A drug-tunable Flt23k gene therapy for controlled intervention in retinal neovascularization.

Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.

Therapeutic applications of chelating drugs in iron metabolic disorders of the brain and retina.

Iron is essential for normal cellular function, however, excessive accumulation of iron in neural tissue has been implicated in both cortical and retinal diseases. The exact role of iron in the pathogenesis of neurodegenerative disorders remains incompletely understood. However, iron-induced damage to the brain and retina is often attributed to the redox ability of iron to generate dangerous free radicals, which exacerbates local oxidative stress and neuronal damage. Iron chelators are compounds designed to scavenge labile iron, aiding to regulate iron bioavailability. Recently there has been growing interest in the application of chelating agents for treatment of diseases including neurodegenerative conditions, characterized by increased oxidative stress. This article reviews both clinical and preclinical evidence relating to the effectiveness of iron chelation therapy in conditions of iron dyshomeostasis linked to neurodegeneration in the brain and retina. The limitations as well as future opportunities iron chelation therapy are discussed.

AAV-mediated gene delivery of the calreticulin anti-angiogenic domain inhibits ocular neovascularization.

Ocular neovascularization is a common pathological feature in diabetic retinopathy and neovascular age-related macular degeneration that can lead to severe vision loss. We evaluated the therapeutic efficacy of a novel endogenous inhibitor of angiogenesis, the calreticulin anti-angiogenic domain (CAD180), and its functional 112-residue fragment, CAD-like peptide 112 (CAD112), delivered using a self-complementary adeno-associated virus serotype 2 (scAAV2) in rodent models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. The expression of CAD180 and CAD112 was elevated in human umbilical vein endothelial cells transduced with scAAV2-CAD180 or scAAV2-CAD112, respectively, and both inhibited angiogenic activity in vitro. Intravitreal gene delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly inhibited ischemia-induced retinal neovascularization in rat eyes (CAD180: 52.7% reduction; CAD112: 49.2% reduction) compared to scAAV2-mCherry, as measured in retinal flatmounts stained with isolectin B4. Moreover, the retinal structure and function were unaffected by scAAV2-CAD180 or scAAV2-CAD112, as measured by optical coherence tomography and electroretinography. Moreover, subretinal delivery of scAAV2-CAD180 or scAAV2-CAD112 significantly attenuated laser-induced choroidal neovascularization in mouse eyes compared to scAAV2-mCherry, as measured by fundus fluorescein angiography (CAD180: 62.4% reduction; CAD112: 57.5% reduction) and choroidal flatmounts (CAD180: 40.21% reduction; CAD112: 43.03% reduction). Gene delivery using scAAV2-CAD180 or scAAV2-CAD112 has significant potential as a therapeutic option for the management of ocular neovascularization.

Dietary ω-3 deficiency and IOP insult are additive risk factors for ganglion cell dysfunction.

AIM: Dietary deficiencies in ω-3 polyunsaturated fatty acids are known to effect retinal function including retinal ganglion cell (RGC) activity, which may have implications for glaucoma. In this study we consider retinal function after dietary manipulation and intraocular pressure (IOP) stress designed to compromise RGCs. METHODS: Sprague-Dawley dams were fed either ω-3 sufficient (ω-3, n=15) or deficient (ω-3, n=16) diets 5 weeks before conception with pups subsequently weaned onto their mothers diets. At 20 weeks of age, acute IOP elevation was induced repeatedly through anterior chamber cannulation to 70 mm Hg for 1 hour on 3 separate occasions separated by 1 week. Electroretinograms were recorded 1 week after each IOP elevation to assay the photoreceptors (PIII), ON-bipolar cells (PII), and ganglion/amacrine cells (STR). RESULTS: Repeat IOP insult results in a specific RGC dysfunction (pSTR -14.5%, P<0.035) as does ω-3 deficiency (-26.4%, P<0.01). However, the combination of both causes an even larger RGC functional loss (-40.1%, P<0.001) than does either diet or IOP insult in isolation (P<0.001). CONCLUSIONS: Both ω-3 deficiency and repeat acute IOP insult cause RGC dysfunction and the combination of these factors results in a cumulative effect. Our data indicate that sufficient dietary ω-3 improves RGC function making it less susceptible to IOP insult.

Dietary omega-3 fatty acids and ganglion cell function.

PURPOSE: Diet-induced deficiencies in Omega-3 (omega-3) fatty acids are well known to alter photoreceptor function. In this study, the broader functional changes in a diversity of retinal neurons were considered. METHODS: Sprague-Dawley dams were fed either omega-3-sufficient (omega-3(+), n = 21) or -deficient (omega-3(-), n = 19) diets 5 weeks before conception, with the pups continued on the mothers' diet. After 20 weeks of age, electroretinograms (ERGs) were recorded by using protocols that isolate separate cellular generators, including; photoreceptors (PIII), ON-bipolar cells (PII), and ganglion/amacrine cells (STR). At the brightest energies, rod and cone responses were isolated with a paired-flash paradigm. Retinal tissue (omega-3(+), n = 5; omega-3(-), n = 5) was harvested at 23 weeks of age for fatty acid assays with thin layer and gas liquid chromatography. RESULTS: Omega-3 deficiency caused a 48.6% decrease in total retinal docosahexaenoic acid (DHA). This change induced significant amplitude decreases only in the rod PII (-8.2%) and positive (p)STR components (-27.4%), with widespread delays in all signals (PIII 5.7%, PII 13.6%, pSTR 7.6%, and negative [n]STR 8.3%). Omega-3 deficiency exerted its greatest effects on signals originating in the inner retina (pSTR). CONCLUSIONS: Increasing dietary omega-3 has beneficial effects across the retina, with the greatest improvement occurring in ganglion cell function.