Anti-nucleolin aptamer, iSN04, inhibits the inflammatory responses in C2C12 myoblasts by modulating the ß-catenin/NF-κB signaling pathway.

A myogenetic oligodeoxynucleotide, iSN04, is the 18-base single-stranded DNA that acts as an anti-nucleolin aptamer. iSN04 has been reported to restore myogenic differentiation by suppressing inflammatory responses in myoblasts isolated from patients with diabetes or healthy myoblasts exposed to cancer-releasing factors. Thus, iSN04 is expected to be a nucleic acid drug for the muscle wasting associated with chronic diseases. The present study investigated the anti-inflammatory mechanism of iSN04 in the murine myoblast cell line C2C12. Tumor necrosis factor-α (TNF-α) or Toll-like receptor (TLR) ligands (Pam3CSK4 and FSL-1) induced nuclear translocation and transcriptional activity of nuclear factor-κB (NF-κB), resulting in upregulated expression of TNF-α and interleukin-6. Pre-treatment with iSN04 significantly suppressed these inflammatory responses by inhibiting the nuclear accumulation of ß-catenin induced by TNF-α or TLR ligands. These results demonstrate that antagonizing nucleolin with iSN04 downregulates the inflammatory effect mediated by the ß-catenin/NF-κB signaling pathway in C2C12 cells. In addition, the anti-inflammatory effects of iSN04 were also observed in the rat smooth muscle cell line A10 and the murine adipocyte-like fibroblast cell line 3T3-L1, suggesting that iSN04 may be useful in preventing inflammation induced by metabolic disorders.

Immunotherapy for lung cancer combining the oligodeoxynucleotides of TLR9 agonist and TGF-ß2 inhibitor.

Tumor immunotherapies have shown promising antitumor effects, especially immune checkpoint inhibitors (ICIs). However, only 12.46% of the patients benefit from the ICIs, the rest of them shows limited effects on ICIs or even accelerates the tumor progression due to the lack of the immune cell infiltration and activation in the tumor microenvironment (TME). In this study, we administrated a combination of Toll-like receptor 9 (TLR9) agonist CpG ODN and Transforming growth factor-ß2 (TGF-ß2) antisense oligodeoxynucleotide TIO3 to mice intraperitoneally once every other day for a total of four injections, and the first injection was 24 h after LLC cell inoculation. We found that the combination induced the formation of TME toward the enrichment and activation of CD8+ T cells and NK cells, accompanied with a marked decrease of TGF-ß2. The combined therapy also effectively inhibited the tumor growth and prolonged the survival of the mice, even protected the tumor-free mice from the tumor re-challenge. Both of CpG ODN and TIO3 are indispensable, because replacing CpG ODN with TLR9 inhibitor CCT ODN showed no antitumor effect, CpG ODN or TIO3 alone did not lead to ideal antitumor results. This effect was possibly initiated by the activation of dendritic cells at the tumor site. This systemic antitumor immunotherapy with a combination of the two oligonucleotides (an immune stimulant and an immunosuppressive cytokine inhibitor) before the tumor formation may provide a novel strategy for clinical prevention of the postoperative tumor recurrence.

BAX as the mediator of C-MYC sensitizes acute lymphoblastic leukemia to TLR9 agonists.

BACKGROUND: The prognosis of B-cell acute lymphoblastic leukemia (B-ALL) has improved significantly with current first-line therapy, although the recurrence of B-ALL is still a problem. Toll-like receptor 9 (TLR9) agonists have shown good safety and efficiency as immune adjuvants. Apart from their immune regulatory effect, the direct effect of TLR9 agonists on cancer cells with TLR9 expression cannot be ignored. However, the direct effect of TLR9 agonists on B-ALL remains unknown. METHODS: We discussed the relationship between TLR9 expression and the clinical characteristics of B-ALL and explored whether CpG 685 exerts direct apoptotic effect on B-ALL without inhibiting normal B-cell function. By using western blot, co-immunoprecipitation, immunofluorescence co-localization, and chromatin immunoprecipitation, we explored the mechanism of the apoptosis-inducing effect of CpG 685 in treating B-ALL cells. By exploring the mechanism of CpG 685 on B-ALL, the predictive biomarkers of the efficacy of CpG 685 in treating B-ALL were explored. These efficiencies were also confirmed in mouse model as well as clinical samples. RESULTS: High expression of TLR9 in B-ALL patients showed good prognosis. C-MYC-induced BAX activation was the key to the effect of CpG oligodeoxynucleotides against B-ALL. C-MYC overexpression promoted P53 stabilization, enhanced Bcl-2 associated X-protein (BAX) activation, and mediated transcription of the BAX gene. Moreover, combination therapy using CpG 685 and imatinib, a BCR-ABL kinase inhibitor, could reverse resistance to CpG 685 or imatinib alone by promoting BAX activation and overcoming BCR-ABL1-independent PI3K/AKT activation. CONCLUSION: TLR9 is not only a prognostic biomarker but also a potential target for B-ALL therapy. CpG 685 monotherapy might be applicable to Ph- B-ALL patients with C-MYC overexpression and without BAX deletion. CpG 685 may also serve as an effective combinational therapy against Ph+ B-ALL.

Site-Specific Tritium Labeling at the Predefined Internal Position of the Chemically-Modified RNA.

In nucleic acid drug discovery, it is extremely important to develop a technology to understand the distribution in target organs and to trace the degradation process in the body in order to optimize the structure and improve the efficiency of the clinical trial process. Since nucleic acid drugs are essentially metabolically degraded into numerous fragments, labeling at the internal position is preferable to that at the terminus. Due to the high molar specific activity of tritium, various approaches for tritium-labeling have been studied for nucleic acid drugs. Nevertheless, a generally-applicable method for tritium labeling of the internal position of a nucleic acid has not been established. In this study, we have demonstrated a new and efficient method for site-specific tritium labeling of the cytosine base at a predefined internal position in nucleic acid drugs. This method was developed by the chemical modification of the cytosine 4-amino group with the pyridinyl vinyl keto group by the functionality-transfer reaction using the reactive oligodeoxynucleotide (ODN), followed by reduction with NaBT4. Applicability to a variety of chemical structures, such as 5-methyl cytosine, 2'-O-methyl, 2'-fluoro ribose derivatives, Locked/Bridged nucleic acid (LNA/BNA) derivatives, as well as phosphorothioate bonds, has been evidenced using nine oligoribonucleic acid (ORN) substrates. It has been clearly demonstrated that this method is an excellent method for tritium-labeling of nucleic acid with an average conversion efficiency of 74%, an average isolated labeling yield of 60%, and an average specific activity of 61 GBq/mmol. This method is expected to contribute to the preclinical absorption, distribution, metabolism, excretion (ADME) studies of nucleic acid drug candidates.

Myogenetic Oligodeoxynucleotide Induces Myocardial Differentiation of Murine Pluripotent Stem Cells.

An 18-base myogenetic oligodeoxynucleotide (myoDN), iSN04, acts as an anti-nucleolin aptamer and induces myogenic differentiation of skeletal muscle myoblasts. This study investigated the effect of iSN04 on murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In the undifferentiated state, iSN04 inhibited the proliferation of ESCs and iPSCs but did not affect the expression of pluripotent markers. In the differentiating condition, iSN04 treatment of ESCs/iPSCs from day 5 onward dramatically induced differentiation into Nkx2-5+ beating cardiomyocytes with upregulation of Gata4, Isl1, and Nkx2-5, whereas iSN04 treatment from earlier stages completely inhibited cardiomyogenesis. RNA sequencing revealed that iSN04 treatment from day 5 onward contributes to the generation of cardiac progenitors by modulating the Wnt signaling pathway. Immunostaining showed that iSN04 suppressed the cytoplasmic translocation of nucleolin and restricted it to the nucleoli. These results demonstrate that nucleolin inhibition by iSN04 facilitates the terminal differentiation of cardiac mesoderm into cardiomyocytes but interferes with the differentiation of early mesoderm into the cardiac lineage. This is the first report on the generation of cardiomyocytes from pluripotent stem cells using a DNA aptamer. Since iSN04 did not induce hypertrophic responses in primary-cultured cardiomyocytes, iSN04 would be useful and safe for the regenerative therapy of heart failure using stem cell-derived cardiomyocytes.

NF-κB Decoy ODN-Loaded Poly(Lactic-co-glycolic Acid) Nanospheres Inhibit Alveolar Ridge Resorption.

Residual ridge resorption combined with dimensional loss resulting from tooth extraction has a prolonged correlation with early excessive inflammation. Nuclear factor-kappa B (NF-κB) decoy oligodeoxynucleotides (ODNs) are double-stranded DNA sequences capable of downregulating the expression of downstream genes of the NF-κB pathway, which is recognized for regulating prototypical proinflammatory signals, physiological bone metabolism, pathologic bone destruction, and bone regeneration. The aim of this study was to investigate the therapeutic effect of NF-κB decoy ODNs on the extraction sockets of Wistar/ST rats when delivered by poly(lactic-co-glycolic acid) (PLGA) nanospheres. Microcomputed tomography and trabecular bone analysis following treatment with NF-κB decoy ODN-loaded PLGA nanospheres (PLGA-NfDs) demonstrated inhibition of vertical alveolar bone loss with increased bone volume, smoother trabecular bone surface, thicker trabecular bone, larger trabecular number and separation, and fewer bone porosities. Histomorphometric and reverse transcription-quantitative polymerase chain reaction analysis revealed reduced tartrate-resistant acid phosphatase-expressing osteoclasts, interleukin-1ß, tumor necrosis factor-α, receptor activator of NF-κB ligand, turnover rate, and increased transforming growth factor-ß1 immunopositive reactions and relative gene expression. These data demonstrate that local NF-κB decoy ODN transfection via PLGA-NfD can be used to effectively suppress inflammation in a tooth-extraction socket during the healing process, with the potential to accelerate new bone formation.

Sharpening up tumor microenvironment to enhance the efficacy of immune checkpoint blockade on head and neck cancer using a CpG-oligodeoxynucleotide.

Head and neck cancers are a type of life-threatening cancers characterized by an immunosuppressive tumor microenvironment. Only less than 20% of the patients respond to immune checkpoint blockade therapy, indicating the need for a strategy to increase the efficacy of immunotherapy for this type of cancers. Previously, we identified a type B CpG-oligodeoxynucleotide (CpG-ODN) called CpG-2722, which has the universal activity of eliciting an immune response in grouper, mouse, and human cells. In this study, we further characterized and compared its cytokine-inducing profiles with different types of CpG-ODNs. The antitumor effect of CpG-2722 was further investigated alone and in combination with an immune checkpoint inhibitor in a newly developed syngeneic orthotopic head and neck cancer animal model. Along with other inflammatory cytokines, CpG-2722 induces the gene expressions of interleukin-12 and different types of interferons, which are critical for the antitumor response. Both CpG-2722 and anti-programmed death (PD)-1 alone suppressed tumor growth. Their tumor suppression efficacies were further enhanced when CpG-2722 and anti-PD-1 were used in combination. Mechanistically, CpG-2722 shaped a tumor microenvironment that is favorable for the action of anti-PD-1, which included promoting the expression of different cytokines such as IL-12, IFN-ß, and IFN-γ, and increasing the presence of plasmacytoid dendritic cells, M1 macrophages, and CD8 positive T cells. Overall, CpG-2722 provided a priming effect for CD8 positive T cells by sharpening the tumor microenvironment, whereas anti-PD-1 released the brake for their tumor-killing effect, resulting in an enhanced efficacy of the combined CpG-2722 and anti-PD-1.

CpG ODN (K3)-toll-like receptor 9 agonist-induces Th1-type immune response and enhances cytotoxic activity in advanced lung cancer patients: a phase I study.

BACKGROUND: Cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) (K3)-a novel synthetic single-stranded DNA immune adjuvant for cancer immunotherapy-induces a potential Th1-type immune response against cancer cells. We conducted a phase I study of CpG ODN (K3) in patients with lung cancer to assess its safety and patients' immune responses. METHODS: The primary endpoint was the proportion of dose-limiting toxicities (DLTs) at each dose level. Secondary endpoints included safety profile, an immune response, including dynamic changes in immune cell and cytokine production, and progression-free survival (PFS). In a 3 + 3 dose-escalation design, the dosage levels for CpG ODN (K3) were 5 or 10 mg/body via subcutaneous injection and 0.2 mg/kg via intravenous administration on days 1, 8, 15, and 29. RESULTS: Nine patients (eight non-small-cell lung cancer; one small-cell lung cancer) were enrolled. We found no DLTs at any dose level and observed no serious treatment-related adverse events. The median observation period after registration was 55 days (range: 46-181 days). Serum IFN-α2 levels, but not inflammatory cytokines, increased in six patients after the third administration of CpG ODN (K3) (mean value: from 2.67 pg/mL to 3.61 pg/mL after 24 hours). Serum IFN-γ (mean value, from 9.07 pg/mL to 12.7 pg/m after 24 hours) and CXCL10 levels (mean value, from 351 pg/mL to 676 pg/mL after 24 hours) also increased in eight patients after the third administration. During the treatment course, the percentage of T-bet-expressing CD8+ T cells gradually increased (mean, 49.8% at baseline and 59.1% at day 29, p = 0.0273). Interestingly, both T-bet-expressing effector memory (mean, 52.7% at baseline and 63.7% at day 29, p = 0.0195) and terminally differentiated effector memory (mean, 82.3% at baseline and 90.0% at day 29, p = 0.0039) CD8+ T cells significantly increased. The median PFS was 398 days. CONCLUSIONS: This is the first clinical study showing that CpG ODN (K3) activated innate immunity and elicited Th1-type adaptive immune response and cytotoxic activity in cancer patients. CpG ODN (K3) was well tolerated at the dose settings tested, although the maximum tolerated dose was not determined. TRIAL REGISTRATION: UMIN-CTR number 000023276. Registered 1 September 2016, https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000026649.

Color-changing fluorescent DNA probe containing solvatochromic Dansyl-nucleoside surrogate for sensing local variation of DNA duplex.

A novel Dansyl-nucleoside surrogate (Dns) based on (±)-trans-4-(hydroxymethyl) piperidin-3-ol was designed and synthesized. The Dns exhibited excellent solvatochromic properties. About 90 nm of red-shift accompanied color change from green to orange could be achieved with an increase of solvent polarity. The Dns was incorporated into oligodeoxynucleotide by phosphoroamidite chemistry. Two kinds of Dns-incorporated fluorescent DNA probes were designed and synthesized for sensing variation of DNA duplexes based on color-changing manner. As a result, the color-changing DNA probe not only can detect complementary oligonucleotide, but also can distinguish mismatch flanked in Dansyl/nucleobase pair by naked eye. Moreover, the change of fluorescence color of sample solutions could be captured by smartphone, and the photographs could be digitalized by image-processing software. Thus, the Dns-incorporated fluorescent DNA probe is expected to open the way to point-of-care assays in the future.

Application of the Functionality Transfer Oligodeoxynucleotide for the Site-Selective Modification of RNA with a Divers Molecule.

Due to the importance of the RNA chemical modifications, methods for the selective chemical modification at a predetermined site of the internal position of RNA have attracted much attention. We have developed functional artificial nucleic acids that modify a specific site of RNA in a site- and base-selective manner. In addition, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) has been shown to introduce additional molecules on the alkynes attached to the pyridine ring. However, it was found that some azide compounds produced the cycloadduct in lower yields. Therefore, in this study, we synthesized the pyridinyl transfer group with the alkyne attached via a polyethylene glycol (PEG) linker with a different length and optimized its structure for both the transfer and CuAAC reaction. Three new transfer groups were synthesized by introducing an alkyne group at the end of the triethylene (11), tetraethylene (12) or pentaethylen glycol linker (13) at the 5-position of the pyridine ring of (E)-3-iodo-1-(pyridin-2-yl)prop-2-en-1-one. These transfer groups were introduced to the 6-thioguanine base in the oligodeoxynucleotide (ODN) in high yields. The transfer groups 11 and 12 more efficiently underwent the cytosine modification. For the CuAAC reaction, although 7 showed low adduct yields with the anionic azide compound, the new transfer groups, especially 12 and 13, significantly improved the yields. In conclusion, the transfer groups 12 and 13 were determined to be promising compounds for the modification of long RNAs.

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.

Anti-Fibrotic Effect of Synthetic Noncoding Oligodeoxynucleotide for Inhibiting mTOR and STAT3 via the Regulation of Autophagy in an Animal Model of Renal Injury.

Renal fibrosis is a common process of various kidney diseases. Autophagy is an important cell biology process to maintain cellular homeostasis. In addition, autophagy is involved in the pathogenesis of various renal disease, including acute kidney injury, glomerular diseases, and renal fibrosis. However, the functional role of autophagy in renal fibrosis remains poorly unclear. The mammalian target of rapamycin (mTOR) plays a negative regulatory role in autophagy. Signal transducer and activator of transcription 3 (STAT3) is an important intracellular signaling that may regulate a variety of inflammatory responses. In addition, STAT3 regulates autophagy in various cell types. Thus, we synthesized the mTOR/STAT3 oligodeoxynucleotide (ODN) to regulate the autophagy. The aim of this study was to investigate the beneficial effect of mTOR/STAT3 ODN via the regulation of autophagy appearance on unilateral ureteral obstruction (UUO)-induced renal fibrosis. This study showed that UUO induced inflammation, tubular atrophy, and tubular interstitial fibrosis. However, mTOR/STAT3 ODN suppressed UUO-induced renal fibrosis and inflammation. The autophagy markers have no statistically significant relation, whereas mTOR/STAT3 ODN suppressed the apoptosis in tubular cells. These results suggest the possibility of mTOR/STAT3 ODN for preventing renal fibrosis. However, the role of mTOR/STAT3 ODN on autophagy regulation needs to be further investigated.

Fe3O4 magnetic nanoparticle-enhanced radiotherapy for lung adenocarcinoma via delivery of siBIRC5 and AS-ODN.

BACKGROUND: Radiotherapy is the mainstay treatment for lung adenocarcinoma, yet remains highly susceptible to resistance. Fe3O4 magnetic nanoparticles (MNPs) possess the ability to induce biological therapeutic effects. Herein, the current study set out to explore the effects of Fe3O4 MNPs on radiosensitivity of lung adenocarcinoma cells. METHODS: Fe3O4 MNPs loaded with both negatively-charged small interfering RNA against baculoviral IAP repeat containing 5 (siBIRC5) and oligodeoxynucleotide antisense (AS-ODN) to generate co-delivery NPs, followed by evaluation. Gel retardation assay was further performed to determine the binding ability of Fe3O4 MNPs to AS-ODN/siBIRC5. The radiosensitizing effect of NPs on lung adenocarcinoma cells was determined in the absence or the presence of NPs or radiotherapy. A549 and H460 tumor-bearing mice were established, where tumor tissues were subjected to immunohistochemistry. RESULTS: NPs were successfully prepared and characterized. BIRC5 expression levels were augmented in tissues of lung cancer patients. Fe3O4 MNPs enhanced the uptake of siBIRC5 and AS-ODN by lung adenocarcinoma cells. The presence of NPs under magnetic field reduced the BIRC5 expression and elevated the DR5 expression in lung adenocarcinoma cells. Lung adenocarcinoma cells treated with NPs exhibited inhibited tumor cell migration and increased DNA damage. After magnetic field treatment, tumors were better suppressed in the tumor-bearing mice treated with NPs, followed by radiotherapy. CONCLUSION: Findings obtained in our study indicated that Fe3O4 MNPs-targeted delivery of siBIRC5 and AS-ODN enhances radiosensitivity, providing an innovative solution for the current clinically existing lung adenocarcinoma patients with radiotherapy resistance with a low risk of toxicity.

Blockade of transforming growth factor ß2 by anti-sense oligonucleotide improves immunotherapeutic potential of IL-2 against melanoma in a humanized mouse model.

BACKGROUND AIMS: IL-2 is a potent cytokine that activates natural killer cells and CD8+ cytotoxic T lymphocytes (CTLs) and has been approved for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, the medical use of IL-2 is restricted because of its narrow therapeutic window and potential side effects, including the expansion of regulatory T cells (Tregs). METHODS: In this study, the authors investigated the complementary effects of transforming growth factor-ß2 (TGF-ß2) anti-sense oligodeoxynucleotide (TASO) on the immunotherapeutic potential of IL-2 in a melanoma-bearing humanized mouse model. RESULTS: The authors observed that the combination of TASO and IL-2 facilitated infiltration of CTLs into the tumor, thereby potentiating the tumor killing function of CTLs associated with increased granzyme B expression. In addition, TASO attenuated the increase in Tregs by IL-2 in the peripheral blood and spleen and also inhibited infiltration of Tregs into the tumor, which was partly due to decreased CCL22. Alteration of T-cell constituents at the periphery by TGF-ß2 inhibition combined with IL-2 might be associated with the synergistic augmentation of serum pro-inflammatory cytokines (such as interferon Î³ and tumor necrosis factor α) and decreased ratio of Tregs to CTLs in tumor tissues, which consequently results in significant inhibition of tumor growth CONCLUSIONS: These results indicate that the application of TASO improves IL-2-mediated anti-tumor immunity, thus implying that blockade of TGF-ß2 in combination with IL-2 may be a promising immunotherapeutic strategy for melanoma.

Anti-fibrotic effects of synthetic TGF-ß1 and Smad oligodeoxynucleotide on kidney fibrosis in vivo and in vitro through inhibition of both epithelial dedifferentiation and endothelial-mesenchymal transitions.

Kidney fibrosis is a common process of various kidney diseases leading to end-stage renal failure irrespective of etiology. Myofibroblasts are crucial mediators in kidney fibrosis through production of extracellular matrix (ECM), but their origin has not been clearly identified. Many study proposed that epithelial and endothelial cells become myofibroblasts by epithelial dedifferentiation and endothelial-mesenchymal transition (EndoMT). TGF-ß1/Smad signaling plays a crucial role in partly epithelial-mensencymal transition (EMT) and EndoMT. Thus, we designed the TGF-ß1/Smad oligodeoxynucleotide (ODN), a synthetic short DNA containing complementary sequence for Smad transcription factor and TGF-ß1 mRNA. Therefore, this study investigated the anti-fibrotic effect of synthetic TGF-ß1/Smad ODN on UUO-induced kidney fibrosis in vivo model and TGF-ß1-induced in vitro model. To examine the effect of TGF-ß1/Smad ODN, we performed various experiments to evaluate kidney fibrosis. The results showed that UUO induced inflammation, ECM accumulation, epithelial dedifferentiation and EndoMT processes, and tubular atrophy. However, synthetic TGF-ß1/Smad ODN significantly suppressed UUO-induced fibrosis. Furthermore, synthetic ODN attenuated TGF-ß1-induced epithelial dedifferentiation and EndoMT program via blocking TGF-ß1/Smad signaling. In conclusion, this study demonstrated that administration of synthetic TGF-ß1/Smad ODN attenuates kidney fibrosis, epithelial dedifferentiation, and EndoMT processes. The findings propose the possibility of synthetic ODN as a new effective therapeutic tool for kidney fibrosis.

Suppressing the metastatic properties of the breast cancer cells using STAT3 decoy oligodeoxynucleotides: A promising approach for eradication of cancer cells by differentiation therapy.

Due to the presence of cancer stem cells (CSCs), breast cancer often relapsed after conventional therapies. Strategies that induce differentiation of CSCs will be helpful in eradication of tumor cells, so we designed an oligodeoxynucleotide (ODNs) for targeting of signal transducer and activator of transcription 3 (STAT3) transcription factor which is involved in stemness, and constitutively activated in triple-negative breast cancer. Molecular docking and electrophoretic mobility shift assay analysis showed that decoy ODN bound specifically to the DNA binding site of STAT3 protein. The prevalent uptake of Cy3-labeled ODNs is in the cytoplasm and the nucleus of MDA-MB-231 treated cells. STAT3 decoy ODNs treatment showed cell growth inhibition by decreasing cell viability (17%), increasing the percentage of arrested cells in G0/G1 phases (18%), and triggering apoptosis (29%). Migration and invasion potential decreased from 10.77 to 6.76 µm/hr, by wound closure rate, and migrated/invaded percentage by 26.4% and 15.4% in the transwell assays, respectively. CD44 protein expression level on the cell surface also decreased, while CD24 increased. Mammosphere formation efficiency reduced in terms of tumorsphere size by 47%, while the required time increased. Cells morphology was changed, and lipid droplets were accumulated in the cytoplasm compared to the control and scrambled groups, in all assays (repeated triplicate). Furthermore, the gene expression of all downstream targets significantly decreased owing to suppressing the STAT3 transcription factor. Overall, the results confirmed the antitumor effects of STAT3 decoy in MDA-MB-231 cells. Thus, it seems that STAT3 decoy ODNs might be considered as an auxiliary tool for breast cancer eradicating by the differentiation therapy approach.

Novel Toll-Like Receptor 9 Agonist Derived from Cryptococcus neoformans Attenuates Allergic Inflammation Leading to Asthma Onset in Mice.

INTRODUCTION: The enhanced type 2 helper (Th2) immune response is responsible for the pathogenesis of allergic asthma. To suppress the enhanced Th2 immune response, activation of the Th1 immune response has been an alternative strategy for anti-asthma therapy. In this context, effective Th1-inducing adjuvants that inhibit the development of allergic asthma but do not flare the side effects of the primary agent are required in clinical treatment and preventive medicine. OBJECTIVE: In this study, we aimed to determine the regulation of the Th2 type immune response in asthma by a novel immunostimulatory oligodeoxynucleotide (ODN) derived from Cryptococcus neoformans, termed ODN112, which contains a cytosine-guanine (CG) sequence but not canonical CpG motifs. METHODS: Using an ovalbumin-induced asthma mouse model, we assessed the effect of ODN112 on prototypical asthma-related features in the lung and on the Th1/Th2 profile in the lymph nodes and lung of mice treated with ODN112 during sensitization. RESULTS AND CONCLUSION: ODN112 treatment attenuated asthma features in mice. In the bronchial lymph nodes of the lungs and in the spleen, ODN112 increased interferon-γ production and attenuated Th2 recall responses. In dendritic cells (DCs) after allergen sensitization, ODN112 enhanced cluster of differentiation (CD) 40 and CD80 expression but did not alter CD86 expression. Interleukin-12p40 production from DCs was also increased in a Th2-polarizing condition. Our results suggest that ODN112 is a potential Th1-inducing adjuvant during Th2 cell differentiation in the sensitization phase.

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.

Synthesis of Nucleoside Derivatives of N-Acetyl-7-nitroindoline, Their Incorporation into the DNA Oligomer, and Evaluation of Their Photoreactivity in the DNA/RNA Duplex.

N-Acetyl-7-nitroindoline has a characteristic reaction in that its acetyl group is photo-activated to acetylate amines to form amides. In this study, the N-acetyl-7-nitroindoline part was connected to the 2'-deoxyribose part at the 3- or 5-position or to a glycerol unit at the 3-position through an ethylene linker (1, 2, and 3, respectively). They were incorporated into the oligodeoxynucleotides, and their photo-reactivities toward the complementary RNA were evaluated. The acetyl group of 1 was photo-activated to form the deacelylated nitroso derivative without affecting the RNA strand. The photoreaction with 2 suggested acetylation of the RNA strand. In contrast, compound 3 formed the photo-cross-linked adduct with the RNA. These results have shown the potential application of N-acetyl-7-nitroindoline unit in aqueous solutions.

Beneficial Effects of SREBP Decoy Oligodeoxynucleotide in an Animal Model of Hyperlipidemia.

Hyperlipidemia is a chronic disorder that plays an important role in the development of cardiovascular diseases, type II diabetes, atherosclerosis, hypertension, and non-alcoholic fatty liver disease. Hyperlipidemias have created a worldwide health crisis and impose a substantial burden not only on personal health but also on societies and economies. Transcription factors in the sterol regulatory element binding protein (SREBP) family are key regulators of the lipogenic genes in the liver. SREBPs regulate lipid homeostasis by controlling the expression of a range of enzymes required for the synthesis of endogenous cholesterol, fatty acids, triacylglycerol, and phospholipids. Thereby, SREBPs have been considered as targets for the treatment of metabolic diseases. The aim of this study was to investigate the beneficial functions and the possible underlying molecular mechanisms of SREBP decoy ODN, which is a novel inhibitor of SREBPs, in high-fat diet (HFD)-fed hyperlipidemic mice. Our studies using HFD-induced hyperlipidemia animal model revealed that SREBB decoy ODN inhibited the increased expression of fatty acid synthetic pathway, such as SREBP-1c, FAS, SCD-1, ACC1, and HMGCR. In addition, SREBP decoy ODN decreased pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-8, and IL-6 expression. These results suggest that SREBP decoy ODN exerts its anti-hyperlipidemia effects in HFD-induced hyperlipidemia mice by regulating their lipid metabolism and inhibiting lipogenesis through inactivation of the SREPB pathway.