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In vivo gene editing for genodermatoses

Ongoing
Project lead Dr Thomas Kocher
Organisation EB House Austria, Salzburg, AUSTRIA
Project budget DDK 1,389,845.00
Start date / Duration 01. Sep 2020 / 26 months
Funder(s) / Co-Funder(s) Others
Other funder(s) Leo Foundation
Research area Molecular therapy, Cellular therapy

Project details

Short lay summary

The goal of this project is to evaluate the translational and therapeutic potential of two in vivo CRISPR/Cas9 delivery methods. CRISPR/Cas9 is a gene-editing technology that enables researchers to edit parts of the genome by removing, adding or altering sections of a specific DNA sequence. Although CRISPR/Cas9-based technologies hold great promise as genome editing tools in many genetic diseases, its clinical application, remains a big challenge. To challenge this hurdle, CRISPR/Cas9 molecules will be delivered into the skin of a suitable animal model via two application methods: laser microporation and gene gun bombardment. The first method uses a laser to make micropores into the skin to allow the CRISPR/Cas9 constructs to enter the outer skin barrier and subsequently the target skin cells. The second method uses a “gene gun”, where gold particles covered with CRISPR/Cas9 constructs are shot directly into the skin/cells.  Upon successful entry, constructs can then correct the genetic defect thereby restoring the absent protein.

Scientific summary

CRISPR/Cas9-based technologies hold great promise as genome editing tools in many genetic diseases, including genodermatoses. However, the clinical translation of genome editing, especially when applied in vivo, remains a big challenge. Our aim is the development of highly efficient and easily applicable in vivo treatment options for skin disorders and employ here recessive dystrophic epidermolysis bullosa (RDEB), associated with severe skin blistering and a high mortality rate due to cancer development, as our disease model. Rationally designed CRISPR/Cas9 molecules, previously shown to be highly efficient in restoring collagen type VII expression via COL7A1 reframing, will be delivered into the skin of a suitable animal model via two application methods: laser microporation and gene gun bombardment. Skin equivalents (SEs), expanded from RDEB patient keratinocytes and transplanted onto immunodeficient mice will be treated with the precise laser epidermal system (P.L.E.A.S.E.®) to deliver therapeutic CRISPR/Cas9 ribonucleoproteins (RNPs). We will also evaluate biolistic transfections of Cas9-expressing plasmids using a gene gun-based approach. Both delivery approaches represent minimally invasive treatment options for the local therapy of genodermatoses in general. Efficacy of CRISPR/Cas9-mediated in vivo COL7A1 editing at genomic DNA, RNA, protein and suprastructural level, will disclose the in vivo potential of these technologies for the treatment of genodermatoses.

Strategic relevance

The project will investigate the potential of these two delivery methods in a mouse model using grafted human skin equivalents expanded from recessive dystrophic epidermolysis bullosa (RDEB) patient-derived fibroblasts and keratinocytes. If either delivery method proves efficient, it may hold the potential for development of future treatments of genetic skin diseases. 


Keywords

Kocher
EB House Austria
RDEB
CRISPR/Cas9
predictable repair outcome
in vivo gene therapy
gene therapy
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