Yosuke Takahashi

Inhibition of tumor cell growth in the liver by RNA interference-mediated suppression of HIF-1α expression in tumor cells and hepatocytes

Hypoxia-inducible factor-1 (HIF-1) is a ubiquitously expressed oxygen-regulated transcription factor composed of α and β subunits. HIF-1 activates transcription of various genes including those involved in metastatic tumor growth. In the present study, HIF-1α expression in tumor-bearing mouse liver was examined after inoculation of tumor cells into portal vein. We found that tumor-bearing liver showed greatly increased HIF-1α expression. Plasmid DNA (pDNA) expressing short hairpin RNA targeting HIF-1α (pshHIF-1α) was effective in suppressing protein expression of HIF-1α in vitro. Intravenous injection of pshHIF-1α by hydrodynamics-based procedure reduced the HIF-1α protein expression in both normal and tumor cells and tumor cell number in the liver. Pre-injection of pshHIF-1α to mice, by which pDNA was delivered only to liver cells, not to tumor cells, was also effective in reducing the number of tumor cells inoculated 3 days after pDNA injection. These findings indicate that HIF-1α expression is increased in normal liver cells as well as tumor cells, and HIF-1α expression plays an important role in tumor progression. Use of the RNA interference (RNAi) of HIF-1 is an effective strategy for inhibiting tumor cell growth, and both tumor and normal cells can be the target for RNAi-based anticancer treatment.

Development of safe and effective nonviral gene therapy by eliminating CpG motifs from plasmid DNA vector.

Nonviral gene therapy is expected to become a regular treatment for a variety of difficult-to-treat diseases, such as cancer and virus infection. Plasmid DNA, which is used in most nonviral gene delivery systems, usually contains, unmethylated cytosine-guanine dinucleotides, so called CpG motifs. CpG motifs are recognized by immune cells as a danger signal, leading to an inflammatory response. Such inflammatory responses could affect the safety and effectiveness of nonviral gene therapy. Therefore, reducing the number of CpG motifs in plasmid DNA has been used to increase the potency of plasmid DNA-based gene therapy. Previous studies have demonstrated that CpG reduction can extend the time period of transgene expression from plasmid DNA after in vivo gene transfer. In this review, the biological functions of the CpG motif are briefly summarized. Then, safety issues of nonviral gene therapy are discussed from the viewpoint of the inflammatory response to the CpG motif in plasmid DNA, and the effects of the CpG motif in plasmid DNA on the transgene expression profile of nonviral gene transfer are reviewed.

Regulation of immunological balance by sustained interferon-γ gene transfer for acute phase of atopic dermatitis in mice

Interferon (IFN)-γ, a potent T helper 1 (Th1) cell cytokine, is suggested to suppress Th2 cell responses. Here, we aimed to investigate whether pCpG-Muγ, a plasmid continuously expressing murine IFN-γ, is an effective treatment of atopic dermatitis, a Th2-dominant skin disease. Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) atopic mice with early dermatitis were transfected with pCpG-Muγ by a hydrodynamic tail vein injection at a dose of 0.05 or 0.2 pmol per mouse. The skin lesions improved only in mice receiving the high dose of pCpG-Muγ. IFN-γ gene transfer resulted in a high mRNA expression of IFN-γ and interleukin (IL)-12 and regulatory T cell (Treg) related cytokines, such as IL-10 and transforming growth factor-β, in the spleen, whereas it reduced the IL-4 mRNA expression, and serum levels of immunoglobulin (Ig) G1 and IgE. In addition, the gene transfer markedly inhibited the epidermal thickening, infiltration of inflammatory cells into the skin, the occurrence of dry skin and pruritus. No exacerbating effect on the Th1-mediated contact dermatitis was observed after IFN-γ gene transfer. Taken together, these results indicate that sustained IFN-γ gene transfer induced polarized Th1 immunity under Th2-dominant conditions in NC/Nga mice, leading to an improvement in the symptoms of acute atopic dermatitis without adverse side effects.

Effects of upregulated indoleamine 2, 3-dioxygenase 1 by interferon γ gene transfer on interferon γ-mediated antitumor activity.

Interferon γ (IFN-γ), an anticancer agent, is a strong inducer of indoleamine 2,3-dioxygenase 1 (IDO1), which is a tryptophan-metabolizing enzyme involved in the induction of tumor immune tolerance. In this study, we investigated the IDO1 expression in organs after IFN-γ gene transfer to mice. IFN-γ gene transfer greatly increased the mRNA expression of IDO1 in many tissues with the highest in the liver. This upregulation was associated with reduced L-tryptophan levels and increased L-kynurenine levels in serum, indicating that IFN-γ gene transfer increased the IDO activity. Then, Lewis lung carcinoma (LLC) tumor-bearing wild-type and IDO1-knockout (IDO1 KO) mice were used to investigate the effects of IDO1 on the antitumor activity of IFN-γ. IFN-γ gene transfer significantly retarded the tumor growth in both strains without any significant difference in tumor size between the two groups. By contrast, the IDO1 activity was increased only in the wild-type mice by IFN-γ gene transfer, suggesting that cells other than LLC cells, such as tumor stromal cells, are the major contributors of IDO1 expression in LLC tumor. Taken together, these results imply that IFN-γ gene transfer mediated IDO1 upregulation in cells other than LLC cells has hardly any effect on the antitumor activity of IFN-γ.

Effects of transgene expression level per cell in mice livers on induction of transgene-specific immune responses after hydrodynamic gene transfer

We previously showed that high and sustained transgene expression of antigenic proteins induced transgene-specific immune responses. In the present study, a detailed relationship between the level of transgene expression per cell and immune response after hydrodynamic gene transfer was investigated. Cypridina luciferase (cLuc), a secretory antigenic reporter protein, was selected as a model antigen, and pROSA-cLuc, a plasmid expressing cLuc, was constructed. A fixed dose (30 μg) of pROSA-cLuc was delivered to mice by a single hydrodynamic injection or three injections at 24-h intervals because the number of cells transfected with plasmids is dependent on the number of hydrodynamic injections. Serum cLuc activity, an indicator of the total amount of cLuc transgene expression, was almost equal between these two groups. In contrast, the high-dose single injection induced higher levels of cLuc-specific humoral and cellular immune responses than the three low-dose injections. Moreover, the serum cLuc activity of the high-dose single injection group began to decline ~10 days after injection, whereas the activity remained constant in the three low-dose injection group. These results indicate that it is preferable to reduce the level of transgene expression per cell to avoid induction of the transgene-specific immune response after hydrodynamic gene transfer.

Inhibition of nuclear delivery of plasmid DNA and transcription by interferon γ: hurdles to be overcome for sustained gene therapy

Sustained expression of murine interferon (IFN)-γ (Muγ) was found to be effective in preventing tumor metastasis and atopic dermatitis in mouse models. However, our preliminary experiments suggested that the time-dependent decrease in the Muγ expression was not compensated for by repeated injections of Muγ-expressing plasmid. To identify the mechanism underlying this observation, a reporter plasmid was hydrodynamically injected into mice and the levels of the plasmid, mRNA and reporter protein were measured in mice receiving a pre- or co-administration of Muγ-expressing plasmid. Co-injection of Muγ-expressing plasmid had no significant effects on transgene expression from the reporter plasmid. In contrast, pre-injection of Muγ-expressing plasmid greatly inhibited the expression of the reporter protein. Moreover, pre-injection of Muγ-expressing plasmid also reduced the amount of the reporter plasmid in the nuclear fraction of mouse liver to <10%, and that of reporter mRNA to <1%. The degree of reduction in the expression of reporter protein was comparable with the reduction in mRNA. These results indicate that the difficulty in regaining the expression level of IFN-γ is due to the impaired delivery of plasmid to the nucleus and to the suppression of transcription from the plasmid.

Development of orally-deliverable DNA hydrogel by microemulsification and chitosan coating

Graphical abstract Figure. No caption available. ABSTRACT Self‐gelling DNA hydrogels with cytosine‐phosphate‐guanine (CpG) motifs have been shown to exhibit high potency as vaccine adjuvants. However, their oral use is limited because of their thermodynamic and chemical instability in the gastrointestinal tract. In this study, we aimed to develop DNA hydrogel microspheres (Dgel‐MS) coated with chitosan to improve their stability. Chitosan‐coated Dgel‐MS (Cs‐Dgel‐MS) was prepared by emulsifying Dgel to obtain the D‐gel core, followed by mixing with microemulsions of chitosan for electrostatic coating. Fluorescence imaging of Cs‐Dgel‐MS labeled with fluorescent dyes showed that Dgel‐MS (approximately 30 &mgr;m) was coated with chitosan. The recovery efficiency of Alexa Fluor 488‐DNA was 87.4 ± 7.5%. To load a phosphorothioate CpG oligodeoxynucleotide into Dgel, a modified Dgel (mDgel) was designed and fluorescein isothiocyanate (FITC)‐dextran was loaded into Cs‐mDgel‐MS as a model compound. The recovery efficiency of Alexa Fluor 488‐CpG1668 and FITC‐dextran was 83.3 ± 3.8% and 67.8 ± 4.6%, respectively. The release of Alexa Fluor 488‐CpG1668 from Cs‐mDgel‐MS was slower than that from mDgel under acidic or DNase conditions. Intra‐duodenal administration of FITC‐dextran/Cs‐mDgel‐MS showed prolonged intestinal transition of the encapsulated FITC‐dextran. These results indicate that Cs‐Dgel‐MS can be useful for oral delivery of CpG DNA and other bioactive compounds.

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

Abstract 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.