Background: The eye possesses unique anatomical features that make it a valuable target for gene therapy applications. Objective: The aim of the current study was to compare transduction efficiency, safety and biodistribution of four viral vectors following intravitreal injection. Method: Adenovirus (AdV), Adeno-Associated Virus (AAV), Baculovirus (BV) and Lentivirus (LV) vectors encoding Green Fluorescent Protein (GFP) were injected bilaterally intravitreally into adult C57BL/6OlaHsd mice. Control mice received saline. Eyes and other organs were studied at multiple time points from 3 days to 6 months. Immunohistochemical stainings with retinal cell markers were performed to verify GFP-positive cells. Biodistribution in retina and various non-target tissues was studied using a qPCR method. Inflammatory responses and toxicity were investigated from cryostat eye sections and serum samples. Results: AAV-injected eyes showed GFP expression both in inner and outer retinal cells from 7 days up to 6 months. LV eyes showed long lasting transgene expression mostly in retinal pigment epithelium whereas AdV transiently transduced mainly cells in the anterior chamber. In BV-injected eyes, GFP positivity was very low. qPCR results showed that AdV, AAV and LV spread into the optic nerve, but were below the detection limit in other organs. The strongest immune responses were evoked by intravitreal injections of AdV and BV. The highest concentration of anti-GFP IgG was detected in the AdV-treated group, whereas the AAV group showed the lowest concentration. Neither blood chemistry screen nor the number of apoptotic cells showed any differences between the viral vector and saline injected groups. Conclusion: Our findings show that intravitreal gene delivery is safe and feasible with AAV, AdV and lentivirus vectors.

Pathological vessel growth harms vision and may finally lead to vision loss. Anti-angiogenic gene therapy with viral vectors for ocular neovascularization has shown great promise in preclinical studies. Most of the studies have been conducted with different adeno-associated serotype vectors. In addition, adeno- and lentivirus vectors have been used. Therapy has been targeted towards blocking vascular endothelial growth factors or other pro-angiogenic factors. Clinical trials of intraocular gene therapy for neovascularization have shown the treatment to be safe without severe adverse events or systemic effects. Nevertheless, clinical studies have not proceeded further than Phase 2 trials.

Antiangiogenic and antilymphangiogenic gene therapy with soluble vascular endothelial growth factor receptor-2 (VEGFR-2) and soluble VEGFR-3 in combination with chemotherapy is a potential new treatment for ovarian carcinoma. We evaluated the safety, toxicology, and biodistribution of intravenous AdsVEGFR-2 and AdsVEGFR-3 combined with chemotherapy in healthy rats (n=90) before entering a clinical setting. The study groups were: AdLacZ and AdLacZ with chemotherapy as control groups, low dose AdsVEGFR-2 and AdsVEGFR-3, high dose AdsVEGFR-2 and AdsVEGFR-3, combination of low dose AdsVEGFR-2 and AdsVEGFR-3 with chemotherapy, combination of high dose AdsVEGFR-2 and AdVEGFR-3 with chemotherapy, and chemotherapy only. The follow-up time was 4 weeks. Safety and toxicology were assessed by monitoring the clinical status of the animals and by histological, hematological, and clinical chemistry parameters. For the biodistribution studies, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used. Low dose (2×10¹⁰ vp) AdsVEGFR-2 and AdsVEGFR-3 gene therapy was well tolerated, even when gene therapy was combined with chemotherapy. Notably, only transient elevation of liver enzymes and mild regenerative changes were seen in liver after the gene transfer in the groups that received high doses (2×10¹¹ vp) of AdsVEGFR-2 and AdsVEGFR-3 with or without chemotherapy. No life-threatening adverse effects were noticed in any of the treatment groups. The highest protein concentration of soluble VEGFR-2 (sVEGFR-2) in circulation was seen 1 week after the gene transfer. The combination of chemotherapy to gene therapy seemed to prolong the time of detectable transgene protein at least 1 week in the circulation. The expression of AdsVEGFR-2 and AdsVEGFR-3 transgenes was mainly seen in the liver and spleen as detected by qRT-PCR. According to these results, AdsVEGFR-2 and AdsVEGFR-3 gene therapy combined with chemotherapy is safe and can be brought to clinical testing in ovarian cancer patients.

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.

Pathological vessel growth harms vision and may finally lead to vision loss. Anti-angiogenic gene therapy with viral vectors for ocular neovascularization has shown great promise in pre-clinical studies. Most of the studies has conducted with different adeno-associate serotype vectors. In addition, Adeno and lentivirus vectors have been used. Therapy has targeted to block vascular endothelial growth factors or other pro-angiogenic factors. Clinical trials of intraocular gene therapy for neovascularization have shown the treatment to be safe without severe adverse events or systemic effects. Nevertheless, clinical studies have not proceeded phase 2 trials further.

Aim: Transgenic mice with an elastic fiber mutation (C1039G+/-) in the fibrillin-1 gene and apolipoprotein E deficiency express vulnerable atherosclerotic plaque formation in the aorta and coronary and carotid arteries. The fibrillin-1 gene mutation alone leads to impaired fibrillin-1 function common to Marfan syndrome. The aim was to study for the first time the potential effects of atherosclerosis and vulnerable plaques in mouse eye and spontaneous retinal vessel occlusions in these mice.Methods: Apolipoprotein E-deficient and fibrillin-1 mutated mice (ApoE-/-/Fbn1C1039G+/-) were used for the study. ApoE-/- littermates served as controls. Optical coherence tomography and fluorescein angiography were used to study the retina and retinal vessels before and after the 12-week high-fat diet. Series of staining were performed to study morphology, apoptosis, glial fibrillary acidic protein activation, collagen formation, inflammatory cell infiltration and drusen formation.Results: No pathological changes were found in hypercholesterolemic ApoE-/-/Fbn1C1039G+/- mice in imaging studies nor in the histological stainings. There was neither abnormality in the retinal morphology nor any detectable biomarkers.Conclusion: ApoE-/-/Fbn1C1039G+/- mice did not present the ocular signs of Marfan syndrome.12-week high-fat diet is not sufficient to induce retinal vessel occlusion on 20 to 23 weeks old mice. This indicates that the mechanistic background of retinal vessel occlusion is more complex.