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Topical application of Cas9 ribonucleoproteins inhibits corneal neovascularization in a mouse model of alkali burn injury

Gene Therapy (2026)Cite this article

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Abstract

Corneal neovascularization is a sight-threatening condition for which current treatments such as anti-VEGF agents are limited by invasiveness and side effects. We present the first non-viral, CRISPR/Cas9-based gene therapy delivered via topical eye drops that penetrates the cornea and inhibits pathological neovascularization. Cas9 ribonucleoproteins (RNPs) targeting the Vegfa gene were complexed with a liposomal carrier (lipofectamine) and administered to mice after alkali burn injury to the cornea. This approach achieved approximately 2% gene editing at the Vegfa locus in vivo, which significantly reduced local VEGF-A expression. Consequently, treated corneas showed markedly decreased macrophage infiltration and robust suppression of both hemangiogenesis and lymphangiogenesis compared to untreated controls. These findings demonstrate that even modest in vivo gene editing can yield a strong therapeutic effect, highlighting a clinically relevant strategy for controlling corneal angiogenesis. Our study introduces a feasible and safe topical CRISPR therapy for corneal diseases, offering a potential alternative to invasive or virus-based gene delivery methods.

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Fig. 1: Analysis of Cas9 RNPs distribution in the cornea following in vivo intrastromal injection.
Fig. 2: Cas9 RNPs design and indel confirmation in the mouse Vegfa gene.
Fig. 3: Inhibition of corneal neovascularization and VEGFA protein expression in an alkali burn model through intrastromal Cas9 RNPs injection.
Fig. 4: Inhibition of corneal neovascularization and VEGFA protein expression in an alkali burn model by an eyedrop application of Cas9 RNPs.
Fig. 5: Significant reduction in VEGFA-expressing macrophage infiltration across the central and peripheral corneas after Cas9 RNPs treatment in an alkali burn injury model.

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Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its extended data and supplemental information files. The datasets generated during or analyzed in the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

This research was supported by Seoul National University Hospital Research Grant (18-2023-0010), National Research Council of Science & Technology (NST) grant by the Korea government (MSIT) (GTL24021-000 to J.H.K.), grants from the National Research Foundation of Korea (NRF) (2022M3A9E4017127, RS-2023-00260351, and RS-2024-00467177 to J.H.K.), and Korean Fund for Regenerative Medicine(KFRM) grant (21A0202L1-11 to S.B.).

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Author notes

  1. These authors contributed equally: Seok Jae Lee, Bae-Geun Nam.

Authors and Affiliations

  1. Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea

    Seok Jae Lee & Jeong Hun Kim

  2. Global Excellence Center for Gene & Cell Therapy (GEC-GCT), Seoul National University Hospital, Seoul, Republic of Korea

    Seok Jae Lee, Dong Hyun Jo, Sangsu Bae & Jeong Hun Kim

  3. Department of Ophthalmology, Seoul National University Hospital, Seoul, Republic of Korea

    Seok Jae Lee & Jeong Hun Kim

  4. Genomic Medicine Institute, Seoul National University College of Medicine, Seoul, Republic of Korea

    Bae-Geun Nam & Sangsu Bae

  5. Divison of Bio Research, SK Bioscience, Seongnam-si, Republic of Korea

    Sung-Ah Hong

  6. Department of Anatomy & Cell Biology, Seoul National University College of Medicine, Seoul, Republic of Korea

    Dong Hyun Jo

  7. Department of Cornea, External Disease & Refractive Surgery, HanGil Eye Hospital, Incheon, Republic of Korea

    Sang-Mok Lee

  8. Department of Ophthalmology, Catholic Kwandong University College of Medicine, Gangneung-si, Republic of Korea

    Sang-Mok Lee

  9. Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea

    Sangsu Bae & Jeong Hun Kim

  10. Institute of Reproductive Medicine and Population, Seoul National University College of Medicine, Seoul, Republic of Korea

    Jeong Hun Kim

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Contributions

Seok Jae Lee: in vivo experiments, Writing-Original draft. Bae-Geun Nam: in vitro experiments, Writing-Reviewing and Editing. Sung-A Hong: in vitro experiments, Writing-Reviewing and Editing. Dong Hyun Jo: Writing-Reviewing and Editing. Sang-Mok Lee: Supervision, Writing-Reviewing, and Editing. Sangsu Bae: Funding acquisition, Supervision, Writing- Reviewing and Editing. Jeong Hun Kim: Conceptualization, Funding acquisition, Supervision, Writing- Reviewing and Editing.

Corresponding authors

Correspondence to Sang-Mok Lee, Sangsu Bae or Jeong Hun Kim.

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The authors declare no conflicts of interest in relation to this article.

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All animal procedures and related protocols were conducted in accordance with the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Seoul National University Animal Care and Use Committee. This study was conducted exclusively on mice; therefore, obtaining consent to participate was not applicable.

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Lee, S.J., Nam, BG., Hong, SA. et al. Topical application of Cas9 ribonucleoproteins inhibits corneal neovascularization in a mouse model of alkali burn injury. Gene Ther (2026). https://doi.org/10.1038/s41434-026-00607-3

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