivable that the formation of mature hairs traversing both the dermis and the epidermis of the skin may provide some intrinsic epidermal–dermal adherence. In addition, the incorporation of CHO-rhC7 into the DEJ of the animal’s tongue and esophagus may also contribute to their increased survival by allowing them to eat solid food once they weaned off the mother’s milk. In our previous study using an intradermal injection approach for administering the FB-rhC7, the median survival of the RDEB mice receiving FB-rhC7 was 5 weeks (Remington et al., 2009Remington J. Wang X. Hou Y. et al.Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.Mol Ther. 2009; 17: 26-33Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In the present study, the median survival of the RDEB mice receiving CHO-rhC7 was 12 days. It is important to point out, however, that the experiments are not at all comparable. In the intradermal FB-rhC7 administration experiments, the FB-rhC7 was administered multiple times (once every day for the first week and then weekly thereafter) with cumulative total FB-rhC7 doses between 20 and 300 μg. In contrast, in our present study, herein using an intravenous approach, the RDEB mice only received one single dose of 16 μg of CHO-rhC7. In our previous study, using RDEB mice that were injected intradermally with FB-rhC7, we observed that the mice developed circulating anti-C7 antibodies within 1 month after injection and that the titers of these antibodies remained the same for more than 6 months (Remington et al., 2009Remington J. Wang X. Hou Y. et al.Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.Mol Ther. 2009; 17: 26-33Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Nevertheless, these mouse anti-C7 antibodies did not deposit in the DEJ of the skin of the mice, despite an abundance of human C7 at their DEJ. In the present study, which used intravenous administration of CHO-rhC7, however, we did not detect any anti-C7 antibodies in the blood or skin of the mice. In these studies, a single intravenous injection was performed within the neonatal period (within 24 or 48 hours after birth), which is when the developing immune system is particularly susceptible to the induction of tolerance. We speculate that tolerance induction to C7 may be responsible for the lack of the immune responses and the absence of anti-C7 antibodies in these mice. Neonatal gene transfer and neonatal protein injection have the potential to reduce immune responses because newborn immune systems are immature. For example, the induction of immune tolerance by neonatal intravenous injection of human factor VIII into hemophilia A–deficient mice has been reported (Madoiwa et al., 2004Madoiwa S. Yamauchi T. Hakamata Y. et al.Induction of immune tolerance by neonatal intravenous injection of human factor VIII in murine hemophilia A.J Thromb Haemost. 2004; 2: 754-762Crossref PubMed Scopus (33) Google Scholar). Similarly, there were no antibodies detected in hemophilia B dogs after injection of recombinant factor IX protein or gene therapy with retroviral vectors expressing factor IX (Xu et al., 2003Xu L. Gao C. Sands M.S. et al.Neonatal or hepatocyte growth factor-potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B.Blood. 2003; 101: 3924-3932Crossref PubMed Scopus (85) Google Scholar). In general, therapeutic proteins have limited half-lives in vivo and require repeated administration to maintain therapeutic efficacy. For an example, frequent injections or infusions of replacement proteins were needed for patients suffering with hemophilia (Batorova et al., 2010Batorova A. High K.A. Gringeri A. Special lectures in haemophilia management.Haemophilia. 2010; 5: 22-28Crossref Scopus (11) Google Scholar). Nevertheless, in our previous study, using rhC7 purified from gene-corrected RDEB fibroblasts, we showed that intradermally injected FB-rhC7 stably incorporated into the DEJ of RDEB mice and persisted there for at least 2 months (Remington et al., 2009Remington J. Wang X. Hou Y. et al.Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.Mol Ther. 2009; 17: 26-33Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Similarly, our study with human RDEB skin equivalents transplanted onto immunodeficient mice also found that FB-rhC7 administered to skin equivalents endures for at least 3 months (Woodley et al., 2004Woodley D.T. Keene D.R. Atha T. et al.Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa.Nat Med. 2004; 10: 693-695Crossref PubMed Scopus (111) Google Scholar). In the present study, using CHO-rhC7, we were still able to detect low levels of C7 in the tongue and esophagus even at 5 months after injection in a subset of mice. Therefore, it appears that CHO-rhC7 has a comparable in vivo half-life as FB-rhC7, although additional studies are needed to determine the exact tissue half-life of CHO-rhC7. It should be pointed out that there may be an advantage working with collagen as a therapeutic agent, because collagens in general have slow turnover times and are stable long-lived molecules (Pelkonen and Kivirikko, 1970Pelkonen R. Kivirikko K.I. Hydroxyprolinemia: an apparently harmless familial metabolic disorder.N Engl J Med. 1970; 283: 451-456Crossref PubMed Scopus (31) Google Scholar; Burgeson, 1993Burgeson R.E. Type VII collagen, anchoring fibrils, and epidermolysis bullosa.J Invest Dermatol. 1993; 101: 252-255Abstract Full Text PDF PubMed Scopus (236) Google Scholar). In the case of RDEB, it is known that one does not need 100% of the normal levels of C7 or AFs to exhibit physiologically normal epidermal–dermal adherence. In fact, it is estimated that only about 35% of the normal AF complement is required to provide clinically sufficient dermal–epidermal adherence (Fritsch et al., 2008Fritsch A. Loeckermann S. Kern J.S. et al.A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy.J Clin Invest. 2008; 118: 1669-1679Crossref PubMed Scopus (169) Google Scholar). In the present study, quantifying the immunofluorescence intensity by the Image J analysis of RDEB mice intravenously injected with 16 μg of CHO-C7 revealed that one injection restored 25–50% of the normal C7 level at the DEJ in selected areas of tongue and paws. As the maximum volume that can be injected into newborn RDEB mice is 40 μl, this prevents us from evaluating the upper limit of C7 that we can use to achieve maximum C7 levels at the DEJ. In summary, we demonstrated that purified CHO-rhC7 has similar structural and functional properties of FB-rhC7. In addition, we showed that intravenous injection of CHO-rhC7 into neonatal RDEB mice led to restoration of C7 and AFs at the DEJ of murine RDEB skin, tongue, and esophagus with significant beneficial effects including decreased skin fragility and blistering, improved epidermal–dermal adherence, and improved survival. In addition, systemic delivery of extracellular matrix molecules may represent a potential therapeutic strategy for other skin disorders because of defects in genes coding for other structural proteins in the skin. Primary human keratinocytes were purchased from Cascade Biologics (Portland, OR) and cultured in low calcium, serum-free keratinocyte growth medium supplemented with bovine pituitary extract and epidermal growth factor (Gibco BRL, Gaithersburg, MD) as described by Boyce and Ham, 1983Boyce S.T. Ham R.G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.J Invest Dermatol. 1983; 81: 33S-44SAbstract Full Text PDF PubMed Scopus (958) Google Scholar and modified by O’Keefe and Chiu, 1988O’Keefe E.J. Chiu M.L. Stimulation of thymidine incorporation in keratinocytes by insulin, epidermal growth factor, and placental extract: comparison with cell number to assess growth.J Invest Dermatol. 1988; 90: 2-7Abstract Full Text PDF PubMed Scopus (71) Google Scholar. Third or fourth passage keratinocytes were used for cell migration studies. The CHO-rhC7 material for the studies was provided by Shire and produced by cell culture and purification methods known in the art. Some methods are described elsewhere (De Souza and Viswanathan, 2013De Souza M. Viswanathan M. Collagen 7 and Related Methods. Lotus Tissue Repair Inc., 2013Google Scholar). FB-rhC7 was purified as described previously (Chen et al., 2002Chen M. Kasahara N. Keene D.R. et al.Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa.Nat Genet. 2002; 32: 670-675Crossref PubMed Scopus (163) Google Scholar; Woodley et al., 2004Woodley D.T. Keene D.R. Atha T. et al.Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa.Nat Med. 2004; 10: 693-695Crossref PubMed Scopus (111) Google Scholar). The binding of soluble C7 to immobilized fibronectin (purchased from Life Technology, Gaithersburg, MD) was measured using a colorimetric enzyme-linked antibody assay as described previously (Chen et al., 1997Chen M. Marinkovich M.P. Veis A. et al.Interactions of the amino-terminus noncollagenous domain (NC1) of type VII collagen with extracellular matrix components: a potential role in epidermal-dermal adherence in human skin.J Biol Chem. 1997; 272: 14516-14522Crossref PubMed Scopus (160) Google Scholar). Purified recombinant C7 was incubated with chymotrypsin (Sigma, St Louis, MO) at an enzyme-to-substrate ratio of 1:2 by weight in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl at 37 °C for 3 hours and then analyzed by SDS-PAGE, followed by immunoblot analysis with a polyclonal antibody against the collagenous domain of C7, as described previously (Chen et al., 2002Chen M. Costa F.K. Lindvay C.R. et al.The recombinant expression of full-length type VII collagen and characterization of molecular mechanisms underlying dystrophic epidermolysis bullosa.J Biol Chem. 2002; 277: 2118-2124Crossref PubMed Scopus (69) Google Scholar; Woodley et al., 2008Woodley D.T. Hou Y.P. Martin S. et al.Characterization of molecular mechanisms underlying mutations in dystrophic epidermolysis bullosa using site-directed mutagenesis.J Biol Chem. 2008; 283: 17838-17845Crossref PubMed Scopus (29) Google Scholar). Keratinocyte migration was assayed by the method of Albrecht-Buehler, 1977Albrecht-Buehler G. The phagokinetic tracks of 3T3 cells.Cell. 1977; 11: 395-404Abstract Full Text PDF PubMed Scopus (384) Google Scholar, as modified by Woodley et al., 1988Woodley D.T. Bachmann P.M. O’Keefe E.J. Laminin inhibits human keratinocyte migration.J Cell Physiol. 1988; 136: 140-146Crossref PubMed Scopus (144) Google Scholar. Col7a1+/− animals were developed as described previously (Heinonen et al., 1999Heinonen S. Mannikko M. Klement J.F. et al.Targeted inactivation of the type VII collagen gene (Col7a1) in mice results in severe blistering phenotype: a model for recessive dystrophic epidermolysis bullosa.J Cell Sci. 1999; 112: 3641-3648Crossref PubMed Google Scholar) and maintained at the animal facilities of the University of Southern California, Los Angeles, California under guidelines for the care and use of animals in research. All animal studies were conducted using protocols approved by the University of Southern California Institutional Animal Use Committee. For protein therapy, we intravenously injected newborn RDEB mice with either 16 μg of CHO-rhC7 suspended in 40 μl of vehicle buffer (n=53) or 40 μl of vehicle alone (n=23) via the superficial temporal vein. At various times after intravenous administration of rhC7 or vehicle control, the RDEB mice were biopsied at multiple skin mucosal sites including abdomen, back, front leg, rear leg, chest, and tongue and esophagus, and the skin specimens were subjected to immunostaining using a rabbit polyclonal antibody recognizing the NC1 domain of human C7 (Chen et al., 1997Chen M. Petersen P.J. Hai-Li L. et al.Ultraviolet A irradiation up-regulates type VII collagen expression in human dermal fibroblasts.J Invest Dermatol. 1997; 108: 125-128Abstract Full Text PDF PubMed Scopus (34) Google Scholar) as described below. Histological sections of the mouse skin specimens were fixed in 10% buffered formalin and stained with hematoxylin and eosin (H&E). Blood samples were taken at the indicated times from the tail vein and then stored at 4 °C overnight. Immunolabeling of the tissue was performed using standard immunofluorescence methods as described previously (Woodley et al., 2004Woodley D.T. Keene D.R. Atha T. et al.Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa.Nat Med. 2004; 10: 693-695Crossref PubMed Scopus (111) Google Scholar; Remington et al., 2009Remington J. Wang X. Hou Y. et al.Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.Mol Ther. 2009; 17: 26-33Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Transmission electron microscopy was performed on the mouse tongue using a standardized method as described previously (Keene et al., 1987Keene D.R. Sakai L.Y. Lunstrum G.P. et al.Type VII collagen forms an extended network of anchoring fibrils.J Cell Biol. 1987; 104: 611-621Crossref PubMed Scopus (290) Google Scholar; Sakai and Keene, 1994Sakai L.Y. Keene D.R. Fibrillin: monomers and microfibrils.in: Ruoslahti E. Engvall E. Methods in Enzymology. vol 245. Academic Press, New York, NY1994: 47-50Google Scholar). The production of circulating anti-C7 antibodies was evaluated by ELISA with NC1 and NC2 domains of C7 following the manufacturer’s procedure using mouse’s serum at a dilution of 1:100 (MBL, Nagoya, Japan). To evaluate whether there are any anti-C7 antibodies deposited directly in the skin, RDEB mouse skin specimens from CHO-rhC7-positive areas were subjected to direct immunofluorescence staining using FITC–conjugated goat anti-mouse IgG (Sigma), as described previously (Woodley et al., 1984Woodley D.T. Briggaman R.A. O’Keefe E.J. et al.Identification of the skin basement membrane autoantigen in epidermolysis bullosa acquisita.N Engl J Med. 1984; 310: 1007-1013Crossref PubMed Scopus (435) Google Scholar). This work was supported by Sponsored Research Project grant from Shire to MC and DTW. We thank Sara Tufa for technical support of transmission electron microscopy. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid" @default.
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