Traceless Targeting and Isolation of Gene-Edited Immortalized Keratinocytes from Epidermolysis Bullosa Simplex Patients
| Autor: | M. Aushev, Toni Cathomen, Claudio Mussolino, Ulrich Koller, Julia Reichelt |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
intermediate filaments lcsh:QH426-470 Mutant Locus (genetics) epidermolysis bullosa simplex Biology non-homologous end joining dominant negative mutations 03 medical and health sciences Epidermolysis bullosa simplex 0302 clinical medicine Genome editing TALEN Genetics medicine Allele lcsh:QH573-671 Molecular Biology Gene Transcription activator-like effector nuclease integumentary system KRT5 gene editing lcsh:Cytology off-target effects medicine.disease gene therapy 3. Good health lcsh:Genetics 030104 developmental biology medicine.anatomical_structure 030220 oncology & carcinogenesis keratins Molecular Medicine Original Article Keratinocyte |
| Zdroj: | Molecular Therapy: Methods & Clinical Development, Vol 6, Iss C, Pp 112-123 (2017) Molecular Therapy. Methods & Clinical Development |
| ISSN: | 2329-0501 |
| Popis: | Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by dominant-negative mutations in either KRT5 or KRT14, 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 KRT5 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 KRT5 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. |
| Databáze: | OpenAIRE |
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