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.     

Liver- and lobe-selective gene transfection following the instillation of plasmid DNA to the liver surface in mice

The present study has undertaken the liver- and lobe-selective gene transfections following the instillation of plasmid DNA (pDNA) to the liver surface in mice. The luciferase levels produced in the applied (left) liver lobe at 6 h after liver surface instillation of pDNA were significantly higher than those produced in the other tissues assayed, and ranged from 8.5-fold higher in other liver lobes to 320-fold higher in other tissues. After small intestine surface instillation of pDNA, the gene expression was a little detected in the tissues assayed. Following liver surface instillation of pDNA at a time from 2 to 48 h or at a volume from 15 to 120 microl, the gene expressions of the applied liver lobe were always significantly higher than those of other liver lobes and other tissues. We demonstrated the novel liver- and lobe-selective gene transfection utilizing the instillation to the liver surface.     

Preparation and characterization of poly(D,L-lactide-co-glycolide) microspheres for controlled release of human growth hormone

The purpose of this research was to assess the physicochemical properties of a controlled release formulation of recombinant human growth hormone (rHGH) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) composite microspheres. rHGH was loaded in poly(acryloyl hydroxyethyl) starch (acHES) microparticles, and then the protein-containing microparticles were encapsulated in the PLGA matrix by a solvent extraction/evaporation method. rHGH-loaded PLGA microspheres were also prepared using mannitol without the starch hydrogel microparticle microspheres for comparison. The detection of secondary structure changes in protein was investigated by using a Fourier Transfer Infrared (FTIR) technique. The composite microspheres were spherical in shape (44.6±2.47 μm), and the PLGA-mannitol microspheres were 39.7±2.50 μm. Drug-loading efficiency varied from 93.2% to 104%. The composite microspheres showed higher overall drug release than the PLGA/mannitol microspheres. FTIR analyses indicated good stability and structural integrity of HGH localized in the microspheres. The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.     

Poly(D,L-Lactide-Co-Glycolide) microspheres containing 5-fluorouracil: Optimization of process parameters

The objective of this research was to optimize the processing parameters for poly(D,L-lactide-coglycolide) (PLGA) microspheres of 5-fluorouracil (5-FU) and to mathematically relate the process parameters and properties of microspheres. Microspheres were prepared by a water-in-oil-in-water emulsion solvent evaporation technique. A 3² factorial design was employed to study the effect of the volume of the internal phase of the primary emulsion and the volume of the external phase of the secondary emulsion on yield, particle size, and encapsulation efficiency of microspheres. An increase in the volume of the internal phase of the primary emulsion resulted in a decrease in yield and encapsulation efficiency and an increase in particle size of microspheres. When the volume of the external phase of the secondary emulsion was increased, a decrease in yield, particle size, and encapsulation efficiency was observed. Microspheres with good batch-to-batch reproducibility could be produced. Scanning electron microscopic study indicated that microspheres existed as aggregates.     

Identification of chemically modified peptide from poly(D,L-lactide-co-glycolide) microspheres under in vitro release conditions

The purpose of this research was to study the chemical reactivity of a somatostatin analogue octreotide acetate, formulated in microspheres with polymers of varying molecular weight and co-monomer ratio under in vitro testing conditions. Poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide) (PLA) microspheres were prepared by a solvent extraction/evaporation method. The microspheres were characterized for drug load, impurity content, and particle size. Further, the microspheres were subjected to in vitro release testing in acetate buffer (pH 4.0) and phosphate buffered saline (PBS) (pH 7.2). In acetate buffer, 3 microsphere batches composed of low molecular weight PLGA 50∶50, PLGA 85∶15, and PLA polymers (≤10 kDa) showed 100% release with minimal impurity formation (<10%). The high molecular weight PLGA 50∶50 microspheres (28 kDa) displayed only 70% cumulative release in acetate buffer with significant impurity formation (∼24%). In PBS (pH 7.4), on the other hand, only 50% release was observed with the same low molecular weight batches (PLGA 50∶50, PLGA 85∶15, and PLA) with higher percentages of hydrophobic impurity formation (ie, 40%, 26%, and 10%, respectively). In addition, in PBS, the high molecular weight PLGA 50∶50 microspheres showed only 20% drug release with ∼60% mean impurity content. The chemically modified peptide impurities inside microspheres were structurally confirmed through Fourier transform-mass spectrometry (FT-MS) and liquid chromatography/mass spectrometry (LC-MS/MS) analyses after extraction procedures. The adduct compounds were identified as covalently modified conjugates of octreotide with lactic and glycolic acid monomers within polymeric microspheres. The data suggest that due to steric hindrance factors, polymers with greater lactide content were less amenable to the formation of adduct impurities compared with PLGA 50∶50 copolymers.     

Targeted cationic poly(D,L-lactic-co-glycolic acid) nanoparticles for gene delivery to cultured cells

We developed a new targeted cationic nanoparticulate system composed of poly(D,L-lactic-co-glycolic acid) (PLGA), 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and asialofetuin (AF), and found it to be a highly effective formulation for gene delivery to liver tumor cells. The nanoparticles (NP) were prepared by a modified solvent evaporation process that used two protocols in order to encapsulate (NP1 particles) or adsorb (NP2 particles) plasmid DNA. The final particles are in the nanoscale range. pDNA loaded in PLGA/DOTAP/AF particles with high loading efficiency showed a positive surface charge. Targeted asialofetuin-nanoparticles (AF-NP) carrying genes encoding for luciferase and interleukin-12 (IL-12) resulted in increased transfection efficiencies compared to free DNA and to plain (non-targeted) systems, even in the presence of 60% fetal bovine serum (FBS). The results of transfections performed on HeLa cells, defective in asialoglycoprotein receptors (ASGPr-), confirmed the receptor-mediated endocytosis mechanism. In summary, this is the first time that asialoglycoprotein receptor targeting by PLGA/DOTAP/DNA nanoparticles carrying the therapeutic gene IL-12 has been shown to be efficient in gene delivery to liver cancer cells in the presence of a very high concentration of serum, and this could be a potential system for in vivo application.     

Spray-dried poly(D,L-lactide) microspheres containing carboplatin for veterinary use: In vitro and in vivo studies

The aim of this study was the development of a veterinary dosage form constituted by injectable biodegradable microspheres designed for the subcutaneous release of carboplatin, a chemotherapeutic drug. Poly(D,L-lactide) (PDLLA) microspheres were prepared by an emulsification/spray-drying method, using the drug-to-polymer weight ratios 1∶9 and 1∶5; blank microspheres (1% w/v) were prepared as a comparison. Microparticles were characterized in terms of morphology, encapsulation efficiency, and in vitro drug release behavior. In vivo tests were conducted on rats by subcutaneous injection of microsphere aqueous suspensions. Levels of carboplatin were evaluated both in the skin and in serum. The microparticles obtained had a spherical shape; particle size ranged from 5 to 7 μm, dependent on drug loading. Microspheres were able to control the in vitro release of the drug: approximately 90% to 100% of the carboplatin was released over 30 days. In vivo results showed that the microspheres were able to release high drug amounts locally, and sustained serum levels of drug were also achieved. Based on these results, carboplatin-loaded PDLLA microspheres may be useful for local delivery of the antineoplastic drug to the tumor, avoiding tumor recurrence in small animals, and may decrease the formation of distant metastases. Published: September 20, 2005     

Dual bio-responsive gene delivery via reducible poly(amido amine) and survivin-inducible plasmid DNA

A bioreducible poly(amido amine) (SS-PAA) gene carrier, known as poly (amido-butanol) (pABOL), was used to transfect a variety of cancer and non-cancer cell lines. To obtain cancer-specific transgene expression for therapeutic efficiency in cancer treatment, we constructed survivin-inducible plasmid DNA expressing the soluble VEGF receptor, sFlt-1, downstream of the survivin promoter (pSUR-sFlt-1). Cancer-specific expression of sFlt-1 was observed in the mouse renal carcinoma (RENCA) cell line. pABOL enhanced the efficiency of gene delivery compared to traditional carriers used in the past. Thus, a dual bio-responsive gene delivery system was developed by using bioreducible p(ABOL) for enhanced intracellular gene delivery and survivin-inducible gene expression system (pSUR-sFlt-1 or pSUR-Luc reporter gene) that demonstrates increased gene expression in cancer that has advantages over current gene delivery systems.     

Synthesis of biodegradable cross-linked poly(beta-amino ester) for gene delivery and its modification, inducing enhanced transfection efficiency and stepwise degradation

Biodegradable cross-linked poly(beta-amino ester) (CLPAE) was synthesized by Michael addition of pentaerythritol triacrylate and N,N-dimethylethylenediamine and modified with aminohexanoic acid and lysine to CLPAE-Ahx and CLPAE-Lys, respectively, for a gene delivery system. They could self-assemble with plasmid DNA, forming nanosized polyplexes, and CLPAE-Ahx polyplex released plasmid DNA slowly during a week through stepwise degradation. The polymers showed minimal cytotoxicity on 293 cells due to their biodegradability and biocompatibility. Transfection efficiencies of CLPAE-Ahx and CLPAE-Lys were comparable to that of PEI in 293 cells and C2C12 cells. Additionally, high transfection of CLPAE-Ahx on primary rat aorta vascular smooth muscle cells (SMC) and primary mouse embryonic fibroblast cells (MEF) shows a potential for a gene delivery system on primary cells, restenosis treatment of human SMC, and MEF cell function research. In conclusion, CLPAE-Ahx could be used as a nontoxic and highly efficient gene delivery system.     

Preparation and characterization of temperature-sensitive poly(N-isopropylacrylamide)-b-poly(D,L-lactide) microspheres for protein delivery

Temperature-sensitive diblock copolymers, poly(N-isopropylacrylamide)-b-poly(D,L-lactide) (PNIPAAm-b-PLA) with different PNIPAAm contents were synthesized and utilized to fabricate microspheres containing bovine serum albumin (BSA, as a model protein) by a water-in-oil-in-water double emulsion solvent evaporation process. XPS analysis showed that PNIPAAm was a dominant component of the microspheres surface. BSA was well entrapped within the microspheres, and more than 90% encapsulation efficiency was achieved. The in vitro degradation behavior of microspheres was investigated using SEM, NMR, FTIR, and GPC. It was found that the microspheres were erodible, and polymer degradation occurred in the PLA block. Degradation of PLA was completed after 5 months incubation in PBS (pH 7.4) at 37 degrees C. A PVA concentration of 0.2% (w/v) in the internal aqueous phase yielded the microspheres with an interconnected porous structure, resulting in fast matrix erosion and sustained BSA release. However, 0.05% PVA produced the microspheres with a multivesicular internal structure wrapped with a dense skin layer, resulting in lower erosion rate and a biphasic release pattern of BSA that was characterized with an initial burst followed by a nonrelease phase. The microspheres made from PNIPAAm-b-PLA with a higher portion of PNIPAAm provided faster BSA release. In addition, BSA release from the microspheres responded to the external temperature changes. BSA release was slower at 37 degrees C (above the LCST) than at a temperature below the LCST. The microspheres fabricated with PNIPAAm-b-PLA having a 1:5 molar ratio of PNIPAAm to PLA and 0.2% (w/v) PVA in the internal aqueous phase provided a sustained release of BSA over 3 weeks in PBS (pH 7.4) at 37 degrees C.     

Coaxial electrohydrodynamic spraying of plasmid DNA/polyethylenimine (PEI) polyplexes for enhanced nonviral gene delivery

DNA/polyethylenimine (PEI) polyplexes are an important class of nonviral vectors. Although the conventional preparation method, bulk mixing, is straightforward, the formation of the DNA/PEI polyplexes is not well controlled. This work explores coaxial electrohydrodynamic spraying (EHDS) as a novel, alternative method to produce DNA/PEI polyplexes in a more controlled manner. Both pGFP/PEI and pSEAP/PEI polyplexes were produced by EHDS with a coaxial needle setup. The size of the polyplexes was determined using dynamic light scattering, and their ability to transfect NIH 3T3 cells was observed by using an inverted fluorescence microscope (pGFP) or quantified by measuring the activity level of alkaline phosphatase (pSEAP). At nitrogen to phosphate ratio (N/P) of 6.7, the polyplexes produced by coaxial EHDS had delivery efficiencies up to 2.6 times higher than those produced by bulk mixing. The N/P ratio and the structure of the EHDS used to make the polyplexes were crucial factors in determining the delivery efficiency. Biotechnol. Bioeng. 2010. 105: 834–841. © 2009 Wiley Periodicals, Inc.     

Biophysical and transfection studies of an amine-modified poly(vinyl alcohol) for gene delivery

Novel, multifunctional polymers remain an attractive objective for drug delivery, especially for hydrophilic macromolecular drugs candidates such as peptides, proteins, RNA, and DNA. To facilitate intracellular delivery of DNA, new amine-modified poly(vinyl alcohol)s (PVAs) were synthesized by a two-step process using carbonyl diimidazole activated diamines to produce PVAs with different degrees of amine substitution. The resulting polymers were characterized using NMR, thermogravimetric analysis (TGA), and gelpermation chromatography (GPC). Atomic force microscopy (AFM), dynamic light scattering photon correlation spectroscopy (PCS), and zeta-potential were used to investigate polyplexes of DNA with PVA copolymers. These studies suggest an influence of the polycation structure on the morphology of condensed DNA in polyplexes. Significant differences were observed by changing both the degrees of amine substitution and the structure of the PVA backbone, demonstrating that both electrostatic and hydrophobic interactions affect DNA condensation. DNA condensation measured by an ethidium bromide intercalation assay showed a higher degree of condensation with pDNA with increasing degrees of amine substitution and more hydrophobic functional groups. These findings are in line with transfection experiments, in which a good uptake of these polymer DNA complexes was noted, unfortunately, with little endosomal escape. Co-administration of chloroquine resulted in increased endosomal escape and higher transfection efficiencies, due to disruption of the endosomal membrane. In this study, the structural requirements for DNA complexation and condensation were characterized to provide a basis for rational design of nonviral gene delivery systems.     

A radiation target method for size determination of supercoiled plasmid DNA

Supercoiled DNA plasmids were exposed in the frozen state to high-energy electrons. Surviving supercoiled molecules were separated from their degradation products (e.g., open circle and linear forms) by agarose gel electrophoresis and subsequently quantified by staining and image analysis. Complex survival curves were analyzed using radiation target theory, yielding the radiation-sensitive mass of each form. One of the irradiated plasmids was transfected into cells, permitting radiation analysis of gene expression. Loss of this function was associated with a mass much smaller than the entire plasmid molecule, indicating a lack of energy transfer in amounts sufficient to cause structural damage along the DNA polynucleotide. The method of radiation target analysis can be applied to study both structure and function of DNA.     

Nuclear delivery of plasmid DNA determines the efficiency of gene expression

As a cationic non‐viral gene delivery vector, poly(agmatine/ N, N′‐cystamine‐bis‐acrylamide) (AGM‐CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM‐CBA/pDNA polyplexes was found to have a non‐linear relationship with AGM‐CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM‐CBA), we used pGL3‐control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM‐CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate‐limiting step for pLUC transfection expression. Further optimization of the non‐viral gene delivery system can be focused on the improvement of gene intracellular availability.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

Improved efficiency for T-DNA-mediated transformation and plasmid rescue inArabidopsis thaliana

A vector was constructed for the isolation of gene fusions to thelacZ reporter gene following T-DNA integration into the genome ofArabidopsis thaliana. To facilitate the generation of taggedA. thaliana plants, we established a modified method for high-frequency transformation ofA. thaliana byAgrobacterium tumefaciens. The main modification required was to inhibit the methylation of T-DNA in the transformed calli. Apparently, cytosine residues of thenos-nptII gene used as a selectable marker were methylated, and the expression of this gene was suppressed. Treatment of the calli with the cytosine methylation inhibitor 5-azacytidine led to a dramatic increase (from 3% to 96%) in the regeneration of transformed (kanamycin-resistant) shoots. A total of 150 transgenic plants were isolated, and in 17 of these expression of thelacZ reporter was detected byin situ staining. The T-DNA insert together with flanking plant DNA sequences was cloned intoEscherichia coli by plasmid rescue from some of the T₃ transformants that harbored one copy of the integrated T-DNA. Comparison of the rescued DNA with the corresponding DNA of the transgenic plant showed that most of the rescued plasmids had undergone rearrangements. These rearrangements could be totally avoided if anmcrAB (modified cytosine restriction) mutant ofE. coli was used as the recipient in plasmid rescue.     

Scanning force microscopy studies of X-ray-induced double-strand breaks in plasmid DNA

We present an analysis of X-ray-induced damage in ΦX174 plasmid DNA, applying doses between D = 250 and 1,500 Gy. To analyse this damage in detail, the distribution of plasmid fragments after irradiation have been determined by scanning force microscopy. The results show that even for the lowest dose of D = 250 Gy, a significant amount of double-strand breaks are observed. For increasing dose, the percentage of small fragments increases and is accompanied by a shortening of the average fragment length from < L> = 1,400 nm for a dose of D = 250 Gy to < L> = 1,080 nm after irradiation with D = 1,500 Gy. The most crucial parameter, the average number of double-strand breaks per broken plasmid (<DSB_b> ) has been determined for the first time for the applied doses. The results show that the average number of DSBs per broken plasmid <DSB_b> increases almost linearly from a value of <DSB_b> = 1.3 after irradiation with D = 250 Gy to <DSB_b> = 1.7 after exposure to D = 1,500 Gy. The presented results show that the amount of DSBs induced by X-ray radiation in plasmid DNA can be calculated with high accuracy by means of scanning force microscopy, providing relevant information regarding the interaction of X-rays with DNA molecules.

Repair of double-strand breaks in plasmid DNA in the yeast Saccharomyces cerevisiae

Summary We studied the repair of double-strand breaks (DSB) in plasmid DNA introduced into haploid cells of the yeast Saccharomyces cerevisiae. The efficiency of repair was estimated from the frequency of transformation of the cells by an autonomously replicated linearized plasmid. The frequency of lithium transformation of Rad⁺ cells was increased greatly (by 1 order of magnitude and more) compared with that for circular DNA if the plasmid was initially linearized at the XhoI site within the LYS2 gene. This effect is due to recombinational repair of the plasmid DNA. Mutations rad52, rad53, rad54 and rad57 suppress the repair of DSB in plasmid DNA. The kinetics of DSB repair in plasmid DNA are biphasic: the first phase is completed within 1 h and the second within 14–18 h of incubating cells on selective medium.