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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. 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.

Item Type:	Article
Authors/Creators:	Chen, J and Lin, F-L and Leung, JYK and Tu, L and Wang, J-H and Chuang, Y-F and Li, F and Shen, H-H and Dusting, GJ and Wong, VHY and Lisowski, L and Hewitt, AW and Bui, BV and Zhong, J and Liu, G-S
Keywords:	gene therapy, diabetic retinopathy, angiogenesis, VEGF, AAV, destabilizing domain, Flt23k, retinal neovascularization, trimethoprim
Journal or Publication Title:	Angiogenesis
Publisher:	Springer Netherlands
ISSN:	0969-6970
DOI / ID Number:	10.1007/s10456-020-09745-7
Copyright Information:	Copyright 2020 Springer Nature B.V.
Related URLs:	UTAS Profile: Leung, JYK Google Scholar: Hewitt, AW UTAS Profile: Hewitt, AW Google Scholar: Liu, G-S UTAS Profile: Liu, G-S
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