Three-Year Follow-Up of Phase 1 and 2a rAAV.sFLT-1 Subretinal Gene Therapy Trials for Exudative Age-Related Macular Degeneration.

PURPOSE: To assess the safety and the 3-year results of combined phase 1 and 2a randomized controlled trials of rAAV.sFLT-1 gene therapy (GT) for wet age-related macular degeneration. DESIGN: Phase 1/2a clinical trial. METHODS: Patients were prospectively randomized into control (n = 13) and GT (n = 24) groups. GT patients received 1X1011vg rAAV.sFLT-1 and were seen every month for 1 year then as needed every 1 to 2 months. They were given retreatment anti-vascular endothelial growth factor injections according to predetermined criteria. At 12 months, GT patients were divided into 2 groups: HD-1 (n = 14), requiring <2, and HD-2 (n = 10), requiring >2 retreatments. RESULTS: Between 1 year and 3 years there were 3 adverse events (AEs) and 33 serious AEs reported. Of these, 15 occurred in the 13 control subjects and 21 in the 24 GT patients. Except for 1 case of transient choroiditis in a control patient, serious AEs were deemed to be unrelated to the study. Control patients received a median of 7.0 retreatments and lost a median of 7.0 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, HD-1 patients received a median of 2.5 retreatments and lost a median of 4.0 ETDRS letters, and HD-2 patients received a median of 11.0 retreatments and lost a median of 7.0 ETDRS letters over 3 years. Center point thickness fluctuated. Thirty-three percent of control subjects, 44% of HD-2 patients, and 51% of HD-1 patients showed maintenance of baseline visual acuity. Four HD-1 patients (34%) maintained significant visual improvement at 3 years. None of these observations were statistically significant. CONCLUSIONS: Given the small number of patients, this study was unable to unequivocally confirm the existence of a biologic efficacy signal; however, it confirmed that rAAV.sFLT-1 gene delivery was well tolerated among the elderly.

Phase 2a Randomized Clinical Trial: Safety and Post Hoc Analysis of Subretinal rAAV.sFLT-1 for Wet Age-related Macular Degeneration.

BACKGROUND: We present the results of a Phase 2a randomized controlled trial investigating the safety, and secondary endpoints of subretinal rAAV.sFLT-1 gene therapy in patients with active wet age-related macular degeneration (wAMD). METHODS: All patients (n=32), (ClinicalTrials.gov; NCT01494805), received ranibizumab injections at baseline and week 4, and thereafter according to prespecified criteria. Patients in the gene therapy group (n=21) received rAAV.sFLT-1 (1×1011vg). All patients were assessed every 4weeks to the week 52 primary endpoint. FINDINGS: Ocular adverse events (AEs) in the rAAV.sFLT-1 group were mainly procedure related and self-resolved. All 11 phakic patients in the rAAV.sFLT-1 group showed progression of cataract following vitrectomy. No systemic safety signals were observed and none of the serious AEs were associated with rAAV.sFLT-1. AAV2 capsid was not detected and rAAV.sFLT-1 DNA was detected transiently in the tears of 13 patients. ELISPOT analysis did not identify any notable changes in T-cell response. In the rAAV.sFLT-1 group 12 patients had neutralizing antibodies (nAb) to AAV2. There was no change in sFLT-1 levels in bodily fluids. In the rAAV.sFLT-1 group, Best Corrected Visual Acuity (BCVA) improved by a median of 1.0 (IQR: -3.0 to 9.0) Early Treatment Diabetic Retinopathy Study (ETDRS) letters from baseline compared to a median of -5.0 (IQR: -17.5 to 1.0) ETDRS letters change in the control group. Twelve (57%) patients in the rAAV.sFLT-1 group maintained or improved vision compared to 4 (36%) in the control group. The median number of ranibizumab retreatments was 2.0 (IQR: 1.0 to 6.0) for the gene therapy group compared to 4.0 (IQR: 3.5 to 4.0) for the control group. Interpretation rAAV.sFLT-1 combined with the option for co-treatment appears to be a safe and promising approach to the treatment of wAMD. FUNDING: National Health and Medical Research Council of Australia (AP1010405), Lions Eye Institute, Perth Australia, Avalanche Biotechnologies, Menlo Pk, CA, USA.

Biomarkers for Diabetic Retinopathy - Could Endothelin 2 Be Part of the Answer?

PURPOSE: The endothelins are a family of three highly conserved and homologous vasoactive peptides that are expressed across all organ systems. Endothelin (Edn) dysregulation has been implicated in a number of pathophysiologies, including diabetes and diabetes-related complications. Here we examined Edn2 and endothelin receptor B (Endrb) expression in retinae of diabetic mouse models and measured serum Edn2 to assess its biomarker potential. MATERIALS AND METHODS: Edn2 and Ednrb mRNA and Edn2 protein expression were assessed in young (8wk) and mature (24wk) C57Bl/6 (wild type; wt), Kimba (model of retinal neovascularisation, RNV), Akita (Type 1 diabetes; T1D) and Akimba mice (T1D plus RNV) by qRT-PCR and immunohistochemistry. Edn2 protein concentration in serum was measured using ELISA. RESULTS: Fold-changes in Edn2 and Ednrb mRNA were seen only in young Kimba (Edn2: 5.3; Ednrb: 6.0) and young Akimba (Edn2: 7.9, Ednrb: 8.8) and in mature Kimba (Edn2:9.2, Ednrb:11.2) and mature Akimba (Edn2:14.0, Ednrb:17.5) mice. Co-localisation of Edn2 with Müller-cell-specific glutamine synthetase demonstrated Müller cells and photoreceptors as the major cell types for Edn2 expression in all animal models. Edn2 serum concentrations in young Kimba, Akita and Akimba mice were not elevated compared to wt. However, in mature mice, Edn2 serum concentration was increased in Akimba (6.9pg/mg total serum protein) compared to wt, Kimba and Akita mice (3.9, 4.6, and 3.8pg/mg total serum protein, respectively; p<0.05). CONCLUSIONS: These results demonstrated that long-term hyperglycaemia in conjunction with VEGF-driven RNV increased Edn2 serum concentration suggesting Edn2 might be a candidate biomarker for vascular changes in diabetic retinopathy.

Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trial.

BACKGROUND: Neovascular, or wet, age-related macular degeneration causes central vision loss and represents a major health problem in elderly people, and is currently treated with frequent intraocular injections of anti-VEGF protein. Gene therapy might enable long-term anti-VEGF therapy from a single treatment. We tested the safety of rAAV.sFLT-1 in treatment of wet age-related macular degeneration with a single subretinal injection. METHODS: In this single-centre, phase 1, randomised controlled trial, we enrolled patients with wet age-related macular degeneration at the Lions Eye Institute and the Sir Charles Gairdner Hospital (Nedlands, WA, Australia). Eligible patients had to be aged 65 years or older, have age-related macular degeneration secondary to active subfoveal choroidal neovascularisation, with best corrected visual acuity (BCVA) of 3/60-6/24 and 6/60 or better in the other eye. Patients were randomly assigned (3:1) to receive either 1 × 10(10) vector genomes (vg; low-dose rAAV.sFLT-1 group) or 1 × 10(11) vg (high-dose rAAV.sFLT-1 group), or no gene-therapy treatment (control group). Randomisation was done by sequential group assignment. All patients and investigators were unmasked. Staff doing the assessments were masked to the study group at study visits. All patients received ranibizumab at baseline and week 4, and rescue treatment during follow-up based on prespecified criteria including BCVA measured on the Early Treatment Diabetic Retinopathy Study (EDTRS) scale, optical coherence tomography, and fluorescein angiography. The primary endpoint was ocular and systemic safety. This trial is registered with ClinicalTrials.gov, number NCT01494805. FINDINGS: From Dec 16, 2011, to April 5, 2012, we enrolled nine patients of whom eight were randomly assigned to receive either intervention (three patients in the low-dose rAAV.sFLT-1 group and three patients in the high-dose rAAV.sFLT-1 group) or no treatment (two patients in the control group). Subretinal injection of rAAV.sFLT-1 was highly reproducible. No drug-related adverse events were noted; procedure-related adverse events (subconjunctival or subretinal haemorrhage and mild cell debris in the anterior vitreous) were generally mild and self-resolving. There was no evidence of chorioretinal atrophy. Clinical laboratory assessments generally remained unchanged from baseline. Four (67%) of six patients in the treatment group required zero rescue injections, and the other two (33%) required only one rescue injection each. INTERPRETATION: rAAV.sFLT-1 was safe and well tolerated. These results support ocular gene therapy as a potential long-term treatment option for wet age-related macular degeneration. FUNDING: National Health and Medical Research Council of Australia, Richard Pearce Bequest, Lions Save Sight Foundation, Brian King Fellowship, and Avalanche Biotechnologies, Inc.

Gene therapy for eye as regenerative medicine? Lessons from RPE65 gene therapy for Leber's Congenital Amaurosis.

Recombinant virus mediated gene therapy of Leber's Congenital Amaurosis has provided a wide range of data on the utility of gene replacement therapy for recessive diseases. Studies to date demonstrate that gene therapy in the eye is safe and can result in long-term recovery of visual function, but they also highlight that further research is required to identify optimum intervention time-points, target populations and the compatibility of associate therapies. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.

Molecular analysis of blood-retinal barrier loss in the Akimba mouse, a model of advanced diabetic retinopathy.

The molecular mechanisms of vascular leakage in diabetic macular edema and proliferative retinopathy are poorly understood, mainly due to the lack of reliable in vivo models. The Akimba (Ins2(Akita)VEGF(+/-)) mouse model combines retinal neovascularization with hyperglycemia, and in contrast to other models, displays the majority of signs of advanced clinical diabetic retinopathy (DR). To study the molecular mechanism that underlies the breakdown of the blood-retinal barrier (BRB) in diabetic macular edema and proliferative diabetic retinopathy, we investigated the retinal vasculature of Akimba and its parental mice Kimba (trVEGF029) and Akita (Ins2(Akita)). Quantitative PCR, immunohistochemistry and fluorescein angiography were used to characterize the retinal vasculature with special reference to the inner BRB. Correlations between the degree of fluorescein leakage and retinal gene expression were tested by calculating the Spearman's correlation coefficient. Fluorescein leakage demonstrating BRB loss was observed in Kimba and Akimba, but not in Akita or wild type mice. In Kimba and Akimba mice fluorescein leakage was associated with focal angiogenesis and correlated significantly with Plvap gene expression. PLVAP is an endothelial cell-specific protein that is absent in intact blood-retinal barrier, but its expression significantly increases in pathological conditions such as DR. Furthermore, in Akimba mice BRB disruption was linked to decreased expression of endothelial junction proteins, pericyte dropout and vessel loss. Despite fluorescein leakage, no alteration in BRB protein levels or pericyte coverage was detected in retinas of Kimba mice. In summary, our data not only demonstrate that hyperglycemia sensitizes retinal vasculature to the effects of VEGF, leading to more severe microvascular changes, but also confirm an important role of PLVAP in the regulation of BRB permeability.

Influence of endotoxin-mediated retinal inflammation on phenotype of diabetic retinopathy in Ins2 Akita mice.

AIMS: To evaluate the impact of systemic exposure to bacterial lipopolysaccharide (LPS) on a rodent model of background diabetic retinopathy. METHODS: Toll-like receptor 4 (TLR4)-mediated systemic inflammation was induced in Ins2(Akita) heterozygotes and age-matched C57BL6/J-Ins2(+) littermates by single or repeated intraperitoneal injections of the TLR4 ligand LPS (9 µg/g body weight). 24 hours after a single injection in 7-week-old mice retinal Il1b, Tnfa and Vegf transcripts were measured with real-time PCR. Vascular endothelial growth factor (VEGF) protein levels were evaluated with bead-based immunoassay. Leukostasis and endothelial injury were assessed in retinal wholemounts following perfusion with rhodamine or FITC conjugated concanavalin A to label leukocytes and propidium iodide to label dead or injured cells. In mice which had received three fortnightly injections between 10 and 16 weeks of age, retinal thicknesses and vascular structure were evaluated at 17-18 weeks of age using optical coherence tomography (OCT) and fluorescein angiography. Retinal architecture was assesed using resin-based histology. RESULTS: Compared with normoglycaemic controls, systemic LPS exposure in Ins2(Akita) mice was associated with a 3.5-fold increase in endothelial cell injury and attenuated leukostasis in the retinal vasculature. Hyperglycaemia or acute LPS inflammation did not increase retinal VEGF content. Thinning (10-13 µm) of posterior retina was detected with OCT 2 weeks after repeated exposure to LPS in Ins2(Akita) mice but not in normoglycaemic controls. Capillary networks and retinal morphology were unaffected by recurrent LPS inflammation in Ins2(Akita) and control mice. CONCLUSIONS: In hyperglycaemic mice, exposure to systemic LPS was associated with two hallmark pathologies of early background diabetic retinopathy, namely, the injury of capillary endothelium and in vivo thinning of the retina.

Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy.

BACKGROUND: Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy. METHODOLOGY/PRINCIPAL FINDINGS: We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-ß1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection). CONCLUSIONS/SIGNIFICANCE: ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-ß1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.

The effects of age and Cx3cr1 deficiency on retinal microglia in the Ins2(Akita) diabetic mouse.

PURPOSE: Diabetic retinopathy (DR) is a major cause of visual impairment in developed countries. While DR has been described classically as a microvascular disease, recent evidence suggests that changes to retinal microglia are an early feature of retinopathy. In our study, we assessed changes in microglial distribution and morphology in vivo and ex vivo in a mouse model of non-proliferative DR, and further examined effects of age and the absence of the functional chemokine receptor Cx(3)cr1 on the progression of these changes. METHODS: To isolate the effects of the three variables: diabetic status, age, and role of Cx(3)cr1, the Ins2(Akita) mouse was crossed with Cx(3)cr1-eGFP reporter mice. Eyes were assessed clinically in vivo at 10, 20, 30, and 46 weeks of age, and the retinal structure and arrangement of GFP(+) microglia was examined ex vivo using whole mount immunofluorescence staining and confocal microscopy. RESULTS: clinical examination of the fundus, vasculature, or GFP(+) microglial distribution did not reveal any macroscopic changes related to diabetic status: however, ex vivo microscopic analysis revealed alterations in microglial network organization, and evidence of cell shape changes regarded classically as signs of activation, in Ins2(Akita) mice from 10 weeks of age. These changes were exacerbated in older diabetic mice whose microglia lacked Cx(3)cr1 (Ins2(Akita) Cx(3)cr1(gfp/gfp) mice). Diabetic status and Cx(3)cr1 deficiency led to accumulations of Iba-1(+) hyalocytes (vitreal macrophages) and subretinal macrophages. CONCLUSIONS: These data showed that changes to murine retinal microglia occur in response to systemic diabetic status in the absence of overt retinopathy and inflammation. These changes are exaggerated in mice lacking Cx(3)cr1, suggesting fractalkine- Cx(3)cr1 interactions may have a role in early neuronal changes in preproliferative DR.

Changes in murine hyalocytes are valuable early indicators of ocular disease.

PURPOSE: The distribution, density, and phenotype of hyalocytes or vitreous macrophages in mouse eyes was examined during normal aging and in models of background diabetic retinopathy, retinal vascular proliferation, and exposure to TLR4 and TLR9 ligands. METHODS: The phenotype and density of hyalocytes were investigated in retinal and ciliary body wholemounts of normal wild-type (WT; C57BL/6) mice at 7, 17, and 120 weeks of age, Ins2(Akita) mice, transgenic Kimba mice (VEGF-induced retinal neovascularization), and WT mice 24 hours after single intraperitoneal injection with lipopolysaccharide (LPS) or 1 week after three identical doses administered 2 weeks apart. Another group of mice each received a single topical drop of 20 µg CpG-oligodeoxynucleotides (CpG-ODN) to the abraded corneal surface and were euthanized 1 week later. RESULTS: The data revealed an approximately fivefold increase in the density of preretinal hyalocytes in 120-week-old mice. Some hyalocytes in older eyes contained phagocytosed melanin. Hyalocyte density was doubled in Ins2(Akita) mice after only 3 to 4 weeks of hyperglycemia. Kimba mice had an eightfold increase in the density of hyalocytes, and many displayed signs of activation. WT mice exposed to single or multiple systemic doses of LPS showed a twofold to threefold increase in hyalocytes. Topical CpG-ODN treatment led to a very marked (sevenfold) increase in preretinal hyalocyte density. CONCLUSIONS: The present study demonstrated that murine hyalocytes were responsive to aging, hyperglycemia, locally produced VEGF, and both systemic and ocular-derived TLR ligands. Thus hyalocytes or vitreous macrophages may be a valuable and previously unrecognized sensitive indicator of pathologic changes in the eye.

In vivo monitoring of VEGF-induced retinal damage in the Kimba mouse model of retinal neovascularization.

PURPOSE: To monitor retinal and vascular changes associated with neovascularization, which were generated through photoreceptor-specific overexpression of human vascular endothelial growth factor (hVEGF), in transgenic trVEGF029 (Kimba) mice. MATERIALS AND METHODS: The Spectralis Heidelberg Retina Angiography and Optical Coherence Tomography (HRA+OCT) imaging device was used to track changes in the retina and retinal vasculature of Kimba mouse eyes (n = 32) and control C57Bl/6J mouse eyes (n = 20) at 4, 8, 12, 16, and 20 weeks of age. RESULTS: Retinal vascular leakage, focal dilated vessel, vessel tortuosity, attenuated vessel, venous beading, capillary dropout, retinal non-perfusion, neovascularization, and focal retinal detachment were observed in Kimba mouse eyes. Through track changes, we detected edema in the peripheral part of the retina of 2/32 Kimba mouse eyes examined. The retinae of the Kimba mice were significantly thinner than control mice retinae at all ages of the mice assessed (p < 0.01). CONCLUSIONS: In vivo monitoring of retinal vascular and neural retinal changes in the Kimba mice using the Spectralis HRA+OCT imaging device allowed us to assess and track VEGF-induced damages in great detail and in real-time. Real-time monitoring of these changes can be used to study the interplay between VEGF overexpression and other molecular factors and to monitor dynamic retinal changes following therapeutic intervention.

Characterization of a mouse model of hyperglycemia and retinal neovascularization.

One of the limitations of research into diabetic retinopathy is the lack of suitable animal models. To study how the two important factors--hyperglycemia and vascular endothelial growth factor--interact in diabetic retinopathy, the Akimba mouse (Ins2AkitaVEGF+/-) was generated by crossing the Akita mouse (Ins2Akita) with the Kimba mouse (VEGF+/+). C57Bl/6 and the parental and Akimba mouse lines were characterized by biometric measurements, histology, immunohistochemistry, and Spectralis Heidelberg retinal angiography and optical coherence tomography. The Akimba line not only retained the characteristics of the parental strains, such as developing hyperglycemia and retinal neovascularization, but developed higher blood glucose levels at a younger age and had worse kidney-body weight ratios than the Akita line. With aging, the Akimba line demonstrated enhanced photoreceptor cell loss, thinning of the retina, and more severe retinal vascular pathology, including more severe capillary nonperfusion, vessel constriction, beading, neovascularization, fibroses, and edema, compared with the Kimba line. The vascular changes were associated with major histocompatibility complex class II+ cellular staining throughout the retina. Together, these observations suggest that hyperglycemia resulted in higher prevalences of edema and exacerbated the vascular endothelial growth factor-driven neovascular and retinal changes in the Akimba line. Thus, the Akimba line could become a useful model for studying the interplay between hyperglycemia and vascular endothelial growth factor and for testing treatment strategies for potentially blinding complications, such as edema.

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.

Early vascular and neuronal changes in a VEGF transgenic mouse model of retinal neovascularization.

PURPOSE: To investigate early retinal changes in a vascular endothelial growth factor (VEGF) transgenic mouse (tr029VEGF; rhodopsin promoter) with long-term damage that mimics nonproliferative diabetic retinopathy (NPDR) and mild proliferative diabetic retinopathy (PDR). METHODS: Rhodopsin and VEGF expression was assessed up to postnatal day (P)28. Vascular and retinal changes were charted at P7 and P28 using sections and wholemounts stained with hematoxylin and eosin or isolectin IB4 Griffonia simplicifolia Samples were examined using light, fluorescence, and confocal microscopy. RESULTS: Rhodopsin was detected at P5 and reached mature levels by P15; VEGF protein expression was transient, peaking at P10 to P15. In wild-type (wt) mice at P7, vessels had formed in the nerve fiber/retinal ganglion cell layer and showed a centroperipheral maturational gradient; some capillaries had formed a second bed on the vitread side of the inner nuclear layer (INL). By P28, the retinal vasculature had three mature capillary beds, the third abutting the sclerad aspect of the INL. In tr029VEGF mice, capillary bed formation was accelerated compared with that in wt, with abnormal vessels extending to the sclerad side of the INL by P7 and abnormally penetrating the photoreceptors by P28. Compared with P7, vascular lesions were more numerous at P28 when capillary dropout was also evident. At both stages, retinal layers were thinned most where abnormal vessel growth was greatest. CONCLUSIONS: Concomitant damage to the vasculature and neural retina at early stages in tr029VEGF suggest that both tissues are affected, providing opportunities to examine early cellular events that lead to long-term disease.

Synthesis of a library of polycationic lipid core dendrimers and their evaluation in the delivery of an oligonucleotide with hVEGF inhibition.

This article follows on from our previous work in the area of non-viral gene delivery using polycationic dendrimers (PCDs). Herein we report on the synthesis and efficacy of a new library of lipid core PCDs in the delivery of the anti-angiogenic oligonucleotide (ODN-1) to retinal pigment epithelial cells. ELISA was used to monitor hVEGF levels in cells transfected with dendriplexes, Cytofectin GSV and control (non-transfected). At 48 h, hVEGF titres had returned to that of the untransfected control for Cytofectin GSV however, a number of dendriplexes continued to exhibit a marked reduction in hVEGF titres.

Long-term evaluation of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and monkeys.

Vascular endothelial growth factor (VEGF) is one of the major mediators of retinal ischemia-associated neovascularization. We have shown here that adeno-associated virus (AAV)-mediated expression of sFlt-1, a soluble form of the Flt-1 VEGF receptor, was maintained for up to 8 and 17 months postinjection in mice and in monkeys, respectively. The expression of sFlt-1 was associated with the long-term (8 months) regression of neovascular vessels in 85% of trVEGF029 eyes. In addition, it resulted in the maintenance of retinal morphology, as the majority of the treated trVEGF029 eyes (75%) retained high numbers of photoreceptors, and in retinal function as measured by electroretinography. AAV-mediated expression of sFlt-1 prevented the development of laser photocoagulation-induced choroidal neovascularization in all treated monkey eyes. There were no clinically or histologically detectable signs of toxicity present in either animal model following AAV.sFlt injection. These results suggest that AAV-mediated secretion gene therapy could be considered for treatment of retinal and choroidal neovascularizations.

Development and evaluation of the specificity of a cathepsin D proximal promoter in the eye.

PURPOSE: Recombinant adeno-associated virus (rAAV)-mediated gene delivery has emerged as a valuable tool for alternative treatment of ocular diseases. Cellular specificity of transgene expression could be influenced by either the viral capsid or the choice of promoter. The use of cellular promoter, cathepsin D (CatD) proximal promoter, and its potential for application in rAAV-based gene therapy are evaluated in this study. MATERIALS AND METHODS: Different sizes of CatD proximal promoter fragments -769 to -1 (CD768), -366 to -1 (CD365), -253 to -1 (CD252), and -124 to -1 (CD123) were subcloned upstream of the green fluorescent protein (GFP) gene. The specificity and activity of the promoter were tested in vitro using human retinal pigment epithelium (RPE) cell lines (RPE51, D407), with the human fibroblast cell line (F2000) used as control. The promoter fragment that showed higher activity in RPE cells was chosen to generate rAAV vector based on AAV serotype 2. The ability of CatD promoter to target transgene expression to RPE in vivo was determined following subretinal delivery of rAAV particles into nonpigmented RCS/rdy+ rats. RESULTS: In vitro studies showed that the proximal promoter fragment CD365 targeted high GFP expression in RPE cells. This fragment was then used to generate the AAV.CD365.gfp construct. It was shown in vivo that following subretinal injection, the CD365 fragment in AAV.CD365.gfp directed GFP expression preferentially into RPE cells. Relatively lower level of GFP expression was detected in the neuroretina. In contrast, injection of control virus (AAV.CMV.gfp) resulted in equal levels of transduction and fluorescence signal intensity in both the RPE and photoreceptor cells. CONCLUSIONS: The results of our study demonstrate that the promoter fragment CD365 has the potential to target preferential gene expression in the RPE following subretinal injection in rAAV-mediated gene therapy.