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

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Presentation on theme: "Intravenously Administered Recombinant Human Type VII Collagen Derived from Chinese Hamster Ovary Cells Reverses the Disease Phenotype in Recessive Dystrophic."— Presentation transcript:

1 Intravenously Administered Recombinant Human Type VII Collagen Derived from Chinese Hamster Ovary Cells Reverses the Disease Phenotype in Recessive Dystrophic Epidermolysis Bullosa Mice  Yingping Hou, Lin T. Guey, Timothy Wu, Robert Gao, Jon Cogan, Xinyi Wang, Elizabeth Hong, Weihuang Vivian Ning, Douglas Keene, Nan Liu, Yan Huang, Craig Kaftan, Bruce Tangarone, Igor Quinones-Garcia, Jouni Uitto, Omar L. Francone, David T. Woodley, Mei Chen  Journal of Investigative Dermatology  Volume 135, Issue 12, Pages (December 2015) DOI: /jid Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Structural characterization of recombinant human C7 purified from human dermal fibroblasts (FB-rhC7) and Chinese hamster ovary–cells (CHO-rhC7). A measure of 2μg of purified rhC7 was subjected to 6% SDS-PAGE and (a) Coomassie Blue staining and (b) the Pierce Glycoprotein Staining Kit. (c) A measure of 0.2μg of purified rhC7 was subjected to 4–15% SDS-PAGE under non-reducing conditions, followed by immunoblot analysis with a polyclonal antibody to N-terminal noncollagenous domain of type VII collagen (NC1). (d) Purified rhC7 was incubated with chymotrypsin and analyzed by 6% SDS-PAGE, followed by immunoblot analysis with a polyclonal antibody to the triple-helical domain (TH). (e) Schematic shows that NC1 is held by disulfide bonds into a trident-like shape. Within the TH is a “hinge region”, which is non-helical and protease sensitive. Protease digestion of the triple helix of C7 leads to partial cleavage of the “hinge region” and creates two short triple helical fragments. Lane 1: CHO-rhC7; lane 2: FB-rhC7. The positions of molecular weight markers, monomer (M) and homotrimer (T) of C7, the 200kDa intact TH, and the 190kDa carboxyl-terminal half (the P1 fragment), are indicated. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Chinese hamster ovary–derived recombinant type VII collagen (CHO-rhC7) and recombinant human C7 purified from human dermal fibroblasts (FB-rhC7) bind to fibronectin and promote keratinocyte migration. (a) ELISA plates were coated with fibronectin (20μgml−1) and then incubated with the indicated concentrations of CHO-rhC7 or FB-rhC7 at 4°C overnight. Binding was detected using rabbit anti-N-terminal noncollagenous domain of type VII collagen (NC1) polyclonal antibodies (1:1000), followed by an alkaline phosphatase–conjugated secondary antibody (1:300). Error bars, SE of three different experiments. (b) Left: Colloidal gold migration assay: Coverslips were coated with colloidal gold, and keratinocytes were plated on CHO-rhC7, FB-rhC7, laminin 1, or no matrix (NO) at the indicated amounts and incubated for 18h. Representative fields were photographed at × 40 under dark-field optics. Error bars, SE of three different experiments. Right: Migration index—the percentage of total field area consumed by the migration tracks. Error bars, SE of three different experiments. Note that CHO-rhC7 promotes keratinocyte migration as effectively as FB-rhC7, in a dose-dependent manner. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Intravenously injected Chinese hamster ovary–derived recombinant type VII collagen (CHO-rhC7) distributed to the multiple sites of recessive dystrophic epidermolysis bullosa (RDEB) mice. (a) Newborn RDEB mice were intravenously injected with 16μg of CHO-rhC7 or vehicle control via the superficial temporal vein, and various tissue samples were obtained 5 to 14days after mice were injected and subjected to immunofluorescence (IF) staining with an anti-N-terminal noncollagenous domain of type VII collagen (NC1) antibody. Note that intravenously injected CHO-rhC7 readily transported and incorporated into the dermal–epidermal junction (DEJ) of multiple skin sites (top two rows) whereas none was detected in vehicle-injected mice (bottom row). (b) IF staining with an anti-NC1 antibody was performed on mouse tongues 4days following intravenous administration of various doses of CHO-rhC7, as indicated. Note a dose-dependent increase in the deposition of CHO-rhC7 at the mouse DEJ. Error bars, SE of three different injected mice. Bar=200μm. Abd, abdomen; Eso, esophagus; FL, front leg; FP, front paw; RL, rear leg; RP, rear paw; Ton, tongue. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Intravenous Chinese hamster ovary derived recombinant type VII collagen (CHO-rhC7) improved epidermal–dermal adherence and increased survival of recessive dystrophic epidermolysis bullosa (RDEB) mice. (a) Multiple tissue samples, as indicated, from RDEB mice intravenously injected with CHO-rhC7 or vehicle control were obtained 5 to 14days after injection and subjected to hematoxylin and eosin (H&E) staining. Note that the vehicle-injected RDEB sections showed histological evidence of dermal–epidermal separation. In contrast, CHO-rhC7-injected RDEB skin exhibited good dermal–epidermal adherence. (b) Kaplan–Meier curves showing survival comparison between vehicle control (n=23) and CHO-rhC7 (n=53)-injected RDEB mice. There is a significant improvement in the survival of RDEB mice administered CHO-rhC7 in comparison with vehicle control (log-rank P<0.001). Bar=200μm. Abd, abdomen; FL, front leg; FP, front paw; RL, rear leg; RP, rear paw; Ton, tongue. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Intravenously injected CHO-rhC7 formed anchoring fibrils (AFs) in recessive dystrophic epidermolysis bullosa (RDEB) mice. Transmission electron microscopy was performed on the tongues of RDEB mice intravenously injected with either vehicle control (4days) or Chinese hamster ovary–derived recombinant type VII collagen (CHO-rhC7) at 14days after injection. Note that there were no identifiable AFs in vehicle-injected mice. In contrast, intravenously-injected CHO-rhC7 incorporated into the RDEB skin’s dermal–epidermal junction (DEJ) and formed AFs as indicated by arrows. Bar=500nm for the low magnification image and 200nm for the high magnification insert. D, dermis; E, epidermis; HD, hemidesmosome. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

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