The significance of plasmid DNA preparations contaminated with bacterial genomic DNA on inflammatory responses following delivery of lipoplexes to the murine lung
2011
Bazzani, Reto P. | Cai, Ying | Hebel, Henry L. | Hyde, Stephen C. | Gill, Deborah R.
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.
Показать больше [+] Меньше [-]Ключевые слова АГРОВОК
Библиографическая информация
Эту запись предоставил National Agricultural Library