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The diversity of biomolecules present in plasmid DNA (pDNA)-containing extracts and the structural and chemical similarities between pDNA and impurities are some of the main challenges of improving or establishing novel purification procedures. In view of the unequalled specificity of affinity purification, this technique has recently begun to be applied in downstream processing of plasmids. This paper discusses the progress and importance of affinity chromatography (AC) for the purification of pDNA-based therapeutic products. Several affinity approaches have already been successfully developed for a variety of applications, and we will focus here on highlighting their possible contributions to the pDNA purification challenge. Diverse affinity applications and their advantages and disadvantages are discussed, as well as the most significant results and improvements in the challenging task of purifying plasmids. [Copyright &y& Elsevier]

Minicircle DNA (mcDNA) is a smaller and safer version of non-viral DNA vectors that results from a cutting-edge in vivo recombination process to excise prokaryotic sequences from plasmid DNA (pDNA). Considering the molecule's potential and increasing interest as a non-viral DNA-based therapeutic, biomanufacturing methodologies need to be improved, especially in downstream processing. [ABSTRACT FROM AUTHOR]

• Human papillomavirus (HPV) has a primordial role in the development of cervical cancer. • Therapies currently available for cervical cancer treatment are reviewed. • The relevance of nucleic-acid-based therapies targeting HPV functions is discussed. • HPV oncoprotein silencing is a promising path to explore in cervical cancer treatment. Novel nucleic acid-based therapeutic approaches as a promising non-invasive solution for the treatment of cervical cancer caused by Human Papillomavirus. Cervical 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. [ABSTRACT FROM AUTHOR]

Abstract: A mass transfer model is proposed for predicting sieving coefficients, S obs , of supercoiled plasmid DNA (pDNA), in the presence of a salt, in membranes with narrow pores, i.e., pores smaller than the gyration radii of the plasmids to be considered for a certain application. The model assumes that permeation occurs due to plasmid suction at the membrane surface as a result of the convective flow, being the probability of permeation also dependent on the instantaneous molecular conformation of the plasmid, when getting close to the pore. Two different approaches are tested to model plasmid structure, that of a closed segmented chains (CSC) of double stranded DNA, and that of considering the superhelical chain as a freely jointed chains (FJC). Both approaches were used to estimate the radius of gyration, r g , of different plasmids by statistical simulation, and the obtained values were compared with experimental data available in the literature. A 6050bp plasmid, pVAX1-LacZ, was used in the experimental work, in which filtration tests were performed using three different ultrafiltration membranes of known pore size, in a 10ml stirred cell. At constant ionic strength, sieving coefficients were determined as a function of the permeate flux, J v , at two different values of stirring speed, ω. The results are in very good agreement with the model predictions at the highest stirring speed and the observed deviations found at the lowest stirring speed were interpreted with the aid of the developed model by considering the possibility of plasmid adsorption. Then, it was investigated the effect of changing the ionic strength of the medium at constant J v and ω. The obtained results clearly agree with the model predictions. [Copyright &y& Elsevier]

Abstract: A model is proposed for predicting the intrinsic rejections of linear flexible molecules, of high molecular dimensions, in microfiltration and ultrafiltration membranes. The developed model is based on the available theory of hindered transport of solutes through narrow pores, assuming that convection is the only relevant transport mechanism. For the application of the model, partition coefficients of the solutes between the solution and the membrane were estimated by a stochastic simulation, assuming suction of the macrosolutes into the pore. In these simulations, the macromolecules were modeled as freely jointed chains. Different methods for the estimation of the molecular dimensions of the solutes are discussed. The validity of the model was tested using dextran T2000 and plasmid pUC19 in its linear isoform, as model solutes; the tests were performed using membranes of different pore size, that include two ultrafiltration membranes previously characterized, having 4.1 and 10.5nm of pore radius, and two track-etched polycarbonate membranes, having 15 and 40nm of pore radius. Filtration was performed in a stirred cell, at controlled permeate flux. Different conditions of stirring speed and permeate flux were tested to check the consistency of the model. The results show that very satisfactory predictions are obtained, considering the simplicity of the model, especially in the case of the plasmid. [Copyright &y& Elsevier]

• Supercoiled HPV-16 E6/E7 pDNA was successfully purified by two different approaches. • Sample displacement phenomena allowed the elimination of non-functional pDNA isoforms. • CaCO 3 -gelatin complexes showed satisfactory sc pDNA encapsulation. • THP-1 cells were successfully transfected and correct E6 antigen expression was observed. New biotechnological strategies are being explored, aimed at rapid and economic manufacture of large quantities of DNA vaccines with the required purity for therapeutic applications, as well as their correct delivery as biopharmaceuticals to target cells. This report describes the purification of supercoiled (sc) HPV-16 E6/E7 plasmid DNA (pDNA) vaccine from a bacterial lysate, using an arginine-based monolith, presenting a spacer arm in its configuration. To enhance the performance of the purification process, monolith modification with the spacer arm can improve accessibility of the arginine ligand. By using a low NaCl concentration at pH 7.0, a condition to eliminate the RNA impurity directly in the flow through was established. The pH increase to 7.5 allowed the elimination of non-functional pDNA isoforms, the sc pDNA being recovered by increasing the ionic strength. As well as a binding capacity of 2.53 mg/mL obtained with a pre-purified sc pDNA sample, the column also purified sc pDNA from high lysate loading, with capacities above 1 mg/mL. Due to the sample displacement phenomena, non-functional pDNA isoforms were eliminated throughout column loading, favoring the degree of purity of final sc pDNA of 93.3%–98.5%. Thereafter, purified sc pDNA was successfully encapsulated into CaCO 3 -gelatin nano-complexes. Delivery of the pDNA-carriers to THP-1 cells was assessed through pDNA cellular uptake evaluation and correct E6 expression was verified by mRNA and protein detection. A biotechnological platform was established for sc pDNA purification and delivery to dendritic cells, stimulating further in vivo studies. [ABSTRACT FROM AUTHOR]