title: In vitro and in vivo evaluation of intrinsic immunogenicity of reporter and insulin gene therapy plasmids.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Diabetes Research Group, Institute of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
authors.forename: Jarupa
authors.initials: J
authors.lastname: Ratanamart
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Huggins
authors.forename: John A
authors.initials: JA
authors.lastname: Kirby
authors.forename: James A M
authors.initials: JA
authors.lastname: Shaw
mesh_terms: D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D018899:CpG Islands; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology; D004731:Endotoxins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007328:Insulin / Q000235:genetics*; D018664:Interleukin-12 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000276:immunology; D051379:Mice; D009000:Monocytes / Q000276:immunology; D009132:Muscles / Q000276:immunology*; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D019076:Transgenes / Q000502:physiology*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: C408982:CPG-oligonucleotide; D004731:Endotoxins; D007328:Insulin; D008070:Lipopolysaccharides; D009838:Oligodeoxyribonucleotides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D018664:Interleukin-12; D001616:beta-Galactosidase