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A drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization

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Abstract

Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless “switched on” by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization.

Graphic abstract

Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.

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All datasets generated for this study are included in the article/supplementary materials.

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Acknowledgements

The authors thank UTAS CFF animal technicians, Karen Shiels, Keri Smith, Heather Howard, Lisa Harding and Danielle Eastley, for their assistance with rat chamber operation. This work was supported by grants from the National Health and Medical Research Council of Australia (NHMRC; 1061912, 1185600, 1108311 and 1161583), the Ophthalmic Research Institute of Australia, the National Natural Science Foundation of China (8197030485) and the Rebecca L Cooper Medical Research Foundation. A.W.H. received an NHMRC Practitioner Fellowship (1103329). L.L. was supported by the Department of Science and Higher Education of Ministry of National Defense, Republic of Poland (“Kościuszko” k/10/8047/DNiSW/T-WIHE/3) and the National Science Centre, Republic of Poland (UMO-2017/25/B/NZ1/02790). The Centre for Eye Research Australia receives Operational Infrastructure Support from the Victorian Government.

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Author notes

  1. Jingxiang Zhong and Guei-Sheung Liu contributed equally to this work as senior author.

Authors and Affiliations

  1. Department of Ophthalmology, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong, China

    Jinying Chen, Leilei Tu, Jingxiang Zhong & Guei-Sheung Liu

  2. Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia

    Jinying Chen, Fan-Li Lin, Yu-Fan Chuang, Fan Li, Alex W. Hewitt & Guei-Sheung Liu

  3. Shenzhen Key Laboratory of Biomimetic Materials and Cellular Immunomodulation, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong, China

    Fan-Li Lin

  4. Wicking Dementia Research and Education Centre, University of Tasmania, Hobart, TAS, Australia

    Jacqueline Y. K. Leung & Yu-Fan Chuang

  5. Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia

    Jiang-Hui Wang, Gregory J. Dusting & Alex W. Hewitt

  6. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-sen University, Guangzhou, Guangdong, China

    Fan Li

  7. Department of Materials Science and Engineering, Faculty of Engineering, Monash University, Clayton, VIC, Australia

    Hsin-Hui Shen

  8. Department of Biochemistry and Molecular Biology, School of Biomedical Science, Monash University, Clayton, VIC, Australia

    Hsin-Hui Shen

  9. Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia

    Gregory J. Dusting, Alex W. Hewitt & Guei-Sheung Liu

  10. Department of Optometry and Vision Sciences, University of Melbourne, Parkville, VIC, Australia

    Vickie H. Y. Wong & Bang V. Bui

  11. Translational Vectorology Group, Children’s Medical Research Institute, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia

    Leszek Lisowski

  12. Vector and Genome Engineering Facility, Children’s Medical Research Institute, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia

    Leszek Lisowski

  13. Military Institute of Hygiene and Epidemiology, The Biological Threats Identification and Countermeasure Centre, Puławy, Poland

    Leszek Lisowski

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  1. Jinying Chen
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Contributions

Conceptualization—J.C., G-S.L. Methodology—J.C., F-L.L., J.Y.K.L., G-S.L. Formal Analysis—J.C., F-L.L., G-S.L. Investigation—J.C., F-L.L., J.Y.K.L., L.T., Y-F.C., J-H.W., F.L., V.H.Y.W. Resources—G.J.D., H.-H.S., B.V.B., L.L., A.W.H., J.Z., G-S.L. Data Curation—J.C., G-S.L. Writing (Original Draft)—J.C., G-S.L. Writing (Review & Editing)—J.Y.K.L., J-H.W., F-L.L., L.L., G.J.D., V.H.Y.W., B.V.B., J.Z. Visualization—J.C., G-S.L. Supervision—J.Z., G.S.L. Project Administration—J.C., G-S.L. Funding Acquisition—J.Z., G-S.L.

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Correspondence to Guei-Sheung Liu.

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Chen, J., Lin, FL., Leung, J.Y.K. et al. A drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization. Angiogenesis 24, 97–110 (2021). https://doi.org/10.1007/s10456-020-09745-7

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