Generating humanized transgenic zebrafish lines to investigate the pathogenicity of RLBP1 and RAB28 variants

Abstract

Purpose : The visual cycle regenerates the visual chromophore 11-cis-retinaldehyde. Cellular retinaldehyde binding protein (CRALBP), encoded by RLBP1 in humans, facilitates this retinoid cycle in the retinal pigment epithelium (RPE) and Müller glia. RLBP1 is associated with inherited retinal diseases including Newfoundland Rod-Cone Dystrophy and Bothnia dystrophy. RAB28 functions in outer segment phagocytosis and in mutations cause autosomal recessive cone-rod dystrophy. Here, we investigate the potential of humanised transgenic zebrafish to reveal the in vivo consequences of human variants in these genes.

Methods : Zebrafish rlbp1a^-/-, rlbp1b^-/-, and rab28^-/- knockout zebrafish lines were generated by CRISPR-cas9 editing. Transgenic zebrafish lines were generated using the tol2 transgenesis system. Validation of human transgene integration and expression was by Sanger sequencing and confocal microscopy. Visual behaviour was examined by optokinetic response (OKR) and visual motor response (VMR). Retinal histology was examined by transmission electron microscopy.

Results : Transgenic zebrafish lines expressing N-terminal GFP tagged human RAB28 or, p.S23F or p.C217W variants, in cones were generated. Similarly, humanised lines with N-terminal GFP tagged CRALBP or variants p.R151Q or P.A237V expressed in RPE were created. 5 dpf zebrafish rlbp1b^-/- larvae show no OKR defect under standard conditions but show a ~50% reduction in saccades per min under dim light conditions. GFP tagged human RAB28 protein shows identical outer segment localisation as compared to the GFP tagged zebrafish Rab28. The RAB28 patient variant p.C217W is mislocalised to inner segments.

Conclusions : A dim light OKR defect is present in rlbp1b^-/-. Human RAB28 p.C217W patient variant mislocalises within cones. The ability of the human WT RLBP1 transgene to rescue this dim light OKR phenotype is currently being assessed. Work is ongoing to examine if the human WT RAB28 transgene is sufficient to rescue the functional outer segment phagocytosis and retinoid defect in the rab28^-/- line.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.