Gene-Corrected Fibroblast Therapy for Recessive Dystrophic Epidermolysis Bullosa using a Self-Inactivating COL7A1 Retroviral Vector Joanna Jacków, Matthias.

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Gene-Corrected Fibroblast Therapy for Recessive Dystrophic Epidermolysis Bullosa using a Self-Inactivating COL7A1 Retroviral Vector Joanna Jacków, Matthias Titeux, Soizic Portier, Soëli Charbonnier, Clarisse Ganier, Sonia Gaucher, Alain Hovnanian Journal of Investigative Dermatology Volume 136, Issue 7, Pages 1346-1354 (July 2016) DOI: 10.1016/j.jid.2016.02.811 Copyright © 2016 The Authors Terms and Conditions

Figure 1 SIN COL7A1 retroviral transduction induces high expression and secretion of C7 in RDEB fibroblasts. Western blot analysis assessing C7 protein expression and secretion in NHF, RDEBF, and RDEBF+C7. (a) Conditioned medium was collected and directly probed against C7 using LH7.2 antibody. (b) Proteins were isolated from cell extracts and processed for WB using RC1 antibody. (c) Densitrometric quantification of two different Western blot analyses of cells were performed in duplicate and showed similar results. C7 expression in NHF and RDEBF+C7 was normalized to β-actin expression. (d) Thermal stability of C7 was analyzed by limited trypsin digestion of medium from RDEBF+C7 at increasing temperature. Triple helices of recombinant C7 were analyzed by immunoblot using polyclonal antibody against C7. (e) Densitometric quantification of C7 in three independent experiments. a.u., arbitury unit; C7, type VII collagen; NHF, normal human fibroblasts; RDEBF, recessive dystrophic epidermolysis bullosa patient fibroblasts; RDEBF+C7, patient-derived recessive dystrophic epidermolysis fibroblasts transduced with C7; T, temperature; TH, triple helix. Journal of Investigative Dermatology 2016 136, 1346-1354DOI: (10.1016/j.jid.2016.02.811) Copyright © 2016 The Authors Terms and Conditions

Figure 2 C7 re-expression in transduced RDEB fibroblasts improves cell adhesion and maintains essential cell properties in vitro. (a) Trypsin-based cell detachment assay. Three independent measurements were performed in triplicate for each cell type. The graph represents the cell adhesion of NHF, RDEBF, and RDEBF+C7 as a percentage of the remaining cells after indicated time of trypsin treatment. Data are shown as mean ± standard error of the mean; ∗∗∗P > .001; n = 3. (b) Quantitative real-time PCR of ITGs expression in NHF, RDEBF, and RDEBF+C7 fibroblasts with GAPDH as a reference gene. Picture of (c) NHF, (d) RDEBF, and (e) RDEBF+C7 fibroblasts in monolayer culture taken using phase-contrast microscopy at 30% of cells confluence and 70% of cells confluence. C7, type VII collagen; mRNA, messenger RNA; NHF, normal human fibroblasts; RDEBF, recessive dystrophic epidermolysis bullosa patient fibroblasts; RDEBF+C7, patient-derived recessive dystrophic epidermolysis fibroblasts transduced with C7. Journal of Investigative Dermatology 2016 136, 1346-1354DOI: (10.1016/j.jid.2016.02.811) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Epidermal adherence, C7 deposition, and anchoring fibrils are restored in RDEB SE at the DEJ, 2 months after injections of RDEBF+C7. Hematoxylin and eosin staining of grafted SE made of (a) NHK and NHF (noninjected), (b) RDEBK and RDEBF (injected with RDEBF) and (c) RDEBK and RDEBF (injected with RDEBF+C7) analyzed 2 months after injection. Arrows point to dermal-epidermal detachment. Scale bar = 10 μm. (d–f) Immunofluorescence staining of RDEB SE sections using the RC1 antibody 2 months after injections. RDEB SE injected with (d) RDEBF, (e) NHF, and (f) RDEBF+C7. Scale bar = 30 μm. Transmission electron microscopy was performed on human RDEB skin injected with (g) RDEBF, (h) NHF, and (i) RDEBF+C7 fibroblasts. Anchoring fibrils are indicated by red arrows. Scale bar = 500 nm. DAPI, 4',6-diamidino-2-phenylindole; K, keratinocyte; NHF, normal human fibroblasts; RDEBF, recessive dystrophic epidermolysis bullosa patient fibroblasts; RDEBF+C7, patient-derived recessive dystrophic epidermolysis fibroblasts transduced with type VII collagen; SE, skin equivalent. Journal of Investigative Dermatology 2016 136, 1346-1354DOI: (10.1016/j.jid.2016.02.811) Copyright © 2016 The Authors Terms and Conditions