Comparison HEK293T WT vs HEK293T p.G122Afs*65 CRX
Frameshift mutations that disrupt the activity of the transcription factor CRX lead to various phenotypes of inherited retinal dystrophies (IRDs) through a dominant-negative effect. In many cases, these mutations result in increased mRNA and protein levels due to enhanced stability of the mutant transcript. However, some CRX frameshift mutations increase protein levels without altering transcript abundance due to loss of endogenous sites for degradation. This finding allowed us to identify a post-translational regulation of CRX by ubiquitination to control protein abundance via the proteasome. As a potential therapeutic venue, we used camelid ubiquibodies to target novel epitopes of CRX generated by frameshift variants and promote its degradation. We used the truncated protein encoded by the p.G122Afs*65 pathogenic variant as a bait for screening nanobodies, which we then engineered into ubiquibodies. We hereby show that these mutant CRX proteins can be effectively degraded by at least three different ubiquibodies via the proteasome. We performed RNA-seq analysis in order to compare gene expression profiles in HEK293T cells expressing wild-type CRX and HEK293T cells expressing p.G122Afs*65 variant. This analysis shows some retinal altered genes associated with the mutant variant which are restored after its degradation by ubiquibody induction.