Biosafety of the plasmid pcDNA3-1E of Eimeria acervulina in chicken.

To evaluate the biosafety of the plasmid pcDNA3-1E of Eimeria acervulina in chicken, two-week-old chickens were injected intramuscularly with the plasmid pcDNA3-1E at dose of 50 µg/chicken. At the 15 days post-injection, the tissue samples were collected, the total DNA was extracted, and the 3-1E gene was amplified by PCR. Genomic DNA was first purified away from free plasmid using gel electrophoresis, and then assayed for integrated plasmid using PCR amplification of the 3-1E gene. Simultaneously, the environmental dejection samples were collected, the total bacterial DNA was extracted and then transfer of the pcDNA3-1E gene was detected by PCR amplification of the 3-1E gene. Two-week-old chickens were injected intramuscularly with the plasmid pcDNA3-1E with three dosage groups of 100 µg, 500 µg and 2500 µg/chicken for 14 days respectively, and with physiological saline at dose of 2500 µL/chicken as control group for acute toxicity test. A target band of 583 bp was obtained by PCR with chicken genomic DNA as template. If the chicken genomic DNA was purified, no target band could be obtained. It showed that the recombinant plasmid pcDNA3-1E existed in tissues, and no genomic integration of DNA plasmid was detected in the immunized chickens. No target band was found by PCR with environmental dejection bacteria genomic DNA as template. It showed that integration and transfer phenomenon did not exist in environment. The acute toxicity results showed the typical clinical symptoms did not occur in the inoculated chickens, the blood biochemical indices and viscera configuration were not affected significantly in the inoculated group and control group (P>0.05). The results showed that the plasmid pcDNA3-1E was safe and suitable for chicken clinical trials.

Life cycle, growth characteristics and host cell response of Rickettsia helvetica in a Vero cell line.

Rickettsia helvetica, a spotted fever rickettsia and emerging pathogen with Ixodes ricinus ticks as the main vector, is an agent of human disease and may cause febrile illness as well as meningitis. In three parallel series the isolated standard type of R. helvetica, obtained from a PCR-positive I. ricinus tick, was high-passaged and propagated in a Vero cell line. By using quantitative real-time PCR, the generation time from inoculation to stationary phase of growth was calculated to 20-22 h. In the static cultivation system the stationary phase was observed from the seventh day after inoculation, and there was no observed degradation of R. helvetica DNA during the 14 days studied. Microscopy showed that the organisms invaded the host cells rapidly and were primarily found free in the cytoplasm and only occasionally located in the nucleus. Four days after inoculation some of the host cells were broken and many indifferent stages of cytoplasmic organic decomposition were seen. However the R. helvetica organism did not show any morphologic alterations and the number of organisms was stable after the replication peak which may indicate that R. helvetica is adapted to growth in a Vero cell line and/or that the phase of degradation occurs later than the 14 days studied. The findings differ from what has been reported for other rickettsiae of the spotted fever group and may be of importance for invasiveness and virulence of R. helvetica.

Construction of a genetic AND gate under a new standard for assembly of genetic parts.

BACKGROUND: Appropriate regulation of respective gene expressions is a bottleneck for the realization of artificial biological systems inside living cells. The modification of several promoter sequences is required to achieve appropriate regulation of the systems. However, a time-consuming process is required for the insertion of an operator, a binding site of a protein for gene expression, to the gene regulatory region of a plasmid. Thus, a standardized method for integrating operator sequences to the regulatory region of a plasmid is required. RESULTS: We developed a standardized method for integrating operator sequences to the regulatory region of a plasmid and constructed a synthetic promoter that functions as a genetic AND gate. By standardizing the regulatory region of a plasmid and the operator parts, we established a platform for modular assembly of the operator parts. Moreover, by assembling two different operator parts on the regulatory region, we constructed a regulatory device with an AND gate function. CONCLUSIONS: We implemented a new standard to assemble operator parts for construction of functional genetic logic gates. The logic gates at the molecular scale have important implications for reprogramming cellular behavior.

Quick identification of Type I restriction enzyme isoschizomers using newly developed pTypeI and reference plasmids.

Although DNA-recognition sequences are among the most important characteristics of restriction enzymes and their corresponding methylases, determination of the recognition sequence of a Type-I restriction enzyme is a complicated procedure. To facilitate this process we have previously developed plasmid R-M tests and the computer program RM search. To specifically identify Type-I isoschizomers, we engineered a pUC19 derivative plasmid, pTypeI, which contains all of the 27 Type-I recognition sequences in a 248-bp DNA fragment. Furthermore, a series of 27 plasmids (designated 'reference plasmids'), each containing a unique Type-I recognition sequence, were also constructed using pMECA, a derivative of pUC vectors. In this study, we tried those vectors on 108 clinical E. coli strains and found that 48 strains produced isoschizomers of Type I enzymes. A detailed study of 26 strains using these 'reference plasmids' revealed that they produce seven different isoschizomers of the prototypes: EcoAI, EcoBI, EcoKI, Eco377I, Eco646I, Eco777I and Eco826I. One strain EC1344 produces two Type I enzymes (EcoKI and Eco377I).

A cautionary tale of cross-contamination among plasmids from commercial suppliers.

Many researchers have switched to purchasing their desired plasmids from commercial suppliers to save time and resources, as we did for 17 high-risk human papillomavirus plasmids. To our surprise, they were shown to be cross-contaminated with one another. Comparison between the production schedule and the pattern of contaminations proved that this contamination occurred during the production process, which was also shown for another two sets of commercial plasmids. Our experience indicates that the absolute purity of plasmids obtained from external sources cannot be guaranteed. Extreme caution should be exercised, especially when such plasmids are used for human gene therapies and DNA vaccines, where even a minute amount of contamination may pose significant risks to patients.

A Bayesian method for calculating real-time quantitative PCR calibration curves using absolute plasmid DNA standards.

BACKGROUND: In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignored in calibration calculations and in some cases impossible to characterize. A flexible statistical method that can account for uncertainty between plasmid and genomic DNA targets, replicate testing, and experiment-to-experiment variability is needed to estimate calibration curve parameters such as intercept and slope. Here we report the use of a Bayesian approach to generate calibration curves for the enumeration of target DNA from genomic DNA samples using absolute plasmid DNA standards. RESULTS: Instead of the two traditional methods (classical and inverse), a Monte Carlo Markov Chain (MCMC) estimation was used to generate single, master, and modified calibration curves. The mean and the percentiles of the posterior distribution were used as point and interval estimates of unknown parameters such as intercepts, slopes and DNA concentrations. The software WinBUGS was used to perform all simulations and to generate the posterior distributions of all the unknown parameters of interest. CONCLUSION: The Bayesian approach defined in this study allowed for the estimation of DNA concentrations from environmental samples using absolute standard curves generated by real-time qPCR. The approach accounted for uncertainty from multiple sources such as experiment-to-experiment variation, variability between replicate measurements, as well as uncertainty introduced when employing calibration curves generated from absolute plasmid DNA standards.

Arginine homopeptides for plasmid DNA purification using monolithic supports.

Purification 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.

Protocol for Efficient Generation and Characterization of Adeno-Associated Viral Vectors.

Adeno-associated virus vectors are a powerful tool for gene transfer approaches. We have established a simple and fast plasmid-based production system for achieving high adeno-associated virus titers within 6 working days. The same procedure can be used for all serotypes and thus allows direct comparability of different serotypes. In this protocol we describe a step-by-step procedure that results in well-characterized vectors suitable for both in vitro approaches and preclinical studies.

Assessment of UV spectrophotometry for determination of plasmid DNA concentration in vector preparations for human gene therapy products.

The European Pharmacopoeia (Ph. Eur.) general chapter 5.14. Gene transfer medicinal products for human use suggests the use of absorbance measurements at 260 nm to determine the DNA concentration of plasmid vectors used for the preparation of gene therapy products for human use. An international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to confirm the suitability of UV spectrophotometry for the quantification of plasmid vectors used in gene therapy (GT). Three Official Medicine Control Laboratories (OMCLs of the European OMCL Network) and members of the OMCL Working Group for GT products took part in the study, in which various types of spectrophotometers were assessed using common test samples. Results of the study demonstrated that UV spectrophotometry can be considered suitable for the quantification of plasmid DNA in GT products regardless of the instrument used.

Quantitative competitive-PCR assay to measure human parvovirus B19-DNA load in serum samples.

The B19 virus can persist in immunocompromised patients for several months and sometimes even years because of impaired immune response. Viremia in persistent and recurrent infection may range from very low to high titers and may be associated with chronic clinical manifestations, such as chronic anemia. Several recently developed techniques that quantify B19-DNA have improved laboratory diagnosis of the infection and can help guide the choice of treatment in persistent infections (i.e., intravenous immunoglobulin (IVIG) treatment vs immunosuppression reduction). Here we describe the development of a reliable internally controlled quantitative competitive (QC)- polymerase chain reaction (PCR) assay that measures B19-DNA load in serum samples by densitometric analysis of the amplification products for monitoring B19 infection in high-risk patients. A retrospective quantification of B19-DNA in the serum samples from 48 anemic transplanted patients by the QC-PCR assay we developed in our laboratory confirmed the presence of B19-DNA in 11 of 48 samples and showed a viral DNA load between 103 and 108 B19-DNA copies/mL depending on the patients' serostatus (the highest viral load was found in IgM-positive/IgG-negative patients, that is, in patients with active B19 infection at onset). The assay also confirmed B19-DNA negative patients. Our QC-PCR assay may be easily used in monitoring B19 prototype DNA load to follow persistent infections and to better understand the relation between active B19 infection and occurrence of anemia and to assess the efficacy of IVIG therapy or immunosuppression reduction in clearing the virus in high-risk patients.

Topical application of plasmid DNA to mouse and human skin.

Gene expression following direct injection of naked plasmid DNA into the skin has been demonstrated in the past. Topical application of plasmid DNA represents an attractive route of gene delivery. If successful, it would have great prospects in skin gene therapy since it is painless and easy to apply. In this study, we analyzed the expression of plasmid DNA in vivo and in vitro following topical application of plasmid DNA in various liposomal spray formulations. Therefore, different concentrations of plasmid DNA expressing enhanced green fluorescent protein (pEGFP-N1) were sprayed onto mouse or human skin once daily for three consecutive days and compared with direct injection. Gene expression was assessed 24 h after the final topical application of various liposomal DNA formulations. The results showed that EGFP mRNA and protein were detectable by RT-PCR and Western blot, respectively. However, epicutaneously applied EGFP plasmid DNA did not lead to microscopically detectable EGFP protein, when assessed by confocal laser microscopy or fluorescence-activated cell sorting in contrast to about 4% of fluorescent keratinocytes following intradermal injection. In an in vivo mouse model, the application of pEGFP-N1 DNA led to the generation of GFP-specific antibodies. These results indicate that topical spray application of pEGFP-N1 liposomal DNA formulations is a suitable method for plasmid DNA delivery to the skin, yielding limited gene expression. This spray method may thus be useful for DNA vaccination. To increase its attractiveness for skin gene therapy, the improvement of topical formulations with enhanced DNA absorption is desirable.

Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material.

Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments.

Purification of plasmids for gene therapy and DNA vaccination.

This chapter covers the different aspects of the production and purification of plasmids for gene therapy and DNA vaccination. Process issues are extensively covered and complemented with information related to plasmid DNA structure, vector construction, product specifications and quality assurance and control.

Plasmid DNA for pharmaceutical applications.

With the advent and progress of recombinant DNA technology into a variety of fields such as medical therapy, preventive or curative vaccination or the induction of regeneration, the demand for large quantities of highly purified DNA is increasing. Traditional methods of purifying plasmids usually require sophisticated methodology if the DNA is to be separated from RNA and other contaminating organic components. In particular, methods for obtaining supercoiled covalently closed circular (CCC) plasmid DNA in pure form, cope with the requirement that other plasmid topologies also produced have to be separated from the final product. The innovative technology of capillary gel electrophoresis (CGE) contributes a sensitive tool to the short list of applicable quality control assays for clinical grade plasmid DNA.

Capsid modification strategies for detargeting adenoviral vectors.

Adenoviral vectors hold immense potential for a wide variety of gene therapy based applications; however, their efficacy and toxicity is dictated by "off target" interactions that preclude cell specific targeting to sites of disease. A number of "off target" interactions have been described in the literature that occur between the three major capsid proteins (hexon, penton, and fiber) and components of the circulatory system, including cells such as erythrocytes, white blood cells, and platelets, as well as circulatory proteins including complement proteins, coagulation factors, von Willebrand Factor, p-selectin as well as neutralizing antibodies. Thus, to improve efficacious targeting to sites of disease and limit nonspecific uptake of virus to non-target tissues, specifically the liver and the spleen, it is necessary to develop suitable strategies for genetically modifying the capsid proteins to preclude these interactions. To this end we have developed versatile systems based on homologous recombination for modification of each of the major capsid proteins, which are described herein.

Large-scale purification of pharmaceutical-grade plasmid DNA using tangential flow filtration and multi-step chromatography.

The demand for pharmaceutical-grade plasmid DNA in vaccine applications and gene therapy has been increasing in recent years. In the present study, a process consisting of alkaline lysis, tangential flow filtration, purification by anion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography was developed. The final product met the requirements for pharmaceutical-grade plasmid DNA. The chromosomal DNA content was <1 µg/mg plasmid DNA, and RNA was not detectable by agarose gel electrophoresis. Moreover, the protein content was <2 µg/mg plasmid DNA, and the endotoxin content was <10 EU/mg plasmid DNA. The process was scaled up to yield 800 mg of pharmaceutical-grade plasmid DNA from approximately 2 kg of bacterial cell paste. The overall yield of the final plasmid DNA reached 48%. Therefore, we have established a rapid and efficient production process for pharmaceutical-grade plasmid DNA.

A seven-year storage report of good manufacturing practice-grade naked plasmid DNA: stability, topology, and in vitro/in vivo functional analysis.

The great interest for naked plasmid DNA in gene therapy studies is reflected by the fact that it is currently used in 18% of all gene therapy trials. Therefore, validation of topology and functionality of DNA resulting from its long-term stability is an essential requirement for safe and effective gene transfer. To this aim, we analyzed the stability of good manufacturing practice-grade pCMVß reporter plasmid DNA by capillary gel electrophoresis, agarose gel electrophoresis, and atomic force microscopy. The plasmid DNA was produced for a clinical gene transfer study started in 2005 and was stored for meanwhile 7 years under continuously monitored conditions at -20 °C. The stability of plasmid DNA was monitored by LacZ transgene expression functional assays performed in vitro and in vivo on the 7-year-old plasmid DNA samples compared with plasmid batches newly produced in similar experimental conditions and quality standards. The analyses revealed that during the overall storage time and conditions, the proportion of open circular and supercoiled or covalently closed circular forms is conserved without linearization or degradation of the plasmid. The in vitro transfection and the in vivo jet-injection of DNA showed unaltered functionality of the long-stored plasmid. In summary, the 7-year-old and the newly produced plasmid samples showed similar topology and expression performance. Therefore, our stable storage conditions are effective to preserve the integrity of the DNA to be used in clinical studies. This is an important prerequisite for the long-term performance of gene transfer materials used in trials of long duration as well as of the reference material used in standardization procedures and assays.

Successful application of monolithic innovative technology using a carbonyldiimidazole disk to purify supercoiled plasmid DNA suitable for pharmaceutical applications.

The growing demand on plasmid DNA (pDNA) manufacture for therapeutic applications requires a final product with higher quality and quantity, spending the least time. Most of the current processes for pDNA production use at least one chromatographic step, which often constitutes a key-step in the purification sequence. Monolithic stationary phases are new alternatives to the conventional matrices, which offer fast separation of pDNA due to their excellent mass transfer properties and their high binding capacity for large molecules, as pDNA. However, the efficient recovery of pure pDNA focuses on a suitable balance of the feedstock, adsorbent and mobile phase properties. To satisfy the increasing demand for pharmaceutical grade plasmids, we developed a novel downstream process which overcomes the bottlenecks of common lab-scale techniques while complying with all regulatory requirements. This work reports an integrative approach using the carbonyldiimidazole monolith to efficiently purify the supercoiled (sc) pDNA active conformation from other plasmid topologies and Escherichia coli impurities present in a clarified lysate. The monolith specificity and selectivity was also assessed by performing experiments with plasmids of several sizes of 2.7, 6.05 and 7.4 kilo base pairs (kbp), verifying the applicability to purify different plasmids. Hence, the process yield of the pDNA purification step using the CDI monolith was 89%, with an extremely reduced level of impurities (endotoxins and gDNA), which was reflected in good transfection experiments of the sc plasmid DNA sample. Overall, the analytical results and transfection studies performed with the pDNA sample purified with this monolithic enabling technology, confirmed the suitability of this pDNA to be used in pharmaceutical applications.