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1.
Exp Eye Res ; 239: 109758, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38123011

ABSTRACT

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.


Subject(s)
Cytomegalovirus Infections , Retinal Ganglion Cells , Rats , Humans , Animals , Retinal Ganglion Cells/metabolism , Genetic Vectors , Retina/metabolism , Transgenes , Intravitreal Injections , Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/metabolism , Dependovirus/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Transduction, Genetic
2.
Curr Gene Ther ; 21(5): 362-381, 2021.
Article in English | MEDLINE | ID: mdl-33573569

ABSTRACT

Glaucoma leads to irreversible vision loss and current therapeutic strategies are often insufficient to prevent the progression of the disease and consequent blindness. Elevated intraocular pressure is an important risk factor, but not required for the progression of glaucomatous neurodegeneration. The demise of retinal ganglion cells represents the final common pathway of glaucomatous vision loss. Still, lifelong control of intraocular pressure is the only current treatment to prevent severe vision loss, although it frequently fails despite best practices. This scenario calls for the development of neuroprotective and pro-regenerative therapies targeting the retinal ganglion cells as well as the optic nerve. Several experimental studies have shown the potential of gene modulation as a tool for neuroprotection and regeneration. In this context, gene therapy represents an attractive approach as a persistent treatment for glaucoma. Viral vectors engineered to promote overexpression of a broad range of cellular factors have been shown to protect retinal ganglion cells and/or promote axonal regeneration in experimental models. Here, we review the mechanisms involved in glaucomatous neurodegeneration and regeneration in the central nervous system. Then, we point out the current limitations of gene therapy platforms and review a myriad of studies that use viral vectors to manipulate genes in retinal ganglion cells, as a strategy to promote neuroprotection and regeneration. Finally, we address the potential of combining neuroprotective and regenerative gene therapies as an approach to glaucomatous neurodegeneration.


Subject(s)
Glaucoma , Genetic Therapy , Glaucoma/genetics , Glaucoma/therapy , Humans , Intraocular Pressure , Neuroprotection , Retinal Ganglion Cells
3.
Mol Ther ; 25(2): 392-400, 2017 02 01.
Article in English | MEDLINE | ID: mdl-28153090

ABSTRACT

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.


Subject(s)
Brain Ischemia/genetics , Cell Death/genetics , Dependovirus/genetics , Genetic Vectors/genetics , Pyramidal Cells/metabolism , Transduction, Genetic , Ubiquitin-Protein Ligases/genetics , Animals , Brain Ischemia/metabolism , Brain Ischemia/pathology , Dependovirus/classification , Disease Models, Animal , Gene Expression , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors/administration & dosage , Glucose/metabolism , Hypoxia/metabolism , Oxygen/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Pyramidal Cells/pathology , Rats , Ubiquitination
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