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Transfection efficiency of chitosan and thiolated chitosan in retinal pigment epithelium cells: a comparative study

Publication . 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.

2013Journal article

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Non-viral strategies for ocular gene delivery

Publication . V. Oliveira, Ana; Rosa Da Costa, Ana; Silva, Gabriela A.

The success of gene therapy relies on efficient gene transfer and stable transgene expression. The in vivo efficiency is determined by the delivery vector, route of administration, therapeutic gene, and target cells. While some requirements are common to several strategies, others depend on the target disease and transgene product. Consequently, it is unlikely that a single system is suitable for all applications. This review examines current gene therapy strategies, focusing on non-viral approaches and the use of natural polymers with the eye, and particularly the retina, as their gene delivery target. (C) 2017 Elsevier B.V. All rights reserved.

2017-08Journal article

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Efficiency of RAFT-synthesized PDMAEMA in gene transfer to the retina

Publication . Bitoque, Diogo; S, Simão; Oliveira, Ana V.; Machado, S.; Duran, Margarita R.; Lopes, Eduardo; Costa, Ana M. Rosa da; Silva, Gabriela

Gene 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.

2017Journal article

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Human-derived NLS enhance the gene transfer efficiency of chitosan

Publication . Bitoque, Diogo; Morais, Joana; Oliveira, Ana; Sequeira, Raquel L.; Calado, Sofia; Fortunato, Tiago M.; Simão, Sónia; Rosa Da Costa, Ana; Silva, Gabriela A.

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA. In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5. These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.

2021Journal article

Open Access

Sustained gene expression in the retina by improved episomal vectors

Publication . Calado, Sofia; Oliveira, Ana; Machado, Susana; Haase, Rudolf; Silva, Gabriela

Gene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy-low levels of transgene expression, vector loss during mitosis, and gene silencing-need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.

2014-10Journal article

Open Access