In vitro and in vivo evaluation of intrinsic immunogenicity of reporter and insulin gene therapy plasmids

J Gene Med. 2007 Aug;9(8):703-14. doi: 10.1002/jgm.1066.

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CD8-Positive T-Lymphocytes / immunology
  • CpG Islands
  • Dendritic Cells / drug effects
  • Dendritic Cells / immunology
  • Endotoxins / metabolism
  • Fluorescent Antibody Technique
  • Genetic Therapy*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Insulin / genetics*
  • Interleukin-12 / metabolism
  • Lipopolysaccharides / pharmacology
  • Macrophages / immunology
  • Mice
  • Monocytes / immunology
  • Muscles / immunology*
  • Oligodeoxyribonucleotides / pharmacology
  • Plasmids / genetics*
  • Rats
  • Rats, Wistar
  • Transgenes / physiology*
  • beta-Galactosidase / genetics
  • beta-Galactosidase / metabolism

Substances

  • CPG-oligonucleotide
  • Endotoxins
  • Insulin
  • Lipopolysaccharides
  • Oligodeoxyribonucleotides
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Interleukin-12
  • beta-Galactosidase