RESUMEN

Recombinant adeno-associated viral vectors (rAAV) are the safest and most effective gene delivery platform to drive the treatment of many inherited eye disorders in well-characterized animal models. The use in rAAV of ubiquitous promoters derived from viral sequences such as CMV/CBA (chicken ß-actin promoter with cytomegalovirus enhancer) can lead to unwanted side effects such as pro-inflammatory immune responses and retinal cytotoxicity, thus reducing therapy efficacy. Thus, an advance in gene therapy is the availability of small promoters, that potentiate and direct gene expression to the cell type of interest, with higher safety and efficacy. In this study, we used six human mini-promoters packaged in rAAV2 quadruple mutant (Y-F) to test for transduction of the rat retina after intravitreal injection. After four weeks, immunohistochemical analysis detected GFP-labeled cells in the ganglion cell layer (GCL) for all constructs tested. Among them, Ple25sh1, Ple25sh2 and Ple53 promoted a widespread reporter-transgene expression in the GCL, with an increased number of GFP-expressing retinal ganglion cells when compared with the CMV/CBA vector. Moreover, Ple53 provided the strongest levels of GFP fluorescence in both cell soma and axons of retinal ganglion cells (RGCs) without any detectable adverse effects in retina function. Remarkably, a nearly 50-fold reduction in the number of intravitreally injected vector particles containing Ple53 promoter, still attained levels of transgene expression similar to CMV/CBA. Thus, the tested MiniPs show great potential for protocols of retinal gene therapy in therapeutic applications for retinal degenerations, especially those involving RGC-related disorders such as glaucoma.

Asunto(s)

Infecciones por Citomegalovirus , Células Ganglionares de la Retina , Ratas , Humanos , Animales , Células Ganglionares de la Retina/metabolismo , Vectores Genéticos , Retina/metabolismo , Transgenes , Inyecciones Intravítreas , Infecciones por Citomegalovirus/genética , Infecciones por Citomegalovirus/metabolismo , Dependovirus/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Transducción Genética

RESUMEN

Purpose: Based on our preview evidence that reduced nuclear content of the transcription factor Myc-associated protein X (MAX) is an early event associated with degeneration of retinal ganglion cells (RGCs), in the present study, our purpose was to test whether the overexpression of human MAX had a neuroprotective effect against RGC injury. Methods: Overexpression of either MAX or green fluorescent protein (GFP) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs). Lister Hooded rats were used in three models of RGC degeneration: (1) cultures of retinal explants for 30 hours ex vivo from the eyes of 14-day-old rats that had received intravitreal injections of rAAV2-MAX or the control vector rAAV2-GFP at birth; (2) an optic nerve crush model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were operated on; and (3) an ocular hypertension (OHT) glaucoma model, in which 1-month-old rats received intravitreal injection of either rAAV2-MAX or rAAV2-GFP and, 4 weeks later, were subject to cauterization of the limbal plexus. Cell death was estimated by detection of pyknotic nuclei and TUNEL technique and correlated with MAX immunocontent in an ex vivo model of retinal explants. MAX expression was detected by quantitative RT-PCR. In the OHT model, survival of RGCs was quantified by retrograde labeling with DiI or immunostaining for BRN3a at 14 days after in vivo injury. Functional integrity of RGCs was analyzed through pattern electroretinography, and damage to the optic nerve was examined in semithin sections. Results: In all three models of RGC insult, gene therapy by overexpression of MAX prevented RGC death. Also, ON degeneration and electrophysiologic deficits were prevented in the OHT model. Conclusions: Our experiments offer proof of concept for a novel neuroprotective gene therapy for glaucomatous neurodegeneration based on overexpression of MAX.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Regulación de la Expresión Génica , Terapia Genética/métodos , Glaucoma/complicaciones , Regeneración Nerviosa/genética , Enfermedades Neurodegenerativas/terapia , Neuroprotección/genética , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/biosíntesis , Muerte Celular , Modelos Animales de Enfermedad , Femenino , Glaucoma/genética , Glaucoma/patología , Masculino , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/genética , Ratas , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología

RESUMEN

After an injury, axons in the central nervous system do not regenerate over large distances and permanently lose their connections to the brain. Two promising approaches to correct this condition are cell and gene therapies. In the present work, we evaluated the neuroprotective and neuroregenerative potential of pigment epithelium-derived factor (PEDF) gene therapy alone and combined with human mesenchymal stem cell (hMSC) therapy after optic nerve injury by analysis of retinal ganglion cell survival and axonal outgrowth. Overexpression of PEDF by intravitreal delivery of AAV2 vector significantly increased Tuj1-positive cells survival and modulated FGF-2, IL-1ß, Iba-1, and GFAP immunostaining in the ganglion cell layer (GCL) at 4 weeks after optic nerve crush, although it could not promote axonal outgrowth. The combination of AAV2.PEDF and hMSC therapy showed a higher number of Tuj1-positive cells and a pronounced axonal outgrowth than unimodal therapy after optic nerve crush. In summary, our results highlight a synergistic effect of combined gene and cell therapy relevant for future therapeutic interventions regarding optic nerve injury.

Asunto(s)

Proteínas del Ojo/farmacología , Factores de Crecimiento Nervioso/farmacología , Traumatismos del Nervio Óptico/terapia , Células Ganglionares de la Retina/efectos de los fármacos , Serpinas/farmacología , Animales , Axones/fisiología , Línea Celular Tumoral , Supervivencia Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Modelos Animales de Enfermedad , Proteínas del Ojo/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Compresión Nerviosa , Factores de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa , Neuroprotección , Nervio Óptico , Ratas Wistar , Retina , Células Ganglionares de la Retina/metabolismo , Serpinas/metabolismo

RESUMEN

Adeno-associated virus vectors (rAAV) are currently the most common vehicle used in clinical trials of retinal gene therapy, usually delivered through subretinal injections to target cells of the outer retina. However, targeting the inner retina requires intravitreal injections, a simple and safe procedure, which is effective for transducing the rodent retina, but still of low efficiency in the eyes of primates. We investigated whether adjuvant pharmacological agents may enhance rAAV transduction of the retinas of mouse and rat after intravitreal delivery. Tyrosine kinase inhibitors were highly efficient in mice, especially imatinib and genistein, and promoted transduction even of the outer retina. In rats, however, we report that they were not effective. Even with direct proteasomal inhibition in rats, the effects upon transduction were only minimal and restricted to the inner retina. Even tyrosine capsid mutant rAAVs in rats had a transduction profile similar to wtAAV. Thus, the differences between mouse and rat, in both eye size and the inner limiting membrane, compromise the efficiency of AAV vectors penetration from the vitreous into the retina, and impact the efficacy of strategies developed to enhance intravitreal retinal rAAV transduction. Further improvement of strategies, then are required.

Asunto(s)

Adyuvantes Farmacéuticos/administración & dosificación , Dependovirus/genética , Vectores Genéticos/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Retina/virología , Animales , Electrorretinografía , Terapia Genética , Genisteína/administración & dosificación , Mesilato de Imatinib/administración & dosificación , Inyecciones Intravítreas , Ratones , Mutación , Ratas , Transducción Genética

RESUMEN

Brain ischemia is a major cause of adult disability and death, and it represents a worldwide health problem with significant economic burden for modern society. The identification of the molecular pathways activated after brain ischemia, together with efficient technologies of gene delivery to the CNS, may lead to novel treatments based on gene therapy. Recombinant adeno-associated virus (rAAV) is an effective platform for gene transfer to the CNS. Here, we used a serotype 8 rAAV bearing the Y733F mutation (rAAV8-733) to overexpress co-chaperone E3 ligase CHIP (also known as Stub-1) in rat hippocampal neurons, both in an oxygen and glucose deprivation model in vitro and in a four-vessel occlusion model of ischemia in vivo. We show that CHIP overexpression prevented neuronal degeneration in both cases and led to a decrease of both eIF2α (serine 51) and AKT (serine 473) phosphorylation, as well as reduced amounts of ubiquitinated proteins following hypoxia or ischemia. These data add to current knowledge of ischemia-related signaling in the brain and suggest that gene therapy based on the role of CHIP in proteostasis may provide a new venue for brain ischemia treatment.

Asunto(s)

Isquemia Encefálica/genética , Muerte Celular/genética , Dependovirus/genética , Vectores Genéticos/genética , Células Piramidales/metabolismo , Transducción Genética , Ubiquitina-Proteína Ligasas/genética , Animales , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Dependovirus/clasificación , Modelos Animales de Enfermedad , Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/administración & dosificación , Glucosa/metabolismo , Hipoxia/metabolismo , Oxígeno/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Piramidales/patología , Ratas , Ubiquitinación

RESUMEN

Endoplasmic reticulum (ER) stress and protein misfolding are associated with various neurodegenerative diseases. ER stress activates unfolded protein response (UPR), an adaptative response. However, severe ER stress can induce cell death. Here we show that the E3 ubiquitin ligase and co-chaperone Carboxyl Terminus HSP70/90 Interacting Protein (CHIP) prevents neuron death in the hippocampus induced by severe ER stress. Organotypic hippocampal slice cultures (OHSCs) were exposed to Tunicamycin, a pharmacological ER stress inducer, to trigger cell death. Overexpression of CHIP was achieved with a recombinant adeno-associated viral vector (rAAV) and significantly diminished ER stress-induced cell death, as shown by analysis of propidium iodide (PI) uptake, condensed chromatin, TUNEL and cleaved caspase 3 in the CA1 region of OHSCs. In addition, overexpression of CHIP prevented upregulation of both CHOP and p53 both pro-apoptotic pathways induced by ER stress. We also detected an attenuation of eIF2a phosphorylation promoted by ER stress. However, CHIP did not prevent upregulation of BiP/GRP78 induced by UPR. These data indicate that overexpression of CHIP attenuates ER-stress death response while maintain ER stress adaptative response in the central nervous system. These results indicate a neuroprotective role for CHIP upon UPR signaling. CHIP emerge as a candidate for clinical intervention in neurodegenerative diseases associated with ER stress.

Asunto(s)

Dependovirus/genética , Terapia Genética/métodos , Proteínas de Filamentos Intermediarios/genética , Glicoproteínas de Membrana/genética , Proteínas del Tejido Nervioso/genética , ARN Interferente Pequeño/genética , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/terapia , Animales , Modelos Animales de Enfermedad , Electrorretinografía , Genes Dominantes/genética , Células HEK293 , Humanos , Ratones , Periferinas , Células Fotorreceptoras de Vertebrados/fisiología , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología