Ratanamart Jarupa, Huggins Christopher G, Kirby John A, Shaw James A M
Diabetes Research Group, Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
J Gene Med. 2007 Aug;9(8):703-14. doi: 10.1002/jgm.1066.
Plasmid DNA vectors offer the potential of safe gene therapy avoiding viral vector-mediated toxicity and immunogenicity. As plasmid DNA is bacterial in origin, presence of bacterial lipopolysaccharide (LPS) or unmethylated CpG dinucleotides may stimulate host innate immunity.
Primary cultures of mouse and rat dendritic cells were established and incubated with bacterial lipopolysaccharide; immunostimulatory CpG oligodeoxynucleotide; control GpC oligodeoxynucleotide; and a range of (pVR1012) plasmids encoding transgenes with increasing CpG content (wild-type and mutant human preproinsulin; non-mammalian eukaryotic eGFP reporter gene; and bacterial beta-galactosidase reporter gene). IL-12 secretion was assayed to determine in vitro plasmid immunogenicity. Local inflammatory response following intramuscular injection of these plasmids, with or without a non-ionic carrier SP1017, was characterised in vivo.
Dose-responsive LPS and CpG stimulation of IL-12 secretion from dendritic cells was demonstrated. All plasmids induced significant IL-12 secretion in comparison to control unstimulated cells. The beta-galactosidase plasmid had highest CpG content and induced significantly higher IL-12 secretion than constructs containing a eukaryotic transgene. Injection of rat muscle with the beta-galactosidase construct induced greater inflammatory response than human preproinsulin constructs. This was further enhanced by SP1017. At 2 days post-injection, monocyte/macrophage injection site infiltration predominated with CD8-positive lymphocytes predominating at 7 days. There was no evidence of transgene expression in infiltrating immune cells.
Dendritic cell immunostimulation may be employed as an in vitro bioassay of innate immune response to plasmid DNA vectors during evaluation for clinical gene therapy.
质粒DNA载体为安全的基因治疗提供了可能，避免了病毒载体介导的毒性和免疫原性。由于质粒DNA起源于细菌，细菌脂多糖（LPS）或未甲基化的CpG二核苷酸的存在可能刺激宿主先天免疫。
建立小鼠和大鼠树突状细胞的原代培养物，并与细菌脂多糖、免疫刺激CpG寡脱氧核苷酸、对照GpC寡脱氧核苷酸以及一系列编码具有增加CpG含量的转基因的（pVR1012）质粒（野生型和突变型人胰岛素原；非哺乳动物真核增强绿色荧光蛋白报告基因；以及细菌β-半乳糖苷酶报告基因）一起孵育。检测白细胞介素-12（IL-12）分泌以确定体外质粒免疫原性。体内表征了肌肉注射这些质粒（有或没有非离子载体SP1017）后的局部炎症反应。
证明了LPS和CpG对树突状细胞IL-12分泌的剂量反应性刺激。与未刺激的对照细胞相比，所有质粒均诱导显著的IL-12分泌。β-半乳糖苷酶质粒的CpG含量最高，并且比含有真核转基因的构建体诱导显著更高的IL-12分泌。用β-半乳糖苷酶构建体注射大鼠肌肉比人胰岛素原构建体诱导更大的炎症反应。SP1017进一步增强了这种反应。注射后2天，单核细胞/巨噬细胞在注射部位浸润占主导，而CD8阳性淋巴细胞在7天占主导。在浸润的免疫细胞中没有转基因表达的证据。
在临床基因治疗评估期间，树突状细胞免疫刺激可用作质粒DNA载体先天免疫反应的体外生物测定。
