Meliha Brankov

Dendrimer delivery of an anti-VEGF oligonucleotide into the eye : a long-term study into inhibition of laser- induced CNV, distribution, uptake and toxicity

We have performed a long-term study into the use of a lipophilic amino-acid dendrimer to deliver an anti-vascular endothelial growth factor (VEGF) oligonucleotide (ODN-1) into the eyes of rats and inhibit laser-induced choroidal neovascularization (CNV). In addition, the uptake, distribution and retinal tolerance of the dendrimer plus oligonucleotide conjugates were examined. Analysis of fluorescein angiograms of laser photocoagulated eyes revealed that dendrimer plus ODN-1 significantly inhibited (P<0.05) the development of CNV for 4–6 months by up to 95% in the initial stages. Eyes similarly injected with ODN-1 alone showed no significant difference (P>0.05) in mean severity score at 2 months (2.86±0.09), 4 months (2.15±0.17) or 6 months (2.7±0.12) compared to the vehicle-injected controls. Furthermore, we showed that intravitreally injected ODN-1 tagged with 6-fam was absorbed by a wide area of the retina and penetrated all of the retinal cell layers to the retinal pigment epithelium. Ophthalmological examinations indicated that the dendrimers plus ODN-1 conjugates were well tolerated in vivo, which was later confirmed using immunohistochemistry, which showed no observable increase in antigens associated with inflammation. We conclude that the use of such dendrimers may provide a viable mechanism for the delivery of therapeutic oligonucleotides for the treatment of angiogenic eye diseases.

Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy

Neovascularisation (NV) within the eye often results in visual loss. Vascular endothelial growth factor (VEGF) has been implicated in the development of ocular NV. Previous studies have shown that VEGF antagonists successfully suppressed retinal and choroidal NV in animal models. However, the systemic approach and transient nature of the delivery systems used in these studies hinder therapeutic application. To achieve stable and localised ocular anti-angiogenic therapy, we explored the use of recombinant adeno-associated virus (rAAV)-mediated secretion gene therapy (SGT). In this study, we generated a rAAV vector encoding soluble VEGF receptor 1, sFlt-1 (AAV-CMV.sflt) and determined its ability to inhibit cautery-induced corneal NV and laser-induced choroidal NV. Delivery of AAV-CMV.sflt into the anterior chamber resulted in transgene expression in the iris pigment epithelium and corneal endothelium, which reduced the development of corneal NV in the stroma of cauterised rats by 36% compared with cauterised control groups (P = 0.009). Subretinal delivery of AAV-CMV.sflt near the equator of the eye also suppressed choroidal NV at the laser lesions around the optic nerve by 19% (P = 0.002), indicating that there was diffusion of the secreted anti-angiogenic protein across the retina. Both results suggest that the long-term suppression of ocular NV is possible through the use of stable rAAV-mediated SGT.

Inhibition of angiogenesis by adenovirus-mediated sFlt-1 expression in a rat model of corneal neovascularization

Pathological angiogenesis, or the production of new capillary vessels from preexisting vasculature, within the eye is a serious event that often leads to blindness. Upregulation of vascular endothelial growth factor (VEGF) has been linked to neovascularization in the eye, suggesting that it could be a suitable target to inhibit angiogenic changes. This work investigated whether the presence of a proven antiangiogenic factor, the soluble variant of the VEGF receptor, sFlt-1, in the anterior chamber is sufficient to inhibit new vessel formation in the cornea in an animal model of corneal neovascularization. A recombinant adenovirus vector that can mediate efficient in vivo gene transfer and expression in ocular cells was selected as a delivery agent. We have shown that after the injection of Ad.betagal into the anterior chamber of normal and cauterized rat eyes, corneal endothelial cells and cells of the trabecular meshwork were efficiently transduced and that beta-galactosidase (beta-Gal) expression was maintained up to 10 days postinjection. Cauterization significantly increased the amount of immunoreactive VEGF in vehicle- or Ad.null-injected animals (t test, p < 0.001 and p < 0.001, respectively). However, when cauterization was combined with Ad.sflt injection there was no statistically significant increase in the amount of immunoreactive VEGF (p = 0.12). The injection of Ad.sflt into the anterior chamber slowed or inhibited VEGF-induced angiogenic changes. After cauterization, 100% of uninjected and vehicle-injected and 82% of Ad.null-injected animals developed moderate to severe corneal angiogenesis in contrast to 18% of Ad.sflt-injected animals. These in vivo results suggest that the transient presence of antiangiogenic agents in the anterior chamber can be successfully used to inhibit the development of corneal angiogenesis.

Recombinant adeno-associated virus type 2-mediated gene delivery into the Rpe65-/- knockout mouse eye results in limited rescue.

BackgroundLebers congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.MethodsrAAV.RPE65 was injected into the subretinal space of Rpe65-/- knockout mice and control mice. Histological and immunohistological analyses were performed to evaluate any rescue of photoreceptors and to determine longevity and pattern of transgene expression. Electron microscopy was used to examine ultrastructural changes, and electroretinography was used to measure changes in visual function following rAAV.RPE65 injection.ResultsrAAV-mediated RPE65 expression was detected for up to 18 months post injection. The delivery of rAAV.RPE65 to Rpe65-/- mouse retinas resulted in a transient improvement in the maximum b-wave amplitude under both scotopic and photopic conditions (76% and 59% increase above uninjected controls, respectively) but no changes were observed in a-wave amplitude. However, this increase in b-wave amplitude was not accompanied by any slow down in photoreceptor degeneration or apoptotic cell death. Delivery of rAAV.RPE65 also resulted in a decrease in retinyl ester lipid droplets and an increase in short wavelength cone opsin-positive cells, suggesting that the recovery of RPE65 expression has long-term benefits for retinal health.ConclusionThis work demonstrated the potential benefits of using the Rpe65-/- mice to study the effects and mechanism of rAAV.RPE65-mediated gene delivery into the retina. Although the functional recovery in this model was not as robust as in the dog model, these experiments provided important clues about the long-term physiological benefits of restoration of RPE65 expression in the retina.

Generation of transgenic mice with mild and severe retinal neovascularisation

Aim: To generate a mouse model for slow progressive retinal neovascularisation through vascular endothelial growth factor (VEGF) upregulation. Methods: Transgenic mice were generated via microinjection of a DNA construct containing the human VEGF165 (hVEGF) gene driven by a truncated mouse rhodopsin promoter. Mouse eyes were characterised clinically and histologically and ocular hVEGF levels assayed by ELISA. Results: One transgenic line expressing low hVEGF levels showed mild clinical changes such as focal fluorescein leakage, microaneurysms, venous tortuosity, capillary non-perfusion and minor neovascularisation, which remained stable up to 3 months postnatal. Histologically, there were some disturbance and thinning of inner and outer nuclear layers, with occasional focal areas of neovascularisation. By contrast, three other lines expressing high hVEGF levels presented with concomitantly severe phenotypes. In addition to the above, clinical features included extensive neovascularisation, haemorrhage, and retinal detachment; histologically, focal to extensive areas of neovascularisation associated with retinal folds, cell loss in the inner and outer nuclear layers, and partial retinal detachment were common. Conclusions: The authors generated four hVEGF overexpressing transgenic mouse lines with phenotypes ranging from mild to severe neovascularisation. These models are a valuable research tool to study excess VEGF related molecular and cellular changes and provide additional opportunities to test anti-angiogenic therapies.

rAAV.sFlt-1 Gene Therapy Achieves Lasting Reversal of Retinal Neovascularization in the Absence of a Strong Immune Response to the Viral Vector

PURPOSE To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy. METHODS trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA. RESULTS After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1-injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1-injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45(+) leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy. CONCLUSIONS The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye.

Discovery of a Novel Control Element within the 5′-Untranslated Region of the Vascular Endothelial Growth Factor REGULATION OF EXPRESSION USING SENSE OLIGONUCLEOTIDES

The regulation of vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis, is controlled primarily through the interactions of control elements located within the 5′- and 3′-untranslated regions, many of which are yet to be described. In this study we examined the 5′-untranslated region of human VEGF for control elements with the aim of regulating expression both in vitro and in vivo using oligonucleotide gene therapy. A potential control element was located, two sense oligonucleotides (S1 and S2) were designed based on its sequence, and a third oligonucleotide (S3) was designed as a control and mapped to the 16 base pairs immediately upstream. Retinal cells cultured in the presence of S1 and S2 resulted in a 2-fold increase of VEGF protein and a 1.5-fold increase in mRNA 24 h post-transfection whereas S3 had no significant effect (p > 0.05) compared with controls. Subsequent reporter gene studies confirmed the necessity of this element for up-regulation by S1. Further in vivo studies showed that S1 and S2 mediated an increase in VEGF protein in a rodent ocular model that resulted in angiogenesis. In addition to providing insight into the regulation of the vascular endothelial growth factor, the use of these oligonucleotides to stimulate vascular growth may prove useful for the treatment of ischemic tissues such as those found in the heart following infarct.

Assessment of rAAV-mediated gene therapy in the Rpe65-/- mouse.

There have been several reports demonstrating the potential of virus-mediated gene delivery for use in treating congenital retinal degenerations (Bennett and Maguire, 2000; Acland et al., 2001; Campochiaro et al., 2002; Narfstrom et al., 2002). A recent report on the restoration of vision to Briard dogs marked the first successful gene therapy for blindness in a large animal model, bringing the possibility of a human retinal gene therapy trial closer (Bennett and Maguire, 2000; Narfstrom et al., 2002). We wished to pursue this potential, by developing a successful gene therapy protocol for the treatment of retinal degeneration in a small animal model. Once established, this small animal model could be used to assess the clinical benefits and limitations of using gene therapy in treating congenital retinal degeneration.

Virus-mediated secretion gene therapy--a potential treatment for ocular neovascularization.

Ocular neovascularization (NV) is a severe and major complication of many ocular conditions such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity and retinal vein occlusion. The uncontrolled growth of blood vessels and its associated vascular hyperpermeability are the most common pathologic causes of vision loss in developed countries. Current conventional therapies can only provide symptomatic treatment for a limited subset of patients, often induce adverse effects and disease recurrence is common. Therefore, gene therapy aimed at blocking the underlying stimuli for new vessel growth is a potentially more effective treatment.