Transgene expression in mononuclear muscle cells not infiltrating inflammatory cells following intramuscular plasmid gene electrotransfer.

BACKGROUND: In situ electroporation-assisted intramuscular plasmid DNA delivery offers high efficiency for therapeutic protein replacement. Expression may be impaired by an immune response against the plasmid or transgenic protein. Expression of the transgene in non-muscle cells may increase the immune response. Gene transfer efficiency and phenotypic identification of intramuscular transgene-expressing mononuclear cells was studied following electroporation-mediated plasmid delivery. METHODS: Plasmids expressing beta-galactosidase (pVR1012-betagal) or enhanced green fluorescent protein (eGFP) (pVR1012-eGFP) were electrotransferred into rat tibialis anterior muscles. Both transfection efficiency and the inflammatory response were determined in pVR1012-betagal-injected muscles by beta-galactosidase and haematoxylin and eosin staining of muscles 7 days post-plasmid injection. Muscles injected with pVR1012-eGFP were stained for CD3, CD68 and desmin at 24 and 48 h post-injection to determine whether mononuclear cells expressing eGFP were of immune or myogenic origin. RESULTS: With electroporation, beta-galactosidase expression was significantly enhanced by up to ten-fold compared to plasmid injection without electroporation. A large area of regenerating muscle fibres and inflammatory cell infiltration was found in electroporated plasmid-injected muscle. No eGFP expression was found in CD3- or CD68-positive cells. Small mononuclear cells expressing eGFP showed negative staining for CD3 and CD68, but all stained positive for desmin. CONCLUSIONS: In situ electroporation enhanced transfection efficiency of plasmid DNA delivery into muscle. Alongside its advantage for improving gene transfer, electroporation led to an increased inflammatory response and muscle damage. Mononuclear cells in muscle were transfected with plasmid and expressed the transgene. These cells were of myogenic origin with no evidence of transgene expression in infiltrating inflammatory cells.

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

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.