44 research outputs found
Ocular Gene Transfer with Self-Complementary AAV Vectors
PURPOSE. Self-complementary AAV (scAAV) vectors have been developed to circumvent rate-limiting second-strand synthesis in single-stranded AAV vector genomes and to facilitate robust transgene expression at a minimal dose. In this study, the authors investigated the effects of intraocular injections of type 2 scAAV.GFP in mice. METHODS. Dose-response experiments were performed to compare conventional single-strand AAV type 2 (ssAAV2) vectors with scAAV2 vectors encoding an identical expression cassette. RESULTS. Subretinal injection of 5 X 108viral particles (vp) of scAAV.CMV-GFP resulted in green fluorescent protein (GFP) expression in almost all retinal pigment epithelial (RPE) cells within the area of the small detachment caused by the injection by 3 days and strong, diffuse expression by 7 days. Expression was strong in all retinal cell layers by days 14 and 28. In contrast, 3 days after subretinal injection of 5 X 108vp of ssAAV.CMV-GFP, GFP expression was detectable in few RPE cells. Moreover, the ssAAV vector required 14 days for the attainment of expression levels comparable to those observed using scAAV at day 3. Expression in photoreceptors was not detectable until day 28. Dose-response experiments confirmed that onset of GFP expression was more rapid and robust after subretinal injection of scAAV.CMV-GFP than of ssAAV.CMV-GFP, resulting in pronounced expression in photoreceptors and other retinal neurons. Similar results were obtained for intravitreous injections. CONCLUSIONS. These data suggest that scAAV vectors may be advantageous for ocular gene therapy, particularly in retinal diseases that require rapid and robust transgene expression in photoreceptor cells
Regulation of Pathologic Retinal Angiogenesis in Mice and Inhibition of VEGF-VEGFR2 Binding by Soluble Heparan Sulfate
Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis
Nonvirally Modified Autologous Primary Hepatocytes Correct Diabetes and Prevent Target Organ Injury in a Large Preclinical Model
BACKGROUND: Current gene- and cell-based therapies have significant limitations which impede widespread clinical application. Taking diabetes mellitus as a paradigm, we have sought to overcome these limitations by ex vivo electrotransfer of a nonviral insulin expression vector into primary hepatocytes followed by immediate autologous reimplantation in a preclinical model of diabetes. METHODS AND RESULTS: In a single 3-hour procedure, hepatocytes were isolated from a surgically resected liver wedge, electroporated with an insulin expression plasmid ex vivo and reimplanted intraparenchymally under ultrasonic guidance into the liver in each of 10 streptozotocin-induced diabetic Yorkshire pigs. The vector was comprised of a bifunctional, glucose-responsive promoter linked to human insulin cDNA. Ambient glucose concentrations appropriately altered human insulin mRNA expression and C-peptide secretion within minutes in vitro and in vivo. Treated swine showed correction of hyperglycemia, glucose intolerance, dyslipidemia and other metabolic abnormalities for > or = 47 weeks. Metabolic correction correlated significantly with the number of hepatocytes implanted. Importantly, we observed no hypoglycemia even under fasting conditions. Direct intrahepatic implantation of hepatocytes did not alter biochemical indices of liver function or induce abnormal hepatic lobular architecture. About 70% of implanted hepatocytes functionally engrafted, appeared histologically normal, retained vector DNA and expressed human insulin for > or = 47 weeks. Based on structural tissue analyses and transcriptome data, we showed that early correction of diabetes attenuated and even prevented pathological changes in the eye, kidney, liver and aorta. CONCLUSIONS: We demonstrate that autologous hepatocytes can be efficiently, simply and safely modified by electroporation of a nonviral vector to express, process and secrete insulin durably. This strategy, which achieved significant and sustained therapeutic efficacy in a large preclinical model without adverse effects, warrants consideration for clinical development especially as it could have broader future applications for the treatment of other acquired and inherited diseases for which systemic reconstitution of a specific protein deficiency is critical
Regression of macular edema secondary to branch retinal vein occlusion during anti-TNF-α therapy for rheumatoid arthritis
Shu Kachi, Kenshin Kobayashi, Hiroaki Ushida, Yasuki Ito, Mineo Kondo, HirokoTerasakiDepartment of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, JapanAbstract: A patient with macular edema secondary to a branch retinal vein occlusion (BRVO) was treated with intravenous injections of infliximab, an antitumor necrosis factor (TNF)-α antibody, for her rheumatoid arthritis (RA). Before the injection, the thickness of the right fovea, determined by optical coherent tomography, was 629 μm and the best-corrected visual acuity (BCVA) was 20/50. After eight injections of infliximab and 10 months after the first injection, her foveal thickness was decreased to 293 μm and the visual acuity improved to 20/20. There was no recurrence of macular edema during the infliximab injections. However, the infliximab injection was stopped because the patient developed pneumonia. Eight months after stopping the infliximab injection, her foveal thickness increased to 494 μm. To treat the RA, her orthopedists began weekly subcutaneous injections of etanercept, a fusion protein of a section of the TNF receptor and immunoglobulin. Five months later, the foveal thickness had decreased to 260 μm, and the visual acuity remained at 20/25+. Because TNF-α is known to break down the blood–retinal barrier, the improvements in our case suggest that TNF-α plays a role in the pathogenesis of macular edema in some patients with BRVO.Keywords: branch retinal vein occlusion, macular edema, tissue necrosis factor-alpha, rheumatoid arthritis, infliximab, etanercept, foveal thicknes
Non-Descemet Stripping Automated Endothelial Keratoplasty for Bullous Keratopathy in Buphthalmic Eye
Purpose: To report the 2-year follow-up findings in a patient with buphthalmic bullous keratopathy (BK) who was successfully treated with non-Descemet stripping automated endothelial keratoplasty (nDSAEK). Methods: A 39-year-old man had an endothelial graft of 8.0 mm diameter placed uneventfully using the nDSAEK method for phakic BK with buphthalmos of the left eye. He had had a penetrating keratoplasty in the right eye due to aphakic BK 5 years earlier, which, however, resulted in the invasion of blood vessels and graft failure. Since the left eye was phakic, Descemetorhexis was not performed because the instruments might touch the crystalline lens. The best-corrected visual acuity (BCVA), intraocular pressure (IOP), and endothelial cell density (ECD) were determined at 2 weeks, and at 1, 3, 6, 12, 18 and 24 months after nDSAEK. Results: Twenty-four months after nDSAEK, his left cornea and lens remained clear, and the decimal BCVA was 0.8. However, the ECD of the graft had decreased from 2,274 cells/mm2 before nDSAEK to 539 cells/mm2 24 months after the surgery, and the rate of decrease appeared to be slightly faster than that of former reports. An IOP of >30 mm Hg was recorded at around 2 months after the surgery, but was well controlled by tapering the topical steroids and the addition of topical brinzolamide and latanoprost. Conclusion: Our findings show that nDSAEK can be successfully used to treat buphthalmic BK. We recommend that nDSAEK be considered especially in phakic eyes with a smooth posterior surface around the pupillary area
Significant Correlation between Retinal Venous Tortuosity and Aqueous Vascular Endothelial Growth Factor Concentration in Eyes with Central Retinal Vein Occlusion.
To determine whether the degree of venous tortuosity is significantly correlated with the aqueous vascular endothelial growth factor (VEGF) concentration in eyes with a central retinal vein occlusion (CRVO).We reviewed the medical records of 32 eyes of 32 patients who had macular edema due to a CRVO. All of the patients were examined at the Nagoya University Hospital and were scheduled to receive an intravitreal injection of bevacizumab (IVB) or ranibizumab (IVR) within 12 weeks of the onset of the CRVO to treat the macular edema. Aqueous humor was collected just before the IVB or IVR, and the VEGF concentration was determined by enzyme-linked immunosorbent assay (ELISA). The venous tortuosity index was calculated by dividing the length of the retinal veins by the chord length of the same segment. The correlation between the mean tortuosity index of the inferotemporal and supratemporal branches of the retinal vein and the aqueous VEGF concentration was determined.The mean aqueous VEGF concentration was 384 ± 312 pg/ml with a range of 90 to 1077 pg/ml. The degree of venous tortuosity was significantly correlated with the VEGF concentration in the aqueous. (r = 0.49, P = 0.004), with the foveal thickness (r = 0.40, P = 0.02), and with the best-corrected visual acuity (r = 0.38, P = 0.03).The significant correlation between the aqueous VEGF concentration and the venous tortuosity indicates that the degree of retinal venous tortuosity can be used to identify eyes that are at a high risk of developing neovascularization
Data from: Significant correlation between retinal venous tortuosity and aqueous vascular endothelial growth factor concentration in eyes with central retinal vein occlusion
Purpose: To determine whether the degree of venous tortuosity is significantly correlated with the aqueous vascular endothelial growth factor (VEGF) concentration in eyes with a central retinal vein occlusion (CRVO). Methods: We reviewed the medical records of 32 eyes of 32 patients who had macular edema due to a CRVO. All of the patients were examined at the Nagoya University Hospital and were scheduled to receive an intravitreal injection of bevacizumab (IVB) or ranibizumab (IVR) within 12 weeks of the onset of the CRVO to treat the macular edema. Aqueous humor was collected just before the IVB or IVR, and the VEGF concentration was determined by enzyme-linked immunosorbent assay (ELISA). The venous tortuosity index was calculated by dividing the length of the retinal veins by the chord length of the same segment. The correlation between the mean tortuosity index of the inferotemporal and supratemporal branches of the retinal vein and the aqueous VEGF concentration was determined. Results: The mean aqueous VEGF concentration was 384 ± 312 pg/ml with a range of 90 to 1077 pg/ml. The degree of venous tortuosity was significantly correlated with the VEGF concentration in the aqueous. (r = 0.49, P = 0.004), with the foveal thickness (r = 0.40, P = 0.02), and with the best-corrected visual acuity (r = 0.38, P = 0.03). Conclusions: The significant correlation between the aqueous VEGF concentration and the venous tortuosity indicates that the degree of retinal venous tortuosity can be used to identify eyes that are at a high risk of developing neovascularization