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Biopharmaceuticals are gaining high relevance in the pharmaceutical industry and market, due to their specific design for many therapeutic conditions and ability to restore biological functions. As a matter of fact, gene therapy products have been studied and developed in the past several years, and more recently, they arouse real interest as promising alternatives for the establishment of rapid, efficient, and safe treatments for incurable diseases and pandemics. Plasmid DNA (pDNA) is a nonviral vector with great potential in gene therapy due to its biosafety, standard manufacturing, and reduced production cost. Among the different pDNA topologies, the supercoiled (sc) isoform is considered a structure with a higher therapeutic effect. Thus, the production and purification of such a biomolecule must be controlled by suitable analytical methods in order to guarantee the fulfillment of the criteria established by regulatory agencies regarding the purity and homogeneity of sc pDNA for therapeutic applications. Therefore, it is important to ensure that the quality control methods are fast, precise, and specific for the assessment of the target biomolecule. Thus, in the laboratory experiment of a pharmaceutical biotechnology class, a commercial CIMac DEAE pDNA analytical column is used by students for the assessment of the purity level and recovery yield of sc pDNA obtained by recombinant production and a histamine monolith-based purification process. This laboratory class allows students of the Biotechnology or Pharmaceutical Sciences Master degrees to understand the global biotechnological process involved in sc pDNA production and the quality control methods necessary to validate these products for safe therapeutic administration.

AbstractUsing a molecular dynamics approach, the study of the interaction between six different known ligands and a predicted pre-miRNA 149 RNA G-quadruplex (rG4) structure is reported. The stabilization of rG4 structures formed within the pre-miRNA stem-loop regions using small ligands is an attractive anticancer strategy. Particularly, miRNA-149 is upregulated in a variety of cancers such as prostate cancer and is therefore a potential target for drug development. The results show that ligands C8and PhenDC3interact with the rG4 structure via stacking interactions with the end G-quartets. Ligands [16]phenN2, [32]phen2N4and pyridostatin on the other hand bind the loops/groove interface of the rG4 being H-bonding and electrostatic interactions the driving force of the interaction. The C8precursor, C8-NH2, emphasizes the structural nuances of the rG4 short loops as the lack of a large terminal aromatic moiety produced a mixed stacking-groove binding mode. Overall, this study may help the design of specific ligands for pre-miRNA rG4 towards anticancer therapeutics development.Communicated by Ramaswamy H. Sarma

Background: Several gene deviations can be responsible for triggering oncogenic processes. However, mutations in tumour suppressor genes are usually more associated to malignant diseases, with p53 being one of the most affected and studied element. p53 is implicated in a number of known cellular functions, including DNA damage repair, cell cycle arrest in G1/S and G2/M and apoptosis, being an interesting target for cancer treatment. Objective: Considering these facts, the development of gene therapy approaches focused on p53 expression and regulation seems to be a promising strategy for cancer therapy. Results: Several studies have shown that transfection of cancer cells with wild-type p53 expressing plasmids could directly drive cells into apoptosis and/or growth arrest, suggesting that a gene therapy approach for cancer treatment can be based on the re-establishment of the normal p53 expression levels and function. Up until now, several clinical research studies using viral and non-viral vectors delivering p53 genes, isolated or combined with other therapeutic agents, have been accomplished and there are already in the market, therapies based on the use of this gene. Conclusion: This review summarizes the different methods used to deliver and/or target the p53 as well as the main results of therapeutic effect obtained with the different strategies applied. Finally, the ongoing approaches are described, also focusing on the combinatorial therapeutics to show increased therapeutic potential of combining gene therapy vectors with chemo or radiotherapy.

ABSTRACTRNA 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 vitrotranscription using DNA templates or via chemical synthesis. As an alternative to overcome the limitations presented by these methods, in vivorecombinant 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 coliand Rhodovolum sulfidophilummicrofactories 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.

Mitochondrial gene therapy seems to be a valuable and promising strategy to treat mitochondrial disorders. The use of a therapeutic vector based on mitochondrial DNA, along with its affinity to the site of mitochondria, can be considered a powerful tool in the reestablishment of normal mitochondrial function. In line with this and for the first time, we successfully cloned the mitochondrial gene ND1 that was stably maintained in multicopy pCAG-GFP plasmid, which is used to transform E. coli. This mitochondrial-gene-based plasmid was encapsulated into nanoparticles. Furthermore, the functionalization of nanoparticles with polymers, such as cellulose or gelatin, enhances their overall properties and performance for gene therapy. The fluorescence arising from rhodamine nanoparticles in mitochondria and a fluorescence microscopy study show pCAG-GFP-ND1-based nanoparticles’ cell internalization and mitochondria targeting. The quantification of GFP expression strongly supports this finding. This work highlights the viability of gene therapy based on mitochondrial DNA instigating further in vitro research and clinical translation.

ABSTRACTIntroduction: 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.

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 expression is a challenging issue that is of critical importance for a successful therapeutic outcome. The use of miniaturized mcDNA cassettes with additional modifications that increase and prolong expression may contribute to an improved generation of biopharmaceuticals. The unique features of mcDNA render it an attractive alternative to overcome current technical issues and to bridge the significant gap that exists between basic research and clinical applications.

The aim of this work was to provide binding information between the recombinant pre-miR-29b and l-arginine/l-lysine by surface plasmon resonance (SPR) and circular dichroism (CD). This information brings important insights concerning the characterization of the microRNA binding onto chromatographic supports using these amino acids as specific ligands. Moreover, it is possible to determine some particular conditions enabling the improvement of the binding specificity of the amino acid ligands used to purify microRNA, preserving their integrity. The binding responses were detectable and reproducible, allowing the determination of the equilibrium dissociation constant (KD). Considering the binding affinities, it was verified that the pre-miR-29b binds more strongly to l-arginine (KDbetween 10−6and 10−7M) than to l-lysine (KDbetween 10−5and 10−7M). Remarkably, the results revealed that the ligands possess high affinity to RNA molecules using buffers with low salt concentrations and no binding responses were detected with high salt concentrations, suggesting that electrostatic interactions are mainly responsible for the ligand–analyte interaction. Above all, this study showed the importance of SPR for future screening of other ligands that, like the ones described herein, can be used to design novel microRNA purification platforms which will have a significant impact on biopharmaceutical-based therapeutics.

The development of a strategy to plasmid DNA (pDNA) purification has become necessary for the development of gene therapy and DNA vaccine production processes in recent years, since this nucleic acid and most of contaminants, such as RNA, genomic DNA and endotoxins, are negatively charged. An ideal separation methodology may be achieved with the use of affinity interactions between immobilized amino acids and nucleic acids. In this study, the binding behaviour of nucleic acids under the influence of different environmental conditions, such as the composition and ionic strength of elution buffer, and the temperature, is compared with various amino acids immobilized on chromatography resins. Supercoiled (sc) plasmid isoform was isolated with all matrices used, but in some cases preferential interactions with other nucleic acids were found. Particularly, lysine chromatography showed to be an ideal technology mainly on RNA purification using low salt concentration. On the other hand, arginine ligands have shown a greater ability to retain the sc isoform comparatively to the other nucleic acids retention, becoming this support more adequate to sc pDNA purification. The temperature variation, competitive elution and oligonucleotides affinity studies also allowed to recognize the dominant interactions inherent to biorecognition of pDNA molecule and the affinity matrices.

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.

The use of gene-based products, such as DNA or RNA, is increasingly being explored for various innovative therapies. However, the success of these strategies is highly dependent on the effective delivery of these biomolecules to target cells. Therefore, the development of pH-responsive nanoparticles comprises the creation of intelligent delivery systems with high therapeutic efficiency. In this work, the pH-responsiveness of the poly(2-(diisopropylamino)ethyl methacrylate)) (PDPA) block was investigated for the encapsulation and delivery of small RNAs (sRNA) to cancer cells. The pH responsiveness was dependent on the protonation profile of the tertiary amines of PDPA, which directly affected the electrostatic interactions established with RNA. Thus, block copolymers based on poly(oligo(ethylene oxide) methyl ether methacrylate) (POEOMA) and PDPA, POEOMA-b-PDPA, were synthesized by supplemental activator and reducing agent atom transfer radical polymerization (SARA ATRP). The structure of the block copolymers was characterized by size exclusion chromatography and 1H NMR spectroscopy. The copolymers allowed effective complexation of model sRNAs and a pre-miRNA with efficiencies of about 89 % and 91 %, respectively. The characterization by dynamic light scattering revealed that these systems had sizes between 76 and 1375 nm. It was also found that the morphology of the polyplexes depended on the pH, since the preparation at a pH lower than the pKa of the copolymers resulted in spherical but polydisperse particles, while higher pH values resulted in nanoparticles with more homogeneous size, but altered morphology. Moreover, due to pH-responsiveness, it was achieved the release of RNA at pH higher than the pKa of the copolymers, while maintaining its integrity. The polyplexes also showed a high potential to protect RNA from RNases. The transfection of a lung cancer model (A549) and fibroblast cell lines showed that these polyplexes did not cause cell toxicity. In addition, the polyplexes enabled the effective transfection of the A549 cell line with pre-miRNA-29b and miRNA-29b, resulting in a decrease of expression levels of the target DNMT3B gene by approximately 51 % and 47 %, respectively. Overall, the POEOMA-b-PDPA copolymers proved to be a promising strategy for developing responsive delivery systems, that can play a critical role in some diseases, such as cancer, where pH varies between the intra and extracellular environments.

MicroRNAs(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 miRNAtherapeutics 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 miRNAdelivery 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 RNA2017, 8:e1409. doi: 10.1002/wrna.1409 For further resources related to this article, please visit the WIREs website. Barriers to in vivo microRNAs delivery