Overexpression of Nfe2l1 increases proteasome activity and delays vision loss in a preclinical model of human blindness.

Proteasomes are the central proteolytic machines that are critical for breaking down most of the damaged and abnormal proteins in human cells. Although universally applicable drugs are not yet available, the stimulation of proteasomal activity is being analyzed as a proof-of-principle strategy to increase cellular resistance to a broad range of proteotoxic stressors. These approaches have included the stimulation of proteasomes through the overexpression of individual proteasome subunits, phosphorylation, or conformational changes induced by small molecules or peptides. In contrast to these approaches, we evaluated a transcription-driven increase in the total proteasome pool to enhance the proteolytic capacity of degenerating retinal neurons. We show that overexpression of nuclear factor erythroid-2-like 1 (Nfe2l1) transcription factor stimulated proteasome biogenesis and activity, improved the clearance of the ubiquitin-proteasomal reporter, and delayed photoreceptor neuron loss in a preclinical mouse model of human blindness caused by misfolded proteins. The findings highlight Nfe2l1 as an emerging therapeutic target to treat neurodegenerative diseases linked to protein misfolding.

Binocular benefit following monocular subretinal AAV injection in a mouse model of autosomal dominant retinitis pigmentosa (adRP).

Autosomal dominant retinitis pigmentosa (adRP) is frequently caused by mutations in RHO, the gene for rhodopsin. In previous experiments in dogs with the T4R mutation in RHO, an AAV2/5 vector expressing an shRNA directed to human and dog RHO mRNA and an shRNA-resistant human RHO cDNA (AAV-RHO820-shRNA820) prevented retinal degeneration for more than eight months following injection. It is crucial, however, to determine if this RNA replacement vector acts in a mutation-independent and species-independent manner. We, therefore, injected mice transgenic for human P23H RHO with this vector unilaterally at postnatal day 30. We monitored their retinal structure by using spectral-domain optical coherence tomography (SD-OCT) and retinal function using electroretinography (ERG) for nine months. We compared these to P23H RHO transgenic mice injected unilaterally with a control vector. Though retinas continued to thin over time, compared to control injected eyes, treatment with AAV-RHO820-shRNA820 slowed the loss of photoreceptor cells and the decrease in ERG amplitudes during the nine-month study period. Unexpectedly, we also observed the preservation of retinal structure and function in the untreated contralateral eyes of AAV-RHO820-shRNA820 treated mice. PCR analysis and western blots showed that a low amount of vector from injected eyes was present in uninjected eyes. In addition, protective neurotrophic factors bFGF and GDNF were elevated in both eyes of treated mice. Our finding suggests that using this or similar RNA replacement vectors in human gene therapy may provide clinical benefit to both eyes of patients with adRP.

Modulation of Retinal Inflammation Delays Degeneration in a Mouse Model of Geographic Atrophy.

The advanced form of AMD, geographic atrophy, is associated with increased RPE oxidative stress and chronic inflammation. Here we evaluated the effects of delivering an anti-inflammatory viral gene by an AAV-vector in a mouse model of geographic atrophy. We measured changes in retinal function, structure, and morphology over nine months with electroretinography, optical coherence tomography, and fundoscopy, respectively. In addition, we used retinal tissue to quantify changes in markers of inflammation by multiplex ELISA, RT-qPCR, and immunofluorescence staining. Our AAV significantly delayed the loss of retinal function and structure and decreased retinal inflammation compared to the control AAV treatment. Our results suggest that modulating retinal inflammation could significantly slow the progression of geographic atrophy.

Suppressor of cytokine signaling 3-derived peptide as a therapeutic for inflammatory and oxidative stress-induced damage to the retina.

Purpose: Inflammation and oxidative stress contribute to age-related macular degeneration (AMD) and other retinal diseases. We tested a cell-penetrating peptide from the kinase inhibitory region of an intracellular checkpoint inhibitor suppressor of cytokine signaling 3 (R9-SOCS3-KIR) peptide for its ability to blunt the inflammatory or oxidative pathways leading to AMD. Methods: We used anaphylatoxin C5a to mimic the effect of activated complement, lipopolysaccharide (LPS), and tumor necrosis factor alpha (TNFα) to stimulate inflammation and paraquat to induce mitochondrial oxidative stress. We used a human retinal pigment epithelium (RPE) cell line (ARPE-19) as proliferating cells and a mouse macrophage cell line (J774A.1) to follow cell propagation using microscopy or cell titer assays. We evaluated inflammatory pathways by monitoring the nuclear translocation of NF-κB p65 and mitogen-activated protein kinase p38. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the induction of inflammatory markers. In differentiated ARPE-19 monolayers, we evaluated the integrity of tight junction proteins through microscopy and the measurement of transepithelial electrical resistance (TEER). We used intraperitoneal injection of sodium iodate in mice to test the ability of R9-SOC3-KIR to prevent RPE and retinal injury, as assessed by fundoscopy, optical coherence tomography, and histology. Results: R9-SOCS3-KIR treatment suppressed C5a-induced nuclear translocation of the NF-kB activation domain p65 in undifferentiated ARPE-19 cells. TNF-mediated damage to tight junction proteins in RPE, and the loss of TEER was prevented in the presence of R9-SOCS3-KIR. Treatment with the R9-SOCS3-KIR peptide blocked the C5a-induced expression of inflammatory genes. The R9-SOCS3-KIR treatment also blocked the LPS-induced expression of interleukin-6, MCP1, cyclooxygenase 2, and interleukin-1 beta. R9-SOCS3-KIR prevented paraquat-mediated cell death and enhanced the levels of antioxidant effectors. Daily eye drop treatment with R9-SOCS3-KIR protected against retinal injury caused by i.p. administration of sodium iodate. Conclusions: R9-SOCS3-KIR blocks the induction of inflammatory signaling in cell culture and reduces retinal damage in a widely used RPE/retinal oxidative injury model. As this peptide can be administered through corneal instillation, this treatment may offer a convenient way to slow down the progression of ocular diseases arising from inflammation and chronic oxidative stress.

Gene Delivery of a Caspase Activation and Recruitment Domain Improves Retinal Pigment Epithelial Function and Modulates Inflammation in a Mouse Model with Features of Dry Age-Related Macular Degeneration.

Purpose: The NLRP3 inflammasome, a cytoplasmic signal transduction complex that regulates inflammation, has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of visual impairment in industrialized countries. We tested the therapeutic effect of anti-inflammatory gene therapy, delivered preventively, in Liver-X-Receptor alpha knockout (LXRα-/-) mice, which exhibit features of dry AMD. Methods:LXRα-/- mice were treated with an adeno-associated virus (AAV) vector that delivers a secretable and cell-penetrating form of the caspase activation and recruitment domain (CARD). A sGFP-FCS-TatCARD-AAV or sGFP-FCS (control) vector was delivered intravitreally to 3-5 month-old, LXRα-/- mice, who were then aged to 15-18 months (12-13 month treatment). Retinal function and morphology were assessed pre- and post-treatment. Results: TatCARD treated LXRα-/- mice did not show improvement in rod and cone photoreceptor function, measured by dark adapted a- and b-wave amplitudes, and rod-saturated b-wave amplitudes. We found a sex-dependent, significant therapeutic effect in c-wave amplitudes in the TatCARD treated mice, which exhibited maintenance of amplitudes in comparison to the significant decline recorded in the control treated group, indicating a therapeutic effect mediated in part through retinal pigment epithelial (RPE) cells. Additionally, the retinas of the TatCARD treated mice exhibited a significant decline in the concentration of interleukin-1 beta (IL-1ß) concomitant with modulation of several inflammatory cytokines in the retina and RPE-choroid tissues, as measured by ELISA and cytokine array, respectively. Conclusion: Collectively, these results support that anti-inflammatory gene constructs such as AAV-TatCARD may be considered for the treatment of inflammation in AMD and other ocular diseases of the posterior pole in which inflammation may play a role. Furthermore, our findings emphasize the need to carefully consider potential sex-different responses when assessing potential therapies in pre-clinical models.

Corneal Application of R9-SOCS1-KIR Peptide Alleviates Endotoxin-Induced Uveitis.

Purpose: Uveitis is an ocular inflammation that can affect individuals of all ages and is a major cause of blindness. We have tested the therapeutic efficacy of a cell penetrating peptide from the kinase inhibitory region of suppressor of cytokine signaling 1, denoted as R9-SOCS1-KIR. Methods: We stimulated J774A.1 cells with lipopolysaccharide (LPS) in the presence of R9-SOCS1-KIR or its inactive control peptide. Effect on inflammatory pathways was followed by the nuclear translocation of nuclear factor κB p65 subunit and phosphorylated-p38. Synthesis of inflammatory markers induced by LPS was tested by reverse transcriptase polymerase chain reaction, Western blot analysis, and ELISA of cell supernatants. We monitored effects on the barrier properties of a differentiated ARPE-19 monolayer treated with LPS. We treated C57BL/6 mice topically with either R9-SOCS1-KIR or vehicle and injected their eyes intravitreally with LPS. Eyes were analyzed by fundoscopy, fluorescein angiography, optical coherence tomography, histology, Western blotting, multiplex enzyme-linked immunosorbent assay, and flow cytometry. Results: Treatment with R9-SOCS1-KIR resulted in suppression of signaling through nuclear factor κB and p-p38 pathways. R9-SOCS1-KIR suppressed the expression of inflammatory genes, the secretion of inflammatory makers such as nitric oxide, and IL-1ß induced by LPS. Increased permeability of retinal pigment epithelial cell monolayers was prevented. Corneal administration of R9-SOCS1-KIR blocked the acute inflammation observed in LPS-injected mouse eyes. Conclusions: Treatment with R9-SOCS1-KIR alleviated the inflammatory responses in cell culture. Topical delivery of this peptide on mouse eyes protected against LPS-induced damage. Translational Relevance: Topical delivery of R9-SOCS1-KIR peptide allows the patient to self-administer the drug, while preventing any systemic effects on unrelated organs.

Molecular Design and Production of AAV Viral Vectors for Gene Therapy.

Adeno-associated virus (AAV) is a helper-dependent single-stranded DNA parvovirus. Over the years, AAV has become the vector of choice in the gene therapy field due to its safety profile and low immunogenicity. With a carrying capacity of 4.2 kbp, these vectors have demonstrated their clinical value, especially in the field of ophthalmology. Herein we describe methods for the molecular design and packaging of AAV viral vectors. These methods apply to the design of single-stranded or self-complementary AAV vectors.

Sectoral activation of glia in an inducible mouse model of autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of blinding disorders caused by diverse mutations, including in rhodopsin (RHO). Effective therapies have yet to be discovered. The I307N Rho mouse is a light-inducible model of autosomal dominant RP. Our purpose was to describe the glial response in this mouse model to educate future experimentation. I307N Rho mice were exposed to 20,000 lx of light for thirty minutes to induce retinal degeneration. Immunofluorescence staining of cross-sections and flat-mounts was performed to visualize the response of microglia and Müller glia. Histology was correlated with spectral-domain optical coherence tomography imaging (SD-OCT). Microglia dendrites extended between photoreceptors within two hours of induction, withdrew their dendrites between twelve hours and one day, appeared ameboid by three days, and assumed a ramified morphology by one month. Glial activation was more robust in the inferior retina and modulated across the boundary of light damage. SD-OCT hyper-reflectivity overlapped with activated microglia. Finally, microglia transiently adhered to the RPE before which RPE cells appeared dysmorphic. Our data demonstrate the spatial and temporal pattern of glial activation in the I307N Rho mouse, and correlate these patterns with SD-OCT images, assisting in interpretation of SD-OCT images in preclinical models and in human RP.

Inhibitors of metalloprotease, γ-sectretase, protein kinase C and Rho kinase inhibit wild-type adenoviral replication.

Adenoviruses cause upper respiratory infections, conjunctivitis, keratitis, and gastrointestinal illness. These can be fatal in immunocompromised individuals. Adenoviruses have also been engineered into viral vectors to deliver therapeutic genes or induce immunity as vaccine carriers. The success of ocular gene therapy is driven partly by the immunologic and biochemical influences of the intraocular environment. We have shown that versican and hyaluronan modulate adenoviral vector transgene expression through CD44 signaling. Herein we explored the role of these pathways on virus replication and viral protein expression of wild type adenovirus. We report that the addition of vitreous humor (which contains both versican and hyaluronan) increases viral hexon protein levels. Vitreous humor also increased wild type adenovirus DNA replication in vitro. Metalloproteinase and γ-secretase inhibitors, which inhibit CD44 proteolytic activation, blocked adenoviral replication in vitro. Similarly, protein kinase C and RhoA kinase inhibitors, both proteins associated with CD44 mediated pathways, also inhibited wild type adenoviral replication in vitro. Application of metalloproteinase and γ-secretase inhibitors to human conjunctival explants sharply decreased adenoviral vector gene expression. Our results demonstrate that pharmacologic delivery of these inhibitors is easily achievable. The inhibition of these enzymes should be explored as potential therapies of wild type adenoviral infections.

Deriving Immune Modulating Drugs from Viruses-A New Class of Biologics.

Viruses are widely used as a platform for the production of therapeutics. Vaccines containing live, dead and components of viruses, gene therapy vectors and oncolytic viruses are key examples of clinically-approved therapeutic uses for viruses. Despite this, the use of virus-derived proteins as natural sources for immune modulators remains in the early stages of development. Viruses have evolved complex, highly effective approaches for immune evasion. Originally developed for protection against host immune responses, viral immune-modulating proteins are extraordinarily potent, often functioning at picomolar concentrations. These complex viral intracellular parasites have "performed the R&D", developing highly effective immune evasive strategies over millions of years. These proteins provide a new and natural source for immune-modulating therapeutics, similar in many ways to penicillin being developed from mold or streptokinase from bacteria. Virus-derived serine proteinase inhibitors (serpins), chemokine modulating proteins, complement control, inflammasome inhibition, growth factors (e.g., viral vascular endothelial growth factor) and cytokine mimics (e.g., viral interleukin 10) and/or inhibitors (e.g., tumor necrosis factor) have now been identified that target central immunological response pathways. We review here current development of virus-derived immune-modulating biologics with efficacy demonstrated in pre-clinical or clinical studies, focusing on pox and herpesviruses-derived immune-modulating therapeutics.

A C-terminal peptide from type I interferon protects the retina in a mouse model of autoimmune uveitis.

Experimental autoimmune uveitis (EAU) in rodents recapitulates many features of the disease in humans and has served as a useful tool for the development of therapeutics. A peptide from C-terminus of interferon α1, conjugated to palmitoyl-lysine for cell penetration, denoted as IFNα-C, was tested for its anti-inflammatory properties in ARPE-19 cells, followed by testing in a mouse model of EAU. Treatment with IFNα-C and evaluation by RT-qPCR showed the induction of anti-inflammatory cytokines and chemokine. Inflammatory markers induced by treatment with TNFα were suppressed when IFNα-C was simultaneously present. TNF-α mediated induction of NF-κB and signaling by IL-17A were attenuated by IFNα-C. Differentiated ARPE-19 cells were treated with TNFα in the presence or absence IFNα-C and analyzed by immmunhistochemistry. IFNα-C protected against the disruption integrity of tight junction proteins. Similarly, loss of transepithelial resistance caused by TNFα was prevented by IFNα-C. B10.RIII mice were immunized with a peptide from interphotoreceptor binding protein (IRBP) and treated by gavage with IFNα-C. Development of uveitis was monitored by histology, fundoscopy, SD-OCT, and ERG. Treatment with IFNα-C prevented uveitis in mice immunized with the IRBP peptide. Splenocytes isolated from mice with ongoing EAU exhibited antigen-specific T cell proliferation that was inhibited in the presence of IFNα-C. IFNα-C peptide exhibits anti-inflammatory properties and protects mice against damage to retinal structure and function suggesting that it has therapeutic potential for the treatment of autoimmune uveitis.

AAV Mediated Delivery of Myxoma Virus M013 Gene Protects the Retina against Autoimmune Uveitis.

Uveoretinitis is an ocular autoimmune disease caused by the activation of autoreactive T- cells targeting retinal antigens. The myxoma M013 gene is known to block NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) and inflammasome activation, and its gene delivery has been demonstrated to protect the retina against lipopolysaccharide (LPS)-induced uveitis. In this report we tested the efficacy of M013 in an experimental autoimmune uveoretinitis (EAU) mouse model. B10RIII mice were injected intravitreally with AAV (adeno associated virus) vectors delivering either secreted GFP (sGFP) or sGFP-TatM013. Mice were immunized with interphotorecptor retinoid binding protein residues 161-180 (IRBP161-180) peptide in complete Freund's adjuvant a month later. Mice were evaluated by fundoscopy and spectral domain optical coherence tomography (SD-OCT) at 14 days post immunization. Eyes were evaluated by histology and retina gene expression changes were measured by reverse transcribed quantitative PCR (RT-qPCR). No significant difference in ERG or retina layer thickness was observed between sGFP and sGFP-TatM013 treated non-uveitic mice, indicating safety of the vector. In EAU mice, expression of sGFP-TatM013 strongly lowered the clinical score and number of infiltrative cells within the vitreous humor when compared to sGFP treated eyes. Retina structure was protected, and pro-inflammatory genes expression was significantly decreased. These results indicate that gene delivery of myxoma M013 could be of clinical benefit against autoimmune diseases.

Systematic Injection of Low-Dose LPS Transiently Improves the Retina Function and Structure of a Mouse Model of Geographic Atrophy.

Geographic atrophy (GA), the advanced form of AMD, has been linked to oxidative stress within the RPE and with low-grade inflammation. The RPE-specific Sod2 knockout mouse model of GA develops increase oxidative stress and slow retinal degeneration. Mice of the SOD2floxed::VMD2Cre+ genotype were injected subcutaneously with either saline or 3 mg/kg of lipopolysaccharide (LPS) at 8 weeks of age. Mice were evaluated by electroretinography (ERG) and spectral domain optical coherence tomography. Inflammatory cells within the retina were studied by CD45 immunofluorescence. Systemic low-dose LPS transiently, but significantly, improved both function and structure of RPE-specific Sod2 KO mice retina when compared to saline-injected mice. There was no difference in CD45 positive cells between saline and LPS treatment. Low-grade activation of the immune system leads to a preconditioning effect that transiently protects the retina of a mouse model of geographic atrophy.

Expression of a CARD Slows the Retinal Degeneration of a Geographic Atrophy Mouse Model.

Age-related macular degeneration (AMD) has been linked to oxidative damage and para-inflammation, an activation of inflammasome signaling in the retinal pigment epithelium (RPE) and the underlying choriocapillaris. Herein, we tested the efficacy of a gene-delivered caspase-1 inhibitor in controlling the retinal degeneration observed in two models of RPE-choroid oxidative damage. In an acute model of oxidative stress (NaIO3 injection), eyes pre-treated with the sGFP-TatCARD (trans-activator of transcription; caspase activation and recruitment domain) vector demonstrated a recovery of retinal function and partial protection of RPE structure 1 month after damage, in contrast with control-treated eyes. In a model of chronic oxidative stress (RPE-specific deletion of Sod2), eyes treated with the sGFP-TatCARD vector after the onset of degeneration had a significantly slower decline in retinal function when compared to control-treated eyes. Earlier treatment of this model with the same adeno-associated virus (AAV) vector resulted in a greater protection of RPE function in eyes treated with the TatCARD when compared to control-treated eyes. Our results demonstrate that intravitreal delivery of sGFP-TatCARD reduces inflammation and can protect the retina from both acute and sustained oxidative damage within the RPE and choroid. Therefore, gene therapy with a cell-penetrating inflammasome inhibitor such as CARD may stem the progression of AMD.

Mitochondrial oxidative stress in the retinal pigment epithelium (RPE) led to metabolic dysfunction in both the RPE and retinal photoreceptors.

Age-related macular degeneration (AMD) is the leading cause of vision loss in the western world. Recent evidence suggests that RPE and photoreceptors have an interconnected metabolism and that mitochondrial damage in RPE is a trigger for degeneration in both RPE and photoreceptors in AMD. To test this hypothesis, this study was designed to induce mitochondrial damage in RPE in mice to determine whether this is sufficient to cause RPE and photoreceptor damage characteristic of AMD. In this study, we conditionally deleted the gene encoding the mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD encoded by Sod2) in the retinal pigment epithelium (RPE) of albino BALB/cJ mice. VMD2-Cre;Sod2flox/flox BALB/cJ mice were housed in either 12-h dark, 12-h 200 lux white lighting (normal light), or 12-h dark, 12-h <10 lux red lighting (dim light). Electroretinography (ERG) and spectral-domain optical coherence tomography (SD-OCT) were performed to assess retinal function and morphology. Immunofluorescence was used to examine protein expression; quantitative RT-PCR was used to measure gene expression. Sod2 knockout (KO) mice had reduced RPE function with age and increased oxidative stress compared to wild type (WT) controls as expected by the cell-specific deletion of Sod2. This was associated with alterations in RPE morphology and the structure and function of RPE mitochondria. In addition, data show a compensatory increase in RPE glycolytic metabolism. The metabolic shift in RPE correlated with severe disruption of photoreceptor mitochondria including a reduction in TOMM20 expression, mitochondrial fragmentation, and reduced COXIII/ß-actin levels. These findings demonstrate that mitochondrial oxidative stress can lead to RPE dysfunction and metabolic reprogramming of RPE. Secondary to these changes, photoreceptors also undergo metabolic stress with increased mitochondrial damage. These data are consistent with the hypothesis of a linked metabolism between RPE and photoreceptors and suggest a mechanism of retinal degeneration in dry AMD.

Co-Delivery of a Short-Hairpin RNA and a shRNA-Resistant Replacement Gene with Adeno-Associated Virus: An Allele-Independent Strategy for Autosomal-Dominant Retinal Disorders.

Recombinant adeno-associated virus (rAAV) has become an important gene delivery vector for the treatment of inherited retinal degenerative diseases. Many of the mutations leading to retinal degeneration are inherited in an autosomal-dominant pattern and can produce toxic gain-of-function and/or dominant-negative effects. Here we describe an allele-independent gene therapy strategy with rAAV to treat autosomal-dominant retinal degenerative diseases. In this methodology, we co-deliver a short-hairpin RNA (shRNA) to inhibit expression of both the toxic and (WT) copies of the gene as well as an shRNA-resistant cDNA for functional gene replacement with a rAAV.

Clinically Relevant Outcome Measures for the I307N Rhodopsin Mouse: A Model of Inducible Autosomal Dominant Retinitis Pigmentosa.

Purpose: The I307N rhodopsin (Rho) mouse is a light-inducible model of autosomal dominant retinitis pigmentosa (adRP) that may be useful in testing therapies. We investigated the time-course of retinal changes of the I307N Rho mouse with spectral-domain optical coherence tomography (SD-OCT). Methods: SD-OCT was performed up to day 30 after light damage; electroretinography (ERG) was employed to evaluate photoreceptor function. We utilized ImageJ to analyze reflectivity of the retina. We used light and electron microscopy to assess retinal organization. We stained synaptophysin and zonula occludins-1 with immunohistochemistry to determine injury to the plexiform layers and retinal pigment epithelium (RPE). We performed lectin staining to evaluate retinal blood vessels. Results: Retinal degeneration increased with longer exposures to light. An increase in retinal thickness was detected by SD-OCT on day 1 after light challenge followed by loss of the outer nuclear layer (ONL) by day 8. Degeneration was most severe in the nasal and inferior retina. Hyper-reflectivity on SD-OCT developed as early as 1 day after light exposure. Disorganization of the ONL, condensation of photoreceptor chromatin, disruption of the outer limiting membrane, and disarray of outer segments were associated with the hyper-reflectivity. Retraction of the outer plexiform synapses and resorption of the subretinal detachment contributed to retinal thinning. The RPE remained intact, whereas atrophied major retinal vessels were evident after light damage. Conclusions: Our time-course analysis of retinal degeneration in the I307N Rho mouse with SD-OCT and other outcome measures should enable the use of the mouse model in preclinical efficacy studies and mechanistic studies.

Adeno-Associated Virus Delivery of Viral Serpins for Ocular Diseases: Design and Validation.

Adeno-associated virus (AAV) has become the preferred viral gene transfer platform for ocular gene therapy due to its known safety profile in human clinical trials. This viral vector has a 4.7 kbp (kilo base pair) carrying capacity (single-stranded DNA) and only retains the inverted terminal repeats (ITRs) from the original virus. Here we describe the design and testing of AAV vectors capable of delivering an anti-inflammatory serine protease inhibitor (serpin) derived from the myxoma virus. Myxoma is a rabbit species specific virus infection, a Leporipoxvirus. Myxomaviral proteins have been developed as therapeutic stand-alone immune-modulating proteins for inflammation-based disorders and the myxoma virus itself is under development as a viral oncolytic platform for cancer treatment. We fused the Serp2 gene with the GFP reporter gene through a self-cleaving peptide.

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

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 IFNγ, TNFα, or IL-17A. R9-SOCS1-KIR and also protected against TNFα 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.

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.