Genetic reprogramming via acute Nr2e3 knockout prevents photoreceptor degeneration

Abstract

Purpose : Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by progressive death of rod photoreceptors, followed by secondary cone loss. There are currently no therapies for RP. Our lab previously showed that acute knockout of the rod-specific transcription factor (TF) NRL reprograms rods into a cone-like state, thereby delaying rod death and preserving native cones in a mouse model of RP. However, this manipulation also causes changes in photoreceptor gene expression, morphology, and function, and thus a more subtle approach is desirable. To address this, our goal is to evaluate NR2E3, a rod-specific TF immediately downstream of NRL, as a therapeutic target, and to uncover the mechanism(s) of neuroprotection downstream of NRL and NR2E3.

Methods : We use subretinal injection of AAV to deliver anti-Nr2e3 CRISPR gRNA or a non-targeting control gRNA to photoreceptors in three mouse models, including Pde6b^rd10/rd10, Rho^-/-, and Rho^P23H/+. Cas9 is expressed ubiquitously from a transgene in each of these mutants. We then allow treated and control mice to survive until the time when most rod photoreceptors will have died. We evaluate vision-guided behavior in the mice using a Morris water maze, and we then analyze retinas electrophysiologically and histologically.

Results : We found that acute knockout of Nr2e3 in adult rods potently rescues photoreceptor morphology, electrophysiologic responses to light, and vision-guided behavior in the Pde6b^rd10/rd10 mouse model of RP. In contrast, acute Nr2e3 knockout failed to prevent degeneration in Rho^-/- and Rho^P23H/+ mice. To understand the mechanism of this selectivity, we profiled the gene expression changes that occur after acute knockout of Nr2e3 and found only 37 dysregulated genes, which largely consisted of upregulated cone-enriched genes. Among these is Pde6c, which encodes the cone-specific homolog of Pde6b which is mutated in the rd10 mouse model. Thus, these data support the hypothesis that rod preservation after acute Nr2e3 knockout is due, in part, to derepression of cone-specific Pde6c in rods, which compensates for the lack of functional PDE6B in Pde6b^rd10/rd10.

Conclusions : Our findings demonstrate that acute Nr2e3 knockout is highly effective in preventing photoreceptor degeneration in Pde6b^rd10/rd10 mice. These studies also shed light on the downstream mechanisms that contribute to neuroprotection after acute targeting of the NRL-NR2E3 axis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.