RESUMO
A resurgence of interest and investment in the field of gene therapy, driven in large part by advances in viral vector technology, has recently culminated in United States Food and Drug Administration approval of the first gene therapy product targeting a disease caused by mutations in a single gene. This product, LUXTURNA™ (voretigene neparvovec-rzyl; Spark Therapeutics, Inc., Philadelphia, PA), delivers a normal copy of the RPE65 gene to retinal cells for the treatment of biallelic RPE65 mutation-associated retinal dystrophy, a blinding disease. Many additional gene therapy programs targeting both inherited retinal diseases and other ocular diseases are in development, owing to an improved understanding of the genetic basis of ocular disease and the unique properties of the ocular compartment that make it amenable to local gene therapy. Here we review the growing body of literature that describes both the design and development of ocular gene therapy products, with a particular emphasis on target and vector selection, and chemistry, manufacturing, and controls.
Assuntos
Dependovirus/química , Desenvolvimento de Medicamentos/métodos , Técnicas de Transferência de Genes/normas , Terapia Genética/métodos , Doenças Retinianas/terapia , Animais , Dependovirus/genética , Dependovirus/isolamento & purificação , Composição de Medicamentos , Vetores Genéticos/administração & dosagem , Humanos , Degeneração Macular/tratamento farmacológico , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/genética , Doenças Retinianas/patologiaRESUMO
Topical delivery of therapeutics to the posterior segment of the eye remains the "holy grail" of ocular drug delivery. As an example, anti-vascular endothelial growth factor biologics, such as ranibizumab, aflibercept, and bevacizumab, are delivered by intravitreal injection to treat neovascular age-related macular degeneration and, although these drugs have revolutionized treatment of the disease, less invasive alternatives to intravitreal injection are desired. Multiple reports in the literature have demonstrated topical delivery of both small and large molecules to the back of the eye in small animal models. Despite this progress, successful translation to larger species, and ultimately humans, has yet to be demonstrated. Selection of animal models with relevant ocular anatomy and physiology, along with appropriate experimental design, is critical to enable more relevant feasibility assessments and increased probability of successful translation.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Olho/metabolismo , Administração Tópica , Animais , Bevacizumab/administração & dosagem , Bevacizumab/química , Química Farmacêutica , Humanos , Injeções Intravítreas , Degeneração Macular/tratamento farmacológico , Ranibizumab/administração & dosagem , Ranibizumab/química , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Receptores de Fatores de Crescimento do Endotélio Vascular/química , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Pesquisa Translacional Biomédica , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Selective killing of RPE cells in vivo by sodium iodate develops cardinal phenotypes of atrophic age-related macular degeneration. However, the molecular mechanisms are elusive. We tried to search for small cyto-protective molecules against sodium iodate and explore their mechanisms of action. Sodium iodate-mediated RPE cell death was associated with increased levels of reactive oxygen species (ROS) and IL-8. Resveratrol, a natural occurring polyphenol compound, was found to strongly protect RPE cells from sodium iodate with inhibition of production of ROS and IL-8. Resveratrol activated all isoforms of PPARs. Treatment with PPARα and PPARδ agonists inhibited sodium iodate-induced ROS production and protected RPE cells from sodium iodate. A PPARα antagonist significantly reduced resveratrol's protection of RPE cells from sodium iodate. Paradoxically, knocking down PPARδ also rendered RPE cells resistant to sodium iodate. Moreover, PPAR agonists reversed sodium iodate-induced production of IL-8. However, neutralizing extracellular IL-8 failed to protect RPE cells from sodium iodate. Taken together, these observations show that resveratrol protects RPE cells from sodium iodate injury through the activation of PPARα and alteration of PPARδ conformation. PPARα and δ modulators might ameliorate stress-induced RPE degeneration in vivo.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Iodatos/toxicidade , Degeneração Macular/psicologia , PPAR alfa/genética , PPAR delta/genética , RNA/genética , Estilbenos/farmacologia , Inibidores da Angiogênese , Antioxidantes/farmacologia , Células Cultivadas , Citoproteção , Humanos , Degeneração Macular/induzido quimicamente , Degeneração Macular/genética , PPAR alfa/biossíntese , PPAR delta/biossíntese , ResveratrolRESUMO
Efficient phagocytosis of photoreceptor outer segments (POS) by retinal pigment epithelial cells (RPE) plays a key role in biological renewal of these highly peroxidizable structures and in maintenance of retina health. Here, we used an in vitro RPE cell phagocytosis assay to investigate how sub-lethal oxidative stress modifies the key components of the cell phagocytic machinery leading to severe impairment of phagocytosis. Sub-lethal oxidative treatment, induced by hydrogen peroxide (H(2)O(2)), significantly inhibited binding and uptake of POS by RPE cells. However, sub-lethal oxidative stress did not affect cell surface expression of αvß5 or RPE cell adhesion to αvß5. Similarly, the enzymatic activity of mature cathepsin D was not altered upon challenge by oxidative stress. In contrast, studies of signaling molecules in the RPE cell phagocytic machinery revealed that sub-lethal oxidative stress inhibits POS-induced activation of FAK and MerTK. Our data demonstrate that sub-lethal oxidative treatment with H(2)O(2) inhibits phagocytic activity of ARPE-19 cells, in part by inhibiting FAK and MerTK.
Assuntos
Quinase 1 de Adesão Focal/fisiologia , Fagocitose/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Vitronectina/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Catepsina D/metabolismo , Adesão Celular , Linhagem Celular , Sobrevivência Celular , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Peróxido de Hidrogênio/toxicidade , Immunoblotting , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , c-Mer Tirosina QuinaseRESUMO
Dying cells release pro-inflammatory molecules, functioning as cytokines to trigger cell/tissue inflammation that is relevant to disease pathology. Heat-shock protein 90 (HSP90) is believed to act as a danger signal for tissue damage once released extracellularly. Potential roles of HSP90 were explored in retinal pigment epithelial (RPE) inflammatory responses to necrosis. Cellular extracts can trigger ARPE-19 cell inflammatory responses, producing cytokines that lead to an increase in ARPE-19 cell monolayer permeability. Addition of recombinant HSP90ß mimics the induction of chemokines IL-8 and MCP-1 in cultured RPE cells, suggesting that released HSP90 can incite RPE cell sterile inflammatory responses. Consistent with this, classical HSP90 inhibitors were shown to substantially reduce necrosis-induced cytokine production and permeability increases in ARPE-19 cells. Moreover, a cell-impermeable inhibitor, 17-N,N-dimethylaminoethylamino-17-demethoxy-geldanamycin-N-oxide, also efficiently inhibited necrosis-induced cytokine production and TNF-α/IL-1ß-induced increase in ARPE-19 cell permeability in vitro and endotoxin-induced development of uveitis in vivo, suggesting that HSP90 can contribute to necrosis-induced RPE inflammatory responses. Collectively, our data identify HSP90 as a pro-inflammatory molecule in RPE cell sterile inflammatory responses.
Assuntos
Anti-Inflamatórios/farmacologia , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Compostos Heterocíclicos com 2 Anéis/farmacologia , Mediadores da Inflamação/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Pirazóis/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Uveíte/prevenção & controle , Animais , Anti-Inflamatórios/metabolismo , Benzoquinonas/metabolismo , Linhagem Celular , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Compostos Heterocíclicos com 2 Anéis/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lactamas Macrocíclicas/metabolismo , Lipopolissacarídeos , Masculino , Necrose , Permeabilidade , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/metabolismo , Ratos , Ratos Endogâmicos Lew , Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo , Uveíte/induzido quimicamente , Uveíte/imunologia , Uveíte/metabolismo , Uveíte/patologiaRESUMO
Lipid peroxidation products such as 4-hydroxy-2-nonenal (4-HNE) cause dysfunction and death of retinal pigmented epithelial (RPE) cells, thereby leading to retinal degeneration. The molecular mechanisms underlying their action remain elusive however. In this study, the roles of AMP-activated protein kinase (AMPK) in 4-HNE-induced RPE cell dysfunction and viability were addressed. 4-HNE caused RPE cell death and down-regulated basal activity of AMPK as evidenced by decreased Thr(172) phosphorylation of AMPKα. Exposure of RPE cells to the AMPK inhibitor, compound C also led to cell death, indicating that RPE cell death is correlated with 4-HNE modulation of AMPK activity. ARPE19 cells express both AMPKα1 and AMPKα2 with predominant expression of the AMPKα1 isoform. siRNA studies revealed that knockdown of AMPKα1 expression sensitized RPE cells to 4-HNE. Intriguingly, knockdown of AMPKα2 protected RPE cells from 4-HNE injury. Sub-lethal doses of 4-HNE induced an increase in RPE monolayer permeability, as measured by reduction in trans-epithelial resistance (TER). Knockdown of AMPKα2 but not AMPKα1 significantly restored RPE cell barrier function. No further protection was observed by knockdown of both AMPKα1 and AMPKα2. In contrast, knockdown of AMPKα1 and/or AMPKα2 did not reverse the 4-HNE's inhibitory effects on production of IL-8 and MCP-1. These data demonstrate that AMPKα1 and AMPKα2 play distinct roles in regulating 4-HNE effects on RPE function and viability. Therefore, selective modulation of AMPKα activity may benefit patients with retinal degeneration associated with RPE cell atrophy.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Aldeídos/toxicidade , Inibidores de Cisteína Proteinase/toxicidade , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Morte Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Citocinas/metabolismo , Regulação para Baixo , Impedância Elétrica , Inativação Gênica/fisiologia , Humanos , Peróxido de Hidrogênio/toxicidade , Peroxidação de Lipídeos/fisiologia , Fosforilação , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Epitélio Pigmentado da Retina/enzimologia , TransfecçãoRESUMO
Purpose: DARPin molecules are a novel class of small proteins that contain engineered ankyrin repeat domain(s) and bind to target proteins with high specificity and affinity. Abicipar-pegol (abicipar), a DARPin molecule targeting vascular endothelial growth factor-A (VEGF-A), is currently under evaluation in patients with age-related macular degeneration. The pharmacodynamic properties of abicipar were characterized using in vivo and in vitro assays. Methods: The binding affinity of abicipar was assessed using a kinetic exclusion assay (KinExA). In vitro assays evaluated abicipar effects on VEGF-A165-induced calcium mobilization and tube formation in human umbilical vein endothelial cells. Abicipar was tested in vivo in a mouse model of corneal neovascularization and a rabbit model of chronic retinal neovascularization. The efficacies of abicipar and ranibizumab were compared in a rabbit model of VEGF-A165-induced retinal vasculopathy. Results: Abicipar has a high affinity for the soluble isoforms of VEGF-A; binding affinities for human VEGF-A165 are approximately 100-fold greater than those of ranibizumab and bevacizumab and are similar for rat VEGF-A164 but approximately 20-fold lower for rabbit VEGF-A165. Abicipar was effective in cell-based and in vivo models of angiogenesis and vascular leak, blocking neovascularization in a mouse model of corneal neovascularization and vascular permeability in a rabbit model of chronic neovascularization. In a rabbit model of VEGF-A165-induced vasculopathy, the duration of effect of abicipar was longer than ranibizumab when the two compounds were administered at molar-equivalent doses. Conclusions: These data support the testing of abicipar as a treatment for retinal diseases characterized by neovascularization and vascular leak.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Permeabilidade Capilar/efeitos dos fármacos , Neovascularização da Córnea/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Proteínas Recombinantes de Fusão/uso terapêutico , Neovascularização Retiniana/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Bevacizumab/uso terapêutico , Vasos Sanguíneos/fisiopatologia , Cálcio/metabolismo , Neovascularização da Córnea/fisiopatologia , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Coelhos , Ranibizumab/uso terapêutico , Neovascularização Retiniana/fisiopatologiaRESUMO
PURPOSE: To investigate the role of the peroxisome proliferator-activated receptor (PPAR)-γ in modulating retinal pigmented epithelium (RPE) responses to oxidative stress. METHODS: ARPE-19 cells were treated with the oxidant, t-butylhydroperoxide (tBH) to induce apoptosis. Cells pretreated with synthetic PPARγ agonists of the antidiabetic thiazolidinediones class before tBH challenge were assessed for viability and, by microarray analysis, for effects on gene expression. RESULTS: Treatment of ARPE-19 cells with tBH resulted in a loss of viability and global changes in the pattern of gene expression. PPARγ ligands were found to have differential modulatory effects on tBH-induced apoptosis of RPE cells. Whereas rosiglitazone and pioglitazone potentiated cell death, troglitazone acted as a potent cytoprotective agent. Downregulation of PPARγ expression by an siRNA resulted in enhanced cell death in response to tBH treatment and blocked the cytoprotective effect of troglitazone consistent with a role of PPARγ in mediating this response. Microarray analysis revealed that while rosiglitazone and pioglitazone had little effect on gene changes induced by tBH treatment, troglitazone dramatically reduced the number of changes caused by oxidative stress. A unique subset of genes that were deregulated by tBH and selectively normalized by troglitazone were identified. CONCLUSIONS: These findings demonstrate that PPARγ agonists can have differential effects on RPE survival in response to oxidative stress. Oxidative stress leads to deregulation of a large set of genes in ARPE-19 cells. A specific subset of these genes can be selectively modulated by troglitazone and represent potential novel targets for cytoprotective therapies.
Assuntos
Apoptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/metabolismo , Epitélio Pigmentado da Retina/patologia , Tiazolidinedionas/farmacologia , Western Blotting , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular , Sinergismo Farmacológico , Inativação Gênica/fisiologia , Humanos , Ligantes , Análise em Microsséries , PPAR gama/agonistas , Pioglitazona , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rosiglitazona , Transfecção , terc-Butil Hidroperóxido/toxicidadeRESUMO
Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The etiology of AMD remains poorly understood and no treatment is currently available for the atrophic form of AMD. Atrophic AMD has been proposed to involve abnormalities of the retinal pigment epithelium (RPE), which lies beneath the photoreceptor cells and normally provides critical metabolic support to these light-sensing cells. Cumulative oxidative stress and local inflammation are thought to represent pathological processes involved in the etiology of atrophic AMD. Studies of tissue culture and animal models reveal that oxidative stress-induced injury to the RPE results in a chronic inflammatory response, drusen formation, and RPE atrophy. RPE degeneration in turn causes a progressive degeneration of photoreceptors, leading to the irreversible loss of vision. This review describes some of the potential major molecular and cellular events contributing to RPE death and inflammatory responses. In addition, potential target areas for therapeutic intervention will be discussed and new experimental therapeutic strategies for atrophic AMD will be presented.