Araksya Izmiryan, Olivier Danos, Alain Hovnanian

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Meganuclease-Mediated COL7A1 Gene Correction for Recessive Dystrophic Epidermolysis Bullosa Araksya Izmiryan, Olivier Danos, Alain Hovnanian Journal of Investigative Dermatology Volume 136, Issue 4, Pages 872-875 (April 2016) DOI: 10.1016/j.jid.2015.11.028 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Evaluation of MN expression efficiency in HIV-derived lentiviral vectors and MN-mediated genetic correction of COL7A1 Exon 3 in immortalized RDEB keratinocytes (RDEB-K-SV40 cells). (a) MN target site in intron 2 and corresponding sequence. (b) The predicted structure of the MN-i1 isoschizomer. (c) Top: design of the lentiviral vectors (LV-EF1-MN and the promoterless LV-Donor). Bottom: COL7A1 encompassing mutated exon 3 and structure of corrected COL7A1 after MN-mediated HDR. Arrows indicate allele-specific PCR primers. (d) Genomic PCR on the cells cotransduced with MN-i.1- and Donor-encoding IDLVs resulted in a specific 1.5 kb band, corresponding to the corrected allele when using P3/P2 primers. The GAPDH gene was used as the reference transcript. (e) Gene-corrected RDEB-K-SV40 cells were positively stained with the LH7:2 anti-C7 antibody. Untreated cells (mock) show negative staining. Scale bar = 100 μm. (f) Percentage of C7 rescue in bulk-transduced RDEB-K-SV40 cells. HDR, homology-directed repair; IDLVs, integration-deficient lentiviral vectors; MN, meganucleases; RDEB, recessive dystrophic epidermolysis bullosa. Journal of Investigative Dermatology 2016 136, 872-875DOI: (10.1016/j.jid.2015.11.028) Copyright © 2016 The Authors Terms and Conditions

Figure 2 MN-mediated genetic correction of COL7A1 Exon 2 in primary RDEB keratinocytes and fibroblasts. (a) Overview of HDR at the COL7A1 locus harboring the c.189delG mutation. (b, c) Genomic PCR analysis at the COL7A1 locus after HDR in RDEB-K and RDEB-F, respectively. A specific 0.8 kb band corresponding to the corrected allele was amplified using P1/P3 primers, when cells were cotransduced with MN- and Donor-encoding IDLVs. (d) Sequencing of the PCR product generated with the modified MN-site primer from targeted RDEB fibroblasts revealed the genetic correction of the c.189delG mutation through MN-mediated HDR. (e) Percentages of corrected COL7A1 mRNA expression in RDEB-K and RDEB-F as assessed by Taqman qPCR. (f) Gene-corrected RDEB-K (left) and RDEB-F (right) were positively stained with the LH7:2 anti-C7 antibody. Untreated (mock) cells show negative staining for C7 as expected. RDEB-K, scale bar = 100 μm; RDEB-F, scale bar = 75 μm. HDR, homology-directed repair; IDLVs, integration-deficient lentiviral vectors; MN, meganucleases; RDEB, recessive dystrophic epidermolysis bullosa. Journal of Investigative Dermatology 2016 136, 872-875DOI: (10.1016/j.jid.2015.11.028) Copyright © 2016 The Authors Terms and Conditions