Gabriel A. Monteiro

Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks

Gene therapy is a promising process for the prevention, treatment and cure of diseases such as cancer, acquired immunodeficiency syndrome (AIDS) and cystic fibrosis. One of the methods used to administer therapeutic genes is the direct injection of naked or lipid-coated plasmid DNA, but this requires considerable amounts of plasmid DNA. There are several problems and bottlenecks associated with the design and operation of large-scale processes for the production of pharmaceutical-grade plasmid DNA for gene therapy.

Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications

Interest in producing large quantities of supercoiled plasmid DNA has recently increased as a result of the rapid evolution of gene therapy and DNA vaccines. Owing to the commercial interest in these approaches, the development of production and purification strategies for gene-therapy vectors has been performed in pharmaceutical companies within a confidential environment. Consequently, the information on large-scale plasmid purification is scarce and usually not available to the scientific community. This article reviews downstream operations for the large-scale purification of plasmid DNA, describing their principles and the strategy used to attain a final product that meets specifications.

Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography

The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis plasmid vector (pCF1‐CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4‐butanediol‐diglycidylether. The use of HIC took advantage of the more hydrophobic character of single‐stranded nucleic acid impurities as compared with double‐stranded plasmid DNA. RNA, denatured genomic and plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled plasmid, were retained and separated from nonbinding plasmid DNA in a 14‐cm HIC column. Anion‐exchange HPLC analysis proved that >70% of the loaded plasmid was recovered after HIC. RNA and denatured plasmid in the final plasmid preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/μg pDNA and 0.048 EU/μg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000‐fold. Host proteins were not detected in the final preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns.

Production, purification and analysis of an experimental DNA vaccine against rabies

The basic and applied research efforts devoted to the development of DNA vaccines must be accompanied by manufacturing processes capable of being scaled up and delivering a clinical‐grade product. This work describes a rapid process of this kind, based on hydrophobic interaction chromatography (HIC) for the production of milligram quantities of an experimental DNA rabies vaccine. Its properties and protective activity are tested in comparison with the same plasmid DNA purified with a commercial kit.

The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression

Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non‐viral vectors. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease‐rich environments. Homopurine‐rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance.

Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli

BackgroundEfflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems.ResultsEtBr transport across the cell envelope of E. coli K-12 and derivatives was analysed by a semi-automated fluorometric method. Accumulation and efflux of EtBr was studied under limiting energy supply (absence of glucose and low temperature) and in the presence and absence of the efflux pump inhibitor, chlorpromazine. The bulk fluorescence variations were also observed by single-cell flow cytometry analysis, revealing that once inside the cells, leakage of EtBr does not occur and that efflux is mediated by active transport. The importance of AcrAB-TolC, the main efflux system of E. coli, in the extrusion of EtBr was evidenced by comparing strains with different levels of AcrAB expression. An experimental model was developed to describe the transport kinetics in the three strains. The model integrates passive entry (influx) and active efflux of EtBr, and discriminates different degrees of efflux between the studied strains that vary in the activity of their efflux systems, as evident from the calculated efflux rates: = 0.0173 ± 0.0057 min-1; = 0.0106 ± 0.0033 min-1; and = 0.0230 ± 0.0075 min-1.ConclusionThe combined use of a semi-automated fluorometric method and an experimental model allowed quantifying EtBr transport in E. coli strains that differ in their overall efflux activity. This methodology can be used for the early detection of differences in the drug efflux capacity in bacteria accounting for antibiotic resistance, as well as for expedite screening of new drug efflux inhibitors libraries and transport studies across the bacterial cell wall.

The role of polyadenylation signal secondary structures on the resistance of plasmid vectors to nucleases

Nuclease degradation of plasmid DNA (pDNA) vectors after delivery and during trafficking to the nucleus is a barrier to gene expression. This barrier may be circumvented by shielding the pDNA from the nuclease‐rich cell environment with adjuvants or by using nuclease inhibitors. A different alternative that is explored in this work is to make pDNA vectors more nuclease‐resistant a priori.

Structural instability of plasmid biopharmaceuticals: challenges and implications

The global increase in the number of applications involving therapeutic plasmid DNA (pDNA) is creating a need for large amounts of highly stable and purified molecules. One of the main obstacles during the developmental stages of a new therapeutic DNA molecule involves tackling a wide array of structural instability events occurring in/with pDNA and therefore assuring its structural integrity. This review focuses on major instability determinants in pDNA. Their elimination could be considered an important step towards the design of safer and more efficient plasmid molecules. Particular emphasis is given to mutations triggered by the presence of repeated sequences, instability events occurring during plasmid intracellular routing, instability mediated by insertion sequences and host genome integration.

In vivo activation of yeast plasma membrane H+-ATPase by ethanol: effect on the kinetic parameters and involvement of the carboxyl-terminus regulatory domain.

The in vivo activation of Saccharomyces cerevisiae plasma membrane H+-ATPase by ethanol was observed during ethanol-stressed cultivation or following the rapid incubation of cells with ethanol (6% (v/v)). Ethanol activated both the basal and the glucose-activated forms of the enzyme being the H+-ATPase fully activated by glucose (5% (w/v)) still additionally activable by ethanol. The kinetic parameters of ethanol-activated and non-activated H+-ATPase were calculated based directly on Michaëlis-Menten equation (with MgATP concentrations in the range 0. 16-8.18 mM and 7.5 mM of free Mg2+); the rectangular hyperbolic function was solved using iterative procedures. Ethanol-induced stimulation of plasma membrane H+-ATPase activity was associated to the increase of Vmax whereas the Km for MgATP increased. Results obtained with mutants constructed and used in previous studies envisaging the analysis of the molecular mechanisms underlying plasma membrane ATPase activation by glucose, external acidification and nitrogen starvation, suggested that the carboxyl-terminus (C-terminus) regulatory domain may also be involved in the in vivo activation by ethanol.

Rational engineering of Escherichia coli strains for plasmid biopharmaceutical manufacturing

Plasmid DNA (pDNA) has become very attractive as a biopharmaceutical, especially for gene therapy and DNA vaccination. Currently, there are a few products licensed for veterinary applications and numerous plasmids in clinical trials for use in humans. Recent work in both academia and industry demonstrates a need for technological and economical improvement in pDNA manufacturing. Significant progress has been achieved in plasmid design and downstream processing, but there is still a demand for improved production strains. This review focuses on engineering of Escherichia coli strains for plasmid DNA production, understanding the differences between the traditional use of pDNA for recombinant protein production and its role as a biopharmaceutical. We will present recent developments in engineering of E. coli strains, highlight essential genes for improvement of pDNA yield and quality, and analyze the impact of various process strategies on gene expression in pDNA production strains.