Ângela Sousa

Nanoparticle mediated delivery of pure P53 supercoiled plasmid DNA for gene therapy

The translation of non-viral gene replacement therapies for cancer into clinical application is currently hindered due to known issues associated with the effectiveness of plasmid DNA (pDNA) expression vectors and the production of gene delivery vehicles. Herein we report an integrative approach established on the synthesis of nanoparticulated carriers, in association with the supercoiled (sc) isoform purification of a p53 tumor suppressor encoding plasmid, to improve both delivery and transfection. An arginine-based chromatographic matrix with specific recognition for the different topoisoforms was used to completely isolate the biologically active sc pDNA. Our findings showed that the sc topoisoform is recovered under mild conditions with high purity and structural stability. In addition, to further enhance protection and transfection efficiency, the naked sc pDNA was encapsulated within chitosan nanoparticles by ionotropic gelation. The mild conditions for particle synthesis used in the former technique allowed the attainment of a high encapsulation efficiency for sc pDNA (>75%). Moreover, in vitro transfection experiments confirmed the reinstatement of the p53 protein expression and most importantly, the sc pDNA transfected cells exhibited the highest p53 expression levels when compared to other formulations. Overall, given the fact that sc pDNA topoisoform indeed enhances transgene expression rates this approach might have a profound impact on the development of a sustained nucleic acid-based therapy for cancer.

Advances in chromatographic supports for pharmaceutical-grade plasmid DNA purification.

Chromatographic technology is undoubtedly one of the most diverse and powerful purification methods for downstream process applications. The diversity and quantity of biomolecules present in crude extracts as well as the similarities between impurities and the target biomolecule are considered the critical challenges in the extraction and purification steps. Thus, it is important to optimize the purification protocol to achieve maximum recovery of the target sample. The structure of chromatographic supports has been continuously developed to afford rapid and efficient separations, as well as, the application of specific ligands to improve the selectivity for the target molecule. The present review discusses the structural progress and evolution of the chromatographic supports that have been used for plasmid DNA purification. Nowadays, the most desirable form of plasmid for gene therapy and DNA vaccination is the supercoiled isoform, due to its stability and higher transfection efficiency over other plasmid topologies. However, the main challenge is not only to produce high quantities of supercoiled plasmid DNA but also to preserve its quality, meeting the strict requirements recommended by the regulatory agencies. Therefore, this review will focus on the chemical and structural classification of the different media and on some of the specific ligands used for plasmid DNA bioseparation.

Purification of pre-miR-29 by arginine-affinity chromatography

Recently, differential expression of microRNAs, in patients with Alzheimers disease (AD) suggests that they might have key regulatory roles in this neurodegenerative disease. Taking into account this fact, several studies demonstrated that the miR-29 is significantly decreased in AD patients, also displaying abnormally high levels of β-site APP-cleaving enzyme 1. Thus, RNA biochemical or structural studies often require a RNA sample that is chemically pure and biologically active. The present work describes a new affinity chromatography method using an arginine support to specifically purify pre-miR-29 from other Rhodovulum sulfidophilum small RNA species. Nevertheless, in order to achieve higher efficiency and selectivity, it is essential to characterize the behavior of pre-miR-29 binding/elution. Thus, three different strategies based on increased sodium chloride (280-500mM), arginine (25mM) or decreased ammonium sulfate (2-0.1M) stepwise gradients are described to purify pre-miR-29. In this way, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance. As a matter of fact, by employing elution strategies using sodium chloride or arginine, an improvement in the final pre-miR-29 yields (96.5 and 56.7%, respectively) was obtained. Moreover, the quality control analysis revealed high integrity in pre-miR-29 preparations as well as high purity (90 and 98%, respectively), demonstrated by the scarce detection of proteins. This improved method takes advantage of its simplicity, significant cost reduction, due to the elimination of some complex operations, and speed for large-scale purification of pre-miRNAs suitable for biochemical and structural studies.

Selectivity of arginine chromatography in promoting different interactions using synthetic oligonucleotides as model

Arginine has been effectively used in several chromatography methodologies to improve recovery, resolution, and to suppress aggregation. Recently, arginine chromatography was used to fully separate supercoiled and open circular plasmid DNA isoforms. The specific recognition of supercoiled plasmid isoform by arginine was hypothesised to be due to the ability of arginine matrix to be involved in complex interactions that are partly dependent on the conformation of the DNA molecule. In light of these considerations a study was conducted to understand the several interactions that a DNA molecule can promote with the arginine support, in accordance with the chromatographic conditions established. Consequently, knowing the ideal conditions to promote the specific interactions, it could be possible to perform a more targeted and efficient purification. This work describes the chromatography of oligonucleotides with sizes up to 30 bases on the arginine-agarose gel. The effect of several conditions like hydrophobic character of the individual bases, molecular mass of the oligonucleotides, presence of secondary structures, temperature and elution buffer composition (salt and arginine supplemented buffer) was investigated. According to previous atomic data referent to possible interactions between amino acids and DNA nucleotides, arginine can preferentially interact with guanine by hydrogen bond, but other interactions (ionic interactions, van der Waals contacts, water mediated bonds) may also be present and become dominant depending on the conditions used. The results also revealed that the application of arginine in the elution buffer led to an effective elution of oligonucleotides from the arginine chromatographic support by a competition strategy. In general, it was suggested that the affinity interaction promoted by the arginine support is responsible for the specific recognition of particular oligonucleotide bases, involving multiple interactions.

Rapid quantification of supercoiled plasmid deoxyribonucleic acid using a monolithic ion exchanger

The demand for high-purity supercoiled plasmid DNA to be applied as a vector for new therapeutic strategies, such as gene therapy or DNA vaccination has increased in the last years. Thus, it is necessary to implement an analytical technique suitable to control the quality of the supercoiled plasmid as a pharmaceutical product during the manufacturing process. The present study describes a new methodology to quantify and monitor the purity of supercoiled plasmid DNA by using a monolithic column based on anion-exchange chromatography. This analytical method with UV detection allows the separation of the plasmid isoforms by combining a NaCl stepwise gradient. The specificity, linearity, accuracy, reproducibility and repeatability of the method have been evaluated, and the lower quantification and detection limits were also established. The validation was performed according to the guidelines, being demonstrated that the method is precise and accurate for a supercoiled plasmid concentration up to 200μg/mL. The main advantage achieved by using this monolithic column is the possibility to quantify the supercoiled plasmid in a sample containing other plasmid topologies, in a 4min experiment. This column also permits the assessment of the supercoiled plasmid DNA present in more complex samples, allowing to control its quality throughout the bioprocess. Therefore, these findings strengthen the possibility of using this monolithic column associated with a powerful analytical method to control the process development of supercoiled plasmid DNA production and purification for therapeutic applications.

Effect of chromatographic conditions and plasmid DNA size on the dynamic binding capacity of a monolithic support

DNA therapies are becoming recognized alternatives for the treatment and prevention of severe pathologies. Although most current trials have used plasmids <10 kbp, in the future larger plasmids would be required. The purpose of this work was to study the chromatographic behavior of nongrafted carbonyldiimidazole monolithic disks using plasmids with different sizes under hydrophobic conditions. Thereunto, the purification of several plasmids was performed. Higher size plasmids needed lower ammonium sulfate concentration, due to the greater number of interactions between the plasmids and monolith. The dynamic binding capacity experiments for the different plasmids revealed a lower capacity for bigger plasmids. It was also verified that the increase of salt concentration from 2.5 to 3 M of ammonium sulfate increased the capacity. At the highest salt concentration, a slight improvement in the capacity using lower flow rate was observed, possibly due to compaction of plasmid molecules and its better organization on the monolith channels. Finally, a low pH also had a positive effect on the capacity. So, this monolithic support proved to be appropriate to purify the supercoiled isoform of different plasmids with different sizes, providing a valuable instrument as a purification technique.

Purification of pre-miR-29 by a new O-phospho-l-tyrosine affinity chromatographic strategy optimized using design of experiments

MicroRNAs are the most studied small non-coding RNA molecules that are involved in post-transcriptional regulation of target genes. Their role in Alzheimers disease is being studied and explored in order to develop a new therapeutic strategy based on specific gene silencing. This disease is characterized by protein deposits, mainly deposits of extracellular Aβ plaques, produced upon endoproteolytic cleavage of APP by ß-site APP-cleaving enzyme 1 (BACE1). Recent studies have shown that particularly miR-29 cluster can be involved in the decrease of Aβ plaques production, by acting on BACE1 expression silencing. In order to use this microRNA as potential therapeutic it is essential to guarantee its purity, stability and integrity. Hence, the main purpose of this study was the development of a new affinity chromatographic strategy by using an O-phospho-l-tyrosine matrix and applying Box-Behnken design (BBD) to obtain pre-miR-29 with high purity degree and yield, envisioning its application in gene therapy. Thus, after process optimization the best results were achieved with a decreasing ammonium sulfate gradient in 10mM Tris buffer, pH 8 (1.6M (NH4)2SO4, 1.11M (NH4)2SO4 and 0M (NH4)2SO4), at 16°C. These experimental conditions allowed the recovery of pre-miR-29 with 52% of purity and 71% of recovery yield. The O-phospho-l-tyrosine matrix was initially chosen to mimic the natural interactions that occur inside the cell, and in fact it was proved a satisfactory selectivity for pre-miR-29. Also the innovative application of BBD for this strategy was efficient (R(2)=0.98 for % relative recovery and R(2)=0.93 for % relative purity) and essential to achieve best purification results in short time, saving lab resources.

New approach for purification of pre-miR-29 using lysine-affinity chromatography

miRNA-based gene therapy applications require microRNA with high purity degree, good quality and biologically active. Owing to the commercial interest in these approaches, there is a growing interest in the development of innovative procedures to easily and efficiently purify the RNA. Thus, several chromatographic and non-chromatographic methods have been reported to accomplish this purpose, but not all of these strategies allow the efficient separation of miRNAs. The present study describes a new strategy that uses a lysine ligand in affinity chromatography to efficiently separate pre-miR-29 from a small RNAs mixture. The interest on this biomolecule is related to the fact that pre-miR-29 deficiencies or excesses have been associated to a number of clinically important diseases. The retention behaviour of pre-miR-29 was characterized and adjusted to achieve higher specificity in this chromatographic operation, using an ammonium sulfate stepwise gradient. Overall, it was verified that lysine-agarose support can promote a specific interaction with the pre-miR-29 favouring its total separation. The results also suggest that the underlying mechanism involves biorecognition of pre-miR-29 by the lysine ligands, resulting from the occurrence of different elementary interactions, including hydrogen and hydrophobic interactions.

Pharmaceutical-grade pre-miR-29 purification using an agmatine monolithic support

MicroRNA-based therapeutic applications have fostered a growing interest in the development of microRNAs purification processes in order to obtain the final product with high purity degree, good quality and biologically active. The pre-miR-29 deficiency or overexpression has been associated to a number of clinically important diseases, and its therapeutic application can be considered. Monolithic columns emerged as a new class of chromatographic supports used in the plasmid DNA purification platforms, being an interesting alternative to the conventional particle-based columns. Thus, the current work describes, for the first time, a new affinity chromatography method that combines the high selectivity of agmatine ligands with the versatility of monoliths to specifically and efficiently purify pre-miR-29 from other small RNA species and Rhodovulum sulfidophilum impurities. The effect of different flow rates on pre-miR-29 separation was also evaluated. Moreover, breakthrough experiments were designed to study the effect of different RNA concentrations on the modified monolithic support binding capacity, being verified that the dynamic binding capacity for RNA molecules is dependent of the feed concentration. In order to achieve higher efficiency and selectivity, three different binding and elution strategies based on increased sodium chloride (1.75-3M) or arginine (100mM) and decreased ammonium sulfate (2.4-0M) stepwise gradients are described to purify pre-miR-29. As a matter of fact, by employing elution strategies using sodium chloride or arginine, an improvement in the final pre-miR-29 yields (97.33 and 94.88%, respectively) as well as purity (75.21 and 90.11%, respectively) were obtained. Moreover, the quality control analysis revealed that the level of impurities (proteins, endotoxins, sRNA) in the final pre-miR-29 sample was negligible. In fact, this new monolithic support arises as a powerful instrument on the microRNA purification to be used in further clinical applications, providing a more rapid and economical purification platform.

Binding mechanisms for histamine and agmatine ligands in plasmid deoxyribonucleic acid purifications

Histamine and agmatine amino acid derivatives were immobilized into monolithic disks, in order to combine the specificity and selectivity of the ligand with the high mass transfer and binding capacity offered by monolithic supports, to purify potential plasmid DNA biopharmaceuticals. Different elution strategies were explored by changing the type and salt concentration, as well as the pH, in order to understand the retention pattern of different plasmids isoforms The pVAX1-LacZ supercoiled isoform was isolated from a mixture of pDNA isoforms by using NaCl increasing stepwise gradient and also by ammonium sulfate decreasing stepwise gradient, in both histamine and agmatine monoliths. Acidic pH in the binding buffer mainly strengthened ionic interactions with both ligands in the presence of sodium chloride. Otherwise, for histamine ligand, pH values higher than 7 intensified hydrophobic interactions in the presence of ammonium sulfate. In addition, circular dichroism spectroscopy studies revealed that the binding and elution chromatographic conditions, such as the combination of high ionic strength with extreme pH values can reversibly influence the structural stability of the target nucleic acid. Therefore, ascending sodium chloride gradients with pH manipulation can be preferable chromatographic conditions to be explored in the purification of plasmid DNA biopharmaceuticals, in order to avoid the environmental impact of ammonium sulfate.