Recessive dystrophic epidermolysis bullosa is a rare and severe genetic skin disease resulting in blistering of the skin and mucosa. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by a wide variety of mutations in COL7A1-encoding type VII collagen, which is essential for dermal-epidermal adhesion. Here we demonstrate the feasibility of ex vivo COL7A1 editing in primary RDEB cells and in grafted 3D skin equivalents through CRISPR/Cas9-mediated homology-directed repair. We designed five guide RNAs to correct a RDEB causative null mutation in exon 2 (c.189delG; p.Leu64Trpfs*40). Among the site-specific guide RNAs tested, one showed significant cleavage activity in primary RDEB keratinocytes and in fibroblasts when delivered as integration-deficient lentivirus. Genetic correction was detected in transduced keratinocytes and fibroblasts by allele-specific highly sensitive TaqMan-droplet digital PCR (ddPCR), resulting in 11% and 15.7% of corrected COL7A1 mRNA expression, respectively, without antibiotic selection. Grafting of genetically corrected 3D skin equivalents onto nude mice showed up to 26% re-expression and normal localization of type VII collagen as well as anchoring fibril formation at the dermal-epidermal junction. Our study provides evidence that precise genome editing in primary RDEB cells is a relevant strategy to genetically correct COL7A1 mutations for the development of future ex vivo clinical applications.

Ex Vivo COL7A1 Correction for Recessive Dystrophic Epidermolysis Bullosa Using CRISPR/Cas9 and Homology-Directed Repair / Izmiryan, A.; Ganier, C.; Bovolenta, M.; Schmitt, A.; Mavilio, F.; Hovnanian, A.. - In: MOLECULAR THERAPY NUCLEIC ACIDS. - ISSN 2162-2531. - 12:(2018), pp. 554-567. [10.1016/j.omtn.2018.06.008]

Ex Vivo COL7A1 Correction for Recessive Dystrophic Epidermolysis Bullosa Using CRISPR/Cas9 and Homology-Directed Repair

Mavilio F.;
2018

Abstract

Recessive dystrophic epidermolysis bullosa is a rare and severe genetic skin disease resulting in blistering of the skin and mucosa. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by a wide variety of mutations in COL7A1-encoding type VII collagen, which is essential for dermal-epidermal adhesion. Here we demonstrate the feasibility of ex vivo COL7A1 editing in primary RDEB cells and in grafted 3D skin equivalents through CRISPR/Cas9-mediated homology-directed repair. We designed five guide RNAs to correct a RDEB causative null mutation in exon 2 (c.189delG; p.Leu64Trpfs*40). Among the site-specific guide RNAs tested, one showed significant cleavage activity in primary RDEB keratinocytes and in fibroblasts when delivered as integration-deficient lentivirus. Genetic correction was detected in transduced keratinocytes and fibroblasts by allele-specific highly sensitive TaqMan-droplet digital PCR (ddPCR), resulting in 11% and 15.7% of corrected COL7A1 mRNA expression, respectively, without antibiotic selection. Grafting of genetically corrected 3D skin equivalents onto nude mice showed up to 26% re-expression and normal localization of type VII collagen as well as anchoring fibril formation at the dermal-epidermal junction. Our study provides evidence that precise genome editing in primary RDEB cells is a relevant strategy to genetically correct COL7A1 mutations for the development of future ex vivo clinical applications.
2018
26-giu-2018
12
554
567
Ex Vivo COL7A1 Correction for Recessive Dystrophic Epidermolysis Bullosa Using CRISPR/Cas9 and Homology-Directed Repair / Izmiryan, A.; Ganier, C.; Bovolenta, M.; Schmitt, A.; Mavilio, F.; Hovnanian, A.. - In: MOLECULAR THERAPY NUCLEIC ACIDS. - ISSN 2162-2531. - 12:(2018), pp. 554-567. [10.1016/j.omtn.2018.06.008]
Izmiryan, A.; Ganier, C.; Bovolenta, M.; Schmitt, A.; Mavilio, F.; Hovnanian, A.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1199464
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