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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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. Methods: Nuc1 peptide was designed based on sequences known to bind heparan sulfate proteoglycans and nucleolin found on the surface of retinal cells. Nuc1 was injected into the vitreous of mice with a variety of molecules and retinas examined for uptake and function of these molecules. Results: Nuc1 engages the process of macropynocytosis for cell entry. The delivery of functional recombinant X-linked inhibitor of apoptosis protein to photoreceptors via the intravitreal route of injection inhibited retinal apoptosis. Nuc1 was found to enhance the delivery of anti-VEGF antibodies delivered intravitreally or topically in models of age-related macular degeneration (AMD). Nuc1 enhanced delivery of decorin, facilitating significant inhibition of neovascularization and fibrosis in a model of AMD. Finally, Nuc1 was found to enhance penetration of retinal cells and tissues by AAV via both the subretinal and intravitreal routes of injection. Conclusions: Nuc1 shows promise as a novel approach for the delivery of recombinant proteins into retinal cells in vivo.

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.].

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.

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. Animals injected with PEG-POD~GDNF showed a significant reduction (3.9-7.7 fold) in apoptosis relative to control-injected animals. The thickness of the outer nuclear layer (ONL) of the superior retina of PEG-POD~GDNF-injected eyes was significantly greater (23.6-39.3%) than control-injected retina 14 days post-light treatment. PEG-POD~GDNF-injected eyes showed a 27-39% greater functional response relative to controls, as measured by electroretinogram (ERG) 7 days post-light treatment. This is one of only two studies demonstrating histological and functional rescue of a mouse model of retinal degeneration following nonviral administration of a transgene into adult retina. Although rescue is short lived for clinical application, this study represents an important step in the development of nonviral gene therapy for retinal diseases.

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. Transfection efficiency and localization were determined 48 h post-injection into the subretinal space of the mouse eye using luciferase and LacZ, respectively. Efficiency of ocular transfection was compared to two other PEGylated peptides: PEG-TAT and PEG-CK30. RESULTS: PEG-POD can compact DNA and form discrete nanoparticles of approximately 136 nm that can penetrate and transduce the retinal pigment epithelium (RPE) in vivo. PEG-POD significantly increased expression of plasmid DNA by 215-fold, PEG-TAT by 56.52-fold, and PEG-CK30 by 24.73-fold relative to DNA injected alone. In all cases beta-galactosidase was observed primarily in the RPE layer after subretinal injection. Electrophysiological analyses of PEG-POD transduced retina indicates an absence of PEG-POD-mediated toxicity. PEG-POD can protect plasmid DNA from DNaseI digestion, resulting in significant transfection of the lung after intravenous injection in mice. CONCLUSIONS: PEG-POD was found to significantly increase gene delivery relative to both DNA alone and other pegylated peptides. These findings highlight the use of pegylated peptides, and specifically PEG-POD, as novel gene delivery vectors.

Cell-penetrating peptide for enhanced delivery of nucleic acids and drugs to ocular tissues including retina and cornea.

As in other organ systems, gene and drug delivery to ocular tissues such as the retina and cornea is hampered by inefficient penetration of therapeutic molecules across the plasma membrane. We describe the use of a novel peptide for ocular delivery (POD) with protein transduction properties, for delivery of small and large molecules across the plasma membrane. POD enters cells within 5 minutes in a temperature dependent manner. POD can compact and deliver plasmid DNA, achieving transgene expression in >50% of human embryonic retinoblasts. Delivery of small interfering RNA (siRNA) duplexes to cells using POD, allowed for silencing of transgene expression by >50%. POD could also be used to deliver quantum dots in vitro and in vivo. Upon ocular delivery, POD rapidly entered neural retina and localized to retinal pigment epithelium (RPE), photoreceptor, and ganglion cells. Additionally, POD was able to enter corneal epithelium, sclera, choroid, and the dura of the optic nerve via topical application. POD also functions as a bacteriostatic, a useful property for a carrier of molecules to post mitotic neural ocular tissues.

Barriers for retinal gene therapy: separating fact from fiction.

The majority of recent preclinical gene therapy studies targeting the retina have used adeno-associated virus (AAV) as the gene transfer vector. However, AAV has several limitations including the ability to generate innate inflammatory responses, the ability to cause insertional mutagenesis at a frequency of up to 56% in some tissues and a limited cloning capacity of 4.8Kb. Furthermore, AAV is known to generate limiting immune responses in humans despite the absence of similar immune responses in preclinical canine and murine studies. Three clinical trials to treat Leber's congenital amaurosis using AAV are under way. A clinical trial to treat Stargardt's using lentivirus vectors has also been recently announced. However, very limited evidence currently exists that lentivirus vectors can efficiently transduce photoreceptor cells. In contrast, very few preclinical ocular gene therapy studies have utilized adenovirus as the gene therapy vector. Nonetheless, the only two ocular gene therapy clinical trials performed to date have each used adenovirus as the vector and more significantly, in these published trials there has been no observed serious adverse event. These trials appear to be poised for Phase II/III status. Activation of cytotoxic T lymphocytes limits duration of transgene expression in the retina from first generation adenovirus vectors. However, an advanced class of adenovirus vectors referred to as Helper-dependent Adenovirus (Hd-Ad) have recently been shown to be capable of expressing transgenes in ocular tissues for more than one year. Hd-Ad vectors have many properties that potentially warrant their inclusion in the retinal gene therapy toolbox for the treatment of retinal degenerative diseases.

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.

Inhibition of choroidal neovascularization by adenovirus-mediated delivery of short hairpin RNAs targeting VEGF as a potential therapy for AMD.

PURPOSE: Choroidal neovascularization (CNV) is the leading cause of blindness in age-related macular degeneration (AMD). Several lines of evidence implicate increased levels of vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE) from patients with AMD. Current approaches to attenuate VEGF or its receptors, including the use of small interfering (si)RNA, show significant promise, but still have limited efficacy and require repeat administrations, using procedures associated with multiple complications. The goal of this study was to develop an approach for long-term endogenous expression of short hairpin (sh)RNA that would significantly attenuate VEGF and hence act as a potential therapy for AMD. METHODS: Several shRNAs expressed from recombinant adenovirus were developed. These shRNAs were expressed in human RPE cells in the presence of adenovirus vectors overexpressing VEGF, and the amount of VEGF attenuation was evaluated. Adenovirus vectors expressing VEGF were subsequently injected into the subretinal space of mice, and induction of CNV was measured in the presence of adenovirus vectors expressing shRNA targeting VEGF. RESULTS: Potent shRNA sequences were identified that were able to silence VEGF in human RPE cells. When expressed from adenovirus backbones, these shRNA constructs silenced VEGF by 94% at a 1:5 molar ratio (VEGF to shRNA) and 64% at a 1:0.05 molar ratio. Adenovirus vectors expressing high levels of VEGF could induce CNV in mice within 5 days. Co-injection of VEGF-expressing viruses into mice with shRNA targeting VEGF led to a substantial (84%) reduction in CNV. CONCLUSIONS: shRNA targeting VEGF from adenovirus vectors allows potent attenuation of VEGF and prevents CNV. This approach shows promise as a therapy for AMD.

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.