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.     

Transformation of competent Bacillus subtilis cells by chromosomal and plasmid DNA incorporated in liposomes

Transformation with chromosomal and plasmid DNAs comprised in liposomes of different compositions was studied on competent cells of Bacillus subtilis. Transformation with chromosomal DNA comprised in liposomes appeared to constitute 1.1 to 1.5% of the control, and transformation with plasmid DNA in liposomes reaches 8 to 11%, as compared to the control. It has been revealed that absorbtion of chromosomal or plasmid DNA comprised in liposomes by competent cells is 1-2 orders higher than that of chromosomal or plasmid DNAs which are not contained in liposomes. Besides, chromosomal DNA in liposomes was found to be transferred to competent cells in the double-stranded form, while during common transformation without liposomes, the DNA transferred is single-stranded.     

Comparison of DNA fragments as donor DNAs upon sequence conversion of cleaved target DNA

Pinpoint sequence alteration (genome editing) by the combination of the site-specific cleavage of a target DNA and a donor nucleic acid has attracted much attention and the sequence of the target DNA is expected to be changed to that of a donor nucleic acid. In most cases, oligodeoxyribonucleotides (ODNs) and plasmid DNAs have been used as donors. However, a several hundred-base single-stranded (ss) DNA fragment and a 5′-tailed duplex (TD) accomplished the desired sequence changes without DNA cleavage, and might serve as better donors for the cleaved target DNA than ODNs and plasmid DNAs. In this study, sequence conversion efficiencies were compared with various donor DNAs in model sequence alteration experiments, using episomal DNA. The efficiencies with the ss and TD fragments were higher than those with the ODN and plasmid DNA. The sequence change by the TD seemed somewhat less efficient but slightly more accurate than that by the ss DNA fragment. These results suggested that the ss and TD fragments are better donors for targeted sequence alteration.     

Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.     

A novel procedure for selective cloning of NotI linking fragments from mammalian genomes.

A novel procedure has been developed for selective cloning of NotI linking fragments from mammalian genomes. Since the majority of the NotI sites in mammalian genomes are considered to be localized in so-called HTF (HpaII tiny fragment) islands, an HTF library was constructed as an initial step to enrich the NotI sites. The plasmid DNAs were isolated en masse from the HTF library and digested with NotI. Linearized plasmid DNAs derived from NotI linking clones were efficiently separated from undigested circular DNAs by an unique pulsed field polyacrylamide gel electrophoresis (PF-PAGE). The linear DNAs were eluted from the gel, recircularized with T4 DNA ligase and introduced into E. coli cells. About 95% of the transformants were found to contain NotI linking fragments. The procedure will thus provide a simple and useful way of collecting NotI linking fragments for long range physical mapping of mammalian genomes.     

Rapid method for the detection of genetically engineered microorganisms by polymerase chain reaction from soil and sediments

A rapid and sensitive method for the detection of genetically engineered microorganisms in soil and sediments has been devised by in vitro amplification of the target DNAs by a polymerase chain reaction. A cloned catechol 2,3-dioxygenase gene located on the recombinant plasmid pOH101 was transferred to Pseudomonas putida MMB2442 by triparental crossing and used as a target organism. For the polymerase chain reaction from soil and sediment samples, the template DNA was released from a 100-mg soil sample. Bacterial seeded soil samples were washed with Tris-EDTA buffer (pH 8.0) and treated with a detergent lysis solution at 100°C. After addition of 1% polyvinylpolypyrrolidine solution, the samples were boiled for 5 min. Supernatant containing nucleic acid was purified with a PCR purification kit. The purified DNA was subjected to polymerase chain reaction, using two specific primers designed for the amplification of catechol 2,3-dioxygenase gene sequences. The detection limit was 10² cells per gram of soil. This method is rapid and obviates the need for lengthy DNA purification from soil samples. Received 28 February 1997/ Accepted in revised form 23 November 1997     

A procedure for the isolation and purification of plasmid DNA from Rhizobium meliloti

A procedure is described for the isolation and purification of the DNA of plasmids that are indigenous to the agriculturally important nitrogen-fixing bacterium Rhizobium meliloti. The procedure involves the lysis of bacteria with an ionic detergent or a mixture of ionic and nonionic detergents, the extraction of total DNA from precipitated membrane-DNA complexes, the enrichment of supercoiled plasmid DNA by the selective alkaline denaturation of chromosomal DNA, and a further purification of plasmid DNA using cesium chloridepropidium diiodide gradients. This procedure yields pure plasmid DNA in amounts of 30 to 50 μg per liter of a culture of cell density of approximately one A550 unit. The DNA thus obtained has been found to be of sufficient purity to serve as substrate for the most commonly used restriction endonucleases.     

A rapid alkaline extraction procedure for screening recombinant plasmid DNA.

A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.     

Potential application of hydrogel-based strong anion-exchange membrane for plasmid DNA purification

The potential application of a hydrogel-based strong anion-exchange (Q) membrane to purify plasmid DNAs was evaluated. The maximum binding capacity of plasmid DNA was estimated to be 12.4 mg/ml of membrane volume with a plasmid recovery yield of ～90%. The effect of the inherent properties of plasmid DNA, membrane adsorbent, and the ionic environment on membrane performance was systematically investigated. Plasmid DNAs with smaller tertiary structure tended to have a better recovery than those with larger tertiary structure. Environmental Scanning Electron Microscopy (ESEM) revealed that the hydrogel structure is more porous on one side of membrane than the other. Membrane pre-treatment significantly improved pore distribution and increased membrane porosity resulting in a better adsorption, recovery, and higher flux. The selection of proper operating pH led to further improvement. The relative contribution of these factors to improve membrane chromatography of plasmid DNAs was analyzed using statistical modeling. It was found that the adsorption of plasmid DNA was mainly affected by the available adsorptive area associated with membrane porosity, whereas the recovery of plasmid DNAs was mainly affected by the environmental pH.     

Enzymatic purification of plasmid DNA.

A novel method for plasmid DNA purification using enzymes that degrade all major types of contaminating nucleic acids present in crude plasmid DNA mixtures (but not plasmid DNA) has been devised. This method is quick (can be accomplished in two hours), requires no expensive laboratory equipment (an ultracentrifuge is not necessary) and is inexpensive. Plasmid DNA purified by this methodology can be used in a variety of molecular biological techniques including restriction enzyme digestions, subcloning, sequencing, nick translation and end labeling. This plasmid purification technique will be very useful for the molecular biologist performing cloning experiments.     

A rapid and simple method for screening large numbers of recombinant DNA clones

A simple and rapid method has been described for the isolation of plasmid, phagemid and phage DNAs. Hundreds of recombinant clones can be screened in one day employing this method. It takes half an hour to prepare plasmid DNA from ten clones, and the DNA prepared from a single colony using this method is of sufficient quality and in sufficient amount to perform at least five restriction digestions. This method eliminates the need for RNase treatment and phenol chloroform extraction if the plasmids are needed only for the restriction digestion. If needed, RNAs can be removed after restriction digestion by adding RNase and incubating for two minutes at room temperature. After RNase treatment and phenol/chloroform extraction, the plasmid DNA serves as a good template for sequencing. The DNA can be stored at -20 degrees C for over eight weeks.     

A rapid method for purification of organelles for DNA isolation: self-generated percoll gradients

Self-generated Percoll gradients have been used for rapid purification of crude chloroplasts and mitochondria, obtained by common differential centrifugation techniques. Such purified organelles were used for isolating DNA from safflower (Carthamus tinctorius L.), carrot (Daucus carota L.), various Solanaceae, and numerous somatic hybrids. The method is simple, has the advantage of not requiring DNase, and is particularly well suited when only limited amounts of aseptically grown shoots are available. As judged by restriction enzyme analyses and chloroplast DNA cloning experiments, the DNAs are of sufficient purity for many molecular biological applications without CsCl gradient purification.     

Template requirements for in vivo replication of adenovirus DNA.

The adenovirus (Ad) DNA origin of replication was defined through an analysis of the DNA sequences necessary for the replication of plasmid DNAs with purified viral and cellular proteins. Results from several laboratories have shown that the origin consists of two functionally distinct domains: a 10-base-pair sequence present in the inverted terminal repetition (ITR) of all human serotypes and an adjacent sequence constituting the binding site for a cellular protein, nuclear factor I. To determine whether the same nucleotide sequences are necessary for origin function in vivo, we developed an assay for the replication of plasmid DNAs transfected into Ad5-infected cells. The assay is similar to that described by Hay et al. (J. Mol. Biol. 175:493-510, 1984). With this assay, plasmid DNA replication is dependent upon prior infection of cells with virus and only occurs with linear DNA molecules containing viral terminal sequences at each end. Replicated DNA is resistant to digestion with lambda-exonuclease, suggesting that a protein is covalently bound at both termini. A plasmid containing only the first 67 base pairs of the Ad2 ITR replicates as well as plasmids containing the entire ITR. Deletions or point mutations which reduce the binding of nuclear factor I to DNA in vitro reduce the efficiency of plasmid replication in vivo. A point mutation within the 10-base-pair conserved sequence has a similar effect upon replication. These results suggest that the two sequence domains of the Ad origin identified by in vitro studies are in fact important for viral DNA replication in infected cells. In addition, we found that two separate point mutations which lie outside these two sequence domains, and which have little or no effect upon DNA replication in vitro, also reduce the apparent efficiency of plasmid replication in vivo. Thus, there may be elements of the Ad DNA origin of replication which have not yet been identified by in vitro studies.     

Chloroplast DNA cloning in Escherichia coli. II. The properties of the recombinant plasmids bearing the EcoRI fragments of pea chloroplast DNA and the cloning of the DNA sequences with rRNA genes]

Previously a method of selection of colicine-defective recombinant plasmids by mitomycin C was described. A series of recombinant plasmids (CPS) with various EcoRI-fragments of pea chloroplast DNA has been obtained. This paper describes some properties of cloned fragments replicated in Escherichia coli. The alkali stability of recombinant plasmid DNAs has been demonstrated, indicating the absence of ribonucleotides in their structure. Heterogeneity of chloroplast DNA in nucleotide composition was demonstrated using ultracentrifugation analysis of CPS-plasmid DNAs in CsCl-actinomycin D density gradient. Pea chloroplast rDNA was cloned in recombinant plasmids.     

The harnessing of peptide–monolith constructs for single step plasmid DNA purification

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10⁻⁸ M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide–monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach.     

Requirements for the formation of plasmid-transducing particles of Bacillus subtilis bacteriophage SPP1.

We had previously proposed that the production of concatemeric plasmid DNA in plasmid-transducing SPP1 particles is a consequence of phage-directed rolling-circle-type replication of plasmid DNA. The production of such DNA was greatly enhanced when DNA/DNA homology was provided between phage and plasmid DNAs (facilitation of transduction). Here we present evidence that synthesis of concatemeric plasmid DNA can proceed after phage infection under conditions non-permissive for plasmid replication. We also propose that the naturally occurring homology between plasmid and phage is sufficient to account for the frequency of transduction observed in the absence of facilitating homology. Homology of greater than 47 bp gives the maximal facilitation of plasmid transduction. Recombination is not an essential part in the synthesis of concatemeric plasmid DNA.     

An improved method for rapid isolation of plasmid DNA from wild-type Gram-negative bacteria for plasmid restriction profile analysis

An improved method for the isolation of large and small plasmids from wild-type Gram-negative bacteria has been developed. The protocol combines the lysis and purification procedures of two popular plasmid isolation methods, and produces DNA sufficiently pure for restriction enzyme digestion in less than three hours.     

Optimization of electrotransformation conditions for Propionibacterium acnes

Propionibacterium acnes has been known to be involved in the pathology of acne. However, the definite mechanism in the development of acne and the inflammation are unknown. For P. acnes, a transformation method has not been established, although it is believed to be a basic tool for gene manipulation. This study attempted to develop a P. acnes transformation method by using electroporation. Various parameters were used to develop and optimize the transformation of P. acnes. Among them two factors were crucial in the transformation for P. acnes: one was the E. coli strain from which the plasmid DNA had been isolated and the other the growth temperature of P. acnes-competent cells. It was essential to prepare plasmid DNA from a dam(-) E. coli strain, ET12567. When plasmid DNAs isolated from the other E. coli strains such as JM109 and HB101 were tested, transformation efficiency was extremely low. When P. acnes cells were cultivated at 24 degrees C for competent cell preparation, transformation efficiency increased considerably. When plasmid DNA isolated from a dam(-) mutant strain of E. coli was used for transformation of P. acnes which had been grown at 24 degrees C, maximum transformation efficiency of 1.5 x 10(4) transformants per mug of plasmid DNA was obtained at a field strength of 15 kV/cm with a pulse time of 3.2 ms. This is believed to be the first report on the transformation of P. acnes which can be employed for gene manipulations including knock-out of specific genes.     

Large-scale isolation and partial characterization of plasmid DNA fromB. larvae

Large-scale preparation of plasmid DNA from twoBacillus larvae strains 423 and 728, honey-bee pathogens, is described. The isolated plasmid DNAs were analyzed by restriction enzyme mapping. No difference in the resulting maps was found for six restriction enzymes. The plasmid DNAs were also compared by Southern blot hybridization and by electron microscopy. The results confirmed the identity of these plasmid DNAs. All these data suggest thatB. larvae strains harbor the same plasmid.     

Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification

The past several years have witnessed a rapidly increasing number of reports on utilizing plasmid DNA as a vector for the introduction of genes into mammalian cells for use in both gene therapy and vaccine applications. “Naked DNA vaccines” allow the foreign genes to be transiently expressed in transfected cells, mimicking intracellular pathogenic infection and triggering both the humoral and cellular immune responses. While considerable attention has been paid to the potential of such vaccines to mitigate a number of infections, substantially less consideration has been given to the practical challenges of producing large amounts of plasmid DNA for therapeutic use in humans, for both clinical studies and, ultimately, full-scale manufacturing. Doses of naked DNA vaccines are on the order of milligrams, while typical small-scale Escherichia coli fermentations may routinely yield only a few mg/l of plasmid DNA. There have been many investigations towards optimizing production of heterologous proteins over the past three decades, but in these cases, the plasmid DNA was not the final product of interest. This review addresses the current state-of-the-art means for the production of plasmid DNA at large scale in compliance with existing regulatory guidelines. The impact of the nature of the plasmid vector on the choice of fermentation protocols is presented, along with the effect of varying cultivation conditions on final plasmid content. Practical considerations for the large-scale purification of plasmid DNA are also discussed.