Adenine-base Editing Corrects the Most Common ABCA4 Mutation Causing Stargardt Disease

Abstract

Purpose : Stargardt disease (Fig. 1A, B) is an inherited neurodegenerative disease that leads to macular degeneration and blindness caused by loss-of-function mutations in the ABCA4 gene. Currently, there is no approved therapy. In this study, we developed a dual adeno-associated virus (AAV) split-intein adenine base editing approach (Fig. 1C) to correct the most common mutation in ABCA4 (c.5882G>A, p.G1961E).

Methods : To test different split-intein- and base editor versions, we used mutation-carrying 293T cells. To assay base editing in relevant target cells (photoreceptors and retinal pigment epithelial (RPE) cells), we developed an engineered human retinal organoid model and generated a humanized mouse model carrying the ABCA4 c.5882G>A mutation. Furthermore, using the adjacent adenine (c.5883A, wobble base, Fig. 1B), we tested the same base editing strategy on wild-type human retinal explants and in RPE/choroidal explants. Finally, we subretinally injected six adult Cynomolgus macaques and assayed base editing rates at the genomic DNA level, at the RNA level and on the DNA level from sorted rod and cone photoreceptors.

Results : Base editing on the Stargardt target base (c.5882G>A) and the adjacent wobble base (c.5883A) were similar in lentivirus-carrying HEK293T cells and in human retinal organoids. Therefore, the wobble base assay can be used to assess editing rates in wild-type models (human explants and NHPs). Our split-intein adenine base editor AAV vector led to 5-15% editing in retinal organoids and 10-20% editing in human retinal explants, assayed at the genomic DNA level. Next, we injected the optimized vector construct into mutation-carrying mice and achieved high in vivo gene correction, where ~50% of photoreceptors and ~80% of RPE cells showed editing. In injected NHPs, base editing rates were found to be ~50% in cones, ~25% in rods and up to ~99% in RPE cells.

Conclusions : We developed an optimized vector candidate for the correction of the ABCA4 c.5882G>A mutation in human models (retinal organoids, human explants). Using this optimized vector, we achieved high base editing rates after in vivo injection in mice and NHPs. These results demonstrate that AAV-mediated base editing can lead to a precise genetic change in the nervous tissue in non-human primates.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.