Abstract
Purpose :
Mutations in the gene encoding rhodopsin (RHO) are the most frequent cause of autosomal dominant retinitis pigmentosa (RP). Previous work by our group has shown that expression of the human RHO mutant T4K causes light-dependent retinal degeneration (RD) in transgenic X. laevis. T4K-induced RD is mechanistically distinct from that caused by P23H RHO, in that the mutant rhodopsin does not mislocalize. We aim to generate humanized knock-in mice, carrying a human/mouse hybrid RHO gene, as a model of RP for the purpose of developing translational gene-editing therapeutics. In order to ensure that the hybrid gene design does not alter the mechanism of RD, we generated transgenic X. laevis carrying human/mouse hybrid T4K RHO and characterized the light-exacerbated RD phenotype in comparison to human/mouse hybrid wildtype (WT), human T4K and mouse T4K RHO models in transgenic X. laevis.
Methods :
Transgenes were designed wherein expression of the human/mouse hybrid T4K, human/mouse hybrid WT, human T4K or mouse T4K RHO cDNAs were driven by 0.8 Kb of the X. laevis opsin promoter. At 14 days post-fertilization, developed tadpoles were sacrificed. One eye was solubilized for evaluating rhodopsin expression levels using a dot-blot assay while the contralateral eye was fixed, embedded, sectioned, immunohistochemically labelled using anti-mammalian rhodopsin antibodies and imaged for assessing structural differences.
Results :
For tadpoles reared in cyclic light, expression of T4K rhodopsin in rods caused significant RD regardless of whether the transgene was human, mouse, or a human/mouse hybrid RHO. When raised in the dark, no significant RD was detected in tadpoles expressing T4K RHO, and rhodopsin expression levels were not significantly different from WT animals.
Conclusions :
The light-exacerbated RD phenotype associated with the RHO T4K mutation is relatively independent of the underlying RHO cDNA. This raises our confidence that a humanized mouse model based on a hybrid RHO strategy will retain a mechanistically similar RD. Our next step will be to develop and characterize the mouse model expressing the human T4K RHO, which can be used for both mechanistic studies and testing gene editing therapeutics that will be easily transferable to clinical studies.
This is a 2020 ARVO Annual Meeting abstract.