Restoration Of Recombinant Human Type VII Collagen And Correction Of The Disease Phenotype In A Murine Model Of Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa ( DEB ) is a group of heritable mechano-bullous skin diseases characterized by skin fragility,separation of the epidermis from the dermis(blister formation),milia and scarring of varying clinical severity. DEB is transmitted in either a dominant(DDEB)or a recessive(RDEB)mode. All forms of DEB are caused by mutations in COL7A1, the gene encoding for type VII collagen(C7).DEB affects thousands of families and over 300 distinct COL7A1 mutations have been identified in DEB patients. Mutations in COL7A1 result in the abnormal synthesis and assembly of anchoring fibrils and poor adherence between the epidermis and dermis. The most severe form of DEB is recessive dystrophic EB(the Hallopeau-Siemens type,HS-RDEB)in which both C7 and anchoring fibrils are absent from the skin due to null mutations in the COL7A1 gene. As a result , HS-RDEB is characterized by severe skin blistering,extremely fragile skin, mutilating scarring of the hands and feet,joint contractures,and strictures of the esophagus. In the second or third decade of life , HS-RDEB patients develop aggressive squamous cell carcinomas in chronically wounded areas which often lead to metastasis and death.C7 is the major component of anchoring fibrils,attachment structures in the basement membrane zone(BMZ)of skin that serve to adhere the epidermal layer of skin onto the dermis. Type VII collagen is a genetically distinct member of the collagen family of proteins. It is a major component of anchoring fibrils,attachment structures that mediate dermal-epidermal adherence in human skin. The BMZ of patients with DEB is characterized by a paucity or altered morphology of anchoring fibrils. Type VII collagen is composed of three identicalαchains, each consisting of a 145-kDa central collagenous triple-helical segment characterized by repeating Gly-X-Y amino acid sequences,flanked by a large 145-kDa amino-terminal , noncollagenous domain(NC1),and a small 34-kDa carboxyl-terminal noncollagenous domain(NC2). NC1 domains have been suggested to interact at one end with BMZ components and at the other end with type IV collagen in "anchoring plaques".A mouse model has been developed for RDEB in immunocompetent mice by targeted inactivation of the COL7A1 gene. These COL7A1 null (Col7a1-/-) mice have no C7 at the BMZ of their skin and they entirely lack ultrastructurally recognizable anchoring fibrils. Electron microscopy also reveals sub-lamina densa bullae precisely like those in human DEB patients. Clinically, the newborn mice exhibit extensive blisters and die within the first week of life, probably from complications due to the extensive blistering. Thus, these Col7a1-/- mice recapitulate many of the clinical, genetic and ultrastructural features of severe RDEB patients.In this study,we sought to determine if protein therapy with intradermal C7 injections into these mice could reverse their DEB-like disease. We showed that the intradermally injected human C7 translocated and stably incorporated into the mouse's BMZ and formed anchoring fibrils. As a consequence, the DEB murine phenotype was significantly improved with decreased skin fragility and blistering and markedly prolonged survival. Most interestingly,although anti-human C7 antibodies were induced by the injected protein,apparently the antibodies did not exhibit any adverse effects.Objectives1 Recombine human type VII collagen and express in vitro.2 Expression recombinant human type VII collagen in vivo. Methods1 The restoration of recombinant full-length type VII collagen expression in vitro.1.1 Cell Culture Immortalized RDEB fibroblasts(Primary RDEB fibroblasts were gift from S. Herron,Stanford University) were routinely cultured in DMEM/Ham's F12(1:1), supplemented with 10% fetal bovine serum, incubated in culture dish at 37°C with 5% CO2 incubator.1.2 Gene transfer by lentiviral vector We subcloned a 8,850-bp full- length type VII collagen cDNA (excluding its polyadenylation signal and 3' untranslated sequences) into the polylinker sequence just downstream of the MND internal promoter in the SMPU minimal lentiviral transfer vector construct. Then do the lentiviral infection in RDEB fibroblasts.1.3 Immunofluorescence staining Immunofluorescence staining of lentivirally transduced RDEB fibroblasts.1.4 Type VII protein purification and analysis For immunoblot analysis,grew lentivirally transduced fibroblasts to confluency,add conditioned medium containing 150μM ascorbic acid and then maintained the cultures for an additional 24 h. Collected medium,with serine protease inhibitors, then subjected to 6% SDS?PAGE. We detected the presence of type VII collagen by western-blot analysis using a polyclonal antibody to the NC1 domain of type VII collagen. For large scale purification of recombinant type VII collagen, serum-free media were equilibrated to serine protease inhibitors and precipitated with ammonium sulfate stir overnight. Precipitated proteins were collected by centrifuging , resuspended , and dialyzed. Following dialysis , insoluble material was collected by centrifugation,and the pellet redissolved,then passed over a Q-Sepharose column(Amersham Biosciences,Inc.). Elution was then carried out with a linear gradient from varies concentration NaCl of appropriate volume size. The type VII collagen was eluted at 1 M NaCl.2 The restoration of recombinant full-length type VII collagen expression in vivo.2.1 Identify Col7a1 null(Col7a1-/-) mice DEB mice were readily identified at birth by the large fluid-filled blisters developed primarily on the ventral side of the animals and the large hemorrhagic blisters on their paws.2.2 Identify Col7a1 recessive(Col7a1+/-) mice Col7a1+/- mice genotype was further confirmed by Polymerase chain reaction(PCR).2.3 Treat Col7a1 null(Col7a1-/-)mice with type VII collagen Inject certain amount C7 to back skin of Col7a1-/- mice intradermally everyday, dependent on how many days each of these DEB mice survived(range from 4 days to 7 months), the cumulative total amounts of C7 injected into each DEB mouse varied from 20μg to 300μg.2.4 Immunofluorescence staining and ultrastructural analysis of tissue Fiveμm thick sections of the OCT-embedded tissues were cut on a cryostat, fixed in acetone and incubated with a rabbit polyclonal antibody recognizing both mouse and human C7, followed by a Cy3-conjugated goat anti-rabbit IgG . Slides were mounted with 40% glycerol. Pictures from stained sections were taken using a Zeiss Axioplan fluorescence microscope equipped with a Zeiss Axiocam MRM digital camera system.2.5 Enzyme-linked immunosorbent assay(ELISA) using recombinant NC1-- The production of circulating anti-C7 antibodies was evaluated by ELISA. Sera from C7 injected mice(n = 6)were taken at various time points after treatment and then subjected to our NC1-based ELISA analysis to determine the presence of anti-C7 antibodies.2.6 MR1 and control IgG treatment The DEB mice were injected intraperitoneally with either MR1(n = 3)or control hamster Ig G(n =3).The dose of MR1 was determined based on previous reports as well as our dose-response study. We used 2×10-2μg/kg body weight at day 0 and 1×10-2μg/kg body weight per mouse at days 2, 4, 7, 14, 21, and 28. Serum samples from these mice were collected once a month and analyzed with ELISA using human NC1, as described above.Results1 The restoration of recombinant full-length type VII collagen expression in vitro.1.2 Lentiviral vector production and gene transfe The a 8,850-bp full-length type VII collagen cDNA was subcloned into the polylinker sequence just downstream of the MND internal promoter in the SMPU minimal lentiviral transfer vector construct. And RDEB fibroblasts infected by the lentiviral transfer vector which carry type VII collagen cDNA.1.3 Immunofluorescence staining The cells were examined and photographed with a Zeiss Axioplan fluorescence microscope.1.4 Type VII protein purification and analysis Type VII collagen qualify and quantify by Western Blot and coomassie blue.2 The restoration of recombinant full-length type VII collagen expression in vivo.2.1 Identify Col7a1 null (Col7a1-/-) mice Treat with type VII collagen.2.2 Identify Col7a1 recessive (Col7a1+/-) mice Identified Col7a1 recessive (Col7a1+/-) mice were used for reproduce.2.3 Treat Col7a1 null (Col7a1-/-) mice with type VII collagen Blisters abated and no new blisters were observed after 2 or 3 times injections with C7.2.4 Immunofluorescence staining and ultrastructural analysis of tissue The slides were examined and photographed with a Zeiss Axioplan fluorescence microscope.BMZ staining was detected , oriented correctly and formed anchoring fibril structures , thus demonstrating correction of the major ultrastructural abnormality seen in DEB skin.2.5 ELISA using recombinant NC1 Anti-C7 antibodies were detected in all of the six mice examined at 30 days after the initial injection, and the amount increased steadily thereafter.2.6 MR1 and control IgG treatment The production of anti-C7 IgG was detected in all 3 mice treated with control IgG at day 30 and was maintained thereafter. In contrast,the mice receiving MR1 did not show detectable anti-C7 antibodies at any of the time points tested.ConclusionStructural and functional studies of type VII collagen in vitro.Type VII collagen can improve even reverse DEB-like disease on DEB animal model.