Résumé
Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes progressive vision loss. The second most common mutation causing autosomal dominant (ad) RP is the G56R mutation in NR2E3, a transcription factor essential for photoreceptor development. NR2E3 acts to promote rod differentiation while blocking the expression of cone genes. The G56R variant is exclusively responsible for all cases of NR2E3-associated adRP. Thus, a potential treatment for NR2E3-related adRP would be applicable to all patients. Currently, there is no treatment for NR2E3-related, or other adRP, but genome editing holds promise. Although the most straightforward approach to genome editing would be correction of the mutant allele, this is clinically challenging in photoreceptors, because they do not use the homology-directed repair pathway. Therefore, a more pertinent approach would be to specifically knockout the dominant mutant allele so that the wild type allele can perform unhindered.For my thesis project, I reprogrammed fibroblasts from a G56R patient to iPSCs, which I then characterized. I also developed a CRISPR/Cas strategy to specifically knockout the mutant G56R allele of NR2E3. This was achieved by designing gRNAs that span the mutational site and do not recognize the wild type (WT) allele. I performed a proof-of-concept study in G56R iPSCs demonstrating allele-specific knockout of the mutant G56R allele in the absence of off-target events. Furthermore, we validated this knockout strategy in an exogenous overexpression system. Accordingly, the mutant G56R-CRISPR protein was truncated and mis-localized to the cytosol in contrast to the (peri)nuclear localizations of WT NR2E3 protein. Interestingly, G56R NR2E3 was preferentially localized in the nucleus compared to the predominant perinuclear localization of WT NR2E3.In addition, I show for the first time that G56R iPSCs, as well as G56R-CRISPR iPSCs, can differentiate into mature retinal organoids, 3D structures that contain a photoreceptor layer. These organoids express basic photoreceptor markers such as rhodopsin, cone arrestin and cone opsins, and do not show signs of rod apoptosis up to 230 days. However, my preliminary data suggest that G56R organoids have defective expression of GNB1, ARL13B and ABCA4 compared to WT organoids. Interestingly, these defects were not present in G56R-CRISPR organoids. A higher number of retinal organoids is needed to confirm this data.Overall, I demonstrate that G56R allele-specific knockout by CRISPR/Cas could be a clinically relevant approach to treat NR2E3-associated adRP.