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. 2025 Aug 14;66(11):37. doi: 10.1167/iovs.66.11.37

Dual VEGF-Targeting Strategy Via AAV2-Delivered sFLT-1 and shVEGF for Retinal Neovascularization Therapy

Cuiting Chen 1,2,3, Xiuqin Yang 1,2, Zongyue Jiang 1, Mengyuan Wang 1,2,3, Xiang Li 1,2,3, Hongjun Huang 4, Mengdie Li 1,2, Yi Liu 2, Zeyu Liu 1,2, Yabin Deng 1, Yong Zu 4, Nuo Dong 1,2,3,5,6,✉, Cheng Li 1,2,3,7,✉
PMCID: PMC12366864  PMID: 40810574

Abstract

Purpose

Retinal neovascular diseases, often associated with elevated vascular endothelial growth factor (VEGF) levels, are major causes of blindness. Current therapies lack sustained efficacy and safety. This study aimed to evaluate the effectiveness of an adeno-associated virus 2 (AAV2)-based gene therapy vector carrying soluble fms-like tyrosine kinase-1 (sFLT-1) and short hairpin RNA targeting VEGF (shVEGF) in treating retinal neovascularization.

Methods

P12 mice were divided into six groups: normal group, control group, AAV2-EGFP group, AAV2-sFLT group, AAV2-shVEGF group, and AAV2-sFLT-shVEGF group. AAV2 vectors encoding sFLT-1 alone, shVEGF alone, or a combination of both sFLT-1 and shVEGF were administered to oxygen-induced retinopathy (OIR) mice via intravitreal injection. Fundus photography and fluorescein fundus angiography (FFA) were used to evaluate retinal vascular morphology and density. Hematoxylin and eosin (H&E) staining was used to observe the growth characteristics of retinal neovascularization. The expression levels of angiogenic factors, inflammatory mediators, and blood-retina barrier-associated markers were assessed using immunofluorescence and quantitative PCR (qPCR). Electroretinography (ERG) was performed to evaluate retinal function.

Results

All three gene therapy groups effectively suppressed pathological neovascularization in OIR mice, although their therapeutic outcomes showed subtle differences. The AAV2-shVEGF group demonstrated the strongest efficacy in promoting the regression of the central non-perfusion area. The AAV2-sFLT-shVEGF group was more effective in suppressing neovascular growth and inflammatory infiltration. All three groups exhibited comparable effects in reducing angiogenic factor expression and enhancing blood-retina barrier-related markers. Notably, only the AAV2-sFLT-shVEGF group showed a pronounced ability to restore retinal function.

Conclusions

Dual inhibition with AAV2-sFLT-shVEGF offers a promising therapeutic approach for retinal neovascular diseases, demonstrating efficacy in reducing neovascularization and improving retinal health.

Keywords: adeno-associated virus 2 (AAV2), inflammation, retinal neovascularization, soluble fms-like tyrosine kinase-1 (sFLT-1), short hairpin RNA targeting vascular endothelial growth factor (shVEGF)

Angiogenic eye diseases are major causes of blindness in developed countries.1 These conditions are among the most difficult to treat in ophthalmology and often lead to poor prognosis. Retinal neovascular diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and age-related macular degeneration (AMD),2–4 are primarily driven by hypoxia, oxidative stress, and inflammation.5–7 These factors increase the expression of hypoxia-inducible factor-1 alpha (HIF-1α), which promotes vascular endothelial growth factor (VEGF), ultimately leading to pathological neovascularization.8,9

The VEGF family of growth factors plays a crucial role in retinal neovascular diseases by promoting retinal inflammation, neovascularization, and vascular leakage.10 Due to its central role, current treatments often involve repeated intravitreal injections of anti-VEGF fusion proteins to inhibit angiogenesis.11–13 However, frequent injections and poor patient compliance lead to complications like inflammation, retinal detachment, and vitreous hemorrhage,14–17 increasing patient burden and posing challenges to healthcare systems.18 Thus, developing long-lasting and safe therapies is a critical challenge.

Ophthalmic gene therapy has advanced quickly toward clinical application since the US Food and Drug Administration (FDA) approved the first therapy in 2017.19 Various viral vectors have been extensively investigated and used.20 Among them, adeno-associated virus (AAV) is considered a standard gene delivery system for eye therapy due to its non-pathogenic nature, high transduction efficiency, replication deficiency, and ability to provide long-term gene expression.21–23

Soluble fms-like tyrosine kinase-1 (sFLT-1), a soluble form of the VEGF-A receptor,24 is expressed by vascular endothelial cells and acts as a heparin-binding protein, specifically binding to VEGF. It inhibits VEGF activity by forming heterodimers with the extracellular ligand-binding domains of FLT-1 and kinase insert domain receptor (KDR) VEGF receptors.25 Conversely, short hairpin RNA targeting VEGF (shVEGF) is processed by the Dicer enzyme into small interfering RNA (siRNA) in vivo, which suppresses target mRNA expression with high sequence specificity, showing potential for treating retinal neovascularization.26

This study introduces an innovative dual-cargo AAV recombinant gene system. Using AAV2 as a vector, we evaluated the efficacy of AAV2 loaded with sFLT-1 alone, shVEGF alone, and a dual-loaded vector carrying both sFLT-1 and shVEGF in treating neovascularization. This design aims to develop gene therapy with more stable, long-lasting therapeutic effects and improved retinal repair. The research seeks to provide novel strategies and insights for treating retinal neovascular diseases.

Materials and Methods

Experimental Animal Source

C57BL/6J mice (25–35g, 8–12 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. and housed at the Medical School of Xiamen University. The study was approved by the Xiamen University Laboratory Animal Ethics Committee, and all animal protocols followed the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Viral Vector Construction

We developed an rAAV vector for co-expression or separate expression of sFLT-1 and shVEGF using the AAV2 serotype. The coding sequences were obtained from NCBI Reference Sequences NM_001159920.2 and NM_001025366.3. The sFLT-1 was amplified with BamHI and HindIII restriction sites, whereas shVEGF included a U6 promoter flanked by EcoRI, MluI, and XhoI sites. The amplified fragments and the pAAV-CMV backbone (Vector Biolabs, Malvern, PA, USA) were digested, separated via agarose gel, purified (New England Biolabs), and ligated to a dephosphorylated backbone using T4 DNA ligase (New England Biolabs, Ipswich, MA, USA). The ligation mixture was transformed into Escherichia coli via heat shock. Antibiotic-resistant clones were screened by PCR, restriction digestion, and sequencing. Recombinant plasmids were used to produce rAAV particles in HEK293 cells (NEB 5-alpha; New England Biolabs, Ipswich, MA, USA), purified via iodixanol gradient ultracentrifugation, and aliquoted at 1.0 × 10¹² VG/mL for storage at –80°C to maintain stability.

OIR Model Creation

On postnatal day 7 (P7), neonatal mice and their mothers were placed in a 75% oxygen chamber (BioSpherixA, USA, model: BioSpherixA Chamber+ProOx 110) for 5 days. Afterward, the mice were returned to normoxic conditions for further treatment and observation.27

Intravitreal Injection

On P12, neonatal mice were anesthetized with 1.25% tribromoethanol (0.2 ml/10g; Nanjing IBS BioTech Co., Ltd., China), and their eyes were topically anesthetized with Proparacaine Hydrochloride Ophthalmic Solution (Alcon, China). A 10 µL Hamilton microsyringe (Huawei Ruike, China) was used to inject 1 µL of solution into the vitreous chamber, with the viral titer of each rAAV group adjusted to 1.0 × 1012 VG/mL. Based on the injected solutions, P12 mice were stratified into six groups: (1) normal group (no modeling), (2) control group (OIR mice injected with 1 µL of sterile 1 × PBS), (3) AAV2-EGFP (Enhanced Green Fluorescent Protein) group (OIR mice injected with 1 µL of AAV2-EGFP), (4) AAV2-sFLT group (OIR mice injected with 1 µL of AAV2-sFLT), (5) AAV2-shVEGF group (OIR mice injected with 1 µL of AAV2-shVEGF), and (6) AAV2-sFLT-shVEGF group (OIR mice injected with 1 µL of AAV2-sFLT-shVEGF). Tobramycin eye drops (Alcon, China) were applied to the injection site for antibacterial and anti-inflammatory treatment, followed by Carbomer Eye Drops (Bausch + Lomb, USA).

Fundus Photography and Fluorescein-Sodium for Angiography

On P17, mice were anesthetized, and their pupils were dilated using Tropicamide Phenylephrine Eye Drops (Changchun Dier Pharmaceutical Co., Ltd., China). Fundus images were captured using a fundus camera (OPTOPROBE, model: OPIMG-L, UK) in white light mode. Mice were then given an intraperitoneal injection of 2 µL of 10% fluorescein sodium solution (Merun Biotechnology Co., Ltd., China) for angiography. Images of the optic disc were captured, with additional views from the nasal, temporal, superior, and inferior angles.

Cryosectioning

On P17, mice were euthanized by cervical dislocation, and the eyeballs were excised and placed in tissue embedding cassettes containing optimum cutting temperature (OCT) compound. The tissues were flash-frozen in liquid nitrogen. Sections were prepared using a cryostat (Leica, model: CM1850UV, Germany) at a thickness of approximately 8 µm and mounted on slides.

Hematoxylin and Eosin Staining

Cryosections were air-dried, fixed in 4% paraformaldehyde (Baishike Biotechnology, China), and rinsed with 1 × PBS. Nuclei were stained with hematoxylin, and cytoplasm with eosin (Beijing Solarbio Science & Technology Co., Ltd., China). The sections were dehydrated in 80%, 95%, and 100% ethanol for 2 minutes each, followed by xylene (Shanghai Pharmaceutical Group Reagent Co., Ltd., China) for 2 minutes. Sections were mounted with neutral resin (Zhongshan Golden Bridge Biotechnology Co., Ltd., China) and imaged with an optical microscope (Carl ZEISS, Germany).

Retinal Whole-Mount Staining

On P17, the eyeballs were fixed in 4% paraformaldehyde for 50 minutes. The retina was isolated and washed with methanol (Shanghai Pharmaceutical Group Reagent Co., Ltd., China). A permeabilization and blocking solution (Perm/Block Solution) containing 1% Triton X-100 (Sigma, USA) and 2% bovine serum albumin (BSA; Sigma, USA) was prepared. The retinas were blocked for 1 hour at room temperature. Isolectin antibody (Vector Labs, DL-1207-0.5, USA) was diluted 1:100 with antibody dilution solution (Shanghai Yenzyme Biomedical Science and Technology Co., Ltd., China) and incubated overnight at 4°C. The retinas were dissected into quadrants the next day under a surgical microscope (Carl ZEISS, Germany). The samples were mounted in 70% glycerol (Shanghai Aladdin Biochemical Technology Co., Ltd., China) and imaged using a laser scanning confocal microscope (Yi Jing Tong Optical Technology Co., Ltd., FLUOVIEW FV3000, China).

Immunofluorescence Staining

Cryosections were fixed in 4% paraformaldehyde and rinsed with 1 × PBS. After permeabilization with 0.2% Triton X-100 for 20 minutes, the sections were blocked with 2% BSA for 1 hour. Primary antibodies were incubated overnight at 4°C, including rabbit anti-VEGFA (1:200, Abclonal, A12303, China), rat anti-F4/80 (1:200, ZENBIO, 263101, China), rabbit anti-MMP-9 (1:500, Abcam, ab38898, USA), mouse anti-HIF-1α (1:200, Abcam, ab1-250, USA), mouse anti-ZO-1 (1:200, Invitrogen, 339100, China), rabbit anti-E-cadherin (1:500, Santa Cruz Biotechnology, sc-8426, USA), mouse anti-IL-6 (1:200, Cell Signaling, 12912S, USA), and rabbit anti-Phospho-STAT3-Y705 (1:200, Abclonal, AP0705, China). After rinsing off the primary antibodies, sections were incubated for 1 hour with secondary antibodies conjugated to Alexa 488 or 594 (1:300, Invitrogen, China). The slides were mounted with DAPI (Vector, USA), and observations along with fluorescence intensity measurements were performed within 2 days.

RNA Extraction

On P17, eyeballs were quickly dissected to isolate the retina, which was placed in Total RNA Extractor Reagent (Osaka, Japan). Tissues were homogenized using a tissue grinder (Sevier Biotechnology Co., Ltd., China), followed by the addition of chloroform (Shanghai Pharmaceutical Group, China). The mixture was centrifuged at 12,000 rpm for 15 minutes (Beckman Coulter, USA). The supernatant was transferred to a new tube, mixed with isopropanol (Shanghai Pharmaceutical Group, China), and centrifuged at 12,000 rpm for 10 minutes. The RNA pellet was washed twice with 70% ethanol, air-dried in a fume hood (Wuhan Cobetter Technology Co., Ltd., China), and resuspended in 20 µL DEPC-treated water (Shanghai Bioengineering Co., Ltd., China) for quantification.

Reverse Transcription PCR

RNA concentration was measured using a spectrophotometer (Thermo Fisher Scientific, IVD-ONE, USA). Reverse transcription was performed using a PCR kit (Yisheng Biotechnology Co., Ltd., China) with the following conditions in a PCR machine (Bio-Rad, USA): (1) 37°C for 15 minutes, (2) 85°C for 5 seconds, and (3) 4°C indefinitely.

Real-Time Quantitative Polymerase Chain Reaction

The reaction mixture was prepared according to the kit instructions (Yisheng Biotechnology Co., Ltd., China). The tubes were centrifuged at 3000 rpm for 5 minutes, then placed in a LightCycler96 PCR machine (Roche, Switzerland) with the following conditions: (1) 95°C for 2 minutes, (2) 95°C for 5 seconds, and (3) 60°C for 30 seconds for 40 cycles. Relative gene expression levels were analyzed using the 2-ΔΔCt method. Primer sequences were provided in Supplementary Table S1 for a list.

Electroretinogram

P17 mice were placed in a dark room for dark adaptation in advance. The mice were anesthetized and their pupils were dilated. The mice were positioned prone and relaxed on the heated stage of a small animal visual electrophysiology system (Diagnosys LLC, USA). Electrodes and a 3 mm flash stimulator were connected and aligned with the center of the mouse’s pupil. Flash stimuli of 0.01, 0.1, and 1 cd/m² were administered, recording the mouse’s a-wave, b-wave, and oscillatory potentials (OPs), as well as the c-wave under 1 cd/m² flash.

Statistical Analysis

Animals were randomly assigned, and modeling duration and severity were consistent, with each experiment repeated at least five times (n = 5 eyes per group). Data are presented as mean ± SD and analyzed using GraphPad Prism 10.0. Data from fluorescein-sodium for angiography (FFA), retinal whole-mount staining, and immunofluorescence staining were quantified using AngioTool (available in the public domain at https://ccrod.cancer.gov/confluence/display/ROB2/Downloads) and ImageJ software, to assess fluorescence intensity and vascular area. Statistical analysis was performed using 1-way analysis of variance (ANOVA) followed by post hoc pairwise comparisons. A P value < 0.05 was considered significant (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Results

Gene Therapy Significantly Inhibits the Progression of Neovascularization

P12 mice were divided into six groups: (1) normal group (no modeling), (2) control group (OIR mice injected with 1 µL of sterile 1 × PBS), (3) AAV2-EGFP group (OIR mice injected with 1 µL of AAV2-EGFP), (4) AAV2-sFLT group (OIR mice injected with 1 µL of AAV2-sFLT), (5) AAV2-shVEGF group (OIR mice injected with 1 µL of AAV2-shVEGF), and (6) AAV2-sFLT-shVEGF group (OIR mice injected with 1 µL of AAV2-sFLT-shVEGF). To assess gene therapy effects on retinal neovascularization, fundus photography and FFA were performed on P17, focusing on the optic disc and peripheral areas (Fig. 1A). Quantification of vascular area in the central region (Fig. 1B) and peripheral region (Fig. 1C) was performed based on the FFA images. Results showed that all gene therapy groups inhibited peripheral neovascularization, and the AAV2-shVEGF group displayed the smallest central non-perfusion area.

Figure 1.

Figure 1.

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Five days after gene therapy, (A) fundus photography and FFA images centered on the optic disc, extending to the superior, inferior, nasal, and temporal fields; quantitative analysis of FFA images was performed for both the central region (B) and the peripheral region (C). (D) H&E stained retinal frozen sections, with red arrows indicating inward-growing neovascular clusters in the inner retinal layer and the inward trend of the outer layer (scale bar = 50 µm). norm = untreated group; ctrl = control group; EGFP = AAV2-EGFP group; sFLT = AAV2-sFLT group; shVEGF = AAV2-shVEGF group; sf+sh = AAV2-sFLT-shVEGF group.

Hematoxylin and eosin (H&E) staining was performed to evaluate neovascular cluster numbers and retinal layer integrity. In the control group and AAV2-EGFP group, inward-growing neovascular clusters in the inner retinal layer disrupted the outer retinal layer. In contrast, gene-treated groups showed fewer neovascular clusters and more organized retinal structures, with the AAV2-sFLT-shVEGF group showing the lowest number of neovascular clusters (Fig. 1D).

Gene Therapy can Enhance the Regression of Central Retinal Vessels and the Normal Development of Peripheral Vessels in OIR Mice

Mice raised in a 75% oxygen environment exhibited significant impairment in normal retinal vascular development, leading to the formation of a central non-perfused area. Upon returning to room air, the hypoxic conditions triggered an increase in HIF-1α, stimulating VEGF production and promoting retinal neovascularization.28 To assess the therapy effect, retinal samples were collected from mice on P17. Vascular endothelial cells were labeled using Isolectin, and retinal whole-mounts were prepared for staining. Quantitative analysis of the central non-perfused areas and neovascular regions was then performed (Figs. 2A–D).

Figure 2.

Figure 2.

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Whole-mount staining of mouse retina five days after gene therapy, displaying overall images with marked central non-perfused areas (A) and neovascular areas (C), statistical analysis of the central non-perfused area (B) and neovascular area (D), and representative close-up images of the stained flat mounts (E). Scale bars = 100 µm and 50 µm.

Results demonstrated that two groups promoted central vessel regression, as evidenced by a significant reduction in the non-perfused areas. Additionally, the AAV2-EGFP group showed a significant increase in neovascular area, whereas all three gene-treated groups significantly reduced neovascular areas.

Images of the superficial and deep vascular plexuses revealed that vessels in the control group and AAV2-EGFP group were severely twisted, disorganized, and thinned compared to the gene therapy groups, which generally exhibited more regular vessel morphology, orientation, and arrangement (Fig. 2E).

Gene Therapy Significantly Suppresses the Expression of Angiogenic Factors and Promotes the Repair of the Blood-Retinal Barrier

We then measured the expression of angiogenesis-promoting factors. The VEGF family, key regulators of angiogenesis, showed the highest expression in the control group, indicating severe retinal neovascularization and leakage. After gene therapy, VEGF expression was significantly reduced, demonstrating that all three gene therapies effectively inhibited VEGF expression (Figs. 3A, 3B).

Figure 3.

Figure 3.

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Immunofluorescent staining of VEGF and F4/80 in mouse retinal frozen sections 5 days post-gene therapy (A), with statistical analysis of their relative expression levels (B, C). Immunofluorescent staining of MMP-9 and HIF-1α (D) and their relative expression levels (E, F), ZO-1 staining with its expression analysis (G, H), and E-cadherin staining with its expression analysis (I, J). Scale bar = 50 µm.

Macrophages, the main inflammatory cells in the retina, are recruited by chemotactic signals and contribute to neovascularization.29 We measured the expression of the macrophage marker F4/80. Results showed that all treatment groups significantly reduced macrophage infiltration, with the AAV2-sFLT-shVEGF group showing the most pronounced effect (see Figs. 3A, 3C).

Neovascularization depends on extracellular basement membrane degradation and matrix remodeling, with matrix metalloproteinases (MMPs) playing a crucial role. Five days after gene therapy, MMP-9 expression was significantly reduced (Figs. 3D, 3E), indicating that gene therapy effectively inhibited matrix remodeling associated with neovascularization.

Under acute and chronic hypoxia, HIF-1 expression increases, promoting VEGF gene expression and contributing to neovascularization, vascular leakage, and retinal inflammation.30 In our study, although HIF-1α is an upstream regulator of VEGF, its expression was also significantly downregulated following gene therapy (see Figs. 3D, 3F). This suggests that gene therapy may improve the neovascular microenvironment, offering a more comprehensive therapeutic effect.

In retinal neovascular diseases, inflammation and oxidative stress can disrupt the tight junctions of the inner blood-retinal barrier (iBRB), leading to vascular leakage and dysfunction.31 To assess this, we measured the expression of tight junction-related genes ZO-1 and E-cadherin. Results showed significant downregulation of ZO-1 and E-cadherin in the control group and AAV2-EGFP group, indicating iBRB disruption. However, gene therapy restored iBRB function, with ZO-1 and E-cadherin levels markedly upregulated. Among treatments, AAV2-sFLT-shVEGF showed the best recovery (Figs. 3G–J).

Gene Therapy Inhibits the Inflammatory Microenvironment During the Neovascularization Process

We next assessed the expression of inflammatory factors across groups and observed significant upregulation in the control group. After gene therapy, pro-angiogenic factors, such as IL-1β, IL-6, and TNF-α, were significantly downregulated in the treatment groups (Fig. 4A), along with a marked reduction in other inflammatory factors like IL-1α, IL-21, and IL-22 (Fig. 4B). Among the treatments, AAV2-sFLT-shVEGF showed the most consistent inhibition of inflammation.

Figure 4.

Figure 4.

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Five days post-gene therapy in mice, relative mRNA expression levels of angiogenesis-related inflammatory factors IL-1β, IL-6, and TNF-α (A) and other inflammatory factors IL-1α, IL-21, and IL-22 (B); immunofluorescent staining of IL-6 on retinal frozen sections (C) with statistical analysis of its expression (D); immunofluorescent staining of p-STAT3 (E) and statistical analysis of its expression (F). Scale bar = 50 µm.

IL-6 interacts with the transmembrane protein gp130, activating STAT3, which leads to its phosphorylation and nuclear translocation, promoting retinal angiogenesis and inflammation.32 Elevated levels of p-STAT3 were observed in the control group and AAV2-EGFP group, indicating a strong link between inflammation and neovascularization. In contrast, gene therapy significantly reduced IL-6 and p-STAT3 expression in the treatment groups, disrupting this connection and inhibiting both the inflammatory and neovascular microenvironments, thereby achieving a therapeutic effect against neovascular formation (Figs. 4C–F).

Gene Therapy Partially Restores Retinal Function

When the retina receives light or visual stimuli, neural impulses are generated and transmitted through ganglion and bipolar cells to the photoreceptor layer, where photochemical and photoelectric reactions convert light into electrical signals. These signals travel along the optic nerve, crossing the optic chiasm, optic tract, and lateral geniculate nucleus, ultimately reaching the visual cortex in the calcarine fissure. This process can be recorded by visual electrophysiological methods and presented as an electroretinogram (ERG).33 To evaluate retinal function improvement following gene therapy, we performed ERG measurements on mice 5 days post-treatment.

We measured a-waves, b-waves, and OPs using flash stimuli of 0.01 cd/m², 0.1 cd/m², and 1 cd/m² to assess retinal function at different light intensities. Under scotopic conditions with a 0.01 cd/m² flash, the control group and AAV2-EGFP group showed significant decreases in a-wave, b-wave, and OP3 amplitudes. The AAV2-sFLT-shVEGF group partially recovered b-wave amplitude, with no changes in the other treatment groups (Figs. 5A, 5B, Supplementary Fig. S1). At 0.1 cd/m², the control group and AAV2-EGFP group showed decreases in a-wave, b-wave, and OP2, OP3 amplitudes. The AAV2-sFLT-shVEGF and AAV2-shVEGF groups partially recovered b-wave amplitude, with no changes in the AAV2-sFLT group (Figs. 5C, 5D, Supplementary Fig. S2). With a 1 cd/m² flash, the control group and AAV2-EGFP group showed a decrease in all wave amplitudes. The AAV2-sFLT-shVEGF group partially recovered a-wave, b-wave, OP2 and OP3 amplitudes, AAV2-shVEGF partially recovered b-wave amplitude, and the AAV2-sFLT group showed only slight changes (Figs. 5E, 5F, Supplementary Fig. S3).

Figure 5.

Figure 5.

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ERG images of mice under scotopic conditions following gene therapy with light intensities of 0.01 cd/m² (A), 0.1 cd/m² (C), 1 cd/m² (E), and statistical analysis of a-wave and b-wave amplitudes under light intensities of 0.01 cd/m² (B), 0.1 cd/m² (D), 1 cd/m² (F), c-wave ERG images at 1 cd/m² (G), and amplitude statistics (H).

Next, we focused on c-wave changes. Under controlled scotopic conditions and complete silence, a 1 cd/m² flash stimulus was used to observe c-wave responses. The control group and AAV2-EGFP group showed a significant decrease in c-wave amplitude, indicating severe RPE damage due to neovascular lesions. In contrast, the AAV2-sFLT-shVEGF group showed a significant recovery in c-wave amplitude, approaching levels seen in the normal group, suggesting effective restoration of pigment epithelial visual function (Figs. 5G, 5H).

Discussion

Anti-VEGF therapy is a main treatment for retinal diseases with neovascularization.34 AAV-mediated ocular gene therapy, known for its low cost, safety, stability, and long-term efficacy, is becoming increasingly popular for treating retinal neovascular diseases.35 Our study confirmed the anti-VEGF effects of AAV2-sFLT-shVEGF. By injecting recombinant gene vectors into the vitreous cavity of OIR mice, we achieved dual inhibition of VEGF and RNA interference effectively.

sFLT, a heparin-binding protein, binds tightly to VEGF and primarily functions to inhibit VEGF signaling. By binding to VEGF, it blocks receptor tyrosine phosphorylation and prevents the activation of downstream signal transduction.25,36 Over 2 decades ago, sFLT was identified as a promising treatment for retinal neovascularization. Several studies using recombinant viral vectors carrying sFLT-1 have examined its long-term effects and feasibility in treating neovascularization.37,38 Due to the stability and safety of rAAV2-sFLT-1, this therapy advanced to phase I clinical trials, where it demonstrated long-term stability for up to 3 years.39,40 However, a phase II clinical trial revealed that rAAV2-sFLT-1 did not effectively restore visual acuity, leading to the discontinuation of further trials.41 In contrast, our dual-gene therapy partially reverses retinal function damage, with c-wave restoration to near-normal levels, providing stronger support for clinical application.

RNA interference (RNAi) technology specifically silences target genes through post-transcriptional gene silencing, leading to the loss of related functions. In this study, shVEGF inhibits VEGF transcription and translation by targeting VEGF mRNA, blocking downstream signaling pathways.42 However, direct application of RNAi faces challenges in safety and specificity.43 To address these, methods like lentiviral and nanoparticle delivery have been explored for retinal neovascularization treatment.44,45 While promising in laboratory studies, their stability and side effects need further verification. In our study, rAAV delivered shRNA, offering long-term gene inhibition with minimal side effects due to low immunogenicity and good biocompatibility, making it a promising clinical method.46

Retinal diseases often involve multiple pathogenic mechanisms, leading to irreversible tissue damage. Single therapies have limitations, making combination therapies crucial. Studies show that siRNAs targeting VEGF and ranibizumab are effective for AMD treatment, although requiring repeated injections.47 To overcome this, multigene lentiviral and AAV vectors have been developed for AMD and PDR, showing reliable efficacy.48,49 Askou and colleagues demonstrated the potential of multigene AAV vectors by designing a dual-gene vector encoding PEDF and miRNA targeting VEGF, validating the effectiveness of dual-acting antiangiogenic gene therapy.50

During neovascular proliferation in OIR mice, a pathological inflammatory microenvironment forms, with cytokines and growth factors like IL-6 activating the STAT3 signaling pathway. STAT3 is phosphorylated by receptor-associated tyrosine kinases, forms heterodimers, and translocates to the nucleus, where p-STAT3 promotes transcription of angiogenic mediators like VEGF, MMPs, and fibroblast growth factors, accelerating neovascularization.51 Targeting the STAT3 pathway has emerged as a promising therapeutic strategy. Recent studies show that nanomaterials, microRNA, and endoplasmic reticulum stress-related proteins can inhibit neovascularization by blocking the STAT3 pathway, reducing inflammation and necrotic apoptosis.52–54 Our study confirms the efficacy of gene therapy in blocking p-STAT3, supporting the potential of targeting the STAT3 pathway for neovascular treatments.

The retina is essential for vision, converting light into electrochemical signals at photoreceptors and transmitting them via the optic nerve to the brain.55 Pathological conditions can cause abnormal retinal vascular development and increased vascular anastomoses, leading to permanent damage.56,57 Although most anti-VEGF therapies reduce neovascularization and inflammation, they rarely restore retinal function. Bevacizumab intravitreal injections can temporarily reduce retinal function,58 and although repeated intravitreal injection (IVT) of UPARANT may briefly improve function,59 complications like infections and hemorrhages remain common.60,61 Gene therapy, with its potential for long-term expression, offers promise for continuous retinal function restoration. Our study demonstrates the unique advantages of AAV2-sFLT-shVEGF gene therapy for retinal neovascular diseases and highlights the need for further optimization to achieve more effective retinal recovery.

AAV vectors are known for their ability to provide long-term expression.2 In this study, due to the specificity of the OIR model,27 we only observed short-term effects (5 days post-treatment), where the benefits of dual-gene therapy were already evident. However, because the long-term effects, spanning months or years, were not assessed, further research is necessary to evaluate its lasting efficacy and safety. Retinal neovascular diseases involve complex changes, such as inflammation, proliferation, and atrophy.36 To improve clinical translation, developing a broader range of animal models is essential. This would allow for more accurate simulation of different disease stages and help optimize treatment strategies. Enhancing animal model diversity to better reflect the complexity of the disease will be a key focus of future research.

In summary, this study preliminarily demonstrates the feasibility and potential of the dual inhibition with AAV2-sFLT-shVEGF in treating retinal neovascularization. Moving forward, we will investigate its long-term therapeutic effects and expand the range of animal models to comprehensively evaluate its efficacy and safety. These efforts aim to provide novel and effective treatment options for retinal neovascular diseases.

Supplementary Material

Supplement 1
iovs-66-11-37_s001.pdf (407.8KB, pdf)
Supplement 2
iovs-66-11-37_s002.xlsx (9.6KB, xlsx)

Acknowledgments

The authors thank all the reviewers for their valuable comments.

Supported by grants from the National Key R&D Program of China (No. 2020YFA0908100), the National Natural Science Foundation of China (Nos. 82471048 and 82271045), Natural Science Foundation of Fujian Province (No. 2020D027), the Scientific Research Foundation of State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory (2023XAKJ0102044), and the Shenzhen Science and Technology Program (JCYJ20240813145510014).

Author Contributions: C.C. and X.Y. conceived the research idea; H.H., Y.L., and Y.Z. constructed the recombinant A.A.V. C.C., X.Y., Z.Y.J., M.W., X.L., M.L., and Y.D. conducted the experiments and data collecting; C.C., Z.L., and X.L. contributed to drafting and critically revising the work for intellectual content; C.C., X.Y., M.W., M.L., and Z.L. conducted the analysis and created the figures; N.D. and C.L. provided a critical review of the manuscript.

Disclosure and Competing Interests Statement: We, the authors, have a patent application and/or registration related to this work.

Availability of Published Material and Data: All data and materials used to support this study are available to other researchers. In addition to the data presented in the article and supplement, any other required data will be available by the corresponding author upon reasonable request.

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process: During the preparation of this work the authors used ChatGPT in order to polish the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Disclosure: C. Chen, None; X. Yang, None; Z. Jiang, None; M. Wang, None; X. Li, None; H. Huang, None; M. Li, None; Y. Liu, None; Z. Liu, None; Y. Deng, None; Y. Zu, None; N. Dong, None; C. Li, None

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Supplementary Materials

Supplement 1
iovs-66-11-37_s001.pdf (407.8KB, pdf)
Supplement 2
iovs-66-11-37_s002.xlsx (9.6KB, xlsx)

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