Optimal Inhibition of Choroidal Neovascularization by scAAV2 with VMD2 Promoter-driven Active Rap1a in the RPE

Abstract

Age-related macular degeneration (AMD) is a multifactorial chronic disease that requires long term treatment. Gene therapy is being considered as a promising tool to treat AMD. We found that increased activation of Rap1a in the retinal pigment epithelium (RPE) reduces oxidative signaling to maintain barrier integrity of the RPE and resist neural sensory retinal angiogenesis from choroidal endothelial cell invasion. To optimally deliver constitutively active Rap1a (CARap1a) into the RPE of wild type mice, self-complementary AAV2 (scAAV2) vectors driven by two different promoters, RPE65 or VMD2, were generated and tested for optimal active Rap1a expression and inhibition of choroidal neovascularization (CNV) induced by laser injury. scAAV2-VMD2, but not scAAV2-RPE65, specifically and efficiently transduced the RPE to increase active Rap1a protein in the RPE. Mice with increased Rap1a from the scAAV2-VMD2-CARap1a had a significant reduction in CNV compared to controls. Increased active Rap1a in the RPE in vivo or in vitro inhibited inflammatory and angiogenic signaling determined by decreased activation of NF-κB and expression of VEGF without causing increased cell death or autophagy measured by increased LCA3/B. Our study provides a potential future strategy to deliver active Rap1a to the RPE in order to protect against both atrophic and neovascular AMD.

Figure 1

Diagrams of self-complementary adeno-associated virus 2 (scAAV2) to deliver constitutively active Rap1a (CARap1a) or only GFP driven by (A) an RPE65 promoter (scAAV2-RPE65-GFP-CARap1a and scAAV2-RPE65-GFP) or (B) a VMD2 promoter (scAAV2-VMD2-GFP-CARap1a and scAAV2-VMD2-GFP).

Figure 2

In vivo analysis of scAAV2 transduction in RPE of wild type mice. (A) Micron IV retinal imaging of GFP and (B) immunostaining of GFP and RPE65 in retinal cryosections of wild type mice treated with PBS or 5 weeks after injection of scAAV2-RPE65-GFP or scAAV2-VMD2-GFP vectors at dose of 5 × 10⁸ viral particle/µl.

Figure 3

scAAV2-VMD2 vector shows more specific GFP transduction and greater Rap1 expression in the RPE. (A) IHC of GFP in retinal cryosections (Blue: TO-PRO-3; Green: GFP) (B–E) western blots of Rap1a and β-actin in RPE/choroids (B–D), (representative gel images and C and E, quantification of densitometry) of wild type mice injected with either (B,C) scAAV2-RPE65 or (D,E) scAAV2-VMD2 or PBS (*p < 0.05 vs. scAAV2-VMD2-GFP; ^†p < 0.05 vs. PBS n = 5–6.

Figure 4

Expression of active Rap1a in RPE by scAAV2-VMD2-CARap1a reduces choroidal neovascularization (CNV) in wild type mice in a laser induced CNV model. (A) Representative images of RPE/choroid flat mounts and (B) quantification of CNV lesion (*p < 0.05 vs. scAAV2-VMD2, n = 40 spots from 12 mice).

Figure 5

Expression of active Rap1a in RPE by scAAV2-VMD2-CARap1a reduces inflammation and VEGF in RPE/choroids. Western blots of (A,B) phosphorylated NF-κB (p-NF-κB) and (C,D) VEGF in RPE/choroids of scAAV2-VMD2 injected wild type mice 7 days after laser treatment (A–C), representative gel images and (B–D), quantification of densitometry; *p < 0.05, **p < 0.01 vs. scAAV2-VMD2-GFP; n = 6–8.

Figure 6

Expression of active Rap1a in RPE by scAAV2-VMD2-CARap1a does not activate apoptosis and autophagy. Western blots of (A,B) caspase 3 and (C,D) LC3A/B in RPE/choroids of scAAV2-VMD2 injected wild type mice 7 days after laser treatment (A–C), representative gel images and (B–D), quantification of densitometry; *p < 0.05 vs. scAAV2-VMD2-GFP; n = 5–6; CC, cytochrome C treated cell lysate.

Figure 7

Expression of active Rap1a in RPE by adenovirus transduction reduces VEGF and NF-κB activation. Western blots of (A,B) Rap1, (C,D) VEGF protein and (E,F) phosphorylated NF-κB (p-NF-κB) and total NF-κB in human RPE transduced with adenovirus expressing GFP (Ad-GFP) or GFP and constitutively active Rap1a (Ad-63E) (A,C,E), representative gel images and (B,D,F), quantification of densitometry *p < 0.05, **p < 0.01 vs. Ad-GFP; n = 3.

Figure 8

Expression of active Rap1a in RPE by adenovirus transduction does not increase autophagy and cell death. Western blots of (A,B) LC3A/B protein and (C,D) caspase 3 and cleaved caspase 3; and (J-K) TUNEL staining in human RPE transduced with adenovirus expressing GFP (Ad-GFP) or GFP and constitutively active Rap1a (Ad-63E) (A and C), (representative gel images and (B and D), quantification of densitometry *p < 0.05 vs. Ad-GFP; n = 3; CC in C refers to cytochrome C treated cell lysate.