Targeted Exon Skipping Restores Type VII Collagen Expression and Anchoring Fibril Formation in an In Vivo RDEB Model  Sandrina Turczynski, Matthias Titeux, Laure Tonasso, Audrey Décha, Akemi Ishida-Yamamoto, Alain Hovnanian  Journal of Investigative Dermatology  Volume 136, Issue 12, Pages 2387-2395 (December 2016) DOI: 10.1016/j.jid.2016.07.029 Copyright © 2016 The Authors Terms and Conditions

Figure 1 C7 devoid of the sequence encoded by exon 73 or exon 80 is functional. (a) Protein extracts from cell supernatants were immunoblotted using the LH7.2 antibody. Monomers (295 kDa) and homotrimers (∼1,000 kDa) of full-length and deleted C7 were detected, indicating that deleted C7 are able to form homotrimers. (b) In skin equivalents made from transduced RDEB cells (K-RDEBΔ73/F-RDEBΔ73, K-RDEBΔ80/F-RDEBΔ80), the recombinant C7 proteins are secreted, form functional anchoring fibrils (arrows) and allow a normal dermal-epidermal adherence. Skin equivalent made from untransduced RDEB cells. Immunohistochemistry scale bar = 100 μm; TEM scale bar = 250 nm. C7, type VII collagen; COL, collagen; D, denaturing; F-RDEB, primary RDEB fibroblast; H&E, hematoxylin and eosin; K-RDEB, primary RDEB keratinocytes; ND, nondenaturing; NHF, normal human fibroblast; RDEB, recessive dystrophic epidermolysis bullosa; SRSF, serine and arginine rich splicing factor; TEM, transmission electron microscopy; WT, wild type. Journal of Investigative Dermatology 2016 136, 2387-2395DOI: (10.1016/j.jid.2016.07.029) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Detection of targeted exon skipping in RDEB-cultured cells. (a) ESEfinder 3.0 (Smith et al., 2006) analyses displaying the sequences and the binding sites of AON-1 and AON-2 on (top) exon 73 or (bottom) exon 80 of COL7A1. The color code indicates the best putative binding sites for SRSF proteins. Intronic sequences are in lower case. Nucleotides in red indicate recurrent recessive mutations within the AON sequences. (b, c) Reverse transcriptase–PCR and sequencing data showing efficient in-frame exon 73 skipping, 48 hours after transfection of fibroblasts (F-RDEB) and keratinocytes (K-RDEB) from patient RDEB-1 with AON-1. (d, e) Reverse transcriptase–PCR and sequencing data showing efficient in-frame exon 80 skipping, 48 hours after transfection of fibroblasts and keratinocytes from patient RDEB-1 with AON-2. Data are representative of two independent experiments. AON, 2′-O-methyl antisense oligoribonucleotides; bp, base pairs; F-RDEB, primary RDEB fibroblast; K-RDEB, primary RDEB keratinocytes; NT, nontransfected; RDEB, recessive dystrophic epidermolysis bullosa; SRSF, serine and arginine rich splicing factor; wt, wild type. Journal of Investigative Dermatology 2016 136, 2387-2395DOI: (10.1016/j.jid.2016.07.029) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Ex vivo rescue of C7 expression in RDEB cells. (a) Immunocytofluorescence analysis of keratinocytes from patient RDEB-1 (compound heterozygote for null mutations in exons 73 and 80), transfected with 50 nmol/L of AON-1 or AON-2, showed C7 re-expression in 20% of the cell population. Scale bar = 10 μm. (b) Western blot analyses showed a significant level of C7 re-expression after transfection of (left) patient RDEB-1 keratinocytes with 50 nmol/L of AON-1 or AON-2, (middle) patient RDEB-2 fibroblasts with 50 nmol/L of AON-2 and (right) patient RDEB-3 keratinocytes with 50 nmol/L of AON-2. Patients RDEB-2 and RDEB-3 are homozygotes for a null mutation in exon 80. Representative of two independent experiments. β-actin, β-actin loading control; AON, 2′-O-methyl antisense oligoribonucleotides; C7, type VII collagen; NT, nontransfected; NHF, normal human fibroblast; NHK, normal human keratinocyte; RDEB, recessive dystrophic epidermolysis bullosa. Journal of Investigative Dermatology 2016 136, 2387-2395DOI: (10.1016/j.jid.2016.07.029) Copyright © 2016 The Authors Terms and Conditions

Figure 4 In vivo rescue of C7 expression and AF formation in RDEB SEs treated with AONs. (a) Immunohistofluorescence analyses of SEs-RDEB-1 showed C7 re-expression after one injection of 500 μg of AON-1 (1 × 500 μg) and two injections of 1,000 μg (2 × 1,000 μg), leading to AF formation (arrowheads), observed by transmission electron microscopy. (b) A single subcutaneous injection under the graft of 400 μg of AON-2 (1 × 400 μg) restored C7 expression in SEs-RDEB-2. Two injections (2 × 400 μg) led to a stronger staining and more numerous AFs. (c) Mean average number of AFs over 4 × 10-μm areas in each sample ± standard error of the mean. Student's t test, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Control: SE made of normal human keratinocytes and fibroblasts. Scale bars = 50 μm for immunohistofluorescence, 200 nm for transmission electron microscopy. AF, anchoring fibril; AON, 2′-O-methyl antisense oligoribonucleotides; C7, type VII collagen; NT, nontreated; RDEB, recessive dystrophic epidermolysis bullosa; SE, skin equivalent. Journal of Investigative Dermatology 2016 136, 2387-2395DOI: (10.1016/j.jid.2016.07.029) Copyright © 2016 The Authors Terms and Conditions