Retinal Penetrating Adeno-Associated Virus.

Purpose: The most common method of delivery of genes to the outer retina uses recombinant adeno-associated virus (AAV) injected into the subretinal space using a surgical procedure. In contrast, most drugs are delivered to the retina using an intravitreal approach in an office setting. The objective of the current study was to develop AAV vectors that can reach the outer retina via intravitreal injection.

Cell-Penetrating Chaperone Nuc1 for Small- and Large-Molecule Delivery Into Retinal Cells and Tissues.

Purpose: There are currently no means available for the efficient delivery of recombinant proteins into retinal cells in vivo. Although cell-penetrating peptides have been somewhat effective in protein delivery to the retina, they generally require conjugation chemistry with the payload, negatively impacting function of the therapeutic protein. In this study, we developed a novel peptide (Nuc1) that acts as a chaperone for delivery of small and large molecules, including steroids, peptides, antibodies, recombinant proteins, and viruses (adeno-associated viruses [AAVs]) across biological membranes in vivo without the need for conjugation.

G-quartet oligonucleotide mediated delivery of functional X-linked inhibitor of apoptosis protein into retinal cells following intravitreal injection.

There is currently no efficient method available for the delivery of full length functional proteins into the cytoplasm of retinal cells in vivo. Historically, the most successful approach for the treatment of retinal diseases has been intravitreal injection of antibodies or recombinant proteins, but this approach is not yet utilized for the delivery of proteins that require intracellular access for a therapeutic effect. Here we describe a platform for the delivery of functional proteins into ganglion cells, photoreceptors and retinal pigment epithelium via intravitreal injection.

Complement-Mediated Activation of the NLRP3 Inflammasome and Its Inhibition by AAV-Mediated Delivery of CD59 in a Model of Uveitis.

Uveitis is an inflammatory disorder of the eye responsible for approximately 10%-15% of blindness in the US. In this study, we examined the role of the complement membrane attack complex (MAC) and the NLRP3 inflammasome in the pathogenesis of experimental autoimmune uveitis (EAU) in normal and C9 mice that are incapable of assembling the MAC. We discovered that the MAC and the NLRP3 inflammasome and associated production of IL-1β are elevated in EAU mice and that MAC may be involved in regulation of Th1 and Th17 cell differentiation.

Reducible PEG-POD/DNA Nanoparticles for Gene Transfer In Vitro and In Vivo: Application in a Mouse Model of Age-Related Macular Degeneration.

Non-viral gene delivery systems are being developed to address limitations of viral gene delivery. Many of these non-viral systems are modeled on the properties of viruses including cell surface binding, endocytosis, endosomal escape, and nuclear targeting. Most non-viral gene transfer systems exhibit little correlation between in vitro and in vivo efficiency, hampering a systematic approach to their development.

Tumor Necrosis Factor Inhibits Spread of Hepatitis C Virus Among Liver Cells, Independent From Interferons.

Background & Aims: Tumor necrosis factor (TNF) is an inflammatory cytokine expressed by human fetal liver cells (HFLCs) after infection with cell culture-derived hepatitis C virus (HCV). TNF has been reported to increase entry of HCV pseudoparticles into hepatoma cells and inhibit signaling by interferon alpha (IFNα), but have no effect on HCV-RNA replication. We investigated the effects of TNF on HCV infection of and spread among Huh-7 hepatoma cells and primary HFLCs.

G-quartet oligonucleotide mediated delivery of proteins into photoreceptors and retinal pigment epithelium via intravitreal injection.

There is currently no available method to efficiently deliver proteins across the plasma membrane of photoreceptor or retinal pigment epithelium (RPE) cells in vivo. Thus, current clinical application of recombinant proteins in ophthalmology is limited to the use of proteins that perform their biological function extracellularly. The ability to traverse biological membranes would enable the mobilization of a significantly larger number of proteins with previously well characterized properties.

Adenovirus-mediated delivery of Factor H attenuates complement C3 induced pathology in the murine retina: a potential gene therapy for age-related macular degeneration.

Background: Age-related macular degeneration (AMD) is the most common cause of blindness in the elderly, with no therapy available for 90% of patients. Recent genetic evidence implicates activation of complement in the pathogenesis of AMD. We have recently discovered that adenovirus (Ad)-mediated expression of complement component C3 (AdCMVC3) in the murine retina recapitulates many of the pathological features found in human AMD.

Topical application of a G-Quartet aptamer targeting nucleolin attenuates choroidal neovascularization in a model of age-related macular degeneration.

Choroidal neovascularization (CNV) associated with the 'wet' form of age related macular degeneration (AMD) is one of the most common causes of central vision loss among the elderly. The 'wet' form of AMD is currently treated by intravitreal delivery of anti-VEGF agents. However, intravitreal injections are associated with complications and long-term inhibition of VEGF leads to macular atrophy.

Slp-76 is a critical determinant of NK-cell mediated recognition of missing-self targets.

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying "missing-self" recognition, including the involvement of activating receptors, remain poorly understood. Using ethyl-N-nitrosourea mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell mediated recognition and elimination of "missing-self" targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation (Thr428Ile) in the SH2 domain of Slp-76-a critical adapter molecule downstream of ITAM-containing surface receptors.

Adeno-associated virus mediated delivery of an engineered protein that combines the complement inhibitory properties of CD46, CD55 and CD59.

Background: A variety of disorders are associated with the activation of complement. CD46, CD55 and CD59 are the major membrane associated regulators of complement on human cells. Previously, we have found that independent expression of CD55, CD46 or CD59 through gene transfer protects murine tissues against human complement mediated attack.

The Humoral Immune Response to HCV: Understanding is Key to Vaccine Development.

Hepatitis C virus (HCV) remains a global problem, despite advances in treatment. The low cost and high benefit of vaccines have made them the backbone of modern public health strategies, and the fight against HCV will not be won without an effective vaccine. Achievement of this goal will benefit from a robust understanding of virus-host interactions and protective immunity in HCV infection.

Immune control and failure in HCV infection--tipping the balance.

Despite the development of potent antiviral drugs, HCV remains a global health problem; global eradication is a long way off. In this review, we discuss the immune response to HCV infection and particularly, the interplay between viral strategies that delay the onset of antiviral responses and host strategies that limit or even eradicate infected cells but also contribute to pathogenesis. Although HCV can disable some cellular virus-sensing machinery, IFN-stimulated antiviral genes are induced in the infected liver.

Extended duration of transgene expression from pegylated POD nanoparticles enables attenuation of photoreceptor degeneration.

Retinitis pigmentosa (RP) is the most genetically heterogeneous disorder known to cause blindness, involving over 50 different genes. Previously, we have described nanoparticles (NPs) 150 nm in size, comprised of a 3.5 kD peptide (POD) complexed to PEG and DNA (PEGPOD DNA).

Adeno-associated virus mediated delivery of a non-membrane targeted human soluble CD59 attenuates some aspects of diabetic retinopathy in mice.

Diabetic retinopathy is the leading cause of visual dysfunction in working adults and is attributed to retinal vascular and neural cell damage. Recent studies have described elevated levels of membrane attack complex (MAC) and reduced levels of membrane associated complement regulators including CD55 and CD59 in the retina of diabetic retinopathy patients as well as in animal models of this disease. We have previously described the development of a soluble membrane-independent form of CD59 (sCD59) that when delivered via a gene therapy approach using an adeno-associated virus vector (AAV2/8-sCD59) to the eyes of mice, can block MAC deposition and choroidal neovascularization.

Aurintricarboxylic acid inhibits complement activation, membrane attack complex, and choroidal neovascularization in a model of macular degeneration.

Purpose: Immunocytochemical and genetic data implicate a significant role for the activation of complement in the pathology of AMD. Individuals homozygous for a Y402H polymorphism in Factor H have elevated levels of membrane attack complex (MAC) in their choroidal blood vessels and RPE relative to individuals homozygous for the wild-type allele. An R95X polymorphism in C9, a protein necessary for the final assembly of MAC, is partially protective against the formation of choroidal neovascularization (CNV) in AMD patients.

An ENU mutagenesis approach to dissect "self"-induced immune responses: Unraveling the genetic footprint of immunosurveillance.

The immune system exerts a critical function as it recognizes and eliminates transformed or neoplastic cells, a process also referred to as immunosurveillance. NK cells play a particularly important role in that they are able to recognize tumor cells via "missing-self"-i.e.

Soluble CD59 expressed from an adenovirus in vivo is a potent inhibitor of complement deposition on murine liver vascular endothelium.

Inappropriate activation of complement on the vascular endothelium of specific organs, or systemically, underlies the etiology of a number of diseases. These disorders include atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, atherosclerosis, age-related macular degeneration, diabetic retinopathy, and transplant rejection. Inhibition of the terminal step of complement activation, i.

A non membrane-targeted human soluble CD59 attenuates choroidal neovascularization in a model of age related macular degeneration.

Age related macular degeneration (AMD) is the most common cause of blindness amongst the elderly. Approximately 10% of AMD patients suffer from an advanced form of AMD characterized by choroidal neovascularization (CNV). Recent evidence implicates a significant role for complement in the pathogenesis of AMD.

Expression of complement component 3 (C3) from an adenovirus leads to pathology in the murine retina.

Purpose: Activation of complement has been implicated as one of the major causes of age-related macular degeneration (AMD). Evidence is accumulating for a role of complement in other retinal diseases, such as diabetic retinopathy and proliferative vitreoretinopathy. Because of the paucity of animal models that directly investigate the role of complement in retinal pathology, the authors sought to develop a model of increased complement expression and activation, specifically in the murine retina.

Nuclear targeted delivery of macromolecules to retina and cornea.

Background: Cell-penetrating peptides (CPPs) can deliver molecules into cells by binding and penetrating the plasma membrane. However, the majority of CPPs get trapped in endosomes, resulting in degradation of the cargo molecule and inefficient delivery to the nucleus. The present study investigates the potential use of a nucleolin binding peptide (NBP) for the delivery of macromolecules including fluorophores, recombinant protein and DNA to the nuclei of ocular tissues in vivo.

POD nanoparticles expressing GDNF provide structural and functional rescue of light-induced retinal degeneration in an adult mouse.

Peptide for ocular delivery (POD) is a novel cationic cell-penetrating peptide (CPP) which, when conjugated with polyethylene glycol (PEG-POD), can deliver plasmid DNA to the retinal pigment epithelium (RPE) of adult murine retina. PEG-POD nanoparticles containing an expression cassette for glial cell line-derived neurotrophic factor (PEG-POD~GDNF) were investigated for their ability to inhibit light-induced photoreceptor apoptosis. PEG-POD~GDNF, control nanoparticles, or buffer were injected into the subretinal space of adult murine retina and retinal degeneration induced by blue light.

Decay accelerating factor (CD55)-mediated attenuation of complement: therapeutic implications for age-related macular degeneration.

Purpose: Sequence variations in complement proteins are associated with age-related macular degeneration (AMD). The terminal pathway of complement results in the formation of the membrane attack complex (MAC) on the cell surface, resulting in their lysis. MAC has been documented on the retinal pigment epithelium (RPE), choroidal blood vessels, and drusen of AMD eyes.

Engineered zinc finger nuclease-mediated homologous recombination of the human rhodopsin gene.

Purpose: Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment.

Methods: Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America.

A poly(ethylene) glycolylated peptide for ocular delivery compacts DNA into nanoparticles for gene delivery to post-mitotic tissues in vivo.

Background: We have previously shown that a novel synthetic peptide for ocular delivery (POD) can efficiently compact DNA and deliver it to cells in vitro. This observation prompted us to develop use of POD as a nonviral vector in vivo.

Methods: POD peptide was modified using poly(ethylene) glycol (PEG-POD) and used to compact DNA into nanoparticles that were then analysed using electron microscopy, dynamic light scattering, and fluorescent labeling.