Electrochemical Performance of High-Valence Mo6+ and Low-Valence Mn2+ Doped- Na3 V2 (PO4 )3 @C Cathode for Sodium-Ion Batteries.

The sodium superionic conductor (NASICON)-Na3 V2 (PO4 )3 (NVP) is an attractive cathode for sodium-ion batteries, which is still confronted with limited rate performance due to its low electronic conductivity. In this paper, a chemical strategy is adopted to partially replace V3+ of the NVP framework by low-valence Mn2+ and high-valence Mo6+ substitution. The crystal structure, sodium-ion diffusion coefficient and electrochemical performance of Mn-Mo-doped [Na3.94 V0.98 Mo0.02 Mn(PO4 )3 @C] cathode were investigated. X-ray diffraction confirmed the NASICON-type structure and XPS analysis confirmed the oxidation state of Mn and Mo in doped NVP cathode. The Na ion diffusion processes were inferred from Cyclic Voltammetry (CV), Galvanostatic intermittent titration technique (GITT) and Electrochemical Impedance Spectroscopy (EIS) measurement, which clearly show rapid Na-ion diffusion in NASICON-type cathode materials. The Mn-Mo-substituted NVP shows smoother charge-discharge profiles, improved rate performance (64.80 mAh/g at 1 C rate), better energy density (308.61 mWh/g) and superior Na-ion kinetics than that of unsubstituted NVP@C cathode. Their enhanced performance is attributed to large interstitial volume mainly created by high valence Mo6+ and enhanced capacity is attributed to the low valence Mn2+ doping. These results demonstrate that Mn-Mo-doped NVP cathode is strongly promising cathode material for sodium-ion batteries.

Infantile Aphakic Glaucoma: A Proposed Mechanism.

PURPOSE: To review information pertaining to secondary glaucoma following infant lensectomy and provide evidence to support the mechanism responsible for this condition. METHODS: Reported risk factors and proposed mechanisms for infantile aphakic glaucoma are reviewed. Laboratory studies and clinical observations in affected patients with glaucoma are described. Evidence of postoperative anterior chamber fibrosis is reviewed and interpreted. RESULTS: Clinical evidence demonstrated the development of anterior chamber fibrosis following infant cataract surgery. Laboratory studies showed liberated lens epithelial cell transition to fibroblasts. CONCLUSIONS: The review and assessment of laboratory and clinical evidence support the proposal that infantile aphakic glaucoma is caused, in part, by postoperative anterior chamber fibroization related to lens cell dispersion, cytokine activation, and epithelial-mesenchymal transition with resultant filtration angle fibrosis and secondary loss of filtration function. [J Pediatr Ophthalmol Strabismus. 2022;59(4):236-242.].

The role of complement membrane attack complex in dry and wet AMD - From hypothesis to clinical trials.

Data from human dry and wet age-related macular degeneration (AMD) eyes support the hypothesis that constant 'tickover' of the alternative complement pathway results in chronic deposition of the complement membrane attack complex (MAC) on the choriocapillaris and the retinal pigment epithelium (RPE). Sub-lytic levels of MAC lead to cell signaling associated with tissue remodeling and the production of cytokines and inflammatory molecules. Lytic levels of MAC lead to cell death. CD59 is a naturally occurring inhibitor of the assembly of MAC. CD59 may thus be therapeutically efficacious against the pathophysiology of dry and wet AMD. The first gene therapy clinical trial for geographic atrophy - the advanced form of dry AMD has recently completed recruitment. This trial is studying the safety and tolerability of expressing CD59 from an adeno-associated virus (AAV) vector injected once into the vitreous. A second clinical trial assessing the efficacy of CD59 in wet AMD patients is also under way. Herein, the evidence for the role of MAC in the pathophysiology of dry as well as wet AMD and the scientific rationale underlying the use of AAV- delivered CD59 for the treatment of dry and wet AMD is discussed.

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. A nucleolin binding aptamer, AS1411, was biotinylated and complexed with traptavidin and utilized as a platform for the delivery of GFP or X-linked inhibitor of apoptosis (XIAP) proteins by intravitreal injection in BALB/c mice. Retinal sections were analyzed for uptake of proteins in the retina. Apoptosis was induced by intravitreal injection of N-methyl-D-aspartate (NMDA). Retinas were harvested for analysis of TUNEL and caspase 3/7 activity. Intravitreal injection of AS1411-directed GFP or XIAP complexes enabled delivery of these proteins into ganglion cells, photoreceptors and retinal pigment epithelium in vivo. AS1411-XIAP complexes conferred significant protection to cells in the outer and inner nuclear layers following NMDA induced apoptosis. A concomitant decrease in activity of Caspase 3/7 was observed in eyes injected with the AS1411-XIAP complex. In conclusion, AS1411 can be used as a platform for the delivery of therapeutic proteins into retinal cells. This approach can potentially be utilized to introduce a large variety of therapeutically relevant proteins that are previously well characterized to maintain the structural integrity and function of retina, thus, preventing vision loss due to ocular trauma or inherited retinal degeneration.

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-1ß are elevated in EAU mice and that MAC may be involved in regulation of Th1 and Th17 cell differentiation. In contrast, MAC and the NLRP3 inflammasome were not elevated in C9-/- mice. However, EAU-associated pathophysiology including retinal structure and function were not rescued in C9-/- mice. Unexpectedly, AAV-mediated delivery of sCD59, an inhibitor of C9 incorporation into the MAC, successfully attenuated activation of the NLRP3 inflammasome and EAU pathology as well as MAC. Our studies provide an improved understanding of the role of the MAC and the NLRP3 inflammasome in EAU as well as suggest a novel approach for the treatment of uveitis.

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. Previously, we have described a 3.5 kDa peptide (peptide for ocular delivery [POD]) that targets cell surface sialic acid. When functionalized with polyethylene glycol (PEG) via a sulfhydryl group on the N-terminal cysteine of POD, PEG-POD could compact plasmid DNA, forming 120- to 180-nm homogeneous nanoparticles. PEG-POD enabled modest gene transfer and rescue of retinal degeneration in vivo. Systematic investigation of different stages of gene transfer by PEG-POD nanoparticles was hampered by their inability to deliver genes in vitro. Herein, we describe functionalization of POD with PEG using a reducible orthopyridyl disulfide bond. These reducible nanoparticles enabled gene transfer in vitro while retaining their in vivo gene transfer properties. These reducible PEG-POD nanoparticles were utilized to deliver human FLT1 to the retina in vivo, achieving a 50% reduction in choroidal neovascularization in a murine model of age-related macular degeneration.

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. Nucleolin is abundantly present on the surface of rapidly dividing cells including cancer cells. Surprisingly, nucleolin is also present on the surface of photoreceptor cell bodies. Here we investigated whether nucleolin can be utilized as a gateway for the delivery of proteins into retinal cells following intravitreal injection. AS1411 is a G-quartet aptamer capable of targeting nucleolin. Subsequent to intravitreal injection, fluorescently labeled AS1411 localized to various retinal cell types including the photoreceptors and RPE. AS1411 linked to streptavidin (a ∼50 kDa protein) via a biotin bridge enabled the uptake of Streptavidin into photoreceptors and RPE. AS1411-Streptavidin conjugate applied topically to the cornea allowed for uptake of the conjugate into the nucleus and cytoplasm of corneal endothelial cells. Clinical relevance of AS1411 as a delivery vehicle was strongly indicated by demonstration of the presence of cell surface nucleolin on the photoreceptors, inner neurons and ganglion cells of human retina. These data support exploration of AS1411 as a means of delivering therapeutic proteins to diseased retina.

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. Thus, there is currently an unmet need for the development of therapies for CNV that target molecules other than VEGF. Here, we describe nucleolin as a novel target for the 'wet' form of AMD. Nucleolin was found on the surface of endothelial cells that migrate from the choroid into the subretinal space in the laser-induced model of 'wet' AMD. AS1411 is a previously described G-quartet oligonucleotide that has been shown to bind nucleolin. We found that AS1411 inhibited the formation of tubes by human umbilical vein endothelial cells (HUVECs) by approximately 27.4% in vitro. AS1411 co-localized with the site of laser induced CNV in vivo. Intravitreally injected AS1411 inhibited laser-induced CNV by 37.6% and attenuated infiltration of macrophages by 40.3%. Finally, topical application of AS1411 led to a 43.4% reduction in CNV. Our observations have potential implications for the development of therapies for CNV and specifically for the 'wet' form of AMD.

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. In the present study, utilizing a gene therapy approach, we examine whether Ad-mediated expression of complement Factor H (AdCAGfH) attenuates AdCMVC3-mediated retinal pathology. METHODS: AdCMVC3 was co-injected with either AdCAGfH or a negative control virus expressing green fluorescent protein (AdCMVGFP) into the subretinal space of adult mice. The resulting retinal pathology was analyzed by histology and immunocytochemistry and retinal function was quantified by electroretinography. RESULTS: Morphological and functional analyses indicated that AdCMVC3-mediated retinal pathology could be attenuated by AdCAGfH. Specifically, endothelial cell proliferation was reduced by 91% and atrophy of retinal pigment epithelium (RPE) could be attenuated by 69%. AdCAGfH injected eyes exhibited 90-150% greater A-wave and 120-180% greater B-wave amplitudes relative to control eyes. Immunocytochemical analysis of rhodopsin and RPE65 was consistent with the rescue of photoreceptors and RPE in AdCAGfH injected eyes. CONCLUSIONS: C3-induced pathology in murine retina can be attenuated by Ad-mediated expression of Factor H. Expression of Factor H is worthy of further study as a potential gene therapy for AMD.

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. In the present study, we investigated the potential of combining the complement regulatory properties of CD46, CD55 and CD59 into single gene products expressed from an adeno-associated virus (AAV) vector in a soluble non-membrane anchored form. METHODS: Minigenes encoding the complement regulatory domains from CD46, CD55 and CD59 (SACT) or CD55 and CD59 (DTAC) were cloned into an AAV vector. The specific regulatory activity of each component of SACT and DTAC was measured in vitro. The recombinant AAV vectors were injected into the peritoneum of mice and the efficacy of the transgene products for being able to protect murine liver vasculature against human complement, specifically the membrane attack complex (MAC), was measured. RESULTS: SACT and DTAC exhibited properties similar to CD46, CD55 and CD59 or CD55 and CD59, respectively, in vitro. AAV mediated delivery of SACT or DTAC protected murine liver vasculature from human MAC deposition by 63.2% and 56.7%, respectively. CONCLUSIONS: When delivered to mice in vivo via an AAV vector, SACT and DTAC are capable of limiting human complement mediated damage. SACT and DTAC merit further study as potential therapies for complement mediated disorders when delivered via a gene therapy approach.

Domain contributions to signaling specificity differences between Ras-guanine nucleotide releasing factor (Ras-GRF) 1 and Ras-GRF2.

Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2) constitute a family of similar calcium sensors that regulate synaptic plasticity. They are both guanine exchange factors that contain a very similar set of functional domains, including N-terminal pleckstrin homology, coiled-coil, and calmodulin-binding IQ domains and C-terminal Dbl homology Rac-activating domains, Ras-exchange motifs, and CDC25 Ras-activating domains. Nevertheless, they regulate different forms of synaptic plasticity. Although both GRF proteins transduce calcium signals emanating from NMDA-type glutamate receptors in the CA1 region of the hippocampus, GRF1 promotes LTD, whereas GRF2 promotes θ-burst stimulation-induced LTP (TBS-LTP). GRF1 can also mediate high frequency stimulation-induced LTP (HFS-LTP) in mice over 2-months of age, which involves calcium-permeable AMPA-type glutamate receptors. To add to our understanding of how proteins with similar domains can have different functions, WT and various chimeras between GRF1 and GRF2 proteins were tested for their abilities to reconstitute defective LTP and/or LTD in the CA1 hippocampus of Grf1/Grf2 double knock-out mice. These studies revealed a critical role for the GRF2 CDC25 domain in the induction of TBS-LTP by GRF proteins. In contrast, the N-terminal pleckstrin homology and/or coiled-coil domains of GRF1 are key to the induction of HFS-LTP by GRF proteins. Finally, the IQ motif of GRF1 determines whether a GRF protein can induce LTD. Overall, these findings show that for the three forms of synaptic plasticity that are regulated by GRF proteins in the CA1 hippocampus, specificity is encoded in only one or two domains, and a different set of domains for each form of synaptic plasticity.

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). These NPs expressing GDNF enabled rescue of photoreceptor degeneration in mice up to 11 days post injection. In the current study we examine use of scaffold/ matrix attachment regions (S/MARs), CpG depletion and titration of DNA content of PEGPOD DNA NPs to extend the duration of transgene expression. S/MARs and CpGs did not significantly influence the duration of transgene expression, but did influence its stability. These parameters enabled us to extend transgene expression from 48 hours to 10 weeks. At 77 days post injection, we observed a 76% rescue of the thickness of the retinal outer nuclear layer (ONL) and at 37 days post injection we observed 53% and 55% rescue of the A and B wave ERG amplitudes respectively and 60% rescue of the ONL. Our studies suggest that PEGPOD DNA NPs have potential as gene delivery vectors for the retina.

Topical application of PPADS inhibits complement activation and choroidal neovascularization in a model of age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly. AMD patients have elevated levels of membrane attack complex (MAC) in their choroidal blood vessels and retinal pigment epithelium (RPE). MAC forms pores in cell membranes. Low levels of MAC result in an elevation of cytokine release such as vascular endothelial growth factor (VEGF) that promotes the formation of choroidal neovascularization (CNV). High levels of MAC result in cell lysis and RPE degeneration is a hallmark of advanced AMD. The current standard of care for CNV associated with wet AMD is intravitreal injection of anti-VEGF molecules every 4 to 12 weeks. Such injections have significant side effects. Recently, it has been found that membrane pore-forming proteins such as α-haemolysin can mediate their toxic effects through auto- and paracrine signaling and that complement-induced lysis is amplified through ATP release followed by P2X receptor activation. We hypothesized that attenuation of P2X receptor activation may lead to a reduction in MAC deposition and consequent formation of CNV. Hence, in this study we investigated topical application of the purinergic P2X antagonist Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) as a potential treatment for AMD. We found that 4.17 µM PPADS inhibited formation of HUVEC master junctions and master segments by 74.7%. In a human complement mediated cell lysis assay, 104 µM PPADS enabled almost complete protection of Hepa1c1c7 cells from 1% normal human serum mediated cell lysis. Daily topical application of 4.17 mM PPADS for 3 days attenuated the progression of laser induced CNV in mice by 41.8% and attenuated the deposition of MAC at the site of the laser injury by 19.7%. Our data have implications for the future treatment of AMD and potentially other ocular disorders involving CNV such as angioid streaks, choroidal rupture and high myopia.

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. Aurintricarboxylic Acid (ATA) is a small molecule inhibitor of MAC. Our hypothesis was that attenuation of the formation of MAC on ocular tissues by ATA may protect mice against laser-induced CNV. METHODS: The ability of ATA to inhibit human complement-mediated cell lysis, inhibit formation of human MAC, and inhibit formation of tubes by endothelial cells was examined in vitro. Subsequently, the Bruch's membrane of adult mice was damaged using an argon laser, followed by intravitreal injection of ATA. One week later, choroidal flat mounts from these mice were stained for the presence of MAC, endothelial cells, and macrophages. RESULTS: ATA protects cells from human complement-mediated lysis, attenuates assembly of the MAC, and inhibits tube formation by endothelial cells in vitro. ATA also attenuates CNV, MAC deposition, and macrophage infiltration in a murine model of exudative AMD. CONCLUSIONS: ATA warrants further study as a potential drug for the treatment of exudative and nonexudative AMD.

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. Here, we examine AAV2/8-sCD59 mediated attenuation of MAC deposition and ensuing complement mediated damage to the retina of mice following streptozotocin (STZ) induced diabetes. We observed a 60% reduction in leakage of retinal blood vessels in diabetic eyes pre-injected with AAV2/8-sCD59 relative to negative control virus injected diabetic eyes. AAV2/8-sCD59 injected eyes also exhibited protection from non-perfusion of retinal blood vessels. In addition, a 200% reduction in retinal ganglion cell apoptosis and a 40% reduction in MAC deposition were documented in diabetic eyes pre-injected with AAV2/8-sCD59 relative to diabetic eyes pre-injected with the control virus. This is the first study characterizing a viral gene therapy intervention that targets MAC in a model of diabetic retinopathy. Use of AAV2/8-sCD59 warrants further exploration as a potential therapy for advanced stages of diabetic retinopathy.

Acquisition of contextual discrimination involves the appearance of a RAS-GRF1/p38 mitogen-activated protein (MAP) kinase-mediated signaling pathway that promotes long term potentiation (LTP).

RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

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.e. formation of the membrane attack complex, using CD59 has the advantage of retaining the upstream processes of the complement cascade necessary for fighting pathogens and retaining complement's crucial role in tissue homeostasis. Previous studies have shown the necessity of membrane targeting of soluble CD59 in order for it to prove an effective inhibitor of complement deposition both in vitro and in vivo. In this study we have generated an in vivo model of human complement activation on murine liver vascular endothelium. This model should prove useful for the development of anti-complement therapies for complement-induced pathologies of vascular endothelium. Using this model, we have demonstrated the viability of a non membrane-targeted soluble CD59 to significantly inhibit complement deposition on the endothelium of murine liver vasculature when expressed in vivo from an adenovirus. This result, unanticipated based on prior studies, suggests that the use of non membrane-targeted sCD59 as an anti-complement therapy be re-visited.

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. Activation of complement terminates in the incorporation of the membrane attack complex (MAC) in biological membranes and subsequent cell lysis. Elevated levels of MAC have been documented on choroidal blood vessels and retinal pigment epithelium (RPE) of AMD patients. CD59 is a naturally occurring membrane bound inhibitor of MAC formation. Previously we have shown that membrane bound human CD59 delivered to the RPE cells of mice via an adenovirus vector can protect those cells from human complement mediated lysis ex vivo. However, application of those observations to choroidal blood vessels are limited because protection from MAC- mediated lysis was restricted only to the cells originally transduced by the vector. Here we demonstrate that subretinal delivery of an adenovirus vector expressing a transgene for a soluble non-membrane binding form of human CD59 can attenuate the formation of laser-induced choroidal neovascularization and murine MAC formation in mice even when the region of vector delivery is distal to the site of laser induced CNV. Furthermore, this same recombinant transgene delivered to the intravitreal space of mice by an adeno-associated virus vector (AAV) can also attenuate laser-induced CNV. To our knowledge, this is the first demonstration of a non-membrane targeting CD59 having biological potency in any animal model of disease in vivo. We propose that the above approaches warrant further exploration as potential approaches for alleviating complement mediated damage to ocular tissues in 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. METHODS: The authors constructed a recombinant adenovirus-expressing murine complement component 3 (C3, AdcmvC3). Adult mice were injected in the subretinal space with either AdcmvC3 or a control virus, AdcmvGFP. After 1 to 2 weeks of exogenous C3 expression, mice were analyzed by scotopic electroretinography and fluorescein angiography. Eyes were harvested for histologic, immunohistochemical, and quantitative RT-PCR analyses. RESULTS: Mice injected with C3-expressing adenovirus exhibited significantly increased vascular permeability, endothelial cell proliferation and migration, RPE atrophy, loss of photoreceptor outer segments, reactive gliosis, retinal detachment, and reduced retinal function relative to those injected with a control adenovirus. Deposition of the membrane attack complex was observed on endothelial cells and photoreceptor outer segments. CONCLUSIONS: Adenovirus-mediated delivery of C3 to murine RPE induces significant functional and anatomic changes that reproduce many of the features of AMD as well as those of other retinal diseases. This novel model may be useful in assessing the role of complement in retinal pathology and in developing anti-complement therapies for retinal diseases associated with complement activation.

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. METHODS: Fluorescent dyes covalently linked to NBP or NBP-green fluorescent protein fusion protein were injected intravitreally or subretinally or topically applied to the cornea. Frozen sections were prepared for quantification of transduction. Delivery of plasmid DNA was studied using luciferase and LacZ DNA compacted with pegylated NBP. Levels of luciferase were quantified, and LacZ expression was localized in ocular tissues. RESULTS: We found that NBP-directed fluorophores exhibited retinal and corneal transduction. Subretinal injection transduced cell types throughout the retina, including photoreceptors, retinal pigment epithelium and neuronal cells. Intravitreal injection transduced neuronal cells in the retina, as well as cells in the cornea. Topically applied NBP lead to transduction of the superficial epithelial layer of the cornea. NBP localized to the nucleus upon exogenous application in vivo. Pegylated NBP nanoparticles significantly improved delivery and expression of transgenes over DNA alone without any measureable toxicity. CONCLUSIONS: The results obtained in the present study demonstrate that NBP can deliver small and large molecules into retinal and corneal cells and plasmid DNA into retinal cells and hence may be useful for the delivery of therapeutics to the eye.