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Purification of pre-miRNA-29 with ionic liquids-based supports

Publication . Nunes, Sérgio Almeida; Sousa, Fani Pereira de; Bernardo, Sandra

MicroRNAs (miRNAs) have been studied regarding their biological role in gene expression regulation at post-transcriptional level, envisioning the establishment of new treatment strategies. Pre-miRNA-29 is the precursor form of microRNA-29, a small noncoding RNA that plays an important role in gene silencing, and proved to be involved in several cellular mechanisms, namely in neurodegenerative diseases. To consider the therapeutic application, efficient, and economically feasible manufacturing processes are required for obtaining these biopharmaceuticals, assuring their integrity, purity, stability, and bioactivity. Thus, in this work it is proposed an alternative purification method using Ionic Liquids (ILs)-based chromatographic supports. ILs are described as ionic compounds that present a melting point below 100 °C and are normally composed of large and asymmetric organic cations and organic or inorganic anions. These types of compounds can be tailored in terms of their properties, such as polarity, density, and viscosity to achieve their effectiveness regarding the purification and stabilization of nucleic acids. The application of Supported ILs (SILs) in RNA purification is highly innovative and can lead to the establishment of advantageous purification methods. Herein, the SIL under study resulted from the functionalization of silica with the 1-(3- aminopropyl)imidazole chloride, which was named as SilPrImPrACl. First, a screening of the best experimental conditions, in terms of pH and ionic strength, to promote binding and elution of RNA was carried out. Moreover, in order to develop an Experimental Design, key factors were optimized at 3 levels, using the Central Composite Design (CCD) as model. The statistical analysis of the results showed that this model was not the best fit for the optimization of the purification of pre-miRNA-29b, however further analysis was carried out in order to understand the influence of the factors studied in the recovery, purification factor and removal of impurities. Thus, it was possible to establish some conditions conducting to a recovery and purification factor of 35.1% and 1.55, respectively. Furthermore, it was verified that the process was able to remove 78% of proteins and 70% of gDNA present in the initial sample, proving the effectiveness of this SIL for the purification of RNA and particularly the pre-miRNA-29b.

2023-01-20Master thesis

Embargoed access

Novel Excipient Candidates for RNA Stabilization

Publication . Oliveira, Sofia Pereira; Sousa, Fani Pereira de; Martins, Mara Guadalupe Freire

Until recently, the study of therapies using the RNA molecule as a therapeutic agent was considerably low when compared to therapies using DNA and proteins. For a long time, the RNA molecule was considered as a mere intermediary in the production of proteins. More recently, the study of the RNA molecule has led to a better understanding of its function, thus bringing a new perspective to potential therapeutic applications of this molecule. The use of therapeutic RNAs namely, mRNAs and microRNAs has been increasing, especially due to the recent use of mRNA in vaccination against COVID-19. One of the limitations in the use of mRNA vaccines, which has seen a lot of discussion, is the temperature at which they have to be stored, in order to maintain vaccine stability and effectiveness. This reveals the importance and urgency in identifying compounds capable of stabilizing the RNA molecule in order to facilitate, the storage and handling, while maintaining its biological activity. Taking into account the growing research in the therapeutic RNAs field, identifying stabilizers might have an impact when working with other RNA molecules with different applications. In the present work, Circular Dichroism (CD) assays were performed to assess the stability of the RNA structure, after incubation with different categories of excipients, namely amino acids (arginine and glycine), ionic liquids (N-acetyl amino acid N-alkyl cholinium-based ILs), sugars (glucose, sucrose and trehalose) and Deep Eutectic Solvents (DES) (glucose-based DES and trehalose-based DES). These studies also involved the assessment of stability over time (0, 7, 15, 30 and 60 days) at different storage temperatures (4 ° C and room temperature). In general, the results obtained demonstrate the potential of amino acids to stabilize RNA, for 60 days of storage at both temperatures. Moreover, the samples stabilized by the new excipient candidates also demonstrated great potential, taking into account that the samples stabilized by trehalose-based DES not only maintained their stability after 60 days at both temperatures, but also increased the melting temperature by around 18 °C. Data gathered by molecular docking simulations allowed for a better understanding of the interactions that happen between DES and RNA and which of the compounds might have a higher contribution for the RNA stabilization. Results using amino acid-based ionic liquids as stabilizers were also very encouraging, as most of the samples tested maintained stability over the testing period at both temperatures. Considering the results obtained, these confirm the stabilizing capacity of the classes already used as excipients and the potential of new classes as RNA stabilizers. Thus opening the door to new possibilities for formulating therapeutic products.

2021-03-12Master thesis

Open access

Non-coding RNAs: Emerging from the discovery to therapeutic applications

Publication . Baptista, Bruno; Riscado, Micaela; Queiroz, João; Pichon, Chantal; Sousa, F.

The knowledge about non-coding RNAs (ncRNAs) is rapidly increasing with new data continuously emerging, regarding their diverse types, applications, and roles. Particular attention has been given to ncRNA with regulatory functions, which may have a critical role both in biological and pathological conditions. As a result of the diversity of ncRNAs and their ubiquitous involvement in several biologic processes, ncRNA started to be considered in the biomedical field, with immense potential to be exploited either as biomarkers or as therapeutic agents in certain pathologies. Indeed, ncRNA-based therapeutics have been proposed in many disorders and some even reached clinical trials. However, to prepare an RNA product suitable for pharmacological applications, certain criteria must be fulfilled, and it has to be guaranteed RNA purity, stability, and bioactivity. So, in this review, the different types of ncRNAs are identified and characterized, by describing their biogenesis, functions, and applications. A perspective on the main challenges and innovative approaches for the future and broad therapeutic application of RNA is also presented.

2021-02-10Journal article

Restricted access

Multimodal ionic liquid-based chromatographic supports for an effective RNA purification

Publication . Carapito, Ana Rita Mugeiro; Bernardo, Sandra C.; Pereira, Matheus M.; Neves, Márcia C.; Freire, Mara G.; Sousa, Fani

Nucleic acids have been considered interesting molecules to be used as biopharmaceuticals for the treatment of various diseases, in gene therapy strategies. In particular, RNA arises as the most promising approach because it does not require access to the nucleus of cells to exert its function; however, it is quite challenging due to its labile nature. To increase the possibility of translating RNA-based technology to clinical protocols, the bio- manufacturing of RNAs has been intensively exploited in the last few years. However, the standard RNA puri- fication processes remain time-consuming and present limitations regarding recovery yield and purity. This work describes the functionalization of chromatographic silica-based supports with four ionic liquids (ILs) composed of functional moieties that can promote distinct interactions with nucleic acids. After an initial screening to evaluate the binding and elution behavior of nucleic acids in the IL-based supports, SSi[C3C3NH2Im]Cl has shown to be the most promising for further purification assays. This support was studied for the RNA purification from different samples (clarified or more complex) and has shown to be highly effective, for all the conditions studied. Generally, it is here presented a new method for RNA isolation in a single step, using an IL-based chromato- graphic support, able to eliminate the usage of hazardous compounds often included in standard RNA extraction protocols.

2023Journal article

Open access

Brain-Targeted Delivery of Pre-miR-29b Using Lactoferrin-Stearic Acid-Modified-Chitosan/Polyethyleneimine Polyplexes

Publication . Pereira, Patrícia; Barreira, Maria; Cruz, Carla; Tomás, Joana; Luís, Ângelo; Pedro, Augusto; Queiroz, João; Sousa, Fani

The efficacy of brain therapeutics is largely hampered by the presence of the blood-brain barrier (BBB), mainly due to the failure of most (bio) pharmaceuticals to cross it. Accordingly, this study aims to develop nanocarriers for targeted delivery of recombinant precursor microRNA (pre-miR-29b), foreseeing a decrease in the expression of the BACE1 protein, with potential implications in Alzheimer's disease (AD) treatment. Stearic acid (SA) and lactoferrin (Lf) were successfully exploited as brain-targeting ligands to modify cationic polymers (chitosan (CS) or polyethyleneimine (PEI)), and its BBB penetration behavior was evaluated. The intracellular uptake of the dual-targeting drug delivery systems by neuronal cell models, as well as the gene silencing efficiency of recombinant pre-miR-29b, was analyzed in vitro. Labeled pre-miR-29b-CS/PEI-SA-Lf systems showed very strong fluorescence in the cytoplasm and nucleus of RBE4 cells, being verified the delivery of pre-miR-29b to neuronal cells after 1 h transfection. The experiment of transport across the BBB showed that CS-SA-Lf delivered 65% of recombinant pre-miR-29b in a period of 4 h, a significantly higher transport ratio than the 42% found for PEI-SA-Lf in the same time frame. Overall, a novel procedure for the dual targeting of DDS is disclosed, opening new perspectives in nanomedicines delivery, whereby a novel drug delivery system harvests the merits and properties of the different immobilized ligands.

2020Journal article

Open access