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- Chitosan-Hyaluronic acid hybrid vectors for retinal gene therapyPublication . Oliveira, Ana; Bitoque, Diogo; Silva, Gabriela A.
- Cationic polyene phospholipids as DNA carriers for ocular gene therapyPublication . Machado, Susana; Calado, Sofia; Bitoque, Diogo; Oliveira, Ana Vanessa; Øpstad, Christer L.; Zeeshan, Muhammad; Sliwka, Hans-Richard; Partali, Vassilia; Pungente, Michael D.; Silva, GabrielaRecent success in the treatment of congenital blindness demonstrates the potential of ocular gene therapy as a therapeutic approach. The eye is a good target due to its small size, minimal diffusion of therapeutic agent to the systemic circulation, and low immune and inflammatory responses. Currently, most approaches are based on viral vectors, but efforts continue towards the synthesis and evaluation of new nonviral carriers to improve nucleic acid delivery. Our objective is to evaluate the efficiency of novel cationic retinoic and carotenoic glycol phospholipids, designated C20-18, C20-20, and C30-20, to deliver DNA to human retinal pigmented epithelium (RPE) cells. Liposomes were produced by solvent evaporation of ethanolic mixtures of the polyene compounds and coformulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol). Addition of DNA to the liposomes formed lipoplexes, which were characterized for binding, size, biocompatibility, and transgene efficiency. Lipoplex formulations of suitable size and biocompatibility were assayed for DNA delivery, both qualitatively and quantitatively, using RPE cells and a GFP-encoding plasmid. The retinoic lipoplex formulation with DOPE revealed a transfection efficiency comparable to the known lipid references 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) and GeneJuice. The results demonstrate that cationic polyene phospholipids have potential as DNA carriers for ocular gene therapy.
- Deregulation of the retinal renin-angiotensin system precedes the onset of diabetic retinopathyPublication . S, Simão; Bitoque, D. B.; Santos, D. F.; Araújo, Inês; Silva, G. A.The renin-angiotensin system (RAS) is a set of complex pathways with a well-defined function in the regulation of blood pressure and body fluid homeostasis.
- 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.
- Synthesis and evaluation of polymer nanoparticles for delivery in RPE cellsPublication . Bitoque, Diogo B.; Silva, GabrielaOcular pathologies are among the most debilitating medical conditions affecting all segments of the population. Traditional treatment options are often ineffective, and gene therapy has the potential to become an alternative approach for the treatment of several pathologies. Methacrylate polymers have been described as highly biocompatible and are successfully used in medical applications. Due to their cationic nature, these polymers can be used to form polyplexes with DNA for its delivery. This work aims to study the potential of PDMAEMA (poly(2-(N,N’-dimethylamino)ethyl methacrylate)) as a non viral gene delivery system to the retina. The first part of this work aimed to study the potential for gene delivery of a previously synthesized PDMAEMA polymer of high molecular weight (354kDa). In the second part, we synthesized by RAFT a PDMAEMA with a lower molecular weight (103.3kDa) and similarly, evaluated its ability to act as a gene delivery vehicle. PDMAEMA/DNA polyplexes were prepared at 5, 7.5, 10, 12.5 and 20 nitrogen/phosphorous (N/P) ratio for the 354kDa PDMAEMA and at 5 and 7.5 for the 103.3kDa PDMAEMA. Dynamic light scattering and zeta potential measurements confirmed the nanosize and positive charge of polyplexes for all ratios and for both polymers. Both high and low Mw PDMAEMA were able to efficiently complex and protect DNA from DNase I degradation. Their cytotoxicity was evaluated using a non-retinal cell line (HEK293) and a retinal pigment epithelium (RPE) cell line (D407). We have found that cytotoxicity of the free polymer is concentration and time dependent, as expected, and negligible for all the concentrations of the PDMAEMA-DNA polyplexes. Furthermore, for the concentrations to be used in vivo, the 354kDa PDMAEMA showed no signs of inflammation upon injection in the intravitreal space of C57BL/6 mice. The transfection efficiency, as evaluated by fluorescence microscopy and flow cytometry, showed that the D407 retinal cells were transfected by polyplexes of both high and low Mw PDMAEMA, but with varied efficiency, which was dependent on the N/P ratio. Althogether, these results suggest that PDMAEMA is a feasible candidate for non-viral gene delivery to the retina, and this work constitutes the basis of further studies to elucidate the bottleneck in transfection and further optimization of the material.
- Combining hyaluronic acid with chitosan enhances gene deliveryPublication . Oliveira, Ana; Bitoque, Diogo; Silva, GabrielaThe low gene transfer efficiency of chitosan-DNA polyplexes is a consequence of their high stability and consequent slow DNA release. The incorporation of an anionic polymer is believed to loosen chitosan interactions with DNA and thus promote higher transfection efficiencies. In this work, several formulations of chitosan-DNA polyplexes incorporating hyaluronic acid were prepared and characterized for their gene transfection efficiency on both HEK293 and retinal pigment epithelial cells. The different polyplex formulations showed morphology, size, and charge compatible with a role in gene delivery. The incorporation of hyaluronic acid rendered the formulations less stable, as was the goal, but it did not affect the loading and protection of the DNA. Compared with chitosan alone, the transfection efficiency had a 4-fold improvement, which was attributed to the presence of hyaluronic acid. Overall, our hybrid chitosan-hyaluronic acid polyplexes showed a significant improvement of the efficiency of chitosan-based nonviral vectors in vitro, suggesting that this strategy can further improve the transfection efficiency of nonviral vectors.
- 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.
- Human-derived NLS enhance the gene transfer efficiency of chitosanPublication . 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.