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- Affinity chromatography in plasmid DNA purification for therapeutic applicationsPublication . Sousa, Ângela Maria Almeida de; Queiroz, João António de Sampaio Rodrigues; Sousa, Fani Pereira deThe discovery of disease-related genes and the possibility to manipulate the gene set-up in some organisms has fostered the development of innovative human DNA therapeutics over the last years. Although viral vectors are used in the majority of the trials, non-viral vectors, particularly plasmid DNA (pDNA) vectors, are attracting considerable attention as biotherapeutics both in gene therapy or DNA vaccination, due to their lower immunogenicity, toxicity and also the economic, safer and easier manufacture. Nevertheless, it is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the pDNA manufacturing process. The use of pDNA-based therapeutics relies on procedures that efficiently purify the most biologically active and effective topology, the supercoiled (sc) plasmid conformation. However, chromatographic purification of sc pDNA has specific problems which are mostly related to the structural nature of this biomolecule, the resemblance between pDNA topologies and also with some host impurities, as well as the lack of capacity and selectivity of the traditional bead adsorbents. Recently, sc pDNA purification strategies that use amino acids as immobilized ligands have yielded interesting results. Thus, the present work intends to explore and understand the underlying interactions responsible for the biorecognition of sc conformation by the amino acids ligands already used for pDNA purification as well as to establish the elution conditions that favor the prevalence of some interactions on other. By performing some fundamental studies with oligonucleotides it was observed the involvement of several elementary interactions with the amino acids matrices studied, such as hydrophobic, ring-stacking, cation-π, water-mediated bonds, multiple hydrogen bonds, van der Waals and electrostatic interactions. Although hydrophobic interactions easily appear with histidine matrix or ionic interactions with arginine matrix, it was verified the presence of other interactions in function of the elution conditions used. These results were useful for the implementation of a new affinity chromatographic strategy with the amino acid lysine for efficiently and selectively purify the sc pDNA isoform. Lysineaffinity matrix allowed the elimination of the E. coli impurities as well as other ineffective plasmid isoforms present in a complex clarified lysate meeting all the regulatory requirements. The preferential retention of the nucleic acids with higher bases exposure indicates that this matrix can be more suitable for RNA purification. In accordance with the traditional supports limitations, the alternative monolithic chromatography revealed satisfactory affinity properties with excellent mass transfer and capacity characteritics, allowing a fast and efficient plasmid isoforms separation without flow rate dependence. The similar elution conditions with histidine-agarose and the presence of canbonyldiimidazole groups suggested that the imidazole ring present in this monolithic disk is the major responsible in the specific biorecognition of sc pDNA isoform. Integration of monolithic chromatography in the pDNA manufacturing process also increase the global yield of pDNA xviiirecovery for 89%, with a purity degree according to the recommendations of the regulatory agencies that was reflected in the high transfection efficiency of sc pDNA sample on eukaryotic cells (59% in COS-7 cells). Overall, this doctoral research work revealed that amino acid-based affinity chromatography is a powerful and versatile methodology for nucleic acids purification, mainly the sc pDNA topology, guaranteeing suitable purity degree for DNA-based therapies. Besides the selectivity and specificity obtained with amino acids affinity ligands, the application of the innovative monolithic technology in the pDNA purification field brought a great improvement of the speed, resolution and capacity to the chromatographic performance, being a promising association for plasmid purification technology. Hence, this work provided valuable and helpful information concerning amino acid-affinity chromatography and chromatographic supports that can be useful in the future pDNA bioseparation either for preparative and analytical processes.
- Arginine homopeptides for plasmid DNA purification using monolithic supportsPublication . Cardoso, Sara; Sousa, Ângela; Queiroz, João; Azzoni, Adriano; Sousa, FaniPurification of plasmid DNA targeting therapeutic applications still presents many challenges, namely on supports and specific ligand development. Monolithic supports have emerged as interesting approaches for purifying pDNA due to its excellent mass transfer properties and higher binding capacity values. Moreover, arginine ligands were already described to establish specific and preferential interactions with pDNA. Additionally, some studies revealed the ability of arginine based cationic peptides to condense plasmid DNA, which increased lengthening can result in strongest interactions with higher binding capacities for chromatographic purposes of large molecules such as pDNA. In this work, arginine homopeptides were immobilized in monolithic supports and their performance was evaluated and compared with a single arginine monolithic column regarding supercoiled (sc) plasmid DNA purification. Specific interactions of arginine based peptides with several nucleic acids present in a clarified Escherichia coli lysate sample showed potential for the sc pDNA purification. Effectively, the immobilization of the arginine homopeptides became more functional compared with the single arginine amino acid, showing higher binding capacities, which was also reflected in the intensity of the interactions. The combination of structural versatilities of monoliths with the specificity of arginine peptides raised as a promising strategy for sc pDNA purification.
- HPV-16 targeted DNA vaccine expression: The role of purificationPublication . Almeida, Ana Margarida; Tomás, Joana; Pereira, Patrícia; Queiroz, João; Sousa, Fani; Sousa, ÂngelaDNA vaccines have come to light in the last decades as an alternative method to prevent many infectious diseases, but they can also be used for the treatment of specific diseases, such as cervical cancer caused by Human Papillomavirus (HPV). This virus produces E6 and E7 oncoproteins, which alter the cell cycle regulation and can interfere with the DNA repairing system. These features can ultimately lead to the progression of cervical cancer, after cell infection by HPV. Thus, the development of a DNA vaccine targeting both proteins arises as an interesting option in the treatment of this pathology. Nonetheless, before evaluating its therapeutic potential, the purity levels of a biopharmaceutical must meet the regulatory agency specifications. Previously, our research group successfully purified the supercoiled isoform of the recombinant HPV-16 E6/E7 DNA vaccine with virtual 100% purity by affinity chromatography. The present work was designed to evaluate the effect that pDNA sample purity levels may exert in the expression of a target protein. Thus, in vitro studies were performed to assess the vaccine ability to produce the target proteins and to compare the expression efficiency between the pDNA sample obtained by affinity chromatography, which only presents the sc isoform and fulfils the regulatory agency recommendations, and the same DNA vaccine retrieved by a commercial purification kit, which contains different pDNA isoforms. Our achievements suggest that the E6/E7 DNA vaccine purified by affinity chromatography promotes higher E6 and E7 mRNA and protein expression levels than the DNA vaccine purified with the commercial kit. Overall, these results underline the importance that a purification strategy may present in the therapeutic outcome of recombinant DNA vaccines, envisaging their further application as biopharmaceuticals. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:546-551, 2018.
- Minicircle DNA purification: Performance of chromatographic monoliths bearing lysine and cadaverine ligandsPublication . Almeida, Ana Margarida; Queiroz, João; Sousa, Fani; Sousa, ÂngelaMinicircle DNA (mcDNA) technology is in the vanguard of vectors designed for gene therapy, since the absence of prokaryotic sequences confers to mcDNA higher biosafety in comparison to other DNA vectors. However, the presence of other isoforms and non-recombined parental molecules hampers the isolation of supercoiled (sc) mcDNA with the chromatographic methods already established for plasmid purification. In this work, two monolithic supports were modified with lysine and its decarboxylated derivative, cadaverine, to explore their performance in the sc mcDNA purification. Increasing NaCl gradients and different pH values (from 6 to 9) were tested in both modified monoliths. In general, cadaverine modified support established stronger interactions with mcDNA than lysine modified monolith, at acidic pH. For instance, at pH 6.0 the retention time for RNA and DNA molecules in lysine modified monolith was 11.58 and 14.59, respectively, while for cadaverine modified monolith was 20.32 and 27.12, respectively. The lysine modified monolith was able to successfully isolate sc mcDNA from the lysate sample. However, recovery yield was significantly sacrificed to guarantee high purity levels of sc mcDNA. The cadaverine modified monolith showed better selectivity than the previous monolith, achieving the successful sc mcDNA isolation from the lysate sample. The final sc mcDNA sample, obtained by the column that showed the best performance, was characterized by real-time PCR, presenting 98.4% purity and 78.6% recovery yield. The impurities content, namely genomic DNA, proteins and endotoxins, was found within the criteria established by regulatory agencies. Overall, a simple and practical chromatographic strategy to purify sc mcDNA was for the first time implemented by exploring a modified monolithic column, with no significant reduction on the purity and recovery and without resorting to backbone modification or specific enzymatic digestion. Such features will surely be crucial in the industrial scale-up of this chromatographic strategy since it will not be associated with significant cost-increase.
- Separation of different forms of proteose peptone 3 by hydrophobic interaction chromatography with a dual salt systemPublication . Sousa, Ângela Maria Almeida de; Passarinha, Luís António Paulino; Rodrigues, L.R.; Teixeira, José; Mendonça, António; Queiroz, JoãoA panel of four hydrophobic adsorbents (butyl-, octyl-, phenyl- and epoxy-Sepharose) was used to examine the selectivity and fractionation of several proteose peptone 3 (PP3) forms from a freeze-dried extract of whey bovine milk. In particular, the effects of altering the ligand type and salt were investigated. The chromatographic studies suggest that PP3 strongly interacts among the three commercial hydrophobic resins leading to a drop off in selectivity, while a complete binding was achieved at low salt concentrations (below 0.5 M) and total elution only with phosphate buffer and/or water stepwise conditions. Only in epoxy–Sepharose was an appreciably selectivity of the several fractions of PP3 present in the initial feedstock attained. Despite the high salt concentration for a complete binding of PP3 (above 1.5 M ammonium sulfate) onto this support, the dual salt system (ammonium sulfate 1 M and sodium citrate 0.8 M) led to a high separation degree of high and low molecular weight forms of PP3.
- Cervical cancer and HPV infection: ongoing therapeutic research to counteract the action of E6 and E7 oncoproteinsPublication . Almeida, Ana Margarida; Queiroz, João; Sousa, Fani; Sousa, ÂngelaCervical cancer is the fourth most common cancer among women worldwide and its development is mainly associated with human papillomavirus infection, a highly sexually transmissible virus. The expression of E6 and E7 viral oncoproteins deregulates cell repairing mechanisms through impairment of tumor suppressor protein functions, such as p53 or retinoblastoma protein. Although the implementation of new preventive vaccines has decreased the infection rate and cervical cancer progression, there are still many women who are affected by this pathology. Nowadays, the main treatment often requires the use of invasive techniques. From well-established strategies, like DNA vaccines and gene therapy, to innovative gene silencing technologies; different methodologies are currently under scrutiny that target the E6 and E7 oncoproteins and/or their modes of action.
- 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.
- Nanoparticle mediated delivery of pure P53 supercoiled plasmid DNA for gene therapyPublication . Gaspar, Vítor Manuel Abreu; Correia, Ilídio; Sousa, Ângela; Silva, Filomena; Paquete, Catarina; Queiroz, João; Sousa, FaniThe 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.
- Chromatographic HPV-16 E6/E7 plasmid vaccine purification employing L-histidine and 1-benzyl-L-histidine affinity ligandsPublication . Amorim, Lúcia; Gaspar, Rita; Pereira, Patrícia; Černigoj, Urh; Sousa, Fani; Queiroz, João; Sousa, ÂngelaAffinity chromatography based on amino acids as interacting ligands was already indicated as an alternative compared to ion exchange or hydrophobic interaction for plasmid DNA purification. Understanding the recognition mechanisms occurring between histidine-based ligands and nucleic acids enables more efficient purification of a DNA vaccine, as the binding and elution conditions can be adjusted in order to enhance the purification performance. Decreasing pH to slightly acidic conditions increases the positive charge of histidine ligand, what influences the type of interaction between chromatographic support and analytes. This was proven in this work, where hydrophobic effects established in the presence of ammonium sulfate were affected at pH 5.0 in comparison to pH 8.0, while electrostatic and cation-π interactions were intensified. Histidine ligand at pH 5.0 interacts with phosphate groups or aromatic rings of plasmid DNA. Due to different responses of RNA and pDNA on mobile phase changes, the elution order between RNA and pDNA was changed with mobile phase pH decrease from 8.0 to 5.0. The phenomenon was more evident with L-histidine ligand due to more hydrophilic character, leading to an improved selectivity of L-histidine-modified chromatographic monolith, allowing the product recovery with 99% of purity (RNA removal). With the 1-benzyl- L-histidine ligand, stronger and less selective interactions with the nucleic acids were observed due to the additional hydrophobicity associated with the phenyl aromatic ring. Optimization of sample displacement chromatography parameters (especially (NH4 )2 SO4 concentration) at slightly acidic pH enabled excellent isolation of pDNA, by the removal of RNA in a negative mode, with binding capacities above 1.5 mg pDNA per mL of chromatographic support.
- The Effectiveness of Therapeutic Vaccines for the Treatment of Cervical Intraepithelial Neoplasia 3: A Systematic Review and Meta-AnalysisPublication . Ventura, Cathy; Luís, Ângelo; Soares, Christiane P.; Venuti, Aldo; Paolini, Francesca; Pereira, L.; Sousa, ÂngelaCervical cancer (CC) is a disease that affects many women worldwide, especially in low-income countries. The human papilloma virus (HPV) is the main causative agent of this disease, with the E6 and E7 oncoproteins being responsible for the development and maintenance of transformed status. In addition, HPV is also responsible for the appearance of cervical intraepithelial neoplasia (CIN), a pre-neoplastic condition burdened by very high costs for its screening and therapy. So far, only prophylactic vaccines have been approved by regulatory agencies as a means of CC prevention. However, these vaccines cannot treat HPV-positive women. A search was conducted in several databases (PubMed, Scopus, Web of Science, and ClinicalTrials.gov) to systematically identify clinical trials involving therapeutic vaccines against CIN 3. Histopathological regression data, immunological parameters, safety, DNA clearance, and vaccine efficacy were considered from each selected study, and from the 102 articles found, 8 were selected based on the defined inclusion criteria. Histopathological regression from CIN 3 to CIN < 1 was 22.1% (95% CI: 0.627–0.967; p-value = 0.024), showing a vaccine efficacy of 23.6% (95% CI; 0.666–0.876; p-value < 0.001). DNA clearance was assessed, and the risk of persistent HPV DNA was 23.2% (95% CI: 0.667–0.885; p-value < 0.001). Regarding immunological parameters, immune responses by specific T-HPV cells were more likely in vaccinated women (95% CI: 1.245–9.162; p-value = 0.017). In short, these studies favored the vaccine group over the placebo group. This work indicated that therapeutic vaccines are efficient in the treatment of CIN 3, even after accounting for publication bias.