AAV2 and AAV9 tropism and transgene expression in the mouse eye and major tissues after intravitreal and subretinal delivery

Introduction: The eye is an excellent target for gene therapy because of its anatomical features. Gene therapy to treat ocular disorders relies on efficient gene delivery and transgene expression in the target cells. The aim of this study was to compare the biodistribution and safety of two different AAV serotypes after intravitreal (IVT) and subretinal injections.Methods: AAV2 (1 × 1012 vg/mL) and AAV9 (5 × 1012 vg/mL) vectors expressing an enhanced green fluorescent protein (EGFP) and an AAV9-empty (6 × 1011 vg/mL) vector were injected intravitreally or subretinally into both eyes of adult C57Bl/OlaHsd mice. The biodistribution of the viral vectors in the eye and off-target tissues was studied using qPCR. GFP expression was studied from cryosections, and GFP transduction efficacy was verified using immunohistostaining for GFP. In addition, electroretinography (ERG) was used to assess the effect of vectors on retinal function.Results: In addition to the eyes, viral vector copies were found in distant off-target tissues such as the liver, especially after AAV9-EGFP IVT and subretinal injections. AAV9-EGFP injections showed more GFP expression throughout the retina compared to AAV2-EGFP. AAV2-EGFP IVT showed transgene expression mainly in the ganglion cell layer, whereas subretinal injection showed GFP expression in the retinal pigment epithelium. In addition, GFP was expressed at a moderate level in the liver after both injection routes of AAV9 and in parts of the brain after all injection groups except AAV9-empty. Lowered a- and b-amplitude values were seen in ERG in both scotopic and photopic experiments after AAV9-EGFP subretinal injection compared to all other groups.Discussion: This study shows that intraocular injection of AAV2 and AAV9 transduces retinal cells. Although the more efficient transduction of the retina, negative effect on the retinal function, and off-target transgene expression of AAV9 makes AAV2 a more suitable gene delivery vector to treat ocular disorders

Antiangiogenic AAV2 gene therapy with a truncated form of soluble VEGFR-2 reduces the growth of choroidal neovascularization in mice after intravitreal injection

Pathological angiogenesis related to neovascularization in the eye is mediated through vascular endothelial growth factors (VEGFs) and their receptors. Ocular neovascular-related diseases are mainly treated with anti-VEGF agents. In this study we evaluated the efficacy and safety of novel gene therapy using adeno associated virus 2 vector expressing a truncated form of soluble VEGF receptor-2 fused to the Fc-part of human IgG1 (AAV2-sVEGFR-2-Fc) to inhibit ocular neovascularization in laser induced choroidal neovascularization (CNV) in mice. The biological activity of sVEGFR-2-Fc was determined in vitro. It was shown that sVEGFR-2-Fc secreted from ARPE-19 cells was able to bind to VEGF-A165 and reduce VEGF-A165 induced cell growth and survival. A single intravitreal injection (IVT) of AAV2-sVEGFR-2-Fc (1 mu l, 4.7 x 1012 vg/ml) one-month prior laser photocoagu-lation did not cause any changes in the retinal morphology and significantly suppressed fluorescein leakage at 7, 14, 21 and 28 days post-lasering compared to controls. Macrophage infiltration was observed after the injection of both AAV2-sVEGFR-2-Fc and PBS. Our findings indicate that AAV2 mediated gene delivery of the sVEGFR-2-Fc efficiently reduces formation of CNV and could be developed to a therapeutic tool for the treatment of retinal diseases associated with neovascularization.Peer reviewe

Human Vascular Endothelial Growth Factor A165 Expression Induces the Mouse Model of Neovascular Age-Related Macular Degeneration

Vascular endothelial growth factor (VEGF) expression induces age-related macular degeneration (AMD), which is a common vision-threatening disease due to choroidal neovascularization and a fibrovascular membrane. We describe a mouse model of neovascular AMD with the local expression of human VEGF-A165 in the eye. We use a transgenic mouse in which human VEGF-A165 has been silenced with the loxP-STOP fragment. The choroidal neovascularization and human VEGF-A165 expression in the mouse are induced by subretinal adenoviral Cre gene delivery. Cre gene transfer is compared with adenoviral LacZ gene transfer control. We characterize the AMD phenotype and changes in the vasculature by using fluorescein angiography, optical coherence tomography, and immunohistochemistry. At early time points, mice exhibit increases in retinal thickness (348 ± 114 µm vs. 231 ± 32 µm) and choroidal neovascularization area (12000 ± 15174 µm2 vs. 2169 ± 3495 µm2) compared with the control. At later time points, choroidal neovascularization develops into subretinal fibrovascular membrane. Human VEGF-A165 expression lasts several weeks. In conclusion, the retinas display vascular abnormalities consistent with choroidal neovascularization. Together with immunohistochemical findings, these changes resemble clinical AMD-like ocular pathologies. We conclude that this mouse model of Cre-induced choroidal neovascularization is useful for mimicking the pathogenesis of AMD, studying the effects of human VEGF-A165 in the retina, and evaluating anti-VEGF treatments for choroidal neovascularization

Human Vascular Endothelial Growth Factor A165 Expression Induces the Mouse Model of Neovascular Age-Related Macular Degeneration

Vascular endothelial growth factor (VEGF) expression induces age-related macular degeneration (AMD), which is a common vision-threatening disease due to choroidal neovascularization and a fibrovascular membrane. We describe a mouse model of neovascular AMD with the local expression of human VEGF-A165 in the eye. We use a transgenic mouse in which human VEGF-A165 has been silenced with the loxP-STOP fragment. The choroidal neovascularization and human VEGF-A165 expression in the mouse are induced by subretinal adenoviral Cre gene delivery. Cre gene transfer is compared with adenoviral LacZ gene transfer control. We characterize the AMD phenotype and changes in the vasculature by using fluorescein angiography, optical coherence tomography, and immunohistochemistry. At early time points, mice exhibit increases in retinal thickness (348 ± 114 µm vs. 231 ± 32 µm) and choroidal neovascularization area (12000 ± 15174 µm2 vs. 2169 ± 3495 µm2) compared with the control. At later time points, choroidal neovascularization develops into subretinal fibrovascular membrane. Human VEGF-A165 expression lasts several weeks. In conclusion, the retinas display vascular abnormalities consistent with choroidal neovascularization. Together with immunohistochemical findings, these changes resemble clinical AMD-like ocular pathologies. We conclude that this mouse model of Cre-induced choroidal neovascularization is useful for mimicking the pathogenesis of AMD, studying the effects of human VEGF-A165 in the retina, and evaluating anti-VEGF treatments for choroidal neovascularization