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- Advances in time course extracellular production of human pre-miR-29b from Rhodovulum sulfidophilumPublication . Pereira, Patrícia; Pedro, Augusto; Tomás, Joana; Baptista, Cláudio; Queiroz, João; Figueiras, Ana; Sousa, FaniThe present study reports the successful production of human pre-miR-29b both intra- and extracellularly in the marine phototrophic bacterium Rhodovulum sulfidophilum using recombinant RNA technology. In a first stage, the optimal transformation conditions (0.025 μg of plasmid DNA, with a heat-shock of 2 min at 35 °C) were established, in order to transfer the pre-miR-29b encoding plasmid to R. sulfidophilum host. Furthermore, the extracellular recovery of this RNA product from the culture medium was greatly improved, achieving quantities that are compatible with the majority of applications, namely for in vitro or in vivo studies. Using this system, the extracellular human pre-miR-29b concentration was approximately 182 μg/L, after 40 h of bacterial growth, and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 h. At the end of the fermentation, it was verified that almost 87 % of cells were viable, indicating that cell lysis is minimized and that the extracellular medium is not highly contaminated with the host intracellular ribonucleases (RNases) and endotoxins, which is a critical parameter to guarantee the microRNA (miRNA) integrity. These findings demonstrate that pre-miRNAs can be produced by recombinant RNA technology, offering novel clues for the production of natural pre-miRNA agents for functional studies and RNA interference (RNAi)-based therapeutics.
- Purification of pre-miR-29 by arginine-affinity chromatographyPublication . Pereira, Patrícia; Sousa, Ângela; Queiroz, João; Correia, Ilídio; Figueiras, Ana; Sousa, FaniRecently, differential expression of microRNAs, in patients with Alzheimer's 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 (280–500 mM), arginine (25 mM) or decreased ammonium sulfate (2–0.1 M) 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.
- Smart micelleplexes as a new therapeutic approach for RNA deliveryPublication . Pereira, Patrícia; Barreira, Maria; Queiroz, João; Veiga, Francisco; Sousa, Fani; Figueiras, AnaRecent 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.
- Current progress on microRNAs-based therapeutics in neurodegenerative diseasesPublication . Pereira, Patrícia; Queiroz, João; Figueiras, Ana; Sousa, FaniMicroRNAs (miRNAs)-based therapy has recently emerged as a promising strategy in the treatments of neurodegenerative diseases. Thus, in this review, the most recent and important challenges and advances on the development of miRNA therapeutics for brain targeting are discussed. In particular, this review highlights current knowledge and progress in the field of manufacturing, recovery, isolation, purification, and analysis of these therapeutic oligonucleotides. Finally, the available miRNA delivery systems are reviewed and an analysis is presented in what concerns to the current challenges that have to be addressed to ensure their specificity and efficacy. Overall, it is intended to provide a perspective on the future of miRNA-based therapeutics, focusing the biotechnological approach to obtain miRNAs. WIREs RNA 2017, 8:e1409. doi: 10.1002/wrna.1409 For further resources related to this article, please visit the WIREs website.