Sofia C. Ribeiro

Production, purification and analysis of an experimental DNA vaccine against rabies

The basic and applied research efforts devoted to the development of DNA vaccines must be accompanied by manufacturing processes capable of being scaled up and delivering a clinical‐grade product. This work describes a rapid process of this kind, based on hydrophobic interaction chromatography (HIC) for the production of milligram quantities of an experimental DNA rabies vaccine. Its properties and protective activity are tested in comparison with the same plasmid DNA purified with a commercial kit.

The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression

Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non‐viral vectors. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease‐rich environments. Homopurine‐rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance.

Nonviral Gene Delivery to Mesenchymal Stem Cells Using Cationic Liposomes for Gene and Cell Therapy

Mesenchymal stem cells (MSCs) hold a great promise for application in several therapies due to their unique biological characteristics. In order to harness their full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing an efficient and safe methodology to genetically engineer human bone marrow MSC (BM MSC), enhancing their therapeutic efficacy in Regenerative Medicine. The plasmid DNA delivery was optimized using a cationic liposome-based reagent. Transfection efficiencies ranged from ~2% to ~35%, resulting from using a Lipid/DNA ratio of 1.25 with a transgene expression of 7 days. Importantly, the number of plasmid copies in different cell passages was quantified for the first time and ~20,000 plasmid copies/cell were obtained independently of cell passage. As transfected MSC have shown high viabilities (>90%) and recoveries (>52%) while maintaining their multipotency, this might be an advantageous transfection strategy when the goal is to express a therapeutic gene in a safe and transient way.

The role of polyadenylation signal secondary structures on the resistance of plasmid vectors to nucleases

Nuclease degradation of plasmid DNA (pDNA) vectors after delivery and during trafficking to the nucleus is a barrier to gene expression. This barrier may be circumvented by shielding the pDNA from the nuclease‐rich cell environment with adjuvants or by using nuclease inhibitors. A different alternative that is explored in this work is to make pDNA vectors more nuclease‐resistant a priori.

Gene delivery to human bone marrow mesenchymal stem cells by microporation

Electroporation has been considered one of the most efficient non-viral based methods to deliver genes regardless of frequently observed high cell mortality. In this study we used a microporation technique to optimise the delivery of plasmid DNA encoding green fluorescence protein (GFP) to human bone marrow mesenchymal stem cells (BM-MSC). Using resuspension buffer (RB) and as low as 1.5 x 10⁵ cells and 1 μg of DNA, we achieved 40% of cells expressing the transgene, with cell recovery and cell viabilities of 85% and 90%, respectively. An increase in DNA amount did not significantly increase the number of transfected cells but clearly reduced cell recovery. A face-centered composite design was used to unveil the conditions giving rise to optimal plasmid delivery efficiencies when using a sucrose based microporation buffer (SBB). The BM-MSC proliferation kinetics were mainly affected by the presence of plasmid and not due to the microporation process itself although no effect was observed on their immunophenotypic characteristics and differentiative potential. Based on the data shown herein microporation demonstrated to be a reliable and efficient method to genetically modify hard-to-transfect cells giving rise to the highest levels of cell survival reported so far along with superior gene delivery efficiencies.

Purification of plasmids for gene therapy and DNA vaccination

This chapter covers the different aspects of the production and purification of plasmids for gene therapy and DNA vaccination. Process issues are extensively covered and complemented with information related to plasmid DNA structure, vector construction, product specifications and quality assurance and control.

Plasmid DNA Size Does Affect Nonviral Gene Delivery Efficiency in Stem Cells

Genetic modification of stem cells, prior to transplantation, can enhance their survival and can improve their function in cell therapy settings. Mesenchymal stem cells (MSC) are considered one of the most promising tools for cell-based gene therapy, due to their multipotency, ease of isolation, as well as their high ex vivo expansion potential. Neural stem cells (NSC) may also present an ideal route for gene therapy and have been considered for use in cell replacement therapies in various neurodegenerative diseases. Gene therapy-based applications require the transfer of genetic material, either by viral or nonviral gene delivery methods, although the latter has been associated with low efficiencies, especially within hard to transfect cells as stem cells. Herein, we present results on the influence of plasmid size in gene delivery to human MSC and mouse NSC. We used minimized plasmids encoding a fluorescent protein but lacking the antibiotic resistance gene. This work shows that (1) for smaller plasmids the intracellular plasmid copy number can be up to 2.6-fold higher, and (2) the number of cells presenting fluorescence can be twice the number obtained for larger plasmids. Furthermore, by using plasmid constructs containing different polyA signals, we also demonstrated that differences between the plasmids depend largely on the transgene mRNA level. Based on our data we demonstrate that plasmid size severely affects the efficiency of nuclear uptake and we propose that it can also affect the rate of heterochromatin associated gene silencing in stem cells.

A quantitative method to evaluate mesenchymal stem cell lipofection using real-time PCR.

Genetic modification of human mesenchymal stem cells (MSC) is a powerful tool to improve the therapeutic utility of these cells and to increase the knowledge on their regulation mechanisms. In this context, strong efforts have been made recently to develop efficient nonviral gene delivery systems. Although several studies addressed this question most of them use the end product of a reporter gene instead of the DNA uptake quantification to test the transfection efficiency. In this study, we established a method based on quantitative real‐time PCR (RT‐PCR) to determine the intracellular plasmid DNA copy number in human MSC after lipofection. The procedure requires neither specific cell lysis nor DNA purification. The influence of cell number on the RT‐PCR sensitivity was evaluated. The method showed good reproducibility, high sensitivity, and a wide linear range of 75–2.5 × 106 plasmid DNA copies per cell. RT‐PCR results were then compared with the percentage of transfected cells assessed by flow cytometry analysis, which showed that flow cytometry‐based results are not always proportional to plasmid cellular uptake determined by RT‐PCR. This work contributed for the establishment of a rapid quantitative assay to determine intracellular plasmid DNA in stem cells, which will be extremely beneficial for the optimization of gene delivery strategies.

Genetic engineering of stem cells by non-viral vectors

Stem/progenitor cells hold a great promise for application in several therapies due to their unique biological characteristics. With the purpose of harnessing these cells full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing efficient and safe methodologies to genetically engineer stem cells, boosting their therapeutic efficacy in Regenerative Medicine. In our work, delivery of plasmid DNA to human Bone Marrow Mesenchymal Stem Cells (BM-MSC) was optimized by lipofection and by a recently available microporation technique and no effect was observed in their immunophenotypic characteristics or differentiative potential. After lipofection similar number of plasmid copies was determined at different cell passages. Importantly, cell proliferation kinetics slowed down due to the presence of plasmid. Overall, we believe our findings are extremely useful towards the maximization of gene delivery to human MSC, without compromising cell function and viability.