Tuning CpG motif position in nanostructured DNA for efficient immune stimulation.

It was previously demonstrated that polypod-like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine-phosphate-guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single-stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif-containing nanostructured DNAs. In the present study, four series of polypodna were designed, each comprising a CpG ODN with one potent CpG motif at varying positions and 2-5 CpG-free ODNs, and investigated their immunostimulatory activity using Toll-like receptor-9 (TLR9)-positive murine macrophage-like RAW264.7 cells. Polypodnas with the CpG motif in the 5'-overhang induced more tumor necrosis factor-α release than those with the motif in the double-stranded region, even though their cellular uptake were similar. Importantly, the rank order of the immunostimulatory activity of single-stranded CpG ODNs changed after their incorporation into polypodna. These results indicate that the CpG ODN sequence as well as the motif location in nanostructured DNAs should be considered for designing the CpG motif-containing nanostructured DNAs for immune stimulation.

Development of rice bran-derived nanoparticles with excellent anti-cancer activity and their application for peritoneal dissemination.

BACKGROUND: Rice bran a by-product of the rice milling process is currently underutilized. Recent studies have shown that plant-derived nanoparticles (pdNPs) can be mass-produced at a low cost and exhibit biological and therapeutic activities. Rice bran contains various anti-cancer compounds, including γ-oryzanol and γ-tocotrienol, and rice bran-derived nanoparticles (rbNPs) can be employed as novel therapeutic agents for cancer treatment. RESULTS: Koshihikari rice bran was suspended in water, and the suspension was centrifuged and filtered through a 0.45-µm-pore size syringe filter. The filtrate was ultracentrifuged, and the precipitates were suspended to obtain rbNPs. The rbNPs were negatively charged exosome-like nanoparticles with an average diameter of approximately 130 nm. The rbNPs exhibited cytotoxic activities against cancer cells but not against normal cells. The cytotoxic activity of rbNPs to murine colon adenocarcinoma colon26 cells was significantly greater than DOXIL® or other pdNPs. The rbNPs induced cell cycle arrest and apoptosis, and reduced the expression of proliferative proteins, including ß-catenin and cyclin D1. Intraperitoneal injections of rbNPs into mice bearing peritoneal dissemination of colon26 cells significantly suppressed tumor growth with no significant adverse effects. CONCLUSION: These results indicated that rbNPs are promising nanoparticles, hold significant potential for anti-cancer applications, and are expected to play a vital role in cancer treatment.

Efficient delivery of mesenchymal stem/stromal cells to injured liver by surface PEGylation.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) have been used in clinical trials for various diseases. These have certain notable functions such as homing to inflammation sites, tissue repair, and immune regulation. In many pre-clinical studies, MSCs administered into peripheral veins demonstrated effective therapeutic outcomes. However, most of the intravenously administered MSCs were entrapped in the lung, and homing to target sites was less than 1%. This occurred mainly because of the adhesion of MSCs to vascular endothelial cells in the lung. To prevent this adhesion, we modified the surface of MSCs with polyethylene glycol (PEG; a biocompatible polymer) using the avidin-biotin complex (ABC) method. METHODS: The surface of MSCs was modified with PEG using the ABC method. Then, the cell adhesion to mouse aortic endothelial cells and the tissue distribution of PEG-modified MSCs were evaluated. Moreover, the homing to the injured liver and therapeutic effect of PEG-modified MSCs were evaluated using carbon tetrachloride-induced acute liver failure model mice. RESULTS: The PEG modification significantly suppressed the adhesion of MSCs to cultured mouse aortic endothelial cells as well as the entrapment of MSCs in the lungs after intravenous injection in mice. PEG-modified MSCs efficiently homed to the injured liver of carbon tetrachloride-induced acute liver failure model mice. More importantly, the cells significantly suppressed serum transaminase levels and leukocyte infiltration into the injured liver. CONCLUSION: These results indicate that PEG modification to the surface of MSCs can suppress the lung entrapment of intravenously administered MSCs and improve their homing to the injured liver.

Intradermal delivery of Cryj1 loaded in CpG DNA hydrogel for inhibiting allergic reactions in mice.

Immunotherapy for allergic rhinitis alleviates symptoms associated with antigen exposure by administering pathogenic antigens. However, many current immunotherapies fail to induce sufficient immune responses, resulting in frequent and prolonged hospital visits. Consequently, the development of more effective immunotherapies is necessary. In this study, we focused on the skin, which is rich in immune cells, as an administration site for inducing antigen-specific immune responses. To efficiently and sustainably deliver the cedar pollen antigen Cryj1 to immune cells, we attempted to load Cryj1 in an immunostimulatory CpG DNA hydrogel, prepared using self-gelatinizable nucleic acid technology. In this technology, the hydrogel became gelatinized by self-assembly of multiple predesigned DNA units containing potent CpG motifs. Cryj1 loaded in the CpG DNA hydrogel showed sustained release, was taken up by mouse macrophage-like RAW264.7 and mouse dendritic DC2.4 cells, and induced efficient production of interleukin-12 after intradermal injection into mice. Intradermal injection of Cryj1 loaded CpG DNA hydrogel into mice increased the production of Cryj1-specific IgG while suppressing the production of immunoglobulin E (IgE) antibodies. Furthermore, when Cryj1 was resensitized to mice, a stronger induction of IgG production and suppression of IgE production was observed. These results suggest that intradermal administration of Cryj1 loaded CpG DNA hydrogel is a novel immunotherapy for allergic symptoms caused by cedar pollen and can be used as a replacement for current immunotherapies.

Delivery of Corn-Derived Nanoparticles with Anticancer Activity to Tumor Tissues by Modification with Polyethylene Glycol for Cancer Therapy.

PURPOSE: We recently reported that intratumoral injection of corn-derived nanoparticles (cNPs) affords anticancer activity in tumor-bearing mice. To increase their applicability in cancer therapy, we examined the tissue distribution of cNPs after intravenous injection in mice, modified their surface with polyethylene glycol (PEG) to improve tumor delivery, and examined tissue distribution and anticancer activity of PEG-cNPs in tumor-bearing mice. METHODS: N-(Carbonyl-methoxypolyethyleneglycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG) was added to cNPs by sonication to obtain PEG-cNPs, and the ratio of DSPE-PEG to cNPs was optimized by evaluating the modification efficiency. cNPs and PEG-cNPs were labeled with fluorescent dyes DiO or DiR, and their tissue distribution was subsequently examined after intravenous administration to mice. Finally, we determined the anticancer activity and toxicity of PEG-cNPs. RESULTS: No detectable fluorescence intensity was observed in mouse serum after intravenous DiR-cNP injection. DSPE-PEG was successfully modified into cNPs, and a PEG:cNPs ratio of 50 was determined as optimal for preparing PEG-cNPs, based on their size and zeta potential. DiO-PEG-cNPs exhibited significantly higher serum concentrations and lower liver accumulation than DiO-cNPs. Moreover, DiR-PEG-cNPs accumulated in tumor tissues of colon26 tumor-bearing mice. Repeated intravenous PEG-cNP injections significantly retarded tumor growth, with no significant hepatotoxicity or nephrotoxicity. CONCLUSION: Overall, these results indicate that controlling the tissue distribution of cNPs via PEG modification on their surface can be a valuable strategy for developing intravenously injectable cNPs for cancer therapy.

Quality evaluation of cell spheroids for transplantation by monitoring oxygen consumption using an on-chip electrochemical device.

Three-dimensional cell spheroids are superior cell-administration form for cell-based therapy which generally exhibit superior functionality and long-term survival after transplantation. Here, we nondestructively measured the oxygen consumption rate of cell spheroids using an on-chip electrochemical device (OECD) and examined whether this rate can be used as a marker to estimate the quality of cell spheroids. Cell spheroids containing NanoLuc luciferase-expressing mouse mesenchymal stem cell line C3H10T1/2 (C3H10T1/2/Nluc) were prepared. Spheroids of high or low quality were prepared by altering the medium change frequency. After transplantation into mice, the high-quality C3H10T1/2/Nluc spheroids exhibited a higher survival rate than the low-quality ones. The oxygen consumption rate of the high-quality C3H10T1/2/Nluc spheroids was maintained at high levels, whereas that of the low-quality spheroids decreased with time. These results indicate that OECD-based measurement of the oxygen consumption rate can be used to estimate the quality of cell spheroids without destructive analysis of the spheroids.

Targeted Delivery of Immunostimulatory CpG Oligodeoxynucleotides to Antigen-Presenting Cells in Draining Lymph Nodes by Stearic Acid Modification and Nanostructurization.

Polypod-like structured nucleic acids (polypodnas), which are nanostructured DNAs, are useful for delivering cytosine-phosphate guanine oligodeoxynucleotides (CpG ODNs) to antigen-presenting cells (APCs) expressing Toll-like receptor 9 (TLR9) for immune stimulation. Lipid modification is another approach to deliver ODNs to lymph nodes, where TLR9-positive APCs are abundant, by binding to serum albumin. The combination of these two methods can be useful for delivering CpG ODNs to lymph nodes in vivo. In the present study, CpG1668, a phosphodiester-type CpG ODN, was modified with stearic acid (SA) to obtain SA-CpG1668. Tripodna, a polypodna with three pods, was selected as the nanostructured DNA. Tripodnas loaded with CpG1668 or SA-CpG1668 were obtained in high yields. SA-CpG1668/tripodna bound more efficiently to plasma proteins than CpG1668/tripodna and was more efficiently taken up by macrophage-like RAW264.7 cells than CpG1668/tripodna, whereas the levels of tumor necrosis factor-α released from the cells were comparable between the two. After subcutaneous injection into mice, SA-CpG1668/tripodna induced significantly higher interleukin (IL)-12 p40 production in the draining lymph nodes than SA-CpG1668 or CpG1668/tripodna, with reduced IL-6 levels in plasma. These results indicate that the combination of SA modification and nanostructurization is a useful approach for the targeted delivery of CpG ODNs to lymph nodes.

Intravenous injection of mesenchymal stem cell spheroids improves the pulmonary delivery and prolongs in vivo survival.

BACKGROUND: Because of the excellent therapeutic potential, mesenchymal stem cells (MSCs) have been used as cell therapeutics for various diseases. However, the survival rate and duration of MSCs after transplantation are extremely low and short, respectively. To solve these problems, in this study, we prepared multicellular spheroids of MSCs and investigated their survival and function after intravenous injection in mice. METHODS AND RESULTS: The murine adipose-derived MSC line m17.ASC was cultured in agarose-based microwell plates to obtain size-controlled m17.ASC spheroids of an average diameter and cell number of approximately 170 µm and 1100 cells/spheroid, respectively. The intravenously injected m17.ASC spheroids mainly accumulated in the lung and showed a higher survival rate than suspended m17.ASC cells during the experimental period of 7 days. m17.ASC spheroids efficiently reduced the lipopolysaccharide-induced increase in plasma concentrations of interleukin-6 and tumor necrosis factor-α. CONCLUSIONS: These results indicate that spheroid formation improved the pulmonary delivery and survival of MSCs, as well as their therapeutic potential against inflammatory pulmonary diseases.

Development of nanoparticles derived from corn as mass producible bionanoparticles with anticancer activity.

Recent studies showed that plant-derived nanoparticles (NPs) can be easily produced in high yields and have potential applications as therapeutic agents or delivery carriers for bioactive molecules. In this study, we selected corn as it is inexpensive to grow and mass-produced globally. Super sweet corn was homogenized in water to obtain corn juice, which was then centrifuged, filtered through a 0.45-µm-pore size syringe filter, and ultracentrifuged to obtain NPs derived from corn, or corn-derived NPs (cNPs). cNPs obtained were approximately 80 nm in diameter and negatively charged (- 17 mV). cNPs were taken up by various types of cells, including colon26 tumor cells and RAW264.7 macrophage-like cells, with selective reduction of the proliferation of colon26 cells. Moreover, cNPs induced tumor necrosis factor-α release from RAW264.7 cells. cNPs and RAW264.7 in combination significantly suppressed the proliferation of colon26/fluc cells. Daily intratumoral injections of cNPs significantly suppressed the growth of subcutaneous colon26 tumors in mice, with no significant body weight loss. These results indicate excellent anti-tumor activity of cNPs.

Development of Advanced Cell-Based Therapy by Regulating Cell-Cell Interactions.

Cell-based therapy for disease treatment involves the transplantation of cells obtained either from self or others into relevant patients. While cells constituting the body tissues maintain homeostasis by performing remarkable functions through complicated cell-cell interactions, transplanted cells, which are generally cultured as a monolayer, are unable to recapitulate similar interactions in vivo. The regulation of cell-cell interactions can immensely increase the function and therapeutic effect of transplanted cells. This review aims to summarize the methods of regulating cell-cell interactions that could significantly increase the therapeutic effects of transplanted cells. The first method involves the generation of multicellular spheroids by three-dimensional cell culture. Spheroid formation greatly improved the survival and therapeutic effects of insulin-secreting cells in diabetic mice after transplantation. Moreover, mixed multicellular spheroids, composed of insulin-secreting cells and aorta endothelial cells or fibroblasts, were found to significantly improve insulin secretion. Secondly, adhesamine derivatives, which are low-molecular-weight compounds that accelerate cell adhesion and avoid anoikis and anchorage-dependent apoptosis, have been used to improve the survival of bone marrow-derived cells and significantly enhanced the therapeutic effects in a diabetic mouse model of delayed wound healing. Finally, the avidin-biotin complex method, a cell surface modification method, has been applied to endow tumor-homing mesenchymal stem cells with anti-tumor ability by modifying them with doxorubicin-encapsulated liposomes. The modified cells showed excellent effectiveness in cell-based cancer-targeting therapy. The discussed methods can be useful tools for advanced cell-based therapy, promising future clinical applications.

Mesenchymal stem/stromal cells as next-generation drug delivery vehicles for cancer therapeutics.

INTRODUCTION: Drug delivery to solid tumors remains a significant therapeutic challenge. Mesenchymal stem/stromal cells (MSCs) home to tumor tissues and can be employed as tumor targeted drug/gene delivery vehicles. Reportedly, therapeutic gene- or anti-cancer drug-loaded MSCs have shown remarkable anti-tumor effects in preclinical studies, and some clinical trials for assessing therapeutic MSCs in patients with cancer have been registered. AREAS COVERED: In the present review, we first discuss the source and interdonor heterogeneity of MSCs, their tumor-homing mechanism, and the route of MSC administration in MSC-based cancer therapy. We then summarize the therapeutic applications of MSCs as a drug delivery vehicle for therapeutic genes or anti-cancer drugs and the drug delivery mechanism from drug-loaded MSCs to cancer cells. EXPERT OPINION: Although numerous preclinical studies have revealed significant anti-tumor effects, several clinical trials assessing MSC-based cancer gene therapy have failed to demonstrate corroborative results, documenting limited therapeutic effects. Notably, a successful clinical outcome with MSC-based cancer therapy would require the interdonor heterogeneity of administered MSCs to be resolved, along with improved tumor-homing efficiency and optimized drug delivery efficiency from MSCs to cancer cells.

Critical contribution of macrophage scavenger receptor 1 to the uptake of nanostructured DNA by immune cells.

Despite the efficient uptake of polypod-like nanostructured DNA, or polypodna, by macrophage-like RAW264.7 and other immune cells, the detailed mechanism has not been fully elucidated. Our previous study using HEK-Blue hTLR9 cells showed that transfection of macrophage scavenger receptor 1 (MSR1) increased the uptake of tetrapod-like structured DNA. Here, we investigated the involvement of MSR1 in the structure-dependent uptake of polypodna. Transfection of MSR1 to HEK-Blue hTLR9 cells pod number-dependently increased the uptake of polypodna, and its knockout in RAW264.7 cells reduced the uptake and subsequent cytokine release. To examine the binding of DNA with MSR1, biotinylated DNA added to RAW264.7 cells was cross-linked with cell surface proteins. Then, MSR1 cross-linked with polypodna, but not with single-stranded DNA. Similar results were obtained with murine primary immune cells. Taken together, MSR1 discriminates between simple and nanostructured DNAs and plays a dominant role in the efficient uptake of polypodna by immune cells.

Anticancer drug-loaded mesenchymal stem cells for targeted cancer therapy.

Mesenchymal stem cells (MSCs) have a tumor-homing ability-they accumulate inside tumors after systemic injection, and may thus be useful as carriers for tumor-targeting therapy. To use MSCs effectively as an anti-cancer therapy, they must first be functionalized with a large amount of anti-cancer drugs without causing any significant changes to their tumor-tropism. In the present study, we attempted to modify the cell surface of MSCs with doxorubicin-loaded liposomes (DOX-Lips), using the avidin-biotin complex method, and evaluated delivery efficiency and anti-tumor efficacy of DOX-Lip-modified MSCs. The amount of DOX in DOX-Lip-modified C3H10T1/2 cells, a murine mesenchymal stem cell line, was approximately 21.5 pg per cell, with no significant changes to the tumor-tropism of C3H10T1/2 cells. Notably, DOX-Lip-modified C3H10T1/2 cells significantly suppressed the proliferation of firefly luciferase-expressing murine colon adenocarcinoma colon26/fluc cells, compared to DOX-Lips alone. Fluorescent DOX accumulated at the cell contact surface and inside green fluorescence protein-expressing colon26 (colon26/GFP) in co-cultures of DOX-Lip-modified C3H10T1/2 and colon26/GFP cells. This localized distribution was not observed when only DOX-Lips was added to colon26/GFP cells. These results suggest that DOX-Lips are efficiently delivered from DOX-Lip-modified C3H10T1/2 cells to the neighboring colon26 cells. Furthermore, DOX-Lip-modified C3H10T1/2 cells suppressed tumor growth in subcutaneous tumor-bearing mice, and in a lung metastasis mouse model. Taken together, these results indicate that the intercellular delivery of DOX may be enhanced using DOX-Lip-modified MSCs as an efficient carrier system for targeted tumor therapy.

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 Immunostimulatory Activity of CpG Oligodeoxynucleotide by the Combination of Mannose Modification and Incorporation into Nanostructured DNA.

The immunostimulatory activity of unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) could be improved via delivery to immune cells expressing Toll-like receptor 9 (TLR9). Previously, we showed that the polypod-like structured nucleic acid (polypodna), a nanostructured DNA comprised of three or more ODNs, was an efficient system for the delivery of CpG ODNs to immune cells. Because some TLR9-positive immune cells express mannose receptors (MR), the uptake of polypodna by immune cells can be further increased by its modification with mannose. In this study, we selected the phosphodiester CpG ODN, ODN1668, which has a sequence identical to CpG1668, and a hexapodna, a polypodna with six pods, to design a hexapodna that harbored ODN1668 or the mannosylated CpG ODN (Man-ODN1668) synthesized via modification of the 5'-terminal of ODN1668 with a synthesized mannose motif. By mixing ODN1668 or Man-ODN1668 with the hexapodna, ODN1668/hexapodna and Man-ODN1668/hexapodna were successfully formed with high yields. However, Man-ODN1668/hexapodna was found to induce a greater tumor necrosis factor-α release from TLR9- and MR-positive mouse peritoneal macrophages and macrophage-like J774.1 cells than Man-ODN1668 or ODN1668/hexapodna. These results indicate that the combination of mannose modification and incorporation into nanostructured DNA is a useful approach for enhancing the immunostimulatory activity of CpG ODN.

Construction of Monomeric and Dimeric G-Quadruplex-Structured CpG Oligodeoxynucleotides for Enhanced Uptake and Activation in TLR9-Positive Macrophages.

The G-quadruplex (GQ) structure has potential applications in nucleic acid drug delivery because of its superior stability. In this study, we added one G-tract (five guanines) to an unmethylated phosphodiester-linked cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), a potential immune adjuvant, to construct a GQ-structured CpG ODN with precise structural properties, increased biological stability, and efficient delivery to Toll-like receptor 9 (TLR9)-positive immune cells. A G-tract was added to phosphodiester-backboned CpG1668 at the 5'-end [1668(5'-G5)], 3'-end [1668(3'-G5)], or within the sequence [1668(mid-G5)]. Circular dichroism analysis showed that all CpG ODNs with a G-tract formed parallel GQ structures, irrespective of its position. Electrophoresis showed that 1668(5'-G5) formed a GQ dimer, whereas others remained GQ monomers. GQ-structured CpG ODNs induced greater tumor necrosis factor-α and interleukin-6 secretion from TLR9-positive mouse macrophage-like RAW264.7 cells than single-stranded CpG ODNs, with the highest for 1668(3'-G5). GQ structuration increased CpG ODN uptake by RAW264.7 cells, and 1668(3'-G5) decomposed more slowly in serum than 1668(5'-G5). Thus, GQ formation with one G-tract is a simple and efficient strategy for CpG ODN delivery to TLR9-positive cells, and addition of a G-tract to the 3'-end is effective in obtaining monomeric GQ-structured CpG ODN with high biological stability and immunostimulatory activity.

Chemoproteomic Profiling of a Pharmacophore-Focused Chemical Library.

Pharmacophore-focused chemical libraries are continuously being created in drug discovery programs, yet screening assays to maximize the usage of such libraries are not fully explored. Here, we report a chemical proteomics approach to reutilizing a focused chemical library of 1,800 indole-containing molecules for discovering uncharacterized ligand-protein pairs. Gel-based protein profiling of the library using a photo-affinity indole probe 1 enabled us to find new ligands for glyoxalase 1 (Glo1), an enzyme involved in the detoxification of methylglyoxal. Structure optimization of the ligands yielded an inhibitor for Glo1 (9). Molecule 9 increased the cellular methylglyoxal levels in human cells and suppressed the osteoclast formation of mouse bone marrow-derived macrophages. X-ray structure analyses revealed that the molecule lies at a site abutting the substrate binding site, which is consistent with the enzyme kinetic profile of 9. Overall, this study exemplifies how chemical proteomics can be used to exploit existing focused chemical libraries.

Intracellular Delivery of Antisense DNA and siRNA with Amino Groups Masked with Disulfide Units.

Efficient methods for delivery of antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (i.e., disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.

Analysis of Tertiary Structural Features of Branched DNA Nanostructures with Partially Common Sequences Using Small-Angle X-ray Scattering.

We demonstrated that polypod-like structured DNA, composed of 3-8 kinds of oligodeoxynucleotides (ODNs), exhibit accelerated cellular uptake depending on their structural properties. The biological activities, including immunostimulation and unwanted adverse effects of ODNs depend on their sequences. Therefore, as the number of different types of ODNs within a polypod-like structured DNA increases, the possible risks and concerns regarding its future clinical applications also increase. To minimize this risk and to explore the relationship between structural properties and cellular uptake, we designed Tet(id12), a tetrapod-like structured DNA with 12 identical palindrome sequences in the center of ODNs, and compared it with other tetrapod-like structures without palindrome sequences, symmetric Tet(sym) and asymmetric Tet(asym12). The thermal stability analysis of Tet(id12) revealed a two-step dissociation process. Polyacrylamide gel electrophoresis, small-angle X-ray scattering profile, and the Guinier plot also revealed that the conformation of Tet(id12) was close to that of Tet(sym). No significant differences were observed between Tet(id12) and others in terms of cellular uptake. These results indicate that ODNs with palindrome sequences can be used to design polypod-like structured DNAs without significant changes to their physicochemical and biological properties, although their thermal stability is somewhat different from the ones without palindrome sequences.

Cell-based interferon gene therapy using proliferation-controllable, interferon-releasing mesenchymal stem cells.

An important safety concern on cell-based gene therapy is that few methods have been available to control the proliferation and functioning of therapeutic protein-expressing cells after transplantation. We previously reported that the proliferation and functioning of the cells transfected with herpes simplex virus thymidine kinase (HSVtk) gene, a suicide gene, can be controlled by administration of ganciclovir. In this study, we tried to control the amount of murine interferon-γ (IFN-γ) secreted from transplanted murine mesenchymal stem cell line C3H10T1/2 cells to achieve safe cell-based IFN-γ gene therapy for cancer. C3H10T1/2 cells were transfected with HSVtk- and murine IFN-γ-expressing plasmid vectors to obtain C3H10T1/2/HSVtk/IFN-γ cells. C3H10T1/2/HSVtk/IFN-γ cells released IFN-γ and were sensitive to ganciclovir. C3H10T1/2/HSVtk/IFN-γ cells significantly suppressed the proliferation of murine adenocarcinoma cell line colon26 cells both in vitro and in vivo. Moreover, subcutaneous administration of ganciclovir to mice transplanted with NanoLuc luciferase-expressing C3H10T1/2/HSVtk cells for three consecutive days reduced the luminescence signals from the transplanted cells. These results indicate that the cell regulation system using HSVtk gene and ganciclovir can be useful for safe and efficient cell-based IFN-γ gene therapy for cancer.