Elizabeth Rakoczy

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-β1) 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-β1 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.

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.

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.

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.

Syntheses of polycationic dendrimers on lipophilic peptide core for complexation and transport of oligonucleotides

Synthesis of novel polycationic lipophilic peptide core(s) was accomplished and these agents successfully transfected human retinal pigment epithelium cells with ODN1 upon complexation with the oligonucleotide. The level of transfection was indirectly measured by the decreased production of the protein hVEGF (human vascular endothelial growth factor) in comparison to the transfection agent cytofectin GSV.

Preclinical safety evaluation of subretinal AAV2.sFlt-1 in non-human primates

We report on the long-term safety of AAV2.sFlt-1 (a recombinant adeno-associated virus serotype 2 carrying the soluble form of the Flt-1 receptor) injection into the subretinal space of non-human primates. Levels of sFlt-1 protein were significantly higher (P<0.05) in the vitreous of four out of five AAV2.sFlt-1-injected eyes. There was no evidence of damage to the eyes of animals that received subretinal injections of AAV2.sFlt-1; ocular examination showed no anterior chamber flare, normal fundus and electroretinography responses equivalent to those observed before treatment. Notably, immunological analysis demonstrated that gene therapy involving subretinal injection of AAV2.sFlt-1 does not elicit cell-mediated immunity. Biodistribution analysis showed that AAV2.sFlt-1 could be detected only in the eye and not in the other organs tested. These data indicate that gene therapy with subretinal AAV2.sFlt-1 is safe and well tolerated, and therefore promising for the long-term treatment of neovascular diseases of the eye.

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.

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 × 1011 vg). All patients were assessed every 4 weeks 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.

The effects of age and Cx3cr1 deficiency on retinal microglia in the Ins2Akita 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.