SP-1017

Poor nuclear entry, especially into nondividing cells, is a limiting factor in nonviral gene delivery. We have engineered a novel chimeric vector relying on the controlled assembly of a TAT-tagged multisubunit DNA binding protein (EcoR124I) with expression plasmids containing the EcoR124I recognition site. Molecular interactions of this molecular assembly were studied by electrophoretic mobility shift assay and atomic force microscopy. Maintenance of nanocomplexes in an appropriate stoichiometric ratio was both necessary and sufficient to produce a significant (>8-fold) increase in the activity of the therapeutic alpha-galactosidase A enzyme after intramuscular administration in the mouse model of Fabry disease. To our knowledge, this is the first molecular targeting system significantly enhancing plasmid-based expression in skeletal muscle. Coinjection with pluronic SP1017 produced further enhancement of gene expression, demonstrating cumulative effects of the increased nuclear delivery by TAT chimeras and transcription activation by the pluronic. Cell penetration peptides (CPP), such as TAT, have been shown to improve delivery of macromolecules, when linked directly. However, in our system TAT-enhanced targeting took place even though it was linked to the plasmid DNA molecule indirectly via two noncovalent bonds. Therefore, this proof-of principle result indicates that TAT (and potentially other CPP) can be used for targeting modular chimeric vectors and therapeutic nanodevices.

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 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.

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.

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. Copyright © 2007 John Wiley & Sons, Ltd.