Combined use of chemically modified nucleobases and nanostructured DNA for enhanced immunostimulatory activity of CpG oligodeoxynucleotide.

Oligodeoxynucleotide (ODN) containing a cytosine-phosphate-guanine (CpG) motif, or CpG ODN, is considered suitable for treating immune diseases, including allergies. Although the phosphorothioate modification is used to enhance the stability and immunostimulatory activity of CpG ODNs, it is associated with the risk of adverse effects. Construction of nanostructured DNA assemblies, such as tripod- and hexapod-like structured DNAs, tripodna and hexapodna, respectively, were also found to increase this activity. The chemical modification of nucleobases could be another approach for enhancing CpG ODN activity. Here, we examined whether chemically modified nucleobase substitutions can enhance CpG ODN activity by measuring tumor necrosis factor α (TNF-α) release after addition to murine macrophage-like RAW264.7 cells. First, the guanine at the 18th position of phosphodiester CpG 1668 was substituted with several chemically modified guanines, and then the various guanines were substituted. Among all tested substitutions, 15,18-thdG, in which two guanines outside the CpG motif were substituted with the 2-aminothieno[3,4-d]pyrimidine guanine mimic (thdG), was the most effective. Compared to 32P-CpG 1668, 32P-15,18-thdG was taken up more efficiently by the RAW264.7 cells. Then, 15,18-thdG was incorporated into tripodna and hexapodna. 15,18-thdG/tri- or hexapodna induced higher TNF-α release from the RAW264.7 cells than PO CpG 1668/tri- or hexapodna, respectively. These results indicate that the thdG substitution is a useful effective strategy for enhancing the immunostimulatory activity of CpG DNAs in both single stranded and DNA nanostructure forms.

Enhanced Activity of Immunosuppressive Oligodeoxynucleotides by Incorporating Them into Hexapod-Like Nanostructured DNA.

A151 and other immunosuppressive oligodeoxynucleotides that act as Toll-like receptor (TLR) 9 antagonists are candidate agents for the treatment of autoimmune and inflammatory diseases in which TLR9 activation leads to harmful immune responses. Their efficient delivery to TLR9-positive target cells will increase their potency, but few attempts have been made to enhance their delivery. We previously reported that hexapod-like nanostructured DNA (hexapodna) enhanced the activity of immunostimulatory cytosine-phosphate-guanine (CpG) DNA by efficiently delivering it to immune cells. In this study, to enhance the immunosuppressive activity of A151, we designed a hexapodna containing six copies of the complementary sequence to A151. Structural analyses showed that A151-loaded hexapodna (supHexapodna) was obtained as designed. CpG 1668, which is a typical synthetic CpG DNA, induced tumour necrosis factor-α release from mouse macrophage-like RAW264.7 cells, and supHexapodna inhibited this more efficiently than A151. A flow cytometric analysis showed that the uptake of Alexa Fluor 488-labelled A151 by RAW264.7 cells significantly increased when it was incorporated into supHexapodna, whereas the uptake of Alexa Fluor 488-labelled CpG 1668 was hardly affected by A151 or supHexapodna. These results suggest that the hexapodna-mediated delivery of A151 can increase the potency of its TLR9-inhibitory activity towards immune cells.

Construction of nanostructured DNA harbouring phosphorodiamidate morpholino oligonucleotide for controlled tissue distribution in mice.

Phosphorodiamidate morpholino oligonucleotides (PMOs) are a class of antisense oligonucleotides used in the treatment of neuromuscular diseases. Their major drawbacks are high blood clearance and poor cellular delivery. Previously, we demonstrated that tripod-like nanostructured DNA, or tripodna, was efficiently taken up by macrophages and dendritic cells. In this study, we used iodine-125(125I)-labelled PMOs, designed a tripodna harbouring an 125I-PMO (125I-PMO/tripodna), and evaluated whether this tripodna could control the pharmacokinetic properties of PMO. Gel electrophoresis showed that 125I-PMO was almost completely incorporated into the tripodna. Compared to 125I-PMO, 125I-PMO/tripodna was more efficiently taken up by macrophage-like RAW264.7 cells. Moreover, after intravenous injection into mice, the area under the plasma concentration-time curve of 125I-PMO/tripodna was significantly larger than that of 125I-PMO. The distribution of 125I-PMO/tripodna in the liver and spleen at 24 h was 32- and 51-fold higher than that of 125I-PMO, respectively. The fractionation of liver cells revealed that non-parenchymal cells were the major cells contributing to the hepatic uptake of 125I-PMO/tripodna. These results indicate that tripodna has the potential to deliver PMO, particularly to the liver and spleen.

In Vitro and In Vivo Stimulation of Toll-Like Receptor 9 by CpG Oligodeoxynucleotides Incorporated Into Polypod-Like DNA Nanostructures.

Cytosine-phosphate-guanine (CpG) DNA is known to increase the potency of vaccines. Here, in vitro and in vivo stimulation of toll-like receptor 9 by CpG DNA incorporated into polypod-like DNA nanostructures was evaluated by measuring the levels of tumor necrosis factor alpha released from macrophage-like RAW 264.7 cells and plasma interleukin (IL)-12p40 in vivo following intravenous injection into mice. Phosphodiester CpG1668 was selected as the CpG DNA, and tripodna and hexapodna, which were CpG1668-containing tripod and hexapod-like DNA nanostructures, respectively, were designed. CpG-tripodna and CpG-hexapodna induced tumor necrosis factor alpha release from RAW 264.7 cells about 10- and ∼30-fold higher than single-stranded CpG1668 (CpG-SS). Moreover, in all cases examined, plasma IL-12p40 concentrations increased after intravenous injection into mice, with peak levels depending on the samples and the doses. The area under the plasma concentration-time curves indicated that the CpG-hexapodna was approximately 20-fold more efficient in inducing IL-12p40 production than CpG-SS. The efficiency of CpG-tripodna and CpG-hexapodna to increase the potency of CpG-SS in vivo was comparable to that observed in cultured RAW 264.7 cells. These results provide experimental evidence that in vitro studies can be used to estimate the in vivo immunostimulatory activity of CpG DNA incorporated into DNA nanostructures.

Self-assembling DNA hydrogel-based delivery of immunoinhibitory nucleic acids to immune cells.

Immunoinhibitory oligodeoxynucleotides (INH-ODNs) are promising inhibitors of Toll-like receptor 9 (TLR9) activation. To efficiently deliver INH-ODNs to TLR9-positive cells, we designed a Takumi-shaped DNA (Takumi) consisting of two partially complementary ODNs as the main component of a DNA hydrogel. Polyacrylamide gel electrophoresis showed that Takumi-containing INH-ODNs (iTakumi) and iTakumi-based DNA hydrogel (iTakumiGel) were successfully generated. Their activity was examined in murine macrophage-like RAW264.7 cells and DC2.4 dendritic cells by measuring tumor necrosis factor-α and interleukin-6 release after the addition of a TLR9 ligand (CpG ODN). Cytokine release was efficiently inhibited by the iTakumiGel. Flow cytometry analysis and confocal microscopy showed that cellular uptake of INH-ODN was greatly increased by the iTakumiGel. These results indicate that a Takumi-based DNA hydrogel is useful for the delivery of INH-ODNs to immune cells to inhibit TLR9-mediated hyperinduction of proinflammatory cytokines. From the Clinical Editor: Toll-like receptor 9 activation has been reported to be associated with many autoimmune diseases. DNA inhibition using oligodeoxynucleotides is one of the potential treatments. In this article, the authors described hydrogel-based platform for the delivery of the inhibitory oligodeoxynucleotides for enhanced efficacy. The positive findings could indicate a way for the future.