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CRISPR/Cas9-mediated repair of COL17A1

Project lead Dr Ulrich Koller
Organisation EB House Austria, Salzburg, AUSTRIA
Project budget 352,609.57
Start date / Duration 01. May 2017 / 56 months
Funder(s) / Co-Funder(s) Others, DEBRA Austria
Other funder(s) LHF Charitable Trust
Research area Molecular therapy, Cellular therapy

Project details

Short lay summary

The establishment of a designer nuclease-based gene therapy for JEB patients with COL17A1 mutations is the focus of this project. So far, a gene replacement strategy was successfully applied for JEB patients with LAMB3 deficiency. However, because viral delivery systems are compounded by a random transgene integration, 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 compound existing gene editing strategies and hamper the development of translational applications in this field. Here we are working on an efficient gene editing protocol later translatable into a clinical setting.

Scientific summary

We are aiming to develop a CRISPR/Cas9-based gene editing strategy to correct frameshift mutations within COL17A1, which should be later adaptable for similar mutations in EB-associated genes. Using the Cas9 nuclease from Streptococcus pyogenes and/or its mutant nickase variant D10A (Cas9n) we want to induce the error-prone end joining pathway upon DNA cleavage in near proximity to the mutation leading to the formation of indels (insertions or deletions) at the target site. Thereby, re-framed COL17A1 transcripts are generated resulting, in the optimal case, in the expression of restored full-length type XVII collagen in targeted JEB keratinocytes. Using cell electroporation as a superior and safe delivery option, Cas9 ribonucleoproteins (RNPs) are delivered into JEB patient keratinocytes. We obtained high rates of gene disruption accompanied by a reduced window of nuclease activity. Reframed COL17A1 transcripts will be functionally analysed regarding accurate protein expression and localization within basal keratinocytes in cell culture as well as in an established 3D model.

Strategic relevance

The primary goal of the project is the development of an NHEJ-based gene editing protocol to achieve an efficient and potentially safe 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 frameshift mutation aiming at the generation of a toolbox to treat as many patients as possible.


non-homologous end joining (NHEJ)
ex vivo gene therapy
EB House Austria
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