Characterization of a Humanized Mouse Model of P23H Rhodopsin Autosomal Dominant Retinitis Pigmentosa (adRP)

Abstract

Purpose : Gene editing is a powerful tool to eliminate expression of mutant proteins. Before any editing approach goes to the clinic, targeted and "off target" editing must be evaluated, preferably in the same animal in situ. The P23H mutation in rhodopsin (RHO) is a common cause of human adRP and a frequent target for gene editing approaches. To evaluate genome editing of an ARCUS meganuclease, RHO1-2, we created humanized mice that harbor WT and mutant P23H hRHO near the P23H mRho locus. One line harbors two humanized WT hRHO alleles (hRHO KI Hz), the other, two humanized P23H hRHO alleles (P23H hRHO KI Hz). When crossed the progeny harbor one WT and one P23H hRHO allele (hRHO KI/P23H hRho KI). We To characterized retinal structure and function in all three mouse lines to define the optimal time frame for evaluation of RHO1-2 gene editing.

Methods : We compared dark and light adapted full field electroretinogram (ffERG) responses in all three genotypes to C57Bl6/J mice. Responses were evoked using a standard ISCEV protocol. b-wave amplitudes at all flash intensities were compared across genotype x age. Morphological assessments were conducted in immunohistochemically labeled frozen sections. Outer nuclear layer (ONL) thickness - DAPI labeled somata and rod morphology - rhodopsin and REEP6.

Results : Retinal structure/function in hRHO KI Hz is similar to C57Bl6/J through P65. At P40, P23H hRHO KI Hz retina has no scotopic ERG and the ONL is a single layer of nuclei with clumped chromatin, emblematic of cones. hRHO KI/P23H hRho KI retinal structure shows time dependent ONL thinning. At P20, hRHO KI/P23H hRho KI retina is similar to hRHO KI Hz. At P30 and P60, hRHO KI/P23H hRho KI ONL thins significantly, albeit not as severe as P23H hRHO KI Hz. hRHO KI/P23H hRho KI scotopic b-wave amplitudes are reduced compared to hRHO KI (~ 60% at P20 and 50% at P75). Across all genotypes, photopic b-wave amplitudes are similar through P70. We continue to track changes in retinal structure and function in older mice.

Conclusions : hRHO KI/P23H hRho KI retina has a broad window for genome editing. Early treatment (first postnatal week) will determine maximum editing capability and late treatment will define efficacy in late stage disease. P23H hRHO/hRHO mice are a valuable resource for in situ evaluation of targeted and "off target" editing of P23H and WT hRHO alleles.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.