Plasmid Production Research Articles

Lentiviral vector packaging and producer cell lines yield titers equivalent to the industry-standard four-plasmid process

Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing and transfection all contribute to operational risk and the high cost associated with this therapeutic modality. Thus, we developed LVV packaging and producer cell lines, which reduce or eliminate the need for plasmid transfection during LVV manufacture. To develop a packaging cell line, lentiviral packaging genes were stably integrated by random integration of linearised plasmid DNA. Then, to develop EGFP- and anti-CD19 chimeric antigen receptor-encoding producer cell lines, transfer plasmids were integrated by transposase-mediated integration. Single cell isolation and testing were performed to isolate the top-performing clonal packaging and producer cell lines. Production of LVV that encode various cargo genes revealed consistency in the production performance of the packaging and producer cell lines compared to the industry-standard four-plasmid transfection method. By reducing or eliminating the requirement for plasmid transfection, while achieving production performance consistent with the current industry standard, the packaging and producer cell lines developed here can reduce costs and operational risks of LVV manufacture, thus increasing patient access to LVV-mediated cell and gene therapies.

Comparative assessment of simulation-based and surrogate-based approaches to flowsheet optimization using dimensionality reduction

This work proposes a framework for simulation-based and surrogate-based reduced space Bayesian optimization of process flowsheets. The framework uses global sensitivity analysis for dimensionality reduction via the identification of critical process variables that contribute significantly to the variability of the objective function (e.g. productivity and operating costs). Both simulation- and surrogate-based algorithms are applied to a biopharmaceutical and a chemical process simulator for the production of plasmid DNA and dimethyl ether (DME), respectively. Their capabilities are assessed in terms of the trade-off between computational effectiveness and solution accuracy. Results indicate that simulation-based Bayesian optimization achieves better objective function values, while surrogate-based Bayesian optimization is more computationally effective.

From Bioreactor to Bulk Rheology: Achieving Scalable Production of Highly Concentrated Circular DNA.

DNA serves as a model system in polymer physics due to its ability to be obtained as a uniform polymer with controllable topology and nonequilibrium behavior. Currently, a major obstacle in the widespread adoption of DNA is obtaining it on a scale and cost basis that accommodates bulk rheology and high-throughput screening. To address this, recent advancements in bioreactor-based plasmid DNA production is coupled with anion exchange chromatography producing a unified approach to generating gram-scale quantities of monodisperse DNA. With this method, 1.1 grams of DNA is obtained per batch to generate solutions with concentrations up to 116mg mL-1. This solution of uniform supercoiled and relaxed circular plasmid DNA, is roughly 69 times greater than the overlap concentration. The utility of this method is demonstrated by performing bulk rheology measurements at sample volumes up to 1 mL on DNA of different lengths, topologies, and concentrations. The measured elastic moduli are orders of magnitude larger than those previously reported for DNA and allowed for the construction of a time-concentration superposition curve that spans 12 decades of frequency. Ultimately, these results can provide important insights into the dynamics of ring polymers and the nature of highly condensed DNA dynamics.

A machine learning-based approach for improving plasmid DNA production in Escherichia coli fed-batch fermentations.

Artificial Intelligence (AI) technology is spearheading a new industrial revolution, which provides ample opportunities for the transformational development of traditional fermentation processes. During plasmid fermentation, traditional subjective process control leads to highly unstable plasmid yields. In this study, a multi-parameter correlation analysis was first performed to discover a dynamic metabolic balance among the oxygen uptake rate, temperature, and plasmid yield, whilst revealing the heating rate and timing as the most important optimization factor for balanced cell growth and plasmid production. Then, based on the acquired on-line parameters as well as outputs of kinetic models constructed for describing process dynamics of biomass concentration, plasmid yield, and substrate concentration, a machine learning (ML) model with Random Forest (RF) as the best machine learning algorithm was established to predict the optimal heating strategy. Finally, the highest plasmid yield and specific productivity of 1167.74mg L-1 and 8.87mg L-1/OD600 were achieved with the optimal heating strategy predicted by the RF model in the 50 L bioreactor, respectively, which was 71% and 21% higher than those obtained in the control cultures where a traditional one-step temperature upshift strategy was applied. In addition, this study transformed empirical fermentation process optimization into a more efficient and rational self-optimization method. The methodology employed in this study is equally applicable to predict the regulation of process dynamics for other products, thereby facilitating the potential for furthering the intelligent automation of fermentation processes.

The effect of silver nanoparticles and preparations of various pharmacological groups on the bactericidal activity of <i>Staphylococcus aureus</i>

Multidrug-resistant bacteria have become one of the most serious threats to public health worldwide. The abuse of antibiotics has contributed to the emergence and transmission of resistance mechanisms among bacteria, jeopardizing the therapeutic potential of antibiotics. Uncontrolled use of drugs leads to the formation of antibiotic resistance due to mutations in chromosomal DNA, as well as the production of plasmids, integrons from other bacteria during horizontal gene transfer. In 2010, the BRICS countries (Brazil, Russia, India, China and South Africa) accounted for 76% of antibiotic consumption, with India consuming 12.9 billion units and China – 10 billion units. As of 2017, carbapenem-resistant Acetobacter baumannii and Enterobacteriaceae resulted in approximately US$ 281 million in healthcare costs in the United States. According to numerous reports from the Center for Disease Control and Prevention, approximately 2.3 million episodes of multidrug-resistant microbial diseases resulting in 25,000 deaths are recorded annually in the United States alone. In this regard, the world community of scientists has intensified the study of the combined use of various antibacterial drugs to achieve maximum bactericidal activity. Studies have been conducted to determine the synergistic effect when using combinations of drugs of various pharmacological groups and silver nanoparticles. A significant increase in bactericidal activity by 53.87 times (from 2.528 to 0.0098 mcg/ml) was found with the combined use of AgNPs and DSMO against the reference strain Staphylococcus aureus ATCC 25953. While the cultivation of St. aureus isolate with DSMO and silver nanoparticles revealed a sensitivity increase of 128.2 times (from 5.056 to 0.039 mcg/ml).

Enhancing the biosynthesis of taxadien-5α-yl-acetate in Escherichia coli by combinatorial metabolic engineering approaches

Biosynthesis of paclitaxel (Taxol™) is a hot topic with extensive and durable interests for decades. However, it is severely hindered due to the very low titers of intermediates. In this study, Escherichia coli was employed to de novo synthesize a key intermediate of paclitaxel, taxadien-5α-yl-acetate (T5OAc). Plasmid-based pathway reconstruction and optimization were conducted for T5OAc production. The endogenous methylerythritol phosphate pathway was enhanced to increase the precursor supply. Three taxadien-5α-ol O-acetyltransferases were tested to obtain the best enzyme for the acetylation step. Metabolic burden was relieved to restore cell growth and promote production through optimizing the plasmid production system. In order to achieve metabolic balance, the biosynthesis pathway was regulated precisely by multivariate-modular metabolic engineering. Finally, in a 5-L bioreactor, the T5OAc titer was enhanced to reach 10.9 mg/L. This represents an approximately 272-fold increase in production compared to the original strain, marking the highest yield of T5OAc ever documented in E. coli, which is believed to be helpful for promoting the progress of paclitaxel biosynthesis.

Persistent pollution of genetic materials in a typical laboratory environment

From the onset of coronavirus disease (COVID-19) pandemic, there are concerns regarding the disease spread and environmental pollution of biohazard since studies on genetic engineering flourish and numerous genetic materials were used such as the nucleic acid test of the severe acute respiratory syndrome coronavirus (SARS-CoV-2). In this work, we studied genetic material pollution in an institute during a development cycle of plasmid, one of typical genetic materials, with typical laboratory settings. The pollution source, transmission routes, and pollution levels in laboratory environment were examined. The Real-Time quantitative- Polymerase Chain Reaction results of all environmental mediums (surface, aerosol, and liquid) showed that a targeted DNA segment occurred along with routine experimental operations. Among the 79 surface and air samples collected in the genetic material operation, half of the environment samples (38 of 79) are positive for nucleic acid pollution. Persistent nucleic acid contaminations were observed in all tested laboratories and spread in the public area (hallway). The highest concentration for liquid and surface samples were 1.92 × 108 copies/uL and 5.22 × 107 copies/cm2, respectively. Significant amounts of the targeted gene (with a mean value of 74 copies/L) were detected in the indoor air of laboratories utilizing centrifuge devices, shaking tables, and cell homogenizers. Spills and improper disposal of plasmid products were primary sources of pollution. The importance of establishing designated experimental zones, employing advanced biosafety cabinets, and implementing highly efficient cleaning systems in laboratories with lower biosafety levels is underscored. SYNOPSISSTATEMENT. Persistent environmental pollutions of genetic materials are introduced by typical experiments in laboratories with low biosafety level.

Development of a high-throughput scale-down model in Ambr® 250 HT for plasmid DNA fermentation processes.

Recent advances in messenger ribonucleic acid (mRNA) vaccines and gene therapy vectors have increased the need for rapid plasmid DNA (pDNA) screening and production within the biopharmaceutical industry. High-throughput (HT) fermentor systems, such as the Ambr® 250 HT, can significantly accelerate process development timelines of pDNA upstream processes compared to traditional bench-scale glass fermentors or small-scale steam-in-place (SIP) fermentors. However, such scale-down models must be qualified to ensure that they are representative of the larger scale process similar to traditional small-scale models. In the current study, we developed a representative scale-down model of a Biostat® D-DCU 30 L pDNA fermentation process in Ambr® 250 HT fermentors using three cell lines producing three different constructs. The Ambr scale-down model provided comparable process performance and pDNA quality as the 30 L SIP fermentation process. In addition, we demonstrated the predictive value of the Ambr model by two-way qualification, first by accurately reproducing the prior trends observed in a 30 L process, followed by predicting new process trends that were then successfully reproduced in the 30 L process. The representative and predictive scale-down Ambr model developed in this study would enable a faster and more efficient approach to strain/clone/host-cell screening, pDNA process development and characterization studies, process scale-up studies, and manufacturing support.

Anion exchange HPLC monitoring of mRNA in vitro transcription reactions to support mRNA manufacturing process development.

mRNA technology has recently demonstrated the ability to significantly change the timeline for developing and delivering a new vaccine from years to months. The potential of mRNA technology for rapid vaccine development has recently been highlighted by the successful development and approval of two mRNA vaccines for COVID-19. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, e.g., treatments for cancer, metabolic disorders, cardiovascular conditions, and autoimmune diseases. There is currently significant demand for the development of improved manufacturing processes for the production of mRNA therapeutics in an effort to increase their yield and quality. The development of suitable analytical methods for the analysis of mRNA therapeutics is critical to underpin manufacturing development and the characterisation of the drug product and drug substance. In this study we have developed a high-throughput, high-performance liquid chromatography (HPLC) workflow for the rapid analysis of mRNA generated using in vitro transcription (IVT). We have optimised anion exchange (AEX) HPLC for the analysis of mRNA directly from IVT. Chromatography was performed in under 6min enabling separation of all of the key components in the IVT, including nucleoside triphosphates (NTPs), Cap analogue, plasmid DNA and mRNA product. Moreover, baseline separation of the NTPs was achieved, which facilitates accurate quantification of each NTP such that their consumption may be determined during IVT reactions. Furthermore, the HPLC method was used to rapidly assess the purification of the mRNA product, including removal of NTPs/Cap analogue and other contaminants after downstream purification, including solid phase extraction (SPE), oligo deoxythymidine (oligo-dT) affinity chromatography and tangential flow filtration (TFF). Using the developed method excellent precision was obtained with calibration curves for an external mRNA standard and NTPs giving correlation coefficients of 0.98 and 1.0 respectively. Intra- and inter-day studies on retention time stability of NTPs, showed a relative standard deviation ≤ 0.3% and ≤1.5% respectively. The mRNA retention time variability was ≤0.13%. This method was then utilised to monitor the progress of an IVT reaction for the production of Covid spike protein (C-Spike) mRNA to measure the increasing yield of mRNA alongside the consumption of NTPs during the reaction.

An innovative lab-scale production for a novel therapeutic DNA vaccine candidate against rheumatoid arthritis

BackgroundRecent therapeutic-plasmid DNA vaccine strategies for rheumatoid arthritis (RA) have significantly improved. Our pcDNA-CCOL2A1 vaccine is the most prominent and the first antigen-specific tolerising DNA vaccine with potent therapeutic and prophylactic effects compared with methotrexate (MTX), the current “gold standard” treatment for collagen-induced arthritis (CIA). This study developed a highly efficient, cost-effective, and easy-to-operate system for the lab-scale production of endotoxin-free supercoiled plasmids with high quality and high yield. Based on optimised fermentation culture, we obtained a high yield of pcDNA-CCOL2A1 vaccine by PEG/MgCl2 precipitation and TRION-114. We then established a method for quality control of the pcDNA-CCOL2A1 vaccine. Collagen-induced arthritis (CIA) model rats were subjected to intramuscular injection of the pcDNA-CCOL2A1 vaccine (300 μg/kg) to test its biological activity.ResultsAn average yield of 11.81 ± 1.03 mg purified supercoiled plasmid was obtained from 1 L of fermentation broth at 670.6 ± 57.42 mg/L, which was significantly higher than that obtained using anion exchange column chromatography and a commercial purification kit. Our supercoiled plasmid had high purity, biological activity, and yield, conforming to the international guidelines for DNA vaccines.ConclusionThe proposed innovative downstream process for the pcDNA-CCOL2A1 vaccine can not only provide a large-scale high-quality supercoiled plasmid DNA for preclinical research but also facilitate further pilot-scale and even industrial-scale production of pcDNA-CCOL2A1 vaccine.

Improvement of Plasmid Volumetric Yield by Addition of Glycerol and Phosphate Buffer in Escherichia coli TOP10 Batch Culture

The investigation of mRNA development has gained substantial interest, particularly in the ex vivo and in vivo therapy. mRNA is widely used for the development of gene editing-based therapies and mRNA vaccines. The aim of this study was to optimize the medium and harvest time to increase plasmid DNA production as part of mRNA production. This study modified used a medium modification approach to achieve high density culture of Escherichia coli TOP10 pGEMT-N in batch cultivation method. Various media formulations were assessed, including LB; LB with phosphate buffer (K2HPO4 12.549 g/L and KH2PO4 2.31 g/L); LB with glycerol (50 g/L); LB with glycerol and phosphate buffer; LB with phosphate buffer, glycerol, glucose (15 g/L), and galactose (15 g/L). The effect of additional carbon sources and phosphate buffer on culture density was measured through OD600 and wet cell weight analysis. The highest OD600 and wet cell weight was observed when LB with glycerol and phosphate buffer was used, with OD600 of 4.78±0.14 and wet cell weight of 36.00±0.63 mg/ml. Plasmid DNA was subsequently isolated from these cultures following 5- and 7.5-hour incubation periods. The utilization of LB medium with glycerol and phosphate buffer resulted in a substantial increase in the volumetric concentration of plasmid DNA of 1,516.97±385.00 ng/ml after 5 hours of incubation. In conclusion, a remarkable enhancement in plasmid DNA volumetric yield within 5 hours was achieved by addition of glycerol and phosphate buffer to LB medium, leading to incubation period.

Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered E. coli.

The demand of plasmid DNA (pDNA) as a key element for gene therapy products, as well as mRNA and DNA vaccines, is increasing together with the need for more efficient production processes. An engineered E. coli strain lacking the phosphotransferase system and the pyruvate kinase A gene has been shown to produce more pDNA than its parental strain. With the aim of improving pDNA production in the engineered strain, several strategies to increase the flux to the pentose phosphate pathway (PPP) were evaluated. The simultaneous consumption of glucose and glycerol was a simple way to increase the growth rate, pDNA production rate, and supercoiled fraction (SCF). The overexpression of key genes from the PPP also improved pDNA production in glucose, but not in mixtures of glucose and glycerol. Particularly, the gene coding for the glucose 6-phosphate dehydrogenase (G6PDH) strongly improved the SCF, growth rate, and pDNA production rate. A linear relationship between the G6PDH activity and pDNA yield was found. A higher flux through the PPP was confirmed by flux balance analysis, which also estimates relevant differences in fluxes of the tricarboxylic acid cycle. These results are useful for developing further cell engineering strategies to increase pDNA production and quality.

Optimizing Fed-Batch Processes with Dynamic Control Flux Balance Analysis

Fed-batch processes are prevalent in biotechnological industries, but design of experiments often results in sub-optimal conditions due to incomplete solution space characterization. We employ a single-level dynamic control (DC) algorithm for dynamic flux balance analysis (dFBA), enhancing efficiency by reducing Karush-Kuhn-Tucker (KKT) condition constraints and adapting the algorithm for predicting optimal process length. In a growth-decoupled plasmid DNA production case study, we predict the optimal feeding profile and switching time between growth and production phase. Comparing our algorithm to its predecessor shows a speed-up of at least a factor of four. When the process length is part of the objective function the speed-up becomes considerably larger.

Producing Plasmid DNA Template for Clinical Grade RNA Vaccine Manufacture.

A plasmid production process has been established to manufacture plasmid DNA at a large scale in High-Quality grade. This is used as a starting material to produce mRNA vaccines for clinical trials. Recently, the World Health Organization (WHO) has released regulatory guidelines related to the quality, safety, and efficacy for DNA- as well as for mRNA-based vaccines. Following an extraordinary year of scientific, regulatory, and manufacturing developments, the scientific community today stands considerably better equipped to deal with urgent production requirements in large scale for nucleic acid-based vaccinations and therapies. Going forward, work needs to be done in better coordinating the supply and logistics of essential raw materials for biological manufacturing, especially under emergency conditions.

The Effects of Magnesium, Zinc and Calcium Ions on Endotoxin-Plasmid DNA Interaction at Various Cation Concentrations, pH Values and Incubation Times

In plasmid DNA (pDNA) production from Gram-negative bacteria, endotoxin has been known as the major contaminant. The separation becomes difficult due to its ability to form a stable complex with pDNA apart from sharing common properties like surface charge, molecular size, temperature, and pH stability. This study focused on the analysis of the zeta potential values of endotoxin, the theoretical number of cations bound per molecule of endotoxin as well as the binding tendency of cations towards endotoxin in the presence of pDNA. These analyses were conducted under various experimental conditions such as types of divalent metal cation, cation concentration, pH and incubation time. The analysis of zeta potential at different cation concentrations and pH values showed that Mg2+ had the most significant effect on endotoxin surface charge. The zeta potential of endotoxin was reduced by a magnitude of 43.55 mV, from −43.53 mV to 0.02 mV in the presence of 2.0 M Mg2+, and a magnitude of 44.12 mV, from −43.53 mV to 0.59 mV at the lowest pH level. However, in the analysis of the theoretical number of cations bound per molecule of endotoxin, Zn2+ showed the highest number (0.6) compared to Ca2+ (0.12) and Mg2+ (0.05). The tendency of Zn2+ to preferentially bind with endotoxins forming a larger aggregated structure was also evident in the DNA gel electrophoresis and transmission electron microscopic analysis. The manuscript highlights the significance of cations in the binding and aggregation of endotoxins, which ultimately improves the recovery of pDNA and affects its subsequent downstream processing.

A Bioreactor-Based Yellow Fever Virus-like Particle Production Process with Integrated Process Analytical Technology Based on Transient Transfection.

Yellow Fever (YF) is a severe disease that, while preventable through vaccination, lacks rapid intervention options for those already infected. There is an urgent need for passive immunization techniques using YF-virus-like particles (YF-VLPs). To address this, we successfully established a bioreactor-based production process for YF-VLPs, leveraging transient transfection and integrating Process Analytical Technology. A cornerstone of this approach was the optimization of plasmid DNA (pDNA) production to a yield of 11 mg/L using design of experiments. Glucose, NaCl, yeast extract, and a phosphate buffer showed significant influence on specific pDNA yield. The preliminary work for VLP-production in bioreactor showed adjustments to the HEK cell density, the polyplex formation duration, and medium exchanges effectively elevated transfection efficiencies. The additive Pluronic F-68 was neutral in its effects, and anti-clumping agents (ACA) adversely affected the transfection process. Finally, we established the stirred-tank bioreactor process with integrated dielectric spectroscopy, which gave real-time insight in relevant process steps, e.g., cell growth, polyplex uptake, and harvest time. We confirmed the presence and integrity of YF-VLP via Western blot, imaging flow cytometry measurement, and transmission electron microscopy. The YF-VLP production process can serve as a platform to produce VLPs as passive immunizing agents against other neglected tropical diseases.

Utilizing an automation-on-demand analytical tool for cancer immunotherapy

Richard Parker-Manuel, PhD, is a Group Leader for Plasmid Engineering and Production at OXGENE, A WuXi Advanced Therapies Company. He has twenty years’ experience in molecular biology with nearly seven years in the cell and gene therapy space. He oversees the design and engineering of adeno associated virus and lentivirus vector plasmids for a variety of clients. He is also involved in several R&D initiatives with the aim of making OXGENE’s excellent vectors even better.Qian Liu, PhD, joined OXGENE in July 2017 as a Cell Line Engineering Scientist. She has a background in cell and molecular biology, She was then gradually promoted to lead all biomanufacturing services OXGENE provide. Prior to joining OXGENE, Qian was a postdoctoral researcher in the field of Regenerative Medicine, involved in bioartificial liver development and stem cell differentiation mechanism investigation in Loughborough University and the University of Nottingham, respectively. Qian obtained her PhD in Molecular Nutrition from the University of Nottingham.

Improved endotoxin removal using ecofriendly detergents for intensified plasmid capture.

Increasing plasmid demand for both production of viral and gene therapies as well as nucleic acid based vaccines has highlighted bottlenecks in production. One bottleneck is traditional bead-based chromatography as a capture step. To meet the needs of fast-growing markets, new production solutions are needed. These solutions must enable efficient capture of a diverse range of plasmid types and excellent clearance of bacterial host impurities, such as endotoxin. Enhanced endotoxin clearance during chromatographic purification has previously been demonstrated with detergents such as Triton™ X-100. However, degradation products of Triton™ X-100 are known to have a negative environmental impact, and more sustainable, environmentally benign alternatives have been identified. This work establishes an efficient, intensified plasmid capture using convective anion exchange (AEX) chromatography. The feasibility of the intensified capture approach was assessed with different membrane and a monolith AEX supports. Various detergents from different physico-chemical classes were evaluated with different AEX technologies. Purification efficiency evaluated endotoxin and host cell protein (HCP) clearance, plasmid yield, potential interference of the detergents with analytical in-process control assays, and overall process compatibility. This comprehensive screening approach provides valuable insights to intensified plasmid production.

Exploring the Potential of a Genome-Reduced Escherichia coli Strain for Plasmid DNA Production.

The global demand for nucleic acid-based vaccines, including plasmid DNA (pDNA) and mRNA vaccines, needs efficient production platforms. However, conventional hosts for plasmid production have encountered challenges related to sequence integrity due to the presence of insertion sequences (ISs). In this study, we explored the potential of a genome-reduced Escherichia coli as a host for pDNA production. This strain had been constructed by removing approximately 23% of the genome which were unessential genes, including the genomic unstable elements. Moreover, the strain exhibits an elevated level of NADPH, a coenzyme known to increase plasmid production according to a mathematical model. We hypothesized that the combination of genome reduction and the abundance of NADPH would significantly enhance pDNA production capabilities. Remarkably, our results confirmed a three-fold increase in pDNA production compared to the widely employed DH5α strain. Furthermore, the genome-reduced strain exhibited heightened sensitivity to various antibiotics, bolstering its potential for large scale industrial pDNA production. These findings suggest the genome-reduced E. coli as an exciting candidate for revolutionizing the pDNA industry, offering unprecedented efficiency and productivity.

Minimized antibiotic-free plasmid vector for gene therapy utilizing a new toxin-antitoxin system

Approaches to improve plasmid-mediated transgene expression are needed for gene therapy and genetic immunization applications. The backbone sequences needed for the production of plasmids in bacterial hosts and the use of antibiotic resistance genes as selection markers represent biological safety risks. Here, we report the development of an antibiotic-free expression plasmid vector with a minimized backbone utilizing a new toxin-antitoxin (TA) system. The Rs_0636/Rs_0637 TA pair was derived from the coral-associated bacterium Roseivirga sp. The toxin gene is integrated into the chromosome of Escherichia coli host cells, and a recombinant mammalian expression plasmid is constructed by replacing the antibiotic resistance gene with the antitoxin gene Rs_0637 (here named Tiniplasmid). The Tiniplasmid system affords high selection efficiency (∼80%) for target gene insertion into the plasmid and has high plasmid stability in E. coli (at least 9 days) in antibiotic-free conditions. Furthermore, with the aim of reducing the size of the backbone sequence, we found that the antitoxin gene can be reduced to 153 bp without a significant reduction in selection efficiency. To develop its applications in gene therapy and DNA vaccines, the biosafety and efficiency of the Tiniplasmid-based eukaryotic gene delivery and expression were further evaluated in CHO–K1 cells. The results showed that Rs_0636/Rs_0637 has no cell toxicity and that the Tiniplasmid vector has a higher gene expression efficiency than the commercial vectors pCpGfree and pSTD in the eukaryotic cells. Altogether, the results demonstrate the potential of the Rs_0636/Rs_0637-based antibiotic-free plasmid vector for the development and production of safe and efficacious DNA vaccines.