Direct Immunologic activities of CpG DNA and implications for gene therapy

Abstract

Vertebrate immune systems have evolved the ability to detect and be activated by most microbial and viral DNAs by virtue of their content of unmethylated ‘CpG motifs’, which are selectively suppressed in vertebrate DNA. Because their CpGs are also unmethylated, the DNA in gene therapy vectors routinely induces direct immune stimulation through activating this host defense mechanism. Administration of such ‘CpG DNA’ by injection or inhalation triggers rapid activation of B cells, monocytes, macrophages, dendritic cells, and natural killer cells, along with the release of pro‐inflammatory cytokines. These immune stimulatory effects can be prevented by chloroquine and other drugs that interfere with endosomal maturation or by the presence of certain neutralizing DNA sequences, which block the immune stimulatory CpG motifs. Aside from serving as the genetic code, DNA can have direct immune activities. Vertebrate immune systems have evolved a defense mechanism that is able to broadly detect most microbial and viral DNAs because of differences in the frequency and methylation of CpG dinucleotides in particular base contexts. B cells, monocytes, macrophages, and dendritic cells spontaneously take up DNA of any type. If the DNA contains these immune stimulatory ‘CpG‐S motifs’, the cells become activated within minutes and begin producing pro‐inflammatory cytokines such as IL‐6 and IL‐12 and upregulate expression of co‐stimulatory molecules. This results in the activation of both innate and acquired immune responses. The pro‐inflammatory effects of CpG‐S motifs are opposed by CpG dinucleotides in certain distinct base contexts, termed neutralizing or CpG‐N motifs. Increasing the ratio of CpG‐S to CpG‐N motifs enhances the immune stimulatory effects of DNA, even if the total level of CpGs in the DNA is not altered. While this is useful in generating enhanced genetic vaccines, the opposite strategy is likely to become useful for the generation of gene therapy vectors with reduced inflammatory effects. Copyright © 1999 John Wiley & Sons, Ltd.

Poly-L-glutamate, an anionic polymer, enhances transgene expression for plasmids delivered by intramuscular injection with in vivo electroporation

“…30,32 The presence of DNA endonucleases in muscle degraded 99% of unformulated plasmid within 90 min of delivery. 25 During plasmid delivery via EP, poly-L-glutamate may enhance plasmid uptake by neutralizing processes that normally deactivates exogenous DNA, such as degradation by nucleases or the binding to cationic entities in interstitial fluid. It is interesting to note that plasmid formulated with poly-L-glutamate showed similar levels of transgene expression as plasmid in saline when EP was omitted after i.m.…”

“…25,26 Therefore, the optimal delivery system to introduce plasmid into skeletal muscle would ideally allow substantial levels of transgene expression with a minimal amount of DNA administered. In our search for such systems, the effect of excess plasmid mass 27 was assessed to determine if the presence of a 'silent' DNA could increase transgene expression.…”

Transient Depletion of Kupffer Cells Leads to Enhanced Transgene Expression in Rat Liver Following Retrograde Intrabiliary Infusion of Plasmid DNA and DNA Nanoparticles

“…On the other hand, several studies have revealed that lipoplexes (Dow et al, 1999;Yew et al, 1999;Kako et al, 2008) encapsulating bacterially derived plasmid DNA can induce strong activation of macrophages and subsequent production of high levels of proinflammatory cytokines after intravenous administration. The activation of macrophages is mediated by the strong immunostimulatory effect of unmethylated CpG sequences present at high frequency in bacterially derived plasmid DNA (Krieg, 1999(Krieg, , 2000. To reduce the inflammation response induced by plasmid DNA, depletion of macrophages before systematic administration has been proven to be an effective approach to enhance gene transfection efficiency of lipoplexes (Sakurai et al, 2002).…”

Gene therapy as an alternative to liver transplantation

“…This would, of course, reduce the risk of adverse immunologic reactions from both the carrier system, as well as the plasmid itself, especially by immunostimulatory CpG motifs. 45 As with all gene therapy strategies, the SB system is associated with some potential problems. In particular, the observed random genomic integration could result in insertional mutagenesis as well as inactivation of an essential gene(s).…”

“…73,74 Moreover, methylated CpG motifs within the DNA molecules also may increase the likelihood of immune response and subsequent removal of complexes. 45 Interestingly, it has been proposed recently that the liver parenchymal cells internalize naked DNA by receptor-mediated endocytosis, 75 although specific receptor(s) have not been identified. Direct injection of naked plasmid DNA into the liver of cats and rats produced expression of different reporter genes, as well as human ␣1-antitrypsin.…”