Cristhian J. Ildefonso

Systematic Review and Meta-Analysis of the Association between Complement Factor H I62V Polymorphism and Risk of Polypoidal Choroidal Vasculopathy in Asian Populations

Purpose To investigate whether the polymorphism rs800292 (184G>A, I62V) in the complement factor H gene is associated with polypoidal choroidal vasculopathy (PCV) and the genetic difference between PCV and neovascular age-related macular degeneration (nAMD), in Asian populations. Methods A comprehensive literature search was performed in PubMed, Medline, Web of Science, and reference lists. A system review and meta-analysis of the association between I62V and PCV and/or nAMD were performed from 8 studies involving 5,062 subjects. The following data from individual studies were extracted and analyzed: 1) comparison of I62V polymorphisms between PCV and controls; 2) comparison of I62V polymorphisms between PCV and nAMD. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using fixed-effects models. The Q-statistic test was used to assess heterogeneity, and Egger’s test was used to evaluate publication bias. Sensitivity analysis and cumulative meta-analysis were also performed. Results The I62V polymorphism showed a significant summary OR1 for genotype GA+GG versus homozygous genotype AA was 3.18 (95% CI, 2.51–4.04, P<0.00001), the OR2 of heterozygous genotype GA versus AA was 2.29 (95% CI: 1.79–2.94, P<0.00001), the OR3 of homozygous genotype GG versus AA was 4.42 (95% CI: 3.45–5.67, P<0.00001), and the OR4 of allele G versus A was 2.04 (95% CI: 1.85–2.26, P<0.00001). Sensitivity analysis indicated the robustness of our findings, and evidence of publication bias was not observed in our meta-analysis. Cumulative meta-analysis revealed that the summary ORs were stable. There was no significant difference in every genetic model between PCV and nAMD (n = 5, OR1 = 0.92, OR2 = 0.96, OR3 = 0.90, OR4 = 0.94). Conclusions Our analysis provides evidence that the I62V polymorphism is associated with an increased risk of PCV. The variant of I62V could be a promising genetic biomarker of PCV in Asian populations.

Targeting the Nrf2 Signaling Pathway in the Retina With a Gene-Delivered Secretable and Cell-Penetrating Peptide.

Purpose Oxidative stress has been linked to several ocular diseases, initiating an inflammatory response that increases tissue injury. The Nrf2 transcription factor regulates expression of antioxidant genes and is tightly regulated by Kelch-Like ECH-Associated Protein 1 (Keap-1). We evaluate the antioxidant and anti-inflammatory properties of an adeno-associated virus (AAV) vector delivering an Nrf2-derived peptide that binds Keap-1. Methods The sequence of the Nrf2 peptide was fused to a cell-penetrating peptide (Tat-peptide) sequence (TatNrf2mer). The effects of lentiviral-delivered TatNrf2mer were studied in vitro. Transcript (quantitative [q] RT-PCR) and protein levels (ELISA and immunofluorescence) were quantified. Cell viability was measured by MTT and Cell Titer assays. The AAV vectors were packaged with the TatNrf2mer fused to secretable green fluorescent protein (GFP) under the control of the small chicken β actin promoter. The protective effects of this vector were evaluated in a model of RPE oxidative injury and in a mouse model of uveitis after intravitreal injection. Results Expression of TatNrf2mer peptide induced antioxidant gene expression, blocked IL-1β secretion, and protected cells from oxidative injury. In mice, TatNrf2mer expression partially protected photoreceptor function based on ERG responses and optical coherence tomography measurements in the sodium iodate (NaIO3) model. Furthermore, sGFP-TatNrf2mer expression decreased IL-1β and IL-6 in the NaIO3-treated mice, and resulted in a 54% decrease in the number of inflammatory cells in the vitreous body of the endotoxin-induced uveitis mouse model. Conclusions The intravitreally delivered AAV-TatNrf2mer has antioxidant and anti-inflammatory effects in widely-used models of ocular injury, suggesting it also could be useful in ocular diseases associated with oxidative stress and inflammasome activation.

Gene Therapy With the Caspase Activation and Recruitment Domain Reduces the Ocular Inflammatory Response

Inflammation is a key component of chronic and acute diseases of the eye. Our goal is to test anti-inflammatory genes delivered by an adeno-associated virus (AAV) vector as potential treatments for retinal inflammation. We developed a secretable and cell penetrating form of the caspase activation and recruitment domain (CARD) from the apoptosis-associated speck-like protein containing a CARD (ASC) gene that binds caspase-1 and inhibits its activation by the inflammasome. The secretion and cell penetration characteristics of this construct were validated in vitro by measuring its effects on inflammasome signaling in a monocyte cell line and in an retinal pigmented epithelium (RPE) cell line. This vector was then packaged as AAV particles and tested in the endotoxin-induced uveitis mouse model. Gene expression was monitored one month after vector injection by fluorescence fundoscopy. Ocular inflammation was then induced by injecting lipopolysaccharide into the vitreous and was followed by enucleation 24 hours later. Eyes injected with the secretable and cell penetrating CARD AAV vector had both a significantly lower concentration of IL-1β as well as a 64% reduction in infiltrating cells detected in histological sections. These results suggest that anti-inflammatory genes such as the CARD could be used to treat recurring inflammatory diseases like uveitis or chronic subacute inflammations of the eye.

Gene Delivery of a Viral Anti-Inflammatory Protein to Combat Ocular Inflammation

Inflammation of the retina is a contributing factor in ocular diseases such as uveitis, diabetic retinopathy, and age-related macular degeneration (AMD). The M013 immunomodulatory protein from myxoma virus has been shown to interfere with the proinflammatory signaling pathways involving both the NLRP3 inflammasome and NF-κB. We have developed and characterized an adeno-associated viral (AAV) vector that delivers a secretable and cell-penetrating form of the M013 protein (TatM013). The expressed TatM013 protein was secreted and blocked the endotoxin-induced secretion of interleukin (IL)-1β in monocyte-derived cells and the reactive aldehyde-induced secretion of IL-1β in retinal pigment epithelium cells. The local anti-inflammatory effects of AAV-delivered TatM013 were evaluated in an endotoxin-induced uveitis (EIU) mouse model after intravitreal injection of mice with an AAV2-based vector carrying either TatM013 fused to a secreted green fluorescent protein (GFP) tag (sGFP-TatM013) or GFP. Expression of the sGFP-TatM013 transgene was demonstrated by fluorescence funduscopy in living mice. In EIU, the number of infiltrating cells and the concentration of IL-1β in the vitreous body were significantly lower in the eyes injected with AAV-sGFP-TatM013 compared with the eyes injected with control AAV-GFP. These results suggest that a virus-derived inhibitor of the innate immune response, when delivered via AAV, could be a generalized therapy for various inflammatory diseases of the eye.

The NLRP3 Inflammasome and its Role in Age-Related Macular Degeneration.

Age related macular degeneration (AMD) is the most common cause of blindness among people of 65 years and older in developed countries (Klein and Klein, Invest Ophthalmol Vis Sci 54:7395-7401, 2013). Recent advances in dry AMD research points towards an important role of the inflammatory response in the development of the disease. The presence of inflammatory cells, antibodies, complement factors and pro-inflammatory cytokines in AMD retinas and drusen indicates that the immune system could be an important driving force in dry AMD. The NLRP3 inflammasome has been proposed as an integrator of process associated with AMD and the induction of inflammation. Herein we summarize the most recent studies that attempt to understand the role of the NLRP3 inflammasome in AMD.

Conditional Induction of Oxidative Stress in RPE: A Mouse Model of Progressive Retinal Degeneration

An appropriate animal model is essential to screening drugs or designing a treatment strategy for geographic atrophy. Since oxidative stress contributes to the pathological changes of the retinal pigment epithelium (RPE), we are reporting a new mouse AMD model of retinal degeneration by inducing mitochondrial oxidative stress in RPE. Sod2 the gene for manganese superoxide dismutase (MnSOD) was deleted in RPE layer using conditional knockout strategy. Fundus microscopy, SD-OCT and electroretinography were used to monitor retinal structure and function in living animals and microscopy was used to assess pathology post mortem. Tissue specific deletion of Sod2 caused elevated signs of oxidative stress, RPE dysfunction and showed some key features of AMD. Due to induction of oxidative stress, the conditional knockout mice show progressive reduction in ERG responses and thinning of outer nuclear layer (ONL) compared to non-induced littermates.

Timing of Antioxidant Gene Therapy: Implications for Treating Dry AMD

Purpose To investigate whether antioxidant gene therapy protects the structure and function of retina in a murine model of RPE atrophy, and to determine whether antioxidant gene therapy can prevent degeneration once it has begun. Methods We induced mitochondrial oxidative stress in RPE by conditional deletion of Sod2, the gene for manganese superoxide dismutase (MnSOD). These mice exhibited localized atrophy of the RPE and overlying photoreceptors. We restored Sod2 to the RPE of one eye using adeno-associated virus (AAV) by subretinal injection at an early (6 weeks) and a late stage (6 months), injecting the other eye with an AAV vector expressing green fluorescent protein (GFP). Retinal degeneration was monitored over a period of 9 months by electroretinography (ERG) and spectral-domain optical coherence tomography (SD-OCT). Immunohistochemical and histologic analyses were conducted to measure oxidative stress markers and to visualize retinal structure. Results One month after delivery, the AAV-Sod2 injection resulted in production of MnSod in the RPE and negligible expression in the neural retina. Electroretinography and OCT suggested no adverse effects due to increased expression of MnSOD or subretinal injection. Decrease in the ERG response and thinning retinal thickness was significantly delayed in eyes with early treatment with the Sod2 vector, but treatment at 6 months of age did not affect the ERG decline seen in these mice. Conclusions We conclude that antioxidant gene therapy may be effective in preventing the detrimental effects of oxidative stress, but may not be beneficial once substantial tissue damage has occurred.

The liberation of CD44 intracellular domain modulates adenoviral vector transgene expression

Background: Hyaluronan-CD44 interaction enhances expression of adenoviral vector transgenes. Results: Inhibition of CD44 targeting proteases blocks enhancement of vector transgene expression and liberated intracellular domain enhances expression. Conclusion: CD44 proteolysis plays a role in the modulation of adenoviral vector transgene expression. Significance: Understanding the role of CD44 signaling in enhancement of transcription of adenoviral genes will be beneficial in viral gene transfer applications. The success of gene therapy in the ocular environment is partly due to the presence of hyaluronan in vitreous. Here we explore the mechanism of hyaluronan-mediated enhancement of adenoviral vector transgene expression. Introduction of hyaluronan receptor CD44 into CD44-negative cells followed by transduction in the presence of vitreous with an adenoviral vector containing an IL-12-coding transgene increases IL-12 secretion. We demonstrate that sequential CD44 proteolysis is responsible for hyaluronan-mediated enhancement. Metalloproteinase or γ-secretase inhibitors decrease adenoviral-mediated transgene expression. Deletion of these proteolytic sites in CD44 also inhibits transgene expression. Expression of CD44 with a mutation to prevent phosphorylation of serine 325 inhibits the response to vitreous. Expression of the CD44 intracellular domain enhances transgene expression in the absence of vitreous. CD44-mediated enhancement of gene expression was observed with vectors using different promoters and appears because of an increase in mRNA production, not because of an increase in vector transduction as determined by quantitative RT-PCR and quantitative PCR, respectively. These data fit a model where the interaction of hyaluronan in vitreous and CD44 modulates transgene expression by initiating CD44 proteolysis and release of the cytoplasmic domain, resulting in increased transgene transcription.

Neuroinflammation in Retinitis Pigmentosa, Diabetic Retinopathy, and Age-Related Macular Degeneration: A Minireview

The eye is an immuno-privileged organ. However, certain diseases such as uveitis are intrinsically linked to inflammation. In several retinal degenerative diseases, there is a unique damage at the onset of the disease, but evidence suggests that chronic and low-grade inflammatory processes play an important role in their progression. Studies have identified similar signaling pathways and changes in resident immune cells within the retina among these diseases. Herein, we will discuss some of these studies and propose how understanding this inflammatory response could aid in the development of therapies.

A cell penetrating peptide from SOCS-1 prevents ocular damage in experimental autoimmune uveitis

&NA; We describe an immunosuppressive peptide corresponding to the kinase inhibitory region (KIR) of the intracellular checkpoint protein suppressor of cytokine signaling 1 (SOCS‐1) that binds to the phospho‐tyrosine containing regions of the tyrosine kinases JAK2 and TYK2 and the adaptor protein MAL, and thereby inhibits signaling downstream from these signaling mediators. The peptide, SOCS1‐KIR, is thus capable of downregulating overactive JAK/STAT or NF‐kB signaling in somatic cells, including those in many compartments of the eye. Attachment of poly‐arginine to this peptide (R9‐SOCS1‐KIR) allows it to penetrate the plasma membrane in aqueous media. R9‐SOCS1‐KIR was tested in ARPE‐19 cells and was found to attenuate mediators of inflammation by blocking the inflammatory effects of IFN&ggr;, TNF&agr;, or IL‐17A. R9‐SOCS1‐KIR and also protected against TNF&agr; or IL‐17A mediated damage to the barrier properties of ARPE‐19 cells, as evidenced by immunostaining with the tight junction protein, zona occludin 1 (ZO‐1), and measurement of transepithelial electrical resistance (TEER). Experimental autoimmune uveitis (EAU) was generated in B10. RIII mice using a peptide of interphotoreceptor retinal binding protein (IRBP161−180) as immunogen. Topical administration of R9‐SOCS1‐KIR, 2 days before (prophylactic), or 7 days after immunization (therapeutic) protected ocular structure and function as seen by fundoscopy, optical coherence tomography (OCT), and electroretinography (ERG). The ability R9‐SOCS1‐KIR to suppress ocular inflammation and preserve barrier properties of retinal pigment epithelium makes it a potential candidate for treatment of autoimmune uveitis.