Exploring exon-skipping as a therapeutic intervention strategy for the future treatment of ADGRV1-associated retinitis pigmentosa

Abstract

Purpose : Usher syndrome type 2C (USH2C) results from pathogenic variants in the ADGRV1 gene, causing congenital hearing impairment and progressive vision loss due to retinitis pigmentosa (RP). With no treatment available for ADGRV1-associated RP, we aim to develop therapies to stop the progression of the vision loss. We previously presented skipping of USH2A exon 13 as a promising treatment for USH2A-associated RP and hypothesized that extending this approach to ADGRV1 could offer a therapeutic solution.

Methods : Based on the presence of multiple loss-of-function mutations, we selected ADGRV1 exon 9, and ADGRV1 exons 40-42 as targets for exon-skipping. Based on 2D prediction algorithms, skipping these in-frame exon(s) was predicted to have a minimal impact on the overall ADGRV1 protein domain architecture. To evaluate the functional consequences of skipping the indicated target exons, we generated zebrafish mutants in which the exons orthologous to the human ADGRV1 exons were physically removed using CRISPR/Cas9 genome editing. The resulting adgrv1^dExon9 and adgrv1^dExon40-42 zebrafish lines were phenotypically compared with wild-type- and adgrv1^rmc22 mutant zebrafish, that we previously generated as a model for ADGRV1-associated retinal dysfunction.

Results : Sequencing confirmed the correct excision of zebrafish adgrv1 exon 9 and exons 40-42. No Adgrv1^dExon9 protein could be detected in retinal sections of adgrv1^dExon9 larvae, mirroring findings in the adgrv1^rmc22 mutant. Alphafold2 3D protein modeling revealed that, contrary to 2D predictions, removal of adgrv1 exon 9 does have a detrimental effect on the Adgrv1 protein domain architecture, potentially explaining the protein's absence. Conversely, removal of adgrv1 exons 40-42 is predicted to yield a protein in which two partial CalXβ domains fold into a single hybrid domain with a predicted 3D structure highly similar to native CalXβ domains. Indeed, immunohistochemical analysis confirmed correct translation and localization of Adgrv1^dExon40-42 in photoreceptor cells, similar to observations in wild-type zebrafish.

Conclusions : Our study provides the first indications that skipping ADGRV1 exons 40-42 could be a suitable therapeutic intervention strategy for ADGRV1-associated RP. Moreover, our findings emphasize the crucial contribution of 3D protein modeling in the development of personalized exon-skipping therapies.

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