Rod and cone structure and function decline in a humanized mouse model of P23H Rhodopsin Autosomal Dominant Retinitis Pigmentosa (adRP)

Abstract

Purpose : The P23H mutation in rhodopsin (RHO) is a common cause of human adRP. Although there are no treatments, gene editing approaches are being developed to correct this mutation. Most approaches rely on sequence homology with the target region. Many show targeted as well as unwanted "off target" editing. Prior to clinical use specificity must be evaluated, e.g., “off targeting:targeting". The assays must be performed in vivo and in a model that harbors both mutant and wild type (WT) alleles. We created mice (WT and P23H) in which a short recognition site at the P23H locus had been humanized. Prior to using these mice to evaluate genome editing specificity, we characterized rod and cone structure/function to define the optimal time frame to evaluate the gene editing specificity for the WT vs P23H alleles.

Methods : The humanized WT and P23H homozygous (Hz) mouse lines were created on a C57Bl6/J (B6) background. We crossed Hz lines to produce heterozygous (Het) progeny (hRHO KI/P23H hRho KI). We recorded scotopic and photopic full-field electroretinogram (ffERG) responses from postnatal day 20 (P20) to P160 in all lines and in B6. At the last time point, we euthanized the mice and prepared their retinas for immunohistochemistry and confocal imaging. We compare retinal and photoreceptor morphology across time and genotype.

Results : The outer nuclear layer (ONL) thickness of hRHO KI Hz and B6 mice are similar across time. At P30, P23H hRHO Hz mice already show a marked ONL thinning and only a single layer of cone nuclei (clumped chromatin remains). At P20, the outer retina of hRHO KI/P23H hRho KI is similar to hRHO KI Hz and B6. From P30 to P60, their ONL thins significantly, but degeneration is never as severe as in age-matched P23H hRHO KI Hz mice. At all ages, hRHO KI Hz and B6 rod and cone ffERG responses are similar. At P30 P23H hRHO Hz rod responses are almost completely absent and their cone responses diminish as the animals age. At P20, the hRHO KI/P23H hRho KI rod-isolated b-wave is reduced by 70% and declines to 90% by P160.

Conclusions : hRHO KI/P23H hRho KI mice show a gradual degeneration and will be a valuable resource for in situ evaluation of genome editing specificity. The slow degeneration also enhances the evaluation of the therapeutic treatment window.

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