Fani Sousa

Bio: Fani Sousa is an academic researcher from University of Beira Interior. The author has contributed to research in topics: Affinity chromatography & RNA. The author has an hindex of 30, co-authored 130 publications receiving 2476 citations.

Improvement of transfection efficiency by using supercoiled plasmid DNA purified with arginine affinity chromatography

Abstract: Background It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufacturing process. Several challenges encountered in the gene therapy field have emphasized the need for the development of novel platforms that allow the recovery of gene vectors and enable efficient transfection of cells. The use of plasmid DNA-based therapeutics relies on procedures that efficiently purify the supercoiled (sc) plasmid isoform. Plasmid DNA (pDNA) purification strategies that use amino acids as immobilized ligands have recently yielded interesting results. Methods The present study describes a strategy that uses arginine-chromatography to specifically purify sc pDNA from other isoforms and Escherichia coli impurities present in a clarified lysate. Results Control analysis shows that the purity of the sc pDNA is 100% with a homogeneity higher than 97% of sc. Furthermore, no RNA was detectable, the protein content was lower than 10 µg/ml and a 117-fold reduction on genomic DNA contamination and 95% endotoxin removal were accomplished. The chromatographic process demonstrated an impressive performance on sc isoform recovery (79% yield). Furthermore, the sc transfection efficiency of COS-7 cells (62%) was significantly higher compared to the efficiency (25%) achieved with a pDNA control. Conclusions With the simplified sc pDNA purification process, a high yield was achieved, sc pDNA was purified under mild conditions and shown to be extremely efficient with respect to cell transfection. Arginine-chromatography is thus an interesting option for use as a late stage plasmid purification step. Copyright © 2008 John Wiley & Sons, Ltd.

Minicircle DNA vectors for gene therapy: advances and applications.

Abstract: Introduction: Nucleic-acid-based biopharmaceuticals enclose a remarkable potential for treating debilitating or life-threatening diseases that currently remain incurable. This promising area of research envisages the creation of state-of-the-art DNA vaccines, pluripotent cells or gene-based therapies, which can be used to overcome current issues. To achieve this goal, DNA minicircles are emerging as ideal nonviral vectors due to their safety and persistent transgene expression in either quiescent or actively dividing cells.Areas covered: This review focuses on the characteristics of minicircle DNA (mcDNA) technology and the current advances in their production. The possible modifications to further improve minicircle efficacy are also emphasized and discussed in light of recent advances. As a final point, the main therapeutic applications of mcDNA are summarized, with a special focus on pluripotent stem cells production and cancer therapy.Expert opinion: Achieving in-target and persistent transgene expres...

Selective purification of supercoiled plasmid DNA from clarified cell lysates with a single histidine–agarose chromatography step

Abstract: The ability to isolate sc (supercoiled) pDNA (plasmid DNA) isoform should be one of the features of a pDNA purification process, eliminating sample contaminants such as RNA, gDNA (genomic DNA), proteins and endotoxins. A process is described that uses a single histidine-agarose chromatography step to purify sc pDNA from other isoforms and Escherichia coli impurities present in a clarified lysate. The histidine-agarose support combines the mild hydrophobic characteristics of an epoxy spacer arm with a pseudo-affinity histidine ligand. The 6 kb DNA vaccine backbone pVAX1-LacZ was used as a model target. Following loading at high salt [2.3 M (NH4)2SO4], the different species were eluted by a series of reverse salt step gradients (2.0, 1.5 and 0 M (NH4)2SO4). Open circular pDNA and gDNA was eluted at 2.3 M, sc pDNA was isolated as a single peak at 2.0 M and RNA was eluted at 1.5 M (NH4)2SO4 and lower. The underlying mechanism is thought to involve not only hydrophobic interactions between the support and pDNA molecules, but also non-specific biorecognition of nucleic acid bases by the histidine ligand. Control analysis showed that the isolated sc pDNA conforms to specifications in terms of gDNA (3.4 ng/microg of pDNA), endotoxins (0.02 endotoxin unit/microg of pDNA), RNA and proteins (undetectable) and pDNA homogeneity (approximately 100% sc). Furthermore, the transfection efficiency of Chinese-hamster ovary cells (50%) was significantly higher when compared with the efficiency (25%) of a pDNA control. The present study confirms the possibility of using a single histidine-agarose chromatography step to purify sc pDNA from other isoforms and host contaminants present in a clarified E. coli lysate.

Formulation of chitosan-TPP-pDNA nanocapsules for gene therapy applications.

Abstract: The encapsulation of DNA inside nanoparticles meant for gene delivery applications is a challenging process where several parameters need to be modulated in order to design nanocapsules with specific tailored characteristics. The purpose of this study was to investigate and improve the formulation parameters of plasmid DNA (pDNA) loaded in chitosan nanocapsules using tripolyphosphate (TPP) as polyanionic crosslinker. Nanocapsule morphology and encapsulation efficiency were analyzed as a function of chitosan degree of deacetylation and chitosan-TPP ratio. The manipulation of these parameters influenced not only the particle size but also the encapsulation and release of pDNA. Consequently the transfection efficiency of the nanoparticulated systems was also enhanced with the optimization of the particle characteristics. Overall, the differently formulated nanoparticulated systems possess singular properties that can be employed according to the desired gene delivery application.

MicroRNAs: Target Recognition and Regulatory Functions

Abstract: MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers

Abstract: An emerging approach for treating cancer involves programming patient-derived T cells with genes encoding disease-specific chimeric antigen receptors (CARs), so that they can combat tumour cells once they are reinfused. Although trials of this therapy have produced impressive results, the in vitro methods they require to generate large numbers of tumour-specific T cells are too elaborate for widespread application to treat cancer patients. Here, we describe a method to quickly program circulating T cells with tumour-recognizing capabilities, thus avoiding these complications. Specifically, we demonstrate that DNA-carrying nanoparticles can efficiently introduce leukaemia-targeting CAR genes into T-cell nuclei, thereby bringing about long-term disease remission. These polymer nanoparticles are easy to manufacture in a stable form, which simplifies storage and reduces cost. Our technology may therefore provide a practical, broadly applicable treatment that can generate anti-tumour immunity 'on demand' for oncologists in a variety of settings.