Ana V. Oliveira

Non-viral strategies for ocular gene delivery

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.

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

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

Combining hyaluronic acid with chitosan enhances gene delivery

The 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.

Evaluation of cystamine-modified hyaluronic acid/chitosan polyplex as retinal gene vector.

PURPOSE 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. METHODS To modify its charge, HA was modified with cystamine. Several formulations were prepared using modified HA combined with sodium sulfate, sodium triphosphate, K-carrageenan and chitosan. Vectors were characterized with respect to size, charge, DNA load and its protection, and effect on cell viability. The better performing formulations were further evaluated in vitro for their transfection efficiency in HEK293T and ARPE-19 cells. RESULTS Cell viability assays showed low cytotoxicity for both polymers. Gene transfer efficiency depended on cell line and formulation, but no increased transfection efficiency was observed with the modified polymer. CONCLUSIONS HA has great potential as a gene therapy vector, but further optimization, including incorporation of a higher percentage of positive groups in HA, is needed before its use as a gene delivery vector.

Enhancement of chitosan-mediated gene delivery through combination with phiC31 integrase

Gene transfer efficiency and expression stability are key factors to a successful gene therapy approach. In the present work we have developed a combined system for gene transfer that integrates well established non-viral polymeric vectors based on chitosan particles with the properties of phiC31-integrase that promotes a relatively non-immunogenic, site-specific integration, with sustained gene expression. Simultaneously, to overcome one of the major limitations in adeno-associated virus mediated gene transfer--the delivery of large genes--we have tested the capacity of our non-viral vectors to incorporate a large (8 Kb) transgene. Polyplexes were extensively characterized for their size, surface charge, morphology, pDNA complexation, transfection efficiency and transgene expression in vitro using HEK293 cells. Co-transfection with integrase was done by complexation in a single polyplex preparation or the use of two separate polyplex preparations. Transgene expression, GFP and CEP290 (1Kb and 8 Kb, respectively), was evaluated by fluorescence microscopy, flow cytometry and Western blot analysis. DNA complexation efficiency, particle size and morphology were consistent with gene delivery for all formulations. In contrast, transfection efficiency and transgene expression varied with polymer and polyplex size. Following delivery by chitosan polyplexes, high levels of GFP expression were still visible 16 weeks post-transfection and over-expression of the large transgene was detected at least 6 weeks post-transfection. Polyplexes incorporating phiC1 integrase demonstrate prolonged gene expression of both small (GFP, 1 Kb) and large genes (CEP290, 8Kb). This approach, using a combined strategy of polymers and integrase may overcome the size limitation found in commonly used adeno-associated virus mediated gene transfer techniques, while maintaining a high safety profile and prolonged, sustained gene expression, thus constituting an alternative for gene delivery.

Cationic Polyene Phospholipids as DNA Carriers for Ocular Gene Therapy

Recent 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.

FOREIGN LANGUAGE SKILLS: A CHALLENGE FOR THE ECONOMY

Awareness of the fact that multilingualism opens doors to new markets and new business opportunities is not an original phenomenon, but the various works produced with a view to its being acknowledged are still little publicized and granted little practical use also. Showing mastery of communicative skills in several foreign languages is always valuable for the market economy, not only in terms of sales and marketing, but also in order to reach new target groups and establish lasting strategic relationships. The purpose of this article is to provide a general overview of the studies carried out in this context, focusing in particular on aspects such as the increasingly obvious relationship between in force language practices of a diversified nature and the proper functioning of businesses. Equally important is the mastery of language skills, seen as an important driving force of cross-border cooperation, as well as the development of an emotional culture within the business world, propitious to the implementation of efficient language management strategies, which are clearly beneficial inasmuch as the market economy is concerned.