CpG-Depleted Plasmid DNA Vectors with Enhanced Safety and Long-Term Gene Expression in Vivo

Abstract

Systemic delivery of cationic lipid-plasmid DNA (pDNA) complexes induces an acute inflammatory response with adverse hematologic changes and liver damage. Immunostimulatory CpG motifs in the pDNA are known to contribute substantially to this response. Here we constructed a pDNA vector (pGZB) that has been depleted of 80% of the CpG motifs present in the original vector. Compared with the unmodified vector, systemic administration of pGZB induced considerably fewer changes in blood parameters, reduced levels of inflammatory cytokines, and decreased liver damage. Despite the extensive sequence modifications within pGZB, there were still robust levels of transgene expression. Furthermore, in contrast to the transient expression observed from the unmodified vector, we observed sustained or increasing expression for up to 49 days from pGZB in the lung and liver of immunocompetent BALB/c mice. Studies adding CpG motifs in trans or in cis indicate that the reduced CpG content of pGZB was the major determinant for its persistent expression. This combination of decreased toxicity and sustained expression suggests that CpG-depleted pDNA vectors can greatly improve the safety and efficacy of synthetic gene delivery systems.

Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector

“…35 In similar reports, 36,37 it was verified that CpG dinucleotides could negatively affect transgene expression from pDNA in vivo, even with linearization of the transgene expression plasmid before hydrodynamic injection. Finally, other groups have reported the negative effect of CpG methylation in vitro, 38 and recently the extensive methylation of CpG islands of a CMV promoter-driven adenoviral vector following delivery into the muscles of mice was shown, with methylation levels similar to our results.…”

Detection of plasmid DNA vectors following gene transfer to the murine airways

“…Reducing the host inflammatory response to non-viral GTAs by minimizing the CpG content of the plasmid DNA results in a pronounced extension of the duration of transgene expression. 44 Therefore, to treat chronic lung diseases such as CF, non-viral gene transfer must be optimized at the level of plasmid and formulation in order to be as invisible as possible to the host innate immune surveillance systems.…”

Intranuclear disposition of exogenous DNA in vivo: Silencing, methylation and fragmentation

“…Hong et al suggested that the CpG dinucleotides may undergo de novo methylation in plasmid DNA in mammalian cells [6]. Moreover, transgene expression was reportedly enhanced when fewer CpG sequences were present in the plasmid DNA [7], implying that CpG methylation suppressed transgene expression.…”