Detection of mutant RP1 protein by HiBiT-tagging of patient derived retinal organoids

Abstract

Purpose : Nonsense mutation in the terminal exon of the RP1 gene is a common cause of autosomal dominant retinitis pigmentosa. Production of truncated RP1 proteins has yet to be demonstrated in RP1 patients due to the lack of patient-derived retinal tissue and appropriate antibodies. TO enable detection of truncated RP1 proteins in patient-derived retinal tissues we modified the mutant RP1 locus in patient-derived induced pluripotent stem cells (iPSCs) with the luminescent HiBiT tag.

Methods : Paired nickase CRISPR/Cas9 gene editing was used to insert the HiBiT luminescent tag into iPSCs from an 85-year old female donor with RP1 p.E700X. HiBiT tagged iPSCs were characterised and differentiated into retinal organoids. Retinal organoids were treated with the translational readthrough inducing drug PTC124 and HiBiT-tagged RP1 protein detected by luminescence assays and western blotting.

Results : High efficiency insertion of the HiBiT tag into the RP1 locus was demonstrated in RP1 patient-derived iPSC. Western blotting demonstrated expression of full-length RP1 protein (198kDa) as well as a novel 47kDa band in RP1-HiBiT retinal organoids and adult human retinal tissues. HiBiT blotting of RP1-HiBiT retinal organoids revealed four luminescent bands (22kDa, 47kDa, 90kDa and 100kDa). Treatment with PTC124 reduced expression of the 22kDa and 90kDa bands, increased 47kDa expression and induced expression of a new 120kDa band.

Conclusions : We demonstrate the first direct evidence for production of truncated RP1 protein in retinal cells derived from an RP1 patient. Treatment of patient-derived retinal organoids with PTC124 induced limited translational readthrough of mutant RP1 mRNA, but did not result in production of full-length PR1 protein. We also report but detection of a novel 48kDa RP1 protein isoform in HiBiT retinal organoids and adult human retina.

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