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

Lookup NU author(s): Christopher Huggins, Professor John Kirby, Professor James Shaw

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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 lipopolysaccaride; 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 β-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 β-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 β-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 immmune response to plasmid DNA vectors during evaluation for clinical gene theraphy. Copyright © 2007 John Wiley & Sons, Ltd.


Publication metadata

Author(s): Ratanamart J, Huggins CG, Kirby JA, Shaw J

Publication type: Article

Publication status: Published

Journal: Journal of Gene Medicine

Year: 2007

Volume: 9

Issue: 8

Pages: 703-714

ISSN (print): 1099-498X

ISSN (electronic): 1521-2254

Publisher: John Wiley & Sons, Inc.

URL: http://dx.doi.org/10.1002/jgm.1066

DOI: 10.1002/jgm.1066

PubMed id: 17595049


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