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REGAIN Report Summary

Project ID: 311244
Funded under: FP7-IDEAS-ERC
Country: Germany

Final Report Summary - REGAIN (Retinal Gene Alteration in XLRP)

X linked retinitis pigmentosa (XLRP) is an inherited retinal dystrophy caused in 80% of cases by mutations in the RPGR gene. The retina specific isoform of RPGR contains amino acids encoded by a terminal exon ORF15, which is considered to be a mutational hot spot. Therefore, the ORF15 represents the target locus of the therapeutic approaches developed in this project. Therapeutic genome editing is based on the cell’s own capacity to repair a double (DSB) or sinclge (SSB) strand break that was induced by highly specific endonucleases, such as CRISPR-Cas9, TALEN or ISce-I homing endonucleases. The DSB is repaired either in the absence of a template DNA by nonhomologous endjoining (NHEJ), or in the presence of a template DNA by homology directed repair (HDR) or microhomology mediated endjoining (MMEJ) with longer or shorter homology arms, respectively. While in vitro genome editing is a well-established method, in vivo applications are difficult to realize because of limitations with regard to the transfer of the genes, absence of knowledge about DNA repair mechanisms in specialized neurons, and the necessity to avoid off-target toxicity at all costs.
Within this project, we developed two reporter gene systems that allow us to quantify and optimize DNA repair activity, both in vitro and in vivo. The TLR-3 reporter system was introduced into a mouse line, which is currently at the level of backcrossing. The luciferase based reporter system enabled us to define the optimal sequence length of the microhomology arms within the template DNA in vitro. Using wild type and RPGR mouse lines, we analyzed the DNA repair machinery within the retina and observed cell type specific differences with regard to expression profiles of important DNA repair proteins, indicating different repair mechanisms at place. We studied the CRISPR-Cas9 system in vitro at the target locus ORF15 and prepared template and gRNAs for subsequent application in the RPGR mouse line in vivo.
Taken together, we were able to show that the ORF15 can be targeted by genome editing methods and replacement of the mutant ORF15 is possible in vitro. We are currently enlarging the data set to in vivo experiments in the RPGR mouse model. The knowledge about the DNA repair system in photoreceptors, which was gained during the project, will help us to optimize the treatment approach. The data from the project formed inter alia the basis for the establishment of a research priority program at the German research council that will allow the PI to continue working on the project in collaboration with experts from related fields over the next 3-6 years.

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