198 TALEN-mediated gene editing in epidermolytic ichthyosis patient-derived keratinocytes

We are developing an ex vivo gene therapy for epidermolytic ichthyosis (EI) using TALENs to knockout mutant KRT10 alleles in keratinocytes. EI is a skin fragility disorder caused by dominant-negative mutations in either KRT1 or KRT10. Keratin pairs specifically polymerise to build the intermediate filament cytoskeleton of epithelial cells. Mutant keratins integrate into this, resulting in fragility and collapse upon mild stress, leading to blistering of the skin. Heterozygous parents of patients with recessive EI display normal skin function, demonstrating that one wild type allele is sufficient. Elimination of mutant keratins in EI patients should therefore result in curation of the disease. Transcription activator-like effector nucleases (TALENs) are designer nucleases. They introduce double-strand breaks at the target site which can lead to frame shift mutations that inactivate the targeted allele. TALENs targeting KRT10 were constructed. These cleave efficiently at the target site, inducing mutations that knockout KRT10. Clones were screened, with 35% displaying successful modification without selection. These were expanded for immunofluorescent analysis of monolayer and 3D skin equivalent cultures, with and without heat stress. Phenotypic restoration of the keratin network and structure of 3D skin was demonstrated, concurrent with mRNA and Western blot data. Transfected cells were assessed at 22 TALEN off-target sites for activity via next-generation sequencing and T7E1 assay. Modified clones displayed knockout of mutant KRT10 and phenotypic restoration of the cytoskeleton. Little activity at off-target sites comprehensively demonstrated the safety of using TALENs for gene therapy. We aim to take a skin biopsy from a patient, isolate, grow and treat keratinocytes with TALENs to correct the mutation prior to grafting these cells onto the patient.