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- W4285984814 abstract "RDEB is a severe and life-threatening genetic skin disease responsible for blistering of the skin and mucosa after minor trauma. RDEB is caused by a wide variety of mutations in COL7A1 encoding type VII collagen (C7), the major component of anchoring fibrils (AF) which are critical attachment structures that adhere the epidermis to the dermis. We aimed to achieve highly efficient correction of a null mutation (c.6508C>T, p.Gln2170*) in exon 80 of COL7A1 in a patient’s primary RDEB keratinocytes (KC), fibroblasts (FB) and 3D-skin equivalents (SE) through CRISPR/Cas9-mediated HDR. We designed three gRNAs specifically targeting the mutation or adjacent sequences. Chemically modified gRNAs were delivered together with hfCas9 as a ribonucleoprotein complex (RNP) by nucleofection. One gRNA targeting intron 79 achieved up to 73% cleavage activity in primary RDEB-KC and RDEB-FB. The search for off-target activity in RDEB cells found no evidence for non-specific cleavage activity at in-silico predicted sites. For the correction purpose, RDEB-KC and FB were treated with specific RNPs containing the most active gRNA and hfCas9 together with Donor ssODN. Gene correction and C7 rescue were estimated to be up to 58% by allele-specific TaqMan-ddPCR and Western blotting. Grafting of genetically corrected 3D SE onto nude mice induced re-expression and normal localization of C7 as well as AF formation at the dermal-epidermal junction at 5 and 10 weeks post-grafting. Keratin 5, keratin 10 and loricrin showed similar epidermal differentiation patterns in corrected, uncorrected and normal SE. With this approach, we achieved highly efficient and specific gene editing which could be applicable to all mutations in exon 80 of COL7A1 in primary RDEB-KC and FC. This approach has a potential for future ex vivo clinical applications." @default.
- W4285984814 created "2022-07-20" @default.
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- W4285984814 date "2022-08-01" @default.
- W4285984814 modified "2023-09-28" @default.
- W4285984814 title "477 Efficient ex-vivo COL7A1 correction of patient's primary keratinocytes and fibroblasts using RNP-based CRISPR/Cas9 and homology-directed repair to treat recessive dystrophic epidermolysis bullosa" @default.
- W4285984814 doi "https://doi.org/10.1016/j.jid.2022.05.486" @default.
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