Browsing by Author "Oliveira, Ana V."
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- Development of non-viral vectors for gene therapy for pathologies of the retinaPublication . Oliveira, Ana V.; Silva, GabrielaThe success of gene therapy relies on efficient gene transfer and stable transgene expression. Our goal was to develop non-viral vectors optimized for retinal gene therapy with continued gene expression. Polymers, chitosan and hyaluronic acid or the modified polymers (thiolated chitosan and aminated hyaluronic acid) were chosen considering their biocompatibility and biodegradability to prepare several formulations. Vectors were formulated and characterized regarding their physical properties, biocompatibility and gene transfer efficiency in vitro on both retinal pigment epithelial and HEK293 cells and gene expression in the mouse retina. Our results show that our vectors exhibit size and surface charge consistent with gene delivery. They also present long-term stability in both storage and physiological conditions, remain stable after several freeze-thaw cycles and are capable of efficiently protecting DNA from nuclease degradation. Transfection studies show that transfection efficiency and transgene expression is affected by cell type, polymer molecular weight and mode of integrase delivery with the polyplexes. The incorporation of hyaluronic acid affected formulation stability, as expected, but it did not affect DNA loading and protection. The combination of chitosan and hyaluronic acid in polyplexes showed a significant improvement of transfection efficiency compared to chitosan-based vectors. In order to achieve sustained gene transfer vectors were combined with phiC31-integrase to promote transgene integration. The combined strategy of chitosan-based delivery and integrase demonstrate prolonged gene expression of both small (GFP, 1 Kb) and large genes (CEP290, 8Kb) several weeks post-transfection. In vivo sub-retinal administration of our vectors showed efficient transfection and sustained transgene expression in RPE cells at least 6 months post- injection. Our results indicate this chitosan-based approach may overcome size limitations found in commonly used adeno-associated viruses mediated gene transfer, while maintaining a high safety profile and prolonged, sustained gene expression, thus constituting an alternative for retinal gene delivery.
- Efficiency of RAFT-synthesized PDMAEMA in gene transfer to the retinaPublication . Bitoque, Diogo; S, Simão; Oliveira, Ana V.; Machado, S.; Duran, Margarita R.; Lopes, Eduardo; Costa, Ana M. Rosa da; Silva, GabrielaGene therapy has long been heralded as the new hope to evolve from symptomatic care of genetic pathologies to a full cure. Recent successes in using gene therapy for treating several ocular and haematopoietic pathologies have shown the great potential of this approach that, in the early days, relied on the use of viral vectors, which were considered by many to be undesirable for human treatment. Therefore, there is considerable interest and effort in developing non-viral vectors, with efficiency close to that of viral vectors. The aim of this study was to develop suitable non-viral carriers for gene therapy to treat pathologies affecting the retina. In this study poly(2-(N,N-dimethylamino)ethyl methacrylate), PDMAEMA was synthesized by reversible addition-fragmentation chain transfer (RAFT) and the in vitro cytocompatibility and transfection efficiency of a range of polymer:DNA ratios evaluated using a retinal cell line; in vivo biocompatibility was evaluated by ocular injection in C57BL/6 mice. The results showed that through RAFT, it is possible to produce a defined-size polymer that is compatible with cell viability in vitro and capable of efficiently directing gene expression in a polymer-DNA ratio-dependent manner. When injected into the eyes of mice, these vectors induced a transient, mild inflammation, characteristic of the implantation of medical devices. These results form the basis of future studies where RAFT-synthesized PDMAEMA will be used to deliver gene expression systems to the retina of mouse models of retinal pathologies. Copyright © 2014 John Wiley & Sons, Ltd.
- Evaluation of cystamine-modified hyaluronic acid/chitosan polyplex as retinal gene vectorPublication . Oliveira, Ana V.; Marcelo, Adriana; Costa, Ana M. Rosa da; Silva, Gabriela A.A successful gene therapy approach can prevent or treat congenital and acquired diseases. However, there is still no ideal non-viral vector for gene delivery in a safe and timely manner. In this report the anionic polymer hyaluronic acid (HA) was investigated as a potential vector for gene therapy. Due to its intrinsic characteristics it constitutes an excellent candidate to deliver therapeutic genes, pending the modification of its surface charge.
- Study of the antimicrobial and antioxidant effects of propolis extracts, a type of Algarve portuguese originPublication . Oliveira, Ana V.; Miguel, Maria Graça; Ferreira, Ana LuísaIn this work, extracts (aqueous, ethanolic and methanolic) of propolis harvested at two different times (winter and spring) from several locations of the Algarve region (B. N. Arrodeios, B. N. Pé da Serra, B. S. Arneijoafra and T. N. Madeira) were tested for their biological activities and composition. Results showed that propolis extracts were active against both Gram-positive and Gramnegative bacteria. All tested strains of bacteria showed susceptibility to the diluted propolis extracts (1:10) and in the majority of cases in a dose-dependent way. Most propolis samples collected at springtime showed higher antibacterial activity, in comparison with samples harvested at wintertime. There were also observed differences between collection sites and type of extract. These results correlate to HPLC results, where the same differences where observed. Regarding propolis citotoxicity, results showed that aqueous propolis extracts have no effect and ethanolic propolis extracts causes a small decrease in cell viability. Concerning antioxidant enzymatic activities, the superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GP) activities of samples of propolis were determined. Results showed that SOD activity was dependent on the collection time and decreased drastically from winter to spring in samples from B.N. Arrodeios and B.S. Arneijoafra. The opposite was observed in samples from B.N. Pé da Serra and T.N. Madeira. We expected that the samples with higher SOD activities would have higher CAT activities also. Such was not observed, which may suggest that there could be other antioxidant enzymes involved, different to the ones tested. In respect with protein contents, major differences were not observed when comparing samples collected at different times, except for samples from B.S. Arneijoafra, where a decrease occurred from the sample collected at winter to sample collected at spring time. This was the first study of the biological activities of Portuguese propolis from the Algarve region.
- Transfection efficiency of chitosan and thiolated chitosan in retinal pigment epithelium cells: a comparative studyPublication . Silva, Gabriela; Costa, Ana M. Rosa da; Bitoque, Diogo; Oliveira, Ana V.; Silva, Andreia P.Gene therapy relies on efficient vector for a therapeutic effect. Efficient non-viral vectors are sought as an alternative to viral vectors. Chitosan, a cationic polymer, has been studied for its gene delivery potential. In this work, disulfide bond containing groups were covalently added to chitosan to improve the transfection efficiency. These bonds can be cleaved by cytoplasmic glutathione, thus, releasing the DNA load more efficiently. Chitosan and thiolated chitosan nanoparticles (NPs) were prepared in order to obtain a NH3 :PO ratio of 5:1 and characterized for plasmid DNA complexation and release efficiency. Cytotoxicity and gene delivery studies were carried out on retinal pigment epithelial cells. In this work, we show that chitosan was effectively modified to incorporate a disulfide bond. The transfection efficiency of chitosan and thiolated chitosan varied according to the cell line used, however, thiolation did not seem to significantly improve transfection efficiency. The apparent lack of improvement in transfection efficiency of the thiolated chitosan NPs is most likely due to its size increase and charge inversion relatively to chitosan. Therefore, for retinal cells, thiolated chitosan does not seem to constitute an efficient strategy for gene delivery.