ABSTRACT
Non-viral gene transfer using plasmid DNA (pDNA) is generally acknowledged as safe and non-immunogenic compared with the use of viral vectors. However, pre-clinical and clinical studies have shown that non-viral (lipoplex) gene transfer to the lung can provoke a mild, acute inflammatory response, which is thought to be, partly, due to unmethylated CG dinucleotides (CpGs) present in the pDNA sequence. Using a murine model of lung gene transfer, bronchoalveolar lavage fluid was collected following plasmid delivery and a range of inflammatory markers was analysed. The results showed that a Th1-related inflammatory cytokine response was present that was substantially reduced, though not abolished, by using CpG-free pDNA. The remaining minor level of inflammation was dependent on the quality of the pDNA preparation, specifically the quantity of contaminating bacterial genomic DNA, also a source of CpGs. Successful modification of a scalable plasmid manufacturing process, suitable for the production of clinical grade pDNA, produced highly pure plasmid preparations with reduced genomic DNA contamination. These studies help define the acceptable limit of genomic DNA contamination that will impact FDA/EMEA regulatory guidelines defining clinical grade purity of plasmid DNA for human use in gene therapy and vaccination studies.
Subject(s)
DNA Contamination , DNA, Bacterial/metabolism , Drug Delivery Systems/methods , Genome, Bacterial/genetics , Inflammation/pathology , Lung/metabolism , Plasmids/metabolism , Animals , Biomarkers/metabolism , Bronchoalveolar Lavage Fluid , CpG Islands/genetics , Cytokines/metabolism , Electrophoresis, Agar Gel , Female , Humans , Liposomes , Mice , Mice, Inbred BALB C , Toll-Like Receptor 9/deficiency , Toll-Like Receptor 9/metabolismABSTRACT
Nonviral gene therapy utilizing plasmid DNA (pDNA) complexed with cationic lipids (lipoplexes) or cationic polymers (polyplexes) has demonstrated considerable potential for the treatment of a variety of diseases. However, progress toward clinical application is often delayed by the lack of reliable and scalable mixing of components sufficient to guarantee consistent performance in vivo. Attempts to improve and standardize mixing have been limited by the sensitivity of pDNA to shear-related degradation. Here we describe a simple pneumatic mixing device that enables the rapid and reproducible production of large volumes of nonviral gene therapy formulations and demonstrate its suitability for use with shear-sensitive pDNA.
Subject(s)
DNA/administration & dosage , Genetic Therapy/instrumentation , Plasmids/administration & dosage , Animals , Cations/chemistry , DNA/chemistry , DNA/genetics , Equipment Design , Gene Expression , Lipids/chemistry , Mice , Mice, Inbred BALB C , Plasmids/chemistry , Plasmids/geneticsABSTRACT
The increased use of plasmid-based vaccines to replace their more challenging viral counterparts has increased the demand for high purity and high concentration plasmids. Here we report the production of plasmids encoding different transgenes for DNA vaccine candidates at gram scale with an integrated process consisting of batch fermentation and limited steps of purification. Plasmid products encoding for eight smallpox antigens that were combined into a bioterrorism DNA vaccine exhibited high purity with undetectable RNA, protein and endotoxin, concentration of up to 13.6mg/mL and supercoiled percentage of 94.5+/-1.1% after storage at -80 degrees C for over 1 year. The process has been scaled up for the cGMP manufacture of pharmaceutical-grade human papillomavirus and influenza DNA vaccines up to a 50g scale, also demonstrating high purity and high concentration.
Subject(s)
DNA/biosynthesis , Fermentation , Plasmids , Vaccines, DNA/biosynthesis , DNA/isolation & purification , Influenza Vaccines/biosynthesis , Papillomavirus Vaccines/biosynthesis , Quality Control , Smallpox Vaccine/biosynthesisABSTRACT
The demand for plasmid DNA in large quantities at high purity and concentration is expected to escalate as more DNA vaccines are entering clinical trial status and becoming closer to market approval. This review outlines different methods for DNA vaccine manufacture and discusses the challenges that hinder large-scale production. Current technologies are summarized, focusing on novel approaches that have the potential to address downstream bottlenecks and adaptability for large-scale application. Product quality in terms of supercoiled percentage and impurity levels are compared at the different production levels.
Subject(s)
Plasmids/biosynthesis , Plasmids/isolation & purification , Technology, Pharmaceutical/methods , Vaccines, DNA/genetics , Vaccines, DNA/immunology , Animals , Humans , Plasmids/genetics , Quality ControlABSTRACT
We have developed three major technologies that allow plasmid-based products to be used for large-scale vaccination or therapeutic protein applications. Our team has integrated these components into one complete, cost-effective, easy-to-use system capable of rapid implementation under field conditions. The proprietary manufacturing process uses a lysis method and membrane-based chromatography to rapidly produce large-scale batches of plasmid. Plasmid doses are filled into the Becton-Dickinson Uniject container/closure system. The Uniject adapts to the electrode array of our constant current electrokinetic device, such that the plasmid is delivered in the area of tissue defined by the electrodes. Thus, plasmid uptake and expression levels are dramatically improved. This is the first completely integrated delivery system for plasmid-based products.
