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

Jinying Chen, Fan-Li Lin, Jacqueline Y.K. Leung, Leilei Tu, Jiang-Hui Wang, Yu-Fan Chuang, Fan Li, Hsin-Hui Shen, Gregory J. Dusting, Vickie H.Y. Wong, Leszek Lisowski, Alex W. Hewitt, Bang V. Bui, Jingxiang Zhong, Guei Sheung Liu

Research output: Contribution to journalArticleResearchpeer-review

2 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)97-110
Number of pages14
JournalAngiogenesis
Volume24
DOIs
Publication statusPublished - 2021

Keywords

  • AAV
  • Destabilizing domain
  • Flt23k
  • Gene therapy
  • Retinal neovascularization
  • Trimethoprim
  • VEGF

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