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DNA dendrimers consisting of several branched DNA units connected to each other using DNA ligase were quite effective for the delivery of immunostimulatory CpG DNA to immune cells. Therapeutic application of such DNA dendrimers, however, is hampered by the use of the ligase. Here, we report that self-assembling DNA dendrimers with high immunostimulatory potency can be prepared without DNA ligases. Annealing of DNA consisting of DNA units with elongated adhesive ends resulted in the formation of DNA dendrimers. Atomic force microscopy revealed that the several preparations of DNA dendrimers resulted in dendritic structures as designed. The cellular uptake of DNA dendrimers by mouse macrophage-like RAW264.7 cells and subsequent release of tumor necrosis factor-α were dependent on the structural complexity of the dendrimers. These results indicate that the ligation-free, self-assembling DNA dendrimers are a potent system for the delivery of immunostimulatory CpG DNA to immune cells.

Investigation of mouse macrophage-like RAW264.7 cells showed that the immunostimulatory activity of CpG DNA is increased by formation of polypod-like structured DNA (polypodna), an assembly consisting of three or more oligodeoxynucleotides. To apply CpG polypodna to immunotherapy, its activity was examined in murine dendritic DC2.4 cells, splenic macrophages, and bone marrow-derived dendritic cells (BMDCs). In all cell types, increasing the pod number increased the cellular uptake of DNA and cytokine release. No significant release of cytokines was observed in macrophages lacking Toll-like receptor 9. Similar results were obtained after intradermal injection of polypodna. The polypodna preparations produced significantly higher amounts of interferon α in human peripheral blood mononuclear cells (PBMCs) compared with single-stranded DNA. The conditioned medium of hexapodna-treated human PBMCs effectively inhibited the activity of a hepatitis C virus subgenomic replicon reporter system. These results indicate that polypodna preparations are useful as an immunostimulator., From the Clinical Editor: This study demonstrates the utility of polypoid-like structured DNA (polypodna) preparations as potent immunostimulators in a murine model., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Abstract: Investigation of mouse macrophage-like RAW264.7 cells showed that the immunostimulatory activity of CpG DNA is increased by formation of polypod-like structured DNA (polypodna), an assembly consisting of three or more oligodeoxynucleotides. To apply CpG polypodna to immunotherapy, its activity was examined in murine dendritic DC2.4 cells, splenic macrophages, and bone marrow-derived dendritic cells (BMDCs). In all cell types, increasing the pod number increased the cellular uptake of DNA and cytokine release. No significant release of cytokines was observed in macrophages lacking Toll-like receptor 9. Similar results were obtained after intradermal injection of polypodna. The polypodna preparations produced significantly higher amounts of interferon α in human peripheral blood mononuclear cells (PBMCs) compared with single-stranded DNA. The conditioned medium of hexapodna-treated human PBMCs effectively inhibited the activity of a hepatitis C virus subgenomic replicon reporter system. These results indicate that polypodna preparations are useful as an immunostimulator. From the Clinical Editor: This study demonstrates the utility of polypoid-like structured DNA (polypodna) preparations as potent immunostimulators in a murine model. [Copyright &y& Elsevier]

Abstract: Y-shape formation increased the immunostimulatory activity of phosphodiester (PO) oligodeoxynucleotides (ODNs) containing CpG motif. In this study, PO CpG ODN or CpG ODN containing nuclease-resistant phosphorothioate (PS) linkages, i.e., PS CpG ODN or PO CpG ODN with three PS linkages at the both ends (PS3), was mixed with two PO- or PS ODNs to prepare Y-shaped DNA (Y-DNA) containing a potent CpG motif. The melting temperature of Y-DNA decreased with increasing number of PS linkages. Y(PS/PO/PO), which contained PS CpG ODN, showed the greatest activity to induce tumor necrosis factor-α release from macrophage-like RAW264.7 cells, followed by Y(PS3/PO/PO). However, the high activity of Y(PS/PO/PO) was due to that of PS CpG ODN, and Y-shape formation had no significant effect on the activity. Furthermore, PS CpG ODN of Y(PS/PO/PO) was efficiently taken up by cells, but other PO ODNs in the Y-DNA were not, indicating that PS CpG ODN in Y-DNA behave like single stranded PS CpG ODN. In quite contrast, the immunostimulatory activity of PS3 CpG ODN was significantly increased by Y-shape formation. In conclusion, Y-shape formation and PS substitution can be used simultaneously to increase the immunostimulatory activity of CpG ODN, but extensive substitution should be avoided because it diminishes the benefits of Y-shape formation. [ABSTRACT FROM AUTHOR]