A novel X-linked retinitis pigmentosa beagle dog model for ophthalmic research established rapidly by editing the RPGR gene in zygotes.

Abstract

Inherited retinal diseases (IRDs) constitute several ocular disorders leading to progressive and severe visual impairment. While significant progress has been made in understanding the genetic basis of IRDs, preclinical animal models are crucial for advancing therapeutic development. Although well-established mouse models exist, the scarcity of large animal models represents a significant limitation. Dogs (Canis familiaris) exhibit numerous physiological and anatomical similarities to humans, rendering them as potential models. X-linked retinitis pigmentosa (XLRP) is a severe, early-onset form of IRDs, primarily caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. Here, we efficiently generated a stably inherited RPGR-knockout beagle model of XLRP in one-step via CRISPR/Cas9 gene editing tool with multiple gRNAs and zygote microinjection technology. Two F0 generation beagle dogs were successfully obtained with high knockout efficiency in the RPGR gene and the F0 female exhibited characteristic XLRP phenotypes, including progressive deterioration of both rod and cone photoreceptor function, a gradual reduction in the outer nuclear layer thickness from 7 to 35 months, and attenuated retinal arteries with minimal pigmentation. Notably, the F1 male of hemizygous RPGR-knockout beagle dogs displayed more pronounced disease phenotypes by 7 months of age. In conclusion, we successfully generated a stably inherited beagle dog model of XLRP exhibiting an intermediate characteristic disease onset, which is highly suitable for therapeutic research in gene therapy and stem cell transplantation for determining optimal intervention timing and therapeutic dosing.