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Traceless CRISPR/Cas9-based COL17A1 editing

Project lead Dr Ulrich Koller
Organisation EB House Austria, Salzburg, AUSTRIA
Project budget EUR 289,726.00
Start date / Duration 01. Oct 2020 / 58 months
Funder(s) / Co-Funder(s) PMU, Others, DEBRA Austria, Austrian Science Fund (FWF)
Other funder(s) LHF Charitable Trust
Research area Molecular therapy, Cellular therapy

Project details

Short lay summary

The main aspect of this project is the establishment of a designer nuclease-based gene therapy for JEB patients with COL17A1 mutations. Although a gene replacement strategy was successfully applied for JEB patients with LAMB3 deficiency, viral delivery systems are compounded by a random genomic integration of the transgene. Due to safety concerns, a virus-free therapeutic option for genodermatoses should be a research focus. Many gene editing therapeutic approaches employ ex vivo modification, screening and re-transplantation. This enables selection against incorrectly modified cells. Currently, low gene repair efficiencies hinder the application of existing gene editing strategies and the development of translational applications in this field. This project aims to develop an efficient gene editing protocol, later translatable into a clinical setting.

Scientific summary

Currently, we are working on a potentially traceless CRISPR/Cas9-based gene editing strategy to correct a frequent mutation within COL17A1 causing JEB. Designer nucleases, such as CRISPR/Cas9, represent promising tools for the permanent correction of disease-associated mutations in a site-specific manner. A single treatment is often sufficient to obtain permanent restoration of gene function. Homologous recombination comprises the most traceless approach to gene editing. Therefore, we aim to induce a shift from the frequent error prone end joining pathways to this more elegant repair pathway. This should be possible with the use of Cas9 nickases (Cas9n), proximal targeting of nucleases to the mutation, donor template optimization and cell cycle modulation. Normally the Cas9 from Streptococcus pyogenes is preferentially used to efficiently generate double strand DNA breaks (DSBs). However, double nicking via Cas9 nickases (Cas9n) represents a promising alternative for gene disruption. Electroporation of Cas9 ribonucleoproteins (RNPs) comprises a promising and safe method of delivering gene editing agents into cells, associated with reduced off-target effects, high rates of gene disruption and a reduced window of activity. The co-delivery of the RNPs and a donor template for homologous recombination should result in the correction of the disease-associated mutation. In summary, this study aims to develop a method of highly efficient CRISPR/Cas9-mediated HR with clinical applicability. Further, we want to establish the most recent developed gene editing tool, prime editing, to obtain a traceless gene repair. However, at the end we want to develop “traceless” gene editing protocols for COL17A1-associated junctional EB (JEB).

Strategic relevance

The primary goal of the project is the development of an HR- and/or prime editing-based gene editing protocol to achieve a traceless correction of gene function in epidermal stem cells from junctional EB patients. Once established the protocol should be fast and easily adaptable for any EB-associated mutation aiming at the generation of a toolbox to treat as many patients as possible.


EB House Austria
homologous recombination
prime editing
ex vivo gene therapy
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