Dataset Information

In Vitro Gene Delivery in Retinal Pigment Epithelium Cells by Plasmid DNA-Wrapped Gold Nanoparticles.

ABSTRACT: Many rare diseases course with affectation of neurosensory organs. Among them, the neuroepithelial retina is very vulnerable due to constant light/oxidative stress, but it is also the most accessible and amenable to gene manipulation. Currently, gene addition therapies targeting retinal tissue (either photoreceptors or the retinal pigment epithelium), as a therapy for inherited retinal dystrophies, use adeno-associated virus (AAV)-based approaches. However, efficiency and safety of therapeutic strategies are relevant issues that are not always resolved in virus-based gene delivery and alternative methodologies should be explored. Based on our experience, we are currently assessing the novel physical properties at the nanoscale of inorganic gold nanoparticles for delivering genes to the retinal pigment epithelium (RPE) as a safe and efficient alternative approach. In this work, we present our preliminary results using DNA-wrapped gold nanoparticles (DNA-gold NPs) for successful in vitro gene delivery on human retinal pigment epithelium cell cultures, as a proof-of-principle to assess its feasibility for retina in vivo gene delivery. Our results show faster expression of a reporter gene in cells transfected with DNA-gold NPs compared to DNA-liposome complexes. Furthermore, we show that the DNA-gold NPs follow different uptake, internalization and intracellular vesicle trafficking routes compared to pristine NPs.

SUBMITTER: Trigueros S

PROVIDER: S-EPMC6523520 | biostudies-other | 2019 Apr

REPOSITORIES: biostudies-other

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In Vitro Gene Delivery in Retinal Pigment Epithelium Cells by Plasmid DNA-Wrapped Gold Nanoparticles.

Trigueros Sònia S Domènech Elena B EB Toulis Vasileios V Marfany Gemma G

Genes 20190409 4

Many rare diseases course with affectation of neurosensory organs. Among them, the neuroepithelial retina is very vulnerable due to constant light/oxidative stress, but it is also the most accessible and amenable to gene manipulation. Currently, gene addition therapies targeting retinal tissue (either photoreceptors or the retinal pigment epithelium), as a therapy for inherited retinal dystrophies, use adeno-associated virus (AAV)-based approaches. However, efficiency and safety of therapeutic s ...[more]

PMID: 30970664

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Project description:Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

Dataset Information

In Vitro Gene Delivery in Retinal Pigment Epithelium Cells by Plasmid DNA-Wrapped Gold Nanoparticles.

Publications

In Vitro Gene Delivery in Retinal Pigment Epithelium Cells by Plasmid DNA-Wrapped Gold Nanoparticles.

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