Prime editing for dystrophic epidermolysis bullosa (Koller 1)Ongoing
|Project lead||Dr Ulrich Koller|
|Organisation||EB House Austria, Salzburg, AUSTRIA|
|Project budget||EUR 279,653.00|
|Start date / Duration||09. Aug 2021 / 36 months|
|Funder(s) / Co-Funder(s)||DEBRA Austria|
|Research area||Molecular therapy, Cellular therapy|
Short lay summary
Our overarching aim is to develop a prime editing strategy to achieve perfect and traceless wildtype sequence restoration of mutations associated with the severe blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB) that will meet the stringent requirements for both safety and efficiency to enable advancement to clinical trials. Moreover, we envision that the improvements and protocols established within this project will apply to other forms of EB. The recent development of prime editing represents another significant milestone towards the goal of traceless gene editing. Prime editing directly writes new genetic information into a specified DNA site using a Cas9 nickase fused to an engineered reverse transcriptase domain. This “prime editor” is programmed with a prime editing guide RNA (pegRNA) that is both gRNA, specifying the target site, and reverse transcriptase template, encoding the desired edit. This method is reportedly suitable to correct ~89% of all disease-associated mutations.
We will establish prime editing systems for defined COL7A1 mutations associated with a severe RDEB phenotype. Wildtype correction efficiencies and off-target rates will be evaluated by NGS in immortalized patient-derived cell lines and pegRNAs associated with high correction efficiencies and superior safety profile will be advanced to evaluation in primary RDEB cells. Correct collagen type VII (C7) expression, secretion, and localization will be confirmed in in vitro 3D organotypic models. A humanized skin mouse model, in which RDEB skin equivalents are generated in vitro and transplanted onto the backs of immunodeficient mice, will be employed to analyse the stability of the genetically corrected skin upon application of mechanical stress.
Gene therapy represents the only option to cure monogenic hereditary diseases like EB. The protocol we establish in the course of the project can be adapted to any EB-associated mutation and contributes to the generation of a toolbox and workflow. We can rapidly design and develop therapeutic gene-editing molecules to treat as many patients as possible.