Patrícia Pereira

Recombinant pre-miR-29b for Alzheimer´s disease therapeutics

MicroRNAs are arising as the next generation of diagnostic and therapeutic tools for gene silencing. Studies demonstrated that the miR-29 expression is decreased in Alzheimer’s disease (AD) patients displaying high levels of human β-secretase (hBACE1). Recent advances toward an effective therapy for AD intend to employ miR-29 to suppress hBACE1 expression and subsequent Amyloid-β (Aβ) peptide. However, delivery of mature miRNA has demonstrated modest efficacy in vitro; therefore, the preparation of highly pure and biologically active pre-miRNA arises as one of the most important challenges in the development of these therapeutic strategies. Recently, we described a new strategy based arginine-affinity chromatography to specifically purify the recombinant pre-miR-29b. Following this strategy, the purified pre-miR-29b was successfully encapsulated into polyplexes that were further delivered in cytoplasm. It was verified that Chitosan/pre-miR-29b and Polyethylenimine/pre-miR-29b systems efficiently delivered pre-miR-29b to N2a695 cells, thus reducing the hBACE1 protein expression (around 78% and 86%, respectively) and Aβ42 levels (approximately 44% and 47%, respectively). Furthermore, pre-miR-29b downregulates the hBACE1 mRNA expression in 80%. Overall, it was demonstrated that the recombinant pre-miR-29b using polyplexes allowed to decrease the hBACE1 and Aβ42 expression levels, improving the currently available methodologies of miRNA-based therapeutics.

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.

Characterization of polyplexes involving small RNA

The purpose of the present study is to provide a tool for an efficient design and synthesis of non-viral vectors for small RNA delivery. The effects of properties of the polycation, such as molecular weight, charge density and backbone structure, to polyplex structure and physicochemical behavior were systematically evaluated. The condensing agents, polyethylenimine (PEI), chitosan (CS) and poly(allylamine) (PAA) were added to sRNA molecules at different N/P ratio. The efficiency of encapsulation and protection of sRNA, as well as polyplex size, zeta potential and morphology were followed and compared. The results show that PEI/sRNA polyplexes display a small size and positive zeta potential. However, for low molecular weights, this polycation is unable to protect sRNA in the presence of a decompacting agent. With chitosan, sRNA is efficiently compacted at high N/P ratios. The CS/sRNA complexes display small sizes, ca. 200 nm, positive surface charge and also good stability. Finally, the PAA/sRNA polyplexes were found to be the smallest at low N/P ratios, displaying a good encapsulation efficiency and high stability. A rationale for the experimental observations is provided using Monte Carlo simulation for systems with polycations of different length and charge density. The simulations showed that there is an interplay between the size of polycation chains and its charge density that define the degree of condensation for sRNA.

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.

Affinity approaches in RNAi-based therapeutics purification

The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented.

Smart micelleplexes as a new therapeutic approach for RNA delivery

ABSTRACT Introduction: Recent scientific discoveries have revealed the potential of using RNA molecules as therapeutic agents/targets. However, a significant key factor in the success of RNA-based therapeutics is the development of vehicles that allow their efficient delivery in the correct dose, time and location, without causing unwanted side effects. Areas covered: In this review, we provide an overview of the recent approaches proposed to overcome the chemical, biochemical and physiological barriers still present in the delivery of RNA-based therapeutics, in addition we will be discuss their use and drawbacks. This review highlights current knowledge and progress in the field of delivery of these therapeutic oligoribonucleotides, namely the use of micelleplexes as highly promising carriers. Finally, it is presented an analysis in what concerns to the ongoing challenges and future directions that have to be addressed to ensure the specificity and efficacy of RNA-based therapeutics for clinical application. Expert opinion: Micelleplexes can be prepared with different morphologies and stability depending on the block copolymer composition and structure. Besides, micelleplex functionalization with active targeting ligands and/or polymers allows RNA delivery or co-delivery (with chemotherapeutics agents) in a single nanocarrier, improving their therapeutic efficacy, selectivity and RNA release.

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.

New insights for therapeutic recombinant human miRNAs heterologous production: Rhodovolum sulfidophilum vs Escherichia coli

ABSTRACT RNA interference-based technologies have emerged as an attractive and effective therapeutic option with potential application in diverse human diseases. These tools rely on the development of efficient strategies to obtain homogeneous non-coding RNA samples with adequate integrity and purity, thus avoiding non-targeted gene-silencing and related side-effects that impair their application onto pre-clinical practice. These RNAs have been preferentially obtained by in vitro transcription using DNA templates or via chemical synthesis. As an alternative to overcome the limitations presented by these methods, in vivo recombinant production of RNA biomolecules has become the focus in RNA synthesis research. Therefore, using pre-miR-29b as a model, here it is evaluated the time-course profile of Escherichia coli and Rhodovolum sulfidophilum microfactories to produce this microRNA. As the presence of major host contaminants arising from the biosynthesis process may have important implications in the subsequent downstream processing, it is also evaluated the production of genomic DNA and host total proteins. Considering the rapidly growing interest on these innovative biopharmaceuticals, novel, more cost-effective, simple and easily scaled-up technologies are highly desirable. As microRNA recombinant expression fulfills those requirements, it may take the leading edge in the methodologies currently available to obtain microRNAs for clinical or structural studies.