Gálvez, V;
Chacón-Solano, E;
Bonafont, J;
Mencía, Á;
Di, W-L;
Murillas, R;
Llames, S;
... Larcher, F; + view all
(2020)
Efficient CRISPR-Cas9-Mediated Gene Ablation in Human Keratinocytes to Recapitulate Genodermatoses: Modeling of Netherton Syndrome.
Molecular Therapy: Methods & Clinical Development
, 18
pp. 280-290.
10.1016/j.omtm.2020.05.031.
Preview |
Text
1-s2.0-S2329050120301182-main.pdf - Published Version Download (4MB) | Preview |
Abstract
Current efforts to find specific genodermatoses treatments and define precise pathogenesis mechanisms require appropriate surrogate models with human cells. Although transgenic and gene knockout mouse models for several of these disorders exist, they often fail to faithfully replicate the clinical and histopathological features of the human skin condition. We have established a highly efficient method for precise deletion of critical gene sequences in primary human keratinocytes, based on CRISPR-Cas9-mediated gene editing. Using this methodology, in the present study we generated a model of Netherton syndrome by disruption of SPINK5. Gene-edited cells showed absence of LEKTI expression and were able to recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of Netherton syndrome, after grafting to immunodeficient mice and in organotypic cultures. To validate the model as a platform for therapeutic intervention, we tested an ex vivo gene therapy approach using a lentiviral vector expressing SPINK5. Re-expression of SPINK5 in an immortalized clone of SPINK5-knockout keratinocytes was capable of reverting from Netherton syndrome to a normal skin phenotype in vivo and in vitro. Our results demonstrate the feasibility of modeling genodermatoses, such as Netherton syndrome, by efficiently disrupting the causative gene to better understand its pathogenesis and to develop novel therapeutic approaches.
| Type: | Article |
|---|---|
| Title: | Efficient CRISPR-Cas9-Mediated Gene Ablation in Human Keratinocytes to Recapitulate Genodermatoses: Modeling of Netherton Syndrome |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.omtm.2020.05.031 |
| Publisher version: | https://doi.org/10.1016/j.omtm.2020.05.031 |
| Language: | English |
| Additional information: | This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
| Keywords: | CRISPR/Cas9, Netherton syndrome, disease modeling, gene editing, genodermatosis, human keratinocytes, skin equivalents. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Infection, Immunity and Inflammation Dept |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10105518 |
Archive Staff Only
 |
View Item |
