RESUMO
The pathogenic mutation S163R in C1QTNF5 causes a disorder known as autosomal dominant late-onset retinal degeneration (L-ORD), characterized by the presence of thick extracellular sub-RPE deposits, similar histopathologically to those found in AMD patients. We have previously shown that the S163R C1QTNF5 mutant forms globular aggregates within the RPE in vivo following its AAV-mediated expression in the RPE and exhibits a reversely polarized distribution, being routed toward the basal rather than apical RPE. We show here that when both wild-type and mutant S163R C1QTNF5 are simultaneously delivered subretinally to mouse RPE cells, the mutant impairs the wild-type protein secretion from the RPE, and both proteins are dispersed toward the basal and lateral RPE membrane. This result has mechanistic and therapeutic implications for L-ORD disorder.
Assuntos
Degeneração Macular/genética , Mutação de Sentido Incorreto , Mutação Puntual , Agregação Patológica de Proteínas/genética , Epitélio Pigmentado da Retina/metabolismo , Animais , Polaridade Celular , Colágeno/química , Colágeno/genética , Colágeno/metabolismo , Dependovirus/genética , Eletrorretinografia , Genes Dominantes , Vetores Genéticos , Humanos , Injeções Intraoculares , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Agregação Patológica de Proteínas/patologia , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Epitélio Pigmentado da Retina/ultraestrutura , Frações Subcelulares/químicaRESUMO
Adeno-associated virus (AAV) effectively targets therapeutic genes to photoreceptors, pigment epithelia, Müller glia and ganglion cells of the retina. To date, no one has shown the ability to correct, with gene replacement, an inherent defect in bipolar cells (BCs), the excitatory interneurons of the retina. Targeting BCs with gene replacement has been difficult primarily due to the relative inaccessibility of BCs to standard AAV vectors. This approach would be useful for restoration of vision in patients with complete congenital stationary night blindness (CSNB1), where signaling through the ON BCs is eliminated due to mutations in their G-protein-coupled cascade genes. For example, the majority of CSNB1 patients carry a mutation in nyctalopin (NYX), which encodes a protein essential for proper localization of the TRPM1 cation channel required for ON BC light-evoked depolarization. As a group, CSNB1 patients have a normal electroretinogram (ERG) a-wave, indicative of photoreceptor function, but lack a b-wave due to defects in ON BC signaling. Despite retinal dysfunction, the retinas of CSNB1 patients do not degenerate. The Nyx(nob) mouse model of CSNB1 faithfully mimics this phenotype. Here, we show that intravitreally injected, rationally designed AAV2(quadY-F+T-V) containing a novel 'Ple155' promoter drives either GFP or YFP_Nyx in postnatal Nyx(nob) mice. In treated Nyx(nob) retina, robust and targeted Nyx transgene expression in ON BCs partially restored the ERG b-wave and, at the cellular level, signaling in ON BCs. Our results support the potential for gene delivery to BCs and gene replacement therapy in human CSNB1.
Assuntos
Dependovirus/genética , Oftalmopatias Hereditárias/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Vetores Genéticos , Miopia/genética , Cegueira Noturna/genética , Proteoglicanas/genética , Células Bipolares da Retina/metabolismo , Animais , Modelos Animais de Doenças , Oftalmopatias Hereditárias/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Humanos , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Miopia/metabolismo , Cegueira Noturna/metabolismo , Regiões Promotoras Genéticas , Retina/metabolismo , Transfecção , Transgenes , Visão OcularRESUMO
The rd6 mouse is a natural model of an RPE-based (retinal pigment epithelium) autosomal recessive retinitis pigmentosa (RP) caused by mutations in the Mfrp (membrane-type frizzled related protein) gene. Previously, we showed that subretinal delivery of the wild-type mouse Mfrp mediated by a tyrosine-capsid mutant scAAV8 (Y733F) vector prevented photoreceptor cell death, and rescued retinal function as assessed by electroretinography. In this study, we describe the effect of gene therapy on the retinal structure and function in rd6 mice using a quadruple (Y272, 444, 500, 730F) tyrosine-capsid mutant scAAV2 viral vector delivered subretinally at postnatal day 14 (P14). We show that therapy is effective at slowing the photoreceptor degeneration, and in preventing the characteristic accumulation of abnormal phagocytic cells in the subretinal space. MFRP expression as driven by the ubiquitous chicken ß-actin (smCBA) promoter in treated rd6 mice was found predominantly in the RPE apical membrane and the entire length of its microvilli, as well as in the photoreceptor inner segments, suggesting a potential interaction with actin filaments. In spite of preserving retinal morphology, the effects of gene therapy on retinal function were minimal, suggesting that the scAAV8 (Y733F) vector may be more efficient for the treatment of RP caused by Mfrp mutations.
Assuntos
Dependovirus/genética , Proteínas do Olho/genética , Terapia Genética/métodos , Proteínas de Membrana/genética , Degeneração Retiniana/terapia , Retinose Pigmentar/terapia , Animais , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Retinose Pigmentar/genética , Retinose Pigmentar/patologiaRESUMO
Vectors based on adeno-associated virus serotype 2 (AAV2) have been used extensively in many gene-delivery applications, including several successful clinical trials for one type of Leber congenital amaurosis in the retina. Many studies have focused on improving AAV2 transduction efficiency and cellular specificity by genetically engineering its capsid. We have previously shown that vectors-containing single-point mutations of capsid surface tyrosines in serotypes AAV2, AAV8, and AAV9 displayed significantly increased transduction efficiency in the retina compared with their wild-type counterparts. In the present study, we evaluated the transduction characteristics of AAV2 vectors containing combinations of multiple tyrosine to phenylalanine mutations in seven highly conserved surface-exposed capsid tyrosine residues following subretinal or intravitreal delivery in adult mice. The multiply mutated vectors exhibited different in vivo transduction properties, with some having a unique ability of transgene expression in all retinal layers. Such novel vectors may be useful in developing valuable new therapeutic strategies for the treatment of many genetic diseases.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Retina/metabolismo , Tirosina/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Mutação , Mutação Puntual/genética , Retina/patologiaRESUMO
Rod and cone photoreceptors use similar but distinct sets of phototransduction proteins to achieve different functional properties, suitable for their role as dim and bright light receptors, respectively. For example, rod and cone visual pigments couple to distinct variants of the heterotrimeric G protein transducin. However, the role of the structural differences between rod and cone transducin alpha subunits (Talpha) in determining the functional differences between rods and cones is unknown. To address this question, we studied the translocation and signaling properties of rod Talpha expressed in cones and cone Talpha expressed in rods in three mouse strains: rod Talpha knockout, cone Talpha GNAT2(cpfl3) mutant, and rod and cone Talpha double mutant rd17 mouse. Surprisingly, although the rod/cone Talpha are only 79% identical, exogenously expressed rod or cone Talpha localized and translocated identically to endogenous Talpha in each photoreceptor type. Moreover, exogenously expressed rod or cone Talpha rescued electroretinogram responses (ERGs) in mice lacking functional cone or rod Talpha, respectively. Ex vivo transretinal ERG and single-cell recordings from rd17 retinas treated with rod or cone Talpha showed comparable rod sensitivity and response kinetics. These results demonstrate that cone Talpha forms a functional heterotrimeric G protein complex in rods and that rod and cone Talpha couple equally well to the rod phototransduction cascade. Thus, rod and cone transducin alpha-subunits are functionally interchangeable and their signaling properties do not contribute to the intrinsic light sensitivity differences between rods and cones. Additionally, the technology used here could be adapted for any such homologue swap desired.
Assuntos
Proteínas do Olho/química , Proteínas do Olho/fisiologia , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Animais , Eletrorretinografia , Potenciais Evocados Visuais , Proteínas do Olho/genética , Proteínas Heterotriméricas de Ligação ao GTP/deficiência , Proteínas Heterotriméricas de Ligação ao GTP/genética , Camundongos , Camundongos Knockout , Camundongos Mutantes , Estimulação Luminosa , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Modern electronic devices, such as smartphones and electric vehicles, require multilayer ceramic capacitors (MLCCs), which comprise highly pure Cu terminations and Ni electrodes. Vapor-phase synthesis (VPS) is a promising method for synthesizing nanoparticles (NPs) with high purity and crystallinity. However, the agglomeration of the NPs occurs during their synthesis, which degrades the performance of the MLCC electrodes owing to several factors, including electrical shorts and low packing density. This paper proposes a coating-assisted VPS to inhibit agglomeration using potassium chloride (KCl) as the coating agent. The agglomeration ratio of the Cu NPs synthesized by in-flight coating with KCl at 950 °C significantly decreased from 48.20% to 3.80%, compared to without KCl coating. Furthermore, X-ray fluorescence and X-ray diffraction analyses confirmed that the KCl coating agent and residual copper chloride were removed by washing with ammonium hydroxide.
RESUMO
In this study, we investigated the effect of the sulfur content in the NiCl2 precursor on the shape of nickel nanoparticles (Ni-NPs) prepared by chemical vapor synthesis. We obtained spherical Ni-NPs when using anhydrous NiCl2 mixed with NiSO4 or Na2SO4 with a molar ratio of 0.002 as precursors without changing any other process parameters whereas faceted Ni-NPs when using only anhydrous NiCl2 as a precursor. First-principles calculations supported experimental results, which showed that NiSO4-mixed NiCl2 and Na2SO4-mixed NiCl2 precursors favored the growth of spherical NPs.
RESUMO
Nickel (Ni) nanoparticles (NPs) prepared through vapor-phase synthesis (VPS) are preferred for multilayer ceramic capacitor electrodes due to their high purity and crystallinity advantages. Agglomerated Ni NPs are usually generated using VPS but are undesirable because they cause various problems such as low packing density and electrical shorts. This study proposes the use of coating-assisted chemical vapor synthesis (CVS) for agglomerate inhibition using NaCl or KCl as a coating agent. We have found that the agglomeration ratio, 34.40%, for conventional CVS, can be reduced to 4.80% in the proposed method by in-flight coating with KCl at 900 °C by image analysis using field-emission scanning electron microscopy. Furthermore, the X-ray diffraction and X-ray fluorescence analyses confirm that the NaCl and KCl coating agent can be removed by washing with distilled water. We believe that this coating process can be used to inhibit the formation of agglomerates during the CVS of Ni NPs.
RESUMO
Vectors derived from adeno-associated viruses (AAVs) have become important gene delivery tools for the treatment of many inherited ocular diseases in well-characterized animal models. Previous studies have determined that the viral capsid plays an essential role in the cellular tropism and efficiency of transgene expression. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome- mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo. Because the tyrosine residues are highly conserved in other AAV serotypes, in this study we evaluated the intraocular transduction characteristics of vectors containing point mutations in surface- exposed capsid tyrosine residues in AAV serotypes 2, 8, and 9. Several of these novel AAV mutants were found to display a strong and widespread transgene expression in many retinal cells after subretinal or intravitreal delivery compared with their wild-type counterparts. For the first time, we show efficient transduction of the ganglion cell layer by AAV serotype 8 or 9 mutant vectors, thus providing additional tools besides AAV2 for targeting these cells. These enhanced AAV vectors have a great potential for future therapeutic applications for retinal degenerations and ocular neovascular diseases.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Retina/metabolismo , Transgenes/genética , Animais , Dependovirus/classificação , Cistos Glanglionares , Vetores Genéticos/administração & dosagem , Injeções , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Tirosina/genética , Tirosina/metabolismoRESUMO
In this study, the sintering behaviors of Nb-6Mo-20Si-3Cr (at percentage) in situ composite powders were studied. The Nb alloy powder was fabricated by a hydrogenation-dehydrogenation method, and both the alloy ingot and powders consisted of two phases: An Nb metal phase and the α-Nb5Si3 phase. Consolidation of the alloy powders was performed at 1500, 1600, and 1700 °C using spark plasma sintering, and the microstructures and phases formed at various sintering temperatures were analyzed. Micropores were observed in the compact sintered at 1500 °C due to the lack of complete densification at that temperature. The densification was completed at 1600 °C and the microstructure was slightly coarsened at 1700 °C compared to the microstructure of the compact sintered at 1600 °C. The microstructures prepared by the powder metallurgy method were finer than the microstructure of the ingot prepared by the casting method. The phase formation behavior varied according to the sintering temperature. Specifically, the α-Nb5Si3 phase, which is a stable structure of the Nb5Si3 phase at a low temperature, was transformed to the ß-Nb5Si3 phase (which is stable at a high temperature) with an increasing sintering temperature.
RESUMO
Optogenetics methods are rapidly being developed as therapeutic tools for treating neurological diseases, in particular, retinal degenerative diseases. A critical component of the development is testing the safety of the light stimulation used to activate the optogenetic proteins. While the stimulation needs to be sufficient to produce neural responses in the targeted retinal cell class, it also needs to be below photochemical and photothermal limits known to cause ocular damage. The maximal permissible exposure is determined by a variety of factors, including wavelength, exposure duration, visual angle, pupil size, pulse width, pulse pattern, and repetition frequency. In this paper, we develop utilities to systematically and efficiently assess the contributions of these parameters in relation to the limits, following directly from the 2014 American National Standards Institute (ANSI). We also provide an array of stimulus protocols that fall within the bounds of both safety and effectiveness. Additional verification of safety is provided with a case study in rats using one of these protocols.
Assuntos
Córnea/efeitos da radiação , Optogenética/métodos , Estimulação Luminosa , Retina/efeitos da radiação , Degeneração Retiniana/terapia , Próteses Visuais , Animais , Proteínas do Olho/metabolismo , Humanos , Luz , Ratos , Ratos Long-EvansRESUMO
Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken ß-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.
Assuntos
Terapia Genética , Proteínas de Membrana/biossíntese , Degeneração Retiniana/genética , Síndromes de Usher/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Camundongos , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/patologia , Degeneração Retiniana/terapia , Síndromes de Usher/patologia , Síndromes de Usher/terapiaRESUMO
Recombinant adeno-associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of retinal degeneration in a variety of animal models that mimic corresponding human diseases. AAV vectors possess a number of features that render them ideally suited for retinal gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. In the sheltered environment of the retina, AAV vectors are able to maintain high levels of transgene expression in the retinal pigmented epithelium (RPE), photoreceptors, or ganglion cells for long periods of time after a single treatment. Each cell type can be specifically targeted by choosing the appropriate combination of AAV serotype, promoter, and intraocular injection site. The focus of this review is on examples of AAV-mediated gene therapy in those animal models of inherited retinal degeneration caused by mutations directly affecting the interacting unit formed by photoreceptors and the RPE. In each case discussed, expression of the therapeutic gene resulted in significant recovery of retinal structure and/or visual function. Because of the key role of the vasculature in maintaining a healthy retina, a summary of AAV gene therapy applications in animal models of retinal neovascular diseases is also included.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Doenças Retinianas/terapia , Animais , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Células Fotorreceptoras de Vertebrados/metabolismo , Epitélio Pigmentado Ocular/patologia , Epitélio Pigmentado Ocular/fisiologia , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Doenças Retinianas/genética , Doenças Retinianas/patologia , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/fisiologiaRESUMO
BACKGROUND: Adeno associated virus (AAV) is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV's ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC) of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc) genomes in the anterior segment of the eye. METHODOLOGY/PRINCIPLE FINDINGS: AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE), iris and chamber angle including trabecular meshwork, with scAAV2(Y444F) and scAAV2(triple) being the most efficient. CONCLUSIONS/SIGNIFICANCE: This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene-based therapies for glaucoma and acquired or inherited corneal anomalies.
Assuntos
Câmara Anterior/virologia , Capsídeo/metabolismo , Dependovirus/genética , Mutação/genética , Malha Trabecular/metabolismo , Transdução Genética , Animais , Vetores Genéticos , Proteínas de Fluorescência Verde , Injeções , Camundongos Endogâmicos C57BL , Ratos Sprague-DawleyRESUMO
PURPOSE: The mutation S163R in complement C1q tumor necrosis factor-related protein-5 (C1QTNF5) causes an autosomal dominant disorder known as late-onset retinal degeneration (L-ORD). In this study, our goal is to evaluate the consequences of mutant S163R C1QTNF5 expression in mouse RPE following its delivery using an adeno-associated viral (AAV) vector. METHODS: We generated AAV vectors containing either human wild-type C1QTNF5 or mutant S163R C1QTNF5 driven by an RPE-specific BEST1 promoter, and delivered them subretinally into one eye of adult C57BL/6 mice. Transgene expression was detected by immunohistochemistry. Retinal function was assessed by full-field ERG. Pathological changes were further examined by digital fundus imaging and spectral-domain optical coherence tomography (SD-OCT). RESULTS: We show that the AAV-expressed mutant S163R leads to pathological effects similar to some of those found in patients with advanced L-ORD, including RPE thinning, RPE cell loss, and retinal degeneration. In addition, we provide in vivo evidence that mutant S163R C1QTNF5 can form large, transparent, spherical intracellular aggregates throughout the RPE, which are detectable by light microscopy. In contrast to AAV-expressed wild-type C1QTNF5, which is secreted apically from the RPE toward the photoreceptor cells and the outer limiting membrane, the S163R mutant is primarily routed toward the basal side of RPE, where it forms thick, extracellular deposits over time. CONCLUSIONS: Adeno-associated viral-targeted expression of mutant S163R in the RPE represents a useful approach for quickly generating animal models that mimic pathological features of L-ORD and offers the potential to understand disease mechanisms and develop therapeutic strategies.
Assuntos
Proteínas de Membrana/genética , Epitélio Pigmentado da Retina/patologia , Animais , Bestrofinas , Western Blotting , Proteínas do Olho/genética , Fundo de Olho , Expressão Gênica , Canais Iônicos/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mutação de Sentido Incorreto , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestrutura , Tomografia de Coerência ÓpticaRESUMO
Mutations in GUCY2D are the cause of Leber congenital amaurosis type 1 (LCA1). GUCY2D encodes retinal guanylate cyclase-1 (retGC1), a protein expressed exclusively in outer segments of photoreceptors and essential for timely recovery from photoexcitation. Recent clinical data show that, despite a high degree of visual disturbance stemming from a loss of cone function, LCA1 patients retain normal photoreceptor architecture, except for foveal cone outer segment abnormalities and, in some patients, foveal cone loss. These results point to the cone-rich central retina as a target for GUCY2D replacement. LCA1 gene replacement studies thus far have been conducted in rod-dominant models (mouse) or with vectors and organisms lacking clinical translatability. Here we investigate gene replacement in the Nrl(-/-) Gucy2e(-/-) mouse, an all-cone model deficient in retGC1. We show that AAV-retGC1 treatment fully restores cone function, cone-mediated visual behavior, and guanylate cyclase activity, and preserves cones in treated Nrl(-/-) Gucy2e(-/-) mice over the long-term. A novel finding was that retinal function could be restored to levels above that in Nrl(-/-) controls, contrasting results in other models of retGC1 deficiency. We attribute this to increased cyclase activity in treated Nrl(-/-) Gucy2e(-/-) mice relative to Nrl(-/-) controls. Thus, Nrl(-/-) Gucy2e(-/-) mice possess an expanded dynamic range in ERG response to gene replacement relative to other models. Lastly, we show that a candidate clinical vector, AAV5-GRK1-GUCY2D, when delivered to adult Nrl(-/-) Gucy2e(-/-) mice, restores retinal function that persists for at least 6 months. Our results provide strong support for clinical application of a gene therapy targeted to the cone-rich, central retina of LCA1 patients.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas do Olho/genética , Guanilato Ciclase/genética , Amaurose Congênita de Leber/terapia , Receptores de Superfície Celular/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos , Guanilato Ciclase/metabolismo , Injeções Intraoculares , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Opsinas/genética , Opsinas/metabolismo , Receptores de Superfície Celular/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Resultado do Tratamento , Visão OcularRESUMO
Basic transcription element binding (BTEB1) protein is one of at least 20 Sp/KLF family members that function as transcriptional activators or repressors by binding to GC/GT-rich sequences within target genes to influence cellular homeostasis in mammals. Previously, we demonstrated that increased expression of BTEB1 in a human endometrial epithelial cell line Hec-1-A resulted in serum dependent-enhanced proliferation, which was accompanied by heightened expression of cell cycle- and growth-associated genes. In the present study, we examined the mechanism underlying the altered proliferative potential associated with BTEB1 by the identification of additional BTEB1 downstream gene targets and by the demonstration of BTEB1 transactivation of promoters for a number of growth-associated genes. Using mRNA differential display in the analysis of RNA populations from Hec-1-A sublines with high (4S, 9S) and low (2As, 3As) BTEB1 cellular content, we identified 10 distinct differentially expressed transcripts, nine of which had higher levels in S than in As sublines. The expression levels of two of these cDNAs, Axl receptor tyrosine kinase and mitosin, whose encoded products are implicated in cellular proliferation, were modestly induced by serum, albeit in a BTEB1-independent manner. Moreover, insulin-like growth factor-I, a mitogen present in serum, had no significant effect on their expression in either subline. In transient reporter assays, the basal activities of the Axl gene promoter and those for two other growth-regulatory genes, namely p21(WAF1) and IGFBP-2, were increased by serum and were significantly higher in 4S than in 2As lines. However, while BTEB1 and its ubiquitous family member Sp1 increased basal p21(WAF1) and IGFBP-2 transcription when added as expression constructs in the parental Hec-1-A cell line, only Sp1 activated Axl transcription, despite the presence in all three gene promoters of GC-enriched regions that presumably can bind BTEB1 and Sp1 with similar affinities. To elucidate intracellular signaling pathways that might involve BTEB1, inhibitors of specific kinase-dependent transducers were used in transient transfection assays involving the IGFBP-2 gene promoter in 4S and 2As sublines. While inhibitors of the MAPK, PI-3K, and PKA pathways elicited similar effects on the IGFBP-2 gene promoter activity, irrespective of cellular BTEB1 content, that for JNK had a more pronounced effect on Hec-1-A sublines exhibiting higher BTEB1 expression levels. Taken together, the results suggest that BTEB1 mediates the expression of growth-associated genes through direct and indirect transactivation mechanisms, one of which may involve the participation of a JNK family member.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Endométrio/citologia , Fatores de Transcrição/fisiologia , Ativação Transcricional , Animais , Biomarcadores , Carcinoma/patologia , Divisão Celular/genética , Proteínas Cromossômicas não Histona/biossíntese , Proteínas Cromossômicas não Histona/genética , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Neoplasias do Endométrio/patologia , Células Epiteliais/citologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fator de Crescimento Insulin-Like I/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Fatores de Transcrição Kruppel-Like , Proteínas dos Microfilamentos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Família Multigênica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , Ratos , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Transativadores/biossíntese , Transativadores/genética , Fatores de Transcrição/genética , Células Tumorais Cultivadas/citologia , Receptor Tirosina Quinase AxlRESUMO
Tunable threshold resistive switching characteristics of Pt-Fe(2)O(3) core-shell nanoparticle (NP) assembly were investigated. The colloidal Pt-Fe(2)O(3) core-shell NPs with a Pt core diameter of â¼3 nm and a total diameter of â¼15 nm were chemically synthesized by a one-step process. These NPs were assembled as a layer with a thickness of â¼80 nm by repeated dip-coating between Ti and Pt electrodes on a flexible polyethersulfone (PES) substrate. The Ti/NPs/Pt/PES structure exhibited the threshold switching, i.e. volatile transition from high to low resistance state at a high voltage and vice versa at a low voltage. The current-voltage measurements after charging and discharging NPs revealed that the resistance state and threshold switching voltage of the assembly could be tuned by the space charges stored in high density trap sites of Pt cores in Pt-Fe(2)O(3) core-shell NP assembly. These results demonstrated the possible tuning of threshold switching of core-shell NP assembly by the space charge effect, which can be potentially utilized for the tunable selection device element in nonvolatile memory circuits.
RESUMO
Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY-F+T-V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY-F+T-V) -hGRK1-GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.
Assuntos
Capsídeo/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Mutação/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Animais , Linhagem Celular , Galinhas , Dependovirus/fisiologia , Receptor Quinase 1 Acoplada a Proteína G/genética , Expressão Gênica , Humanos , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/metabolismo , Regiões Promotoras Genéticas/genética , Sorotipagem , Transdução Genética , Transgenes/genética , Tropismo ViralRESUMO
Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated adeno-associated virus (AAV)-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical- and subcortical-based visually-guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic wild-type (WT) mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1.