In vivo reprogramming of rods to cone-like cells by Nrl-knockdown using AAV-delivered CRISPR-Cas9 rescues retinal degeneration

Abstract

Purpose : Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy, characterized by an initial loss of rod photoreceptors and secondary cone death. Acute gene knockout of rod determinant Nrl was shown to reprogram adult rods into cone-like cells, rendering them resistant to effects of mutations in rod-specific genes and consequently preventing secondary cone loss (Montana CL, et al. PNAS, 2013; 110: 1732-7). With a goal to develop this approach for treatment of RP, we used adeno-associated virus (AAV)-delivered CRISPR/Cas9 for Nrl-knockdown in rods.

Methods : Two AAV vectors were constructed for CRISPR/Cas9 knockdown strategy, one expressing Cas9 nuclease, and the other carrying a single-guided RNA targeting the Nrl gene. The two vectors were co-delivered into mice by subretinal administration. Potency of the system was validated by EGFP knockdown in a mouse line with eGFP-labeled rods. Nrl knockdown was conducted in wild-type C57/Bl6 or Crxp-Nrl, a mouse line with rod-only photoreceptors. Molecular, histological and functional alterations were examined by next generation sequencing, immunoblot analysis, immunofluorescence and electron microscopy, and electroretinography (ERG). Rescue of retinal degeneration was assessed in three mouse models harboring either recessive or dominant rod-specific mutations.

Results : eGFP and Nrl were efficiently knocked down following AAV-CRISPR/Cas9 treatment. For Nrl knockdown, insertions and deletions were only detected in the targeted Nrl locus, and no mutation was identified in ten potential off-target loci. A majority of the transduced rods acquired characteristics of cone photoreceptors, as demonstrated by reduced expression of rod-specific genes and enhanced expression of cone-specific genes, loss of the unique rod chromatin pattern, and diminished rod ERG response. In all three disease models, the Nrl-CRISPR/Cas9 treated eyes maintained significantly better photoreceptor viability and cone function than control eyes, as revealed by remarkably thicker photoreceptor layer, higher cone cell number, greater cone ERG amplitude and better optomotor behavior.

Conclusions : AAV-CRISPR/Cas9-mediated Nrl knockdown can efficiently reprogram rods into cone-like photoreceptors and prevent secondary cone death in retinal degeneration. We believe NRL-knockdown can be developed into a viable treatment for RP in humans.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.