Suchergebnisse

Purpose: The use of small non-coding nucleic acids, such as siRNA and miRNA, has allowed for a deeper understand-ing of gene functions, as well as for development of gene therapies for complex neurodegenerative diseases, including retinal degeneration. For effective delivery into the ePurpose: The use of small non-coding nucleic acids, such as siRNA and miRNA, has allowed for a deeper understanding of gene functions, as well as for development of gene therapies for complex neurodegenerative diseases, including retinal degeneration. For effective delivery into the eye and transfection of the retina, suitable transfection methods are required. We investigated the use of a lipid-based transfection agent, Invivofectamine® 3.0 (Thermo Fisher Scientific), as a potential method for delivery of nucleic acids to the retina. Methods: Rodents were injected intravitreally with formulations of Invivofectamine 3.0 containing scrambled, Gapdh, Il-1β, and C3 siRNAs, or sterile PBS (control) using a modified protocol for encapsulation of nucleic acids. TdT-mediated dUTP nick-end labeling (TUNEL) and IBA1 immunohistochemistry was used to determine histological cell death and inflammation. qPCR were used to determine the stress and inflammatory profile of the retina. Electroretinography (ERG) and optical coherence tomography (OCT) were employed as clinical indicators of retinal health. Results: We showed that macrophage recruitment, retinal stress, and photoreceptor cell death in animals receiving Invivofectamine 3.0 were comparable to those in negative controls. Following delivery of Invivofectamine 3.0 alone, no statistically significant changes in expression were found in a suite of inflammatory and stress genes, and ERG and OCT analyses revealed no changes in retinal function or morphology. Injections with siRNAs for proinflammatory genes (C3 and Il-1β) and Gapdh, in combination with Invivofectamine 3.0, resulted in statistically significant targeted gene knockdown in the retina for up to 4 days following injection. Using a fluorescent Block-It siRNA, transfection was visualized throughout the neural retina with evidence of transfection observed in cells of the ganglion cell layer, inner nuclear layer, and outer nuclear layer. Conclusions: This work supports the use of Invivofectamine 3.0 as a transfection agent for effective delivery of nucleic acids to the retina for gene function studies and as potential therapeutics. ; This study was partly supported through funding from the Thermo Fisher Scientific Applications and Claims Expansions Grant with two of the authors (NA and XM) members of the Thermo Fisher Scientific team. Other funding bodies are The National Health and Medical Research Council (APP1127705, 2017-2019), the Australian Government Research Training Program Scholarship and the Australian National University Translational Fellowship. The authors do not have any other competing interests or conflicts of interest to disclose with regards to this paper.

PURPOSE. We sought to determine the role and retinal cellular location of microRNA-124 (miR-124) in a neuroinflammatory model of retinal degeneration. Further, we explored the anti-inflammatory relationship of miR-124 with a predicted messenger RNA (mRNA) binding partner, chemokine (C-C motif) ligand 2 (Ccl2), which is crucially involved in inflammatory cell recruitment in the damaged retina. METHODS. Human AMD donor eyes and photo-oxidative damaged (PD) mice were labeled for miR-124 expression using in situ hybridization. PDGFRa-cre RFP mice were used for Müller cell isolation from whole retinas. MIO-M1 immortalized cells and rat primary Müller cells were used for in vitro analysis of miR-124 expression and its relationship with Ccl2. Therapeutic efficacy was tested with intravitreal administration of miR-124 mimic in mice, with electroretinography used to determine retinal function. IBA1 immunohistochemistry and photoreceptor row counts were used for assessment of inflammation and cell death. RESULTS. MiR-124 expression was correlated with progressive retinal damage, inflammation, and cell death in human AMD and PD mice. In addition, miR-124 expression was inversely correlated to Ccl2 expression in mice following PD. MiR-124 was localized to both neuronal-like photoreceptors and glial (Müller) cells in the retina, with a redistribution from neurons to glia occurring as a consequence of PD. Finally, intravitreal administration of miR-124 mimics decreased retinal inflammation and photoreceptor cell death, and improved retinal function. CONCLUSIONS. This study has provided an understanding of the mechanism behind miR-124 in the degenerating retina and demonstrates the usefulness of miR-124 mimics for the modulation of retinal degenerations. ; Supported by the National Health and Medical Research Council in Australia (APP1127705, 2017-2019), the Australian Government Research Training Program, the Gretel and Gordon Bootes Foundation (2013), and the Ophthalmic Research Institute of Australia/Eye Surgeons' Foundation (2015).