The effect of acute knockout of Nr2e3 on mouse photoreceptor cells

Acute knockout of the rod photoreceptor transcription factor Nrl delays retinal degeneration in multiple mouse models of blindness1-3, but the downstream transcriptomic changes that mediate these therapeutic effects are unknown. Here, we show that acute Nrl knockout causes upregulation of a subset of cone genes in rods as well as downregulation of rod genes, including the rod-specific transcriptional repressor Nr2e3. We hypothesized that Nr2e3 downregulation might mediate some of the therapeutic effects of Nrl knockout. Indeed, acute knockout of Nr2e3 prevents photoreceptor degeneration and preserves visual function in mice with mutations in the catalytic subunit of the rod-specific phosphodiesterase (Pde6brd10/rd10). Upregulation of Pde6c, the cone-specific homolog of Pde6b, in Nr2e3-knockout rods is required to prevent degeneration in Pde6brd10/rd10 mice, suggesting that this therapeutic effect is mediated by a gene-replacement mechanism. In contrast, acute Nr2e3 knockout fails to prevent degeneration caused by loss- or gain-of-function mutations in Rhodopsin (Rho-/- and RhoP23H/P23H), whereas acute Nrl knockout delays degeneration in both models. These results suggest that acute Nrl knockout may rescue Pde6brd10/rd10 mice via downregulation of Nr2e3 and consequent upregulation of Pde6c, while rescuing Rho mutants via a distinct mechanism, possibly by downregulating rod genes. These findings indicate that acute NRL knockout may be a promising gene-independent strategy for preventing photoreceptor degeneration in human patients. Overall design: To determine the transcriptomic effects of acute Nr2e3 knockout we subretinally injected an AAV harboring a guide RNA targeting Nr2e3, or control, into Rosa26-Cas9 mice (Jackson labs strain #024858), which express SpCas9 in all cells. The AAV also harbors an mCherry reporter driven by a photoreceptor specific promoter, allowing us to isolate AAV-transduced photoreceptors by Flourescence Activated Cell (FAC) Sorting. Mice were injected at postnatal day 21 and mCherry expressing cells were sorted for RNA isolation 6 months later.