Rescue of mouse RPGR function in vivo using CRISPR/Cas9 gene editing

Abstract

Purpose : X-linked retinitis pigmentosa is a severe form of inherited retinal disease, and the majority of disease-causing mutations have been identified in the retinitis pigmentosa GTPase regulator (RPGR) gene. RPGR undergoes alternative splicing with most patient mutations located within a repetitive region of the retina-specific RPGR-ORF15 transcript. Here we sought to use CRISPR/Cas9 to target RPGR-ORF15 in vivo as a method to rescue disease-causing mutations.

Methods : Two approaches were used to target the RPGR mutation in rd9 mice. First, embryos were injected with CRISPR/Cas9 to generate germline correction of RPGR-ORF15 for further study. CRISPR/Cas9 machinery was also packaged into AAV vectors to enable gene editing in adult rd9 mice following subretinal injection. Retinas were analyzed via immunohistochemistry and Western blotting using RPGR domain-specific antibodies to identify rescued RPGR protein expression. Similar methods were used to test for rescue of retinal function by comparing RPGR-associated disease features in control, rd9, and treated rd9 mice.

Results : Following treatment, RPGR-ORF15 protein was present and retinas appeared healthy. RPGR glutamylation was restored and opsin mislocalization was no longer evident. Gene correction in vivo using AAV delivery resulted in fully functional RPGR protein in a subpopulation of cells with broad distribution throughout the retina.

Conclusions : These results demonstrate that ORF15 can be edited successfully in vivo to restore RPGR expression. As CRISPR technologies continue to advance, such an approach may be useful to treat human mutations in RPGR.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.