Development of novel models and CRISPR-based gene editing therapeutics for retinal degeneration in Xenopus laevis

Mutations in the rhodopsin gene (RHO) are the most common cause of autosomal dominant retinitis pigmentosa (adRP). Previously, our research group has identified two distinct mechanisms of light-exacerbated retinal degeneration (RD) associated with P23H and T4K RHO mutations. Here, we developed a transgenic X. laevis carrying human/mouse hybrid T4K RHO and compared the light-exacerbated RD phenotype to human/mouse wildtype, human-T4K and mouse-T4K RHO transgenic X. laevis models. For animals 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 animals expressing T4K RHO. Therefore, the light-exacerbated RD phenotype associated with the RHO T4K mutation is relatively independent of the underlying RHO cDNA. This hybrid animal model allows us to explore treatment strategies directly on the human gene, streamlining the transfer of therapeutics from lab benches into clinical trials. To date, RP remains an incurable disease. Utilizing a previously developed X. laevis model for adRP, we tested multiple CRISPR/Cas9-based gene-editing strategies to prevent RD in our adRP model. We designed highly specific guide RNAs to 1. Edit the mutant allele and allow for the error-prone non-homologous end joining (NHEJ) repair mechanism to result in a premature stop codon, nullifying the mutant allele 2. Induce simultaneous double-strand breaks on both sides of the start codon, generating large inactivating deletions and 3. Edit the mutant allele and utilize the homology-directed recombination repair mechanism to restore the mutant allele to wildtype. Remarkably, the CRISPR-induced NHEJ repair mechanism appeared to be the most efficient treatment in preventing RD. We postulate that in developing gene editing therapeutics for human RP, similar results are likely to occur, suggesting that the simplest approach may be the most effective. Moreover, our X. laevis models can be used to characterize and understand the pathomechanism of human RP mutations, as well as to develop novel gene-editing treatment strategies. Lastly, our findings demonstrate that CRISPR/Cas9 technology is an effective therapeutic tool for adRP with potential clinical implications for other dominant diseases of the human retina.Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat