Gene Editing–Mediated Disruption of Epidermolytic Ichthyosis–Associated KRT10 Alleles Restores Filament Stability in Keratinocytes

Oliver P. March, Thomas Lettner, Alfred Klausegger, Michael Ablinger, Thomas Kocher, Stefan Hainzl, Patricia Peking, Nina Lackner, Neil Rajan, Josefina Piñón Hofbauer, Christina Guttmann-Gruber, Anette Bygum, Ulrich Koller, Julia Reichelt*

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Abstrakt

Epidermolytic ichthyosis is a skin fragility disorder caused by dominant-negative mutations in KRT1 or KRT10. No definitive restorative therapies exist that target these genetic faults. Gene editing can be used to efficiently introduce frameshift mutations to inactivate mutant genes. This can be applied to counter the effect of dominantly inherited diseases such as epidermolytic ichthyosis. In this study, we used transcription activator-like effector nuclease technology, to disrupt disease-causing mutant KRT10 alleles in an ex vivo cellular approach, with the intent of developing a therapy for patients with epidermolytic ichthyosis. A transcription activator-like effector nuclease was designed to specifically target a region of KRT10, upstream of a premature termination codon known to induce a genetic knockout. This proved highly efficient at gene disruption in a patient-derived keratinocyte cell line. In addition, analysis for off-target effects indicated no promiscuous gene editing–mediated disruption. Reversion of the keratin intermediate filament fragility phenotype associated with epidermolytic ichthyosis was observed by the immunofluorescence analysis of correctly gene-edited single-cell clones. This was in concurrence with immunofluorescence and ultrastructure analysis of murine xenograft models. The efficiency of this approach was subsequently confirmed in primary patient keratinocytes. Our data demonstrate the feasibility of an ex vivo gene-editing therapy for more than 95.6% of dominant KRT10 mutations.

OriginalsprogEngelsk
TidsskriftJournal of Investigative Dermatology
Vol/bind139
Udgave nummer8
Sider (fra-til)1699-1710.e6
ISSN0022-202X
DOI
StatusUdgivet - aug. 2019

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Citationsformater

March, O. P., Lettner, T., Klausegger, A., Ablinger, M., Kocher, T., Hainzl, S., Peking, P., Lackner, N., Rajan, N., Hofbauer, J. P., Guttmann-Gruber, C., Bygum, A., Koller, U., & Reichelt, J. (2019). Gene Editing–Mediated Disruption of Epidermolytic Ichthyosis–Associated KRT10 Alleles Restores Filament Stability in Keratinocytes. Journal of Investigative Dermatology, 139(8), 1699-1710.e6. https://doi.org/10.1016/j.jid.2019.03.1146