RESUMO
The supply of plasmid DNA has become a major bottle neck in the ever-expanding genetic medicine sector. Therefore, the development of new, scalable, faster DNA production technologies is vital for this sector going forward.
Assuntos
DNARESUMO
Vaccination with DNA that encodes cancer antigens is a simple and convenient way to raise immunity against cancer and has already shown promise in the clinical setting. Conventional plasmid DNA is commonly used which together with the encoded antigen also includes bacterial immunostimulatory CpG motifs to target the DNA sensor Toll-like receptor 9. Recently DNA vaccines using doggybone DNA (dbDNA™), have been developed without the use of bacteria. The cell-free process relies on the use of Phi29 DNA polymerase to amplify the template followed by protelomerase TelN to complete individual closed linear DNA. The resulting DNA contains the required antigenic sequence, a promoter and a poly A tail but lacks bacterial sequences such as an antibiotic resistance gene, prompting the question of immunogenicity. Here we compared the ability of doggybone DNA vaccine with plasmid DNA vaccine to induce adaptive immunity using clinically relevant oncotargets E6 and E7 from HPV. We demonstrate that despite the inability to trigger TLR9, doggybone DNA was able to induce similar levels of cellular and humoral immunity as plasmid DNA, with suppression of established TC-1 tumours.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Neoplasias Pulmonares/imunologia , Plasmídeos/imunologia , Receptor Toll-Like 9/imunologia , Vacinas de DNA/imunologia , Animais , Modelos Animais de Doenças , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/administração & dosagem , Plasmídeos/genética , Células Tumorais Cultivadas , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
The applicability and potential of using elevated temperatures and sub 2-microm porous particles in chromatography for metabonomics/metabolomics was investigated using, for the first time, solvent temperatures higher than the boiling point of water (up to 180 degrees C) and thermal gradients to reduce the use of organic solvents. Ultra performance liquid chromatography, combined with mass spectrometry, was investigated for the global metabolite profiling of the plasma and urine of normal and Zucker (fa/fa) obese rats (a well established disease animal model). "Isobaric" high temperature chromatography, where the temperature and flow rate follow a gradient program, was developed and evaluated against a conventional organic solvent gradient. LC-MS data were first examined by established chromatographic criteria in order to evaluate the chromatographic performance and next were treated by special peak picking algorithms to allow the application of multivariate statistics. These studies showed that, for urine (but not plasma), chromatography at elevated temperatures provided better results than conventional reversed-phase LC with higher peak capacity and better peak asymmetry. From a systems biology point of view, better group clustering and separation was obtained with a larger number of variables of high importance when using high temperature-ultra performance liquid chromatography (HT-UPLC) compared to conventional solvent gradients.
