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.
Highlights
Dystrophic epidermolysis bullosa (DEB) is a rare and severe genetic skin disease of children and adults, responsible for blistering of the skin and mucosa
We developed an ex vivo gene therapy approach based on CRISPR/Cas9-mediated COL7A1 editing without antibiotic- or fluorescence-based selection
Design of Guide RNAs Targeting Exon 2 of COL7A1 To demonstrate the feasibility of COL7A1 editing in primary recessive DEB (RDEB) cells using the CRISPR/Cas[9] approach, we designed five guide RNAs in order to correct a causative null mutation in exon 2 seen in several RDEB subjects (c.189delG; p.Leu64Trpfs*40)
Summary
Dystrophic epidermolysis bullosa (DEB) is a rare and severe genetic skin disease of children and adults, responsible for blistering of the skin and mucosa. DEB is caused by a wide variety of mutations in the COL7A1 gene-encoding type VII collagen.[1] Type VII collagen is the major component of anchoring fibrils (AFs), which are key attachment structures for dermal-epidermal adhesion.[2] COL7A1 mutations lead to defective AF formation, resulting in a loss of adhesion between the epidermis and the dermis.[3] Depending on the nature and position of COL7A1 mutations, the disease is either dominantly or recessively inherited,[4] dominant forms (DDEB) being usually less severe than recessive forms. No specific cure is currently available for RDEB and proposed treatments are mainly symptomatic.[7]
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