Abstract
Purpose :
Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause severe X-linked retinitis pigmentosa (XLRP). More than 80% of the mutations are located in the terminal exon ORF15 of the RPGR gene. The aim of the project is to develop gene therapeutic approaches to treat XLRP through genomic editing. The common strategy for targeted gene editing is based on highly specific nucleases which cleave only once within the complex genome at a chosen position. The DNA gets repaired by two different repair pathways, error prone non-homologous end joining (NHEJ) or homologous recombination (HR) with the help of a donor template. The aim of this study is to develop a gene therapy strategy based on HR for XLRP.
Methods :
Eleven sequences within or around the ORF15 gene have been targeted for the induction of double or single strand breaks of the DNA. Ten target sites for CRISPR/Cas9-FokI were chosen, three before, within and four behind the exon, respectively, and one target site for TALE-MutH within the exon. These sequences have been cloned into the traffic light reporter (TLR) gene expression system at the homing endonuclease I-SceI site. The TLR system has been modified to express either GFP in case of successful HR or BFP in case of NHEJ. Plasmids containing substrate, nucleases and template DNA were transfected into HEK293T cells. Efficiency of DNA modification was measured by FACS analysis and T7 surveyor assay, and toxicity was assessed by cell survival assay. In addition to the episomal TLR system, the genome of murine C2C12 cells were targeted and analysed via the T7 surveyor assay.
Results :
Toxicity of Cas9-FokI and TALE-MutH are comparable to the golden standard I-SceI while standard CRISPR/Cas9 nuclease showed slightly increased toxicity in HEK293T cells. Two different concentrations of the nucleases were used in a toxicity assay and were equally tolerated. Cas9-FokI showed preferences in its activity within the nine target sites with activities well above I-SceI activity, while the one target site of TALE-MutH is as efficient as I-SceI. The results were confirmed in the murine cell line C2C12.
Conclusions :
The characterization of the activity and toxicity of the tested endonucleases helped us to identify the most promising tailored nuclease and its target sequence in our gene targeting approach to treat XLRP. With the help of mouse retinal explants we are studying the efficacy of our strategy in photoreceptors.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.