Traceless Targeting and Isolation of Gene-Edited Immortalized Keratinocytes from Epidermolysis Bullosa Simplex Patients

• Published in: Molecular therapy. Methods & clinical development Vol. 6; no. C; pp. 112 - 123
• Main Authors: Aushev, Magomet, Koller, Ulrich, Mussolino, Claudio, Cathomen, Toni, Reichelt, Julia
• Format: Journal Article
• Language: English
• Published: United States Elsevier Limited 15.09.2017; American Society of Gene & Cell Therapy; Elsevier
• Subjects: Alleles; Binding sites; Cloning; CRISPR; Deoxyribonucleic acid; DNA; dominant negative mutations; Epidermolysis bullosa; epidermolysis bullosa simplex; Flow cytometry; gene editing; Gene expression; Gene therapy; Genetic engineering; Genome editing; Genomes; intermediate filaments; Keratin; Keratinocytes; keratins; KRT5; Mutation; non-homologous end joining; Nuclease; off-target effects; Original; Proteins; Skin diseases; Stem cells; TALEN; Transcription activator-like effector nucleases; United Kingdom > UK; off-target effects; TALEN; dominant negative mutations; gene therapy; non-homologous end joining; KRT5; keratins; gene editing; intermediate filaments; epidermolysis bullosa simplex
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2017.06.008

Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by dominant-negative mutations in either or , resulting in impairment of keratin filament structure and epidermal fragility. Currently, nearly 200 mutations distributed across the entire length of these genes are known to cause EBS. Genome editing using programmable nucleases enables the development of ex vivo gene therapies for dominant-negative genetic diseases. A clinically feasible strategy involves the disruption of the mutant allele while leaving the wild-type allele unaffected. Our aim was to develop a traceless approach to efficiently disrupt alleles using TALENs displaying unbiased monoallelic disruption events and devise a strategy that allows for subsequent screening and isolation of correctly modified keratinocyte clones without the need for selection markers. Here we report on TALENs that efficiently disrupt the locus in immortalized patient-derived EBS keratinocytes. Inactivation of the mutant allele using a TALEN working at sub-optimal levels resulted in restoration of intermediate filament architecture. This approach can be used for the functional inactivation of any mutant keratin allele regardless of the position of the mutation within the gene and is furthermore applicable to the treatment of other inherited skin disorders.