Bystander editing by adenine base editors impairs vision restoration in a mouse model of Leber congenital amaurosis

Creators: Lee, Seok-Hoon¹; Wu, Jun^{1, 2}; Im, Dongjoon^{3, 4}; Hwang, Gue-ho¹; Jeong, You Kyeong¹; Jiang, Hui^{1, 2}; Lee, Seok Jae²; Jo, Dong Hyun¹; Goddard, William A., III⁴; Kim, Jeong Hun^{1, 2}; Bae, Sangsu¹

1. Seoul National University
2. Seoul National University Hospital
3. Korea University
4. California Institute of Technology

Abstract

Base editors (BEs) have emerged as a powerful tool for gene correction with high activity. However, bystander base editing, a byproduct of BEs, presents challenges for precise editing. Here, we investigated the effects of bystander edits on phenotypic restoration in the context of Leber congenital amaurosis (LCA), a hereditary retinal disorder, as a therapeutic model. We observed that in rd12 of LCA model mice, the highest editing activity version of an adenine base editors (ABEs), ABE8e, generated substantial bystander editing, resulting in missense mutations despite RPE65 expression, preventing restoration of visual function. Through AlphaFold-based mutational scanning and molecular dynamics simulations, we identified that the ABE8e-driven L43P mutation disrupts RPE65 structure and function. Our findings underscore the need for more stringent requirements in developing precise BEs for future clinical applications.

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Acknowledgement

This work was supported by grants from the National Research Foundation of Korea (NRF) (nos. 2022M3A9E4017127 and RS-2023-00260351 to J.H.K.; nos. 2021M3A9H3015389, RS-2024-00451880, and SRC - NRF2022R1A5A102641311 to S.B.; no. RS-2023-00274504 to D.I., and no. RS-2023-00246813 to Y.K.J.); the Korean Fund for Regenerative Medicine (KFRM), Republic of Korea (nos. RS-2024-00332601 and 21A0202L1-11 to S.B.); National Research Council of Science & Technology (NST) grant by the Korea government (GTL24021-520 to J.H.K.); Kun-hee Lee Child Cancer & Rare Disease Project, Republic of Korea (no. 202200004004 to J.H.K. and no. 25B-001-0700 to S.B.); Seoul National University Hospital Research Grant, Republic of Korea (nos. 18-2023-0010 and 03-2023-3020); a grant from the Ministry of Food and Drug Safety in 2025, Republic of Korea (no. 25202MFDS003 to S.B.); and grants from the National Institutes of Health, United States (nos. R01HL155532 and R35HL150807) to W.A.G.

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