Objectives: Retinitis Pigmentosa (RP) is characterized by progressive degeneration of photoreceptors leading to vision loss. Variants in over 80 genes cause RP with all modes of inheritance. Currently, there is no universal cure and there is a therapeutic impasse regarding dominant variants as, in addition to haploinsufficiency, they can promote gain-of-function or dominant-negative effects. The NR2E3 variant c.166G>A (G56R) is the second most common variant causing autosomal dominant RP (adRP). Interestingly, this variant is responsible for all the cases of adRP related to NR2E3. Thus, one gene therapy approach could be beneficial for all patients carrying this variant.

Methods: Currently, it has not been elucidated how the G56R variant promotes photoreceptor degeneration. Thus, we are testing different therapeutic approaches to treat this variant in patient-derived retinal organoids. We developed a genome editing approach using CRISPR/Cas9 strategy to specifically invalidate the G56R allele. In parallel, we developed an adenine base editing (ABE) approach to correct the c.166G>A variant in NR2E3 in order to convert directly the mutant allele to wild-type sequence. Lastly, we are testing the effect of NR2E3 gene supplementation.

Results: We previously demonstrated a specific invalidation of the G56R allele in NR2E3 G56R iPSC lines using CRISPR/Cas9 system without off-target events (Diakatou et al., 2021). To identify a possible phenotype rescue, we generated retinal organoids from treated and untreated NR2E3 G56R iPSCs using a 3D-2D-3D differentiation protocol. We performed immunostaining of cryosections from organoids and initially observed a reduced number of cells expressing NR2E3 and NRL in the outer nuclear layer (ONL) of G56R-CRISPR organoids compared to the control and G56R organoids, which is consistent with the loss of one NR2E3 allele. As a step closer to clinical translation, we successfully transduced G56R retinal organoids with an AAV vector expressing the CRISPR/Cas9 system. Finally, we designed an ABE base editing strategy and transfected G56R iPSC lines with our system. The GFP-positive cells were single cell sorted allowing the generation of a correctly edited clone harboring a biallelic wild-type sequence without off-target events.

Conclusion: We aim to identify the most relevant therapeutic approach (genome editing and gene supplementation) to treat adRP due to NR2E3 in a human context and to further understand disease pathogenesis.