Base editing restores retinal and visual function in the mouse model of Leber congenital amaurosis

Abstract

Purpose : CRISPR-Cas9 genome editing has great potential for the treatment of numerous inherited retinal diseases, but its clinical application has been hampered by low precision and substantial indel formation. Cytosine and adenine base editors (CBEs and ABEs) enable conversion of a point mutation in a predictable manner independent of Cas9-induced double-stranded DNA breaks and homology-directed repair. We hypothesized that base editors can be used to target the mutations associated with inherited retinal diseases. To test our hypothesis, we delivered ABE to the rd12 mouse model, which harbors a de novo mutation in the Rpe65 gene, and evaluated the therapeutic efficacy of ABE.

Methods : We first screened for a sgRNA enabling mutation correction using an rd12 mutation-harboring stable cell line. Next, we packaged the selected sgRNA and ABE into a lentivirus, and subretinally delivered to the rd12 mice at 4 weeks old. After 5 weeks, we assessed the DNA correction and RPE65 rescue by deep targeted amplicon sequencing, off-target analysis, immunoblot and retinoid analysis. Furthermore, we evaluated visual function in treated mice with electroretinography, optomotor response assay and visual cortex recordings. Cone density was examined with retinal wholemount stained with cone opsin-specific antibodies.

Results : A subretinal injection of a lentivirus expressing the ABE and a sgRNA corrected a pathogenic mutation in the Rpe65 gene up to 29% with minimal indel and off-target mutations despite the absence of a canonical “NGG” protospacer-adjacent motif (PAM) sequence. The ABE-treated mice show restored RPE65 expression, retinoid isomerase activity, and retinal and visual function at near-normal levels. Treated mice also revealed a higher cone density on retinal flatmounts compared to untreated mice.

Conclusions : In this proof-of-concept study, we provide evidence of the clinical potential of base editors for the correction of mutations causing inherited retinal diseases and for restoring visual function. Base editing technology can provide an alternative treatment model of gene augmentation therapy to permanently rescue the function of a key vision-related protein disabled by mutations.

This is a 2021 ARVO Annual Meeting abstract.