Novel strategy of liver cancer treatment with modified antisense oligonucleotides targeting human vasohibin-2.

Vasohihibin-2 (VASH2) is a homolog of vasohibin-1 (VASH1) and is overexpressed in various cancers. Vasohihibin-2 acts on both cancer cells and cancer microenvironmental cells. Previous analyses have shown that VASH2 promotes cancer progression and abrogation of VASH2 results in significant anticancer effects. We therefore propose VASH2 to be a practical molecular target for cancer treatment. Modifications of antisense oligonucleotide (ASO) such as bridged nucleic acids (BNA)-based modification increases the specificity and stability of ASO, and are now applied to the development of a number of oligonucleotide-based drugs. Here we designed human VASH2-ASOs, selected an optimal one, and developed 2',4'-BNA-based VASH2-ASO. When systemically administered, naked 2',4'-BNA-based VASH2-ASO accumulated in the liver and showed its gene-silencing activity. We then examined the effect of 2',4'-BNA-based VASH2-ASO in liver cancers. Intraperitoneal injection of naked 2',4'-BNA-based VASH2-ASO exerted a potent antitumor effect on orthotopically inoculated human hepatocellular carcinoma cells. The same manipulation also showed potent antitumor activity on the splenic inoculation of human colon cancer cells for liver metastasis. These results provide a novel strategy for the treatment of primary as well as metastatic liver cancers by using modified ASOs targeting VASH2.

Lasso peptide microcin J25 variant containing RGD motif as a PET probe for integrin a v ß 3 in tumor imaging.

Microcin J25 (MccJ25), a lasso peptide, has a unique 3-D interlocked structure that provides high stability under acidic conditions, at high temperatures, and in the presence of proteases. In this study, we generated a positron emission tomography (PET) probe based on MccJ25 analog with an RGD motif and investigated their pharmacokinetics and utility for integrin αvß3 imaging in tumors. The MccJ25 variant with an RGD motif in the loop region and a lysine substitution at the C-terminus (MccJ25(RGDF)GtoK) was produced in E. coli transfected with plasmid DNA containing the MccJ25 biosynthetic gene cluster (mcjABCD). [64Cu]Cu-MccJ25(RGDF)GtoK was synthesized using the C-terminal lysine labeled with copper-64 (t1/2 = 12.7 h) via a bifunctional chelator; it showed stability in 90% mouse plasma for 45 min. Using PET imaging for integrin αvß3 positive U87MG tumor bearing mice, [64Cu]Cu-MccJ25(RGDF)GtoK could clearly distinguish the tumor, and its accumulation was significantly higher than that of MccJ25(GIGT)GtoK without the binding motif for integrin αvß3. Furthermore, MccJ25(RGDF)GtoK enabled visualization of only U87MG tumors but not MCF-7 tumors with low integrin αvß3 expression in double tumor-bearing mice. In ex vivo biodistribution analysis, the integrin αvß3 non-specific accumulation of [64Cu]Cu-MccJ25(RGDF)GtoK was significantly lower in various tissues, except for the kidneys, as compared to the control probe ([64Cu]Cu-cyclic RGD peptide). These results of the present study indicate that 64Cu-labeling methods are appropriate for the synthesis of MccJ25-based PET probes, and [64Cu]Cu-MccJ25 variants are useful tools for cancer molecular imaging.

Development of p53 knockout U87MG cell line for unbiased drug delivery testing system using CRISPR-Cas9 and transcriptomic analysis.

Antibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in the TP53 gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Further in vitro and in vivo assays of cell morphology, proliferation rate and target antigen-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected the best qualifying mutant clone. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes.

Polypod-like structured guanine-rich oligonucleotide aptamer as a selective and cytotoxic nanostructured DNA to cancer cells.

Guanine-rich oligonucleotide (GRO) can be developed as an effective anticancer agent owing to its high selectivity, affinity and antiproliferative activity in cancer cells. In this study, to increase the potency of GRO29A, a 29-mer GRO aptamer against nucleolin, an overexpressed protein in cancer cells, GRO29A was incorporated into three or six pods of polypod-like structured DNA (polypodna), tripodna or hexapodna, respectively. The polypod-like structured GROs, tri-G3, consisting of one tripodna and three GRO29A, or hexa-G1, hexa-G3 or hexa-G6, each of which comprises one hexapodna and one, three or six GRO29A, respectively, were designed. Tri-G3, hexa-G1 and hexa-G3 were prepared in high yield, except for hexa-G6. Polypod-like structured GROs had quadruplex structures under physiological salt conditions, and degraded at a slower rate in buffer containing serum. Cellular interaction experiments using fluorescently labelled DNA samples showed that the uptake of hexa-G3 by nucleolin-positive MCF-7 cells was more than 2-fold higher than GRO29A, and the interaction was increasingly dependent on the number of GRO29A in the structures. Hexa-G3 inhibited the proliferation of MCF-7 cells in more than 40%, but not of CHO cells. These results indicate that polypod-like structured GROs are useful DNA aptamers with high selectivity and cytotoxicity against nucleolin-positive cancer cells.

Improved Immuno-PET Imaging of HER2-Positive Tumors in Mice: Urokinase Injection-Triggered Clearance Enhancement of 64Cu-Trastuzumab.

Immuno-positron emission tomography (immuno-PET) is expected to improve the specificity of small chemical tracers such as 18F-fluorodeoxyglucose. Whole antibodies significantly accumulate in target molecule-expressing tumors but frequently persist too long in the blood circulation for imaging purposes. We investigated the utility of whole antibodies, 64Cu-labeled via a urokinase-substrate linker, and their exogenous urokinase-responsive cleavage to enhance clearance of immuno-PET probes from the blood and shorten the time required to develop adequate imaging contrast. Specifically, we used 64Cu-labeled trastuzumab in human epidermal growth factor receptor 2 (HER2)-positive tumor-bearing mice. 64Cu-labeled trastuzumab with a urokinase-cleavage site (64Cu-CB-TE1A1P-USL-trastuzumab) was synthesized using a bifunctional chelator incorporating an urokinase substrate peptide. Urokinase cleavage was analyzed in vitro by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and radio-gel permeation-high-performance liquid chromatography. Improvements in radioisotope clearance and HER2-imaging by urokinase injection were evaluated by PET imaging and ex vivo biodistribution studies in A431 tumor-bearing mice. 64Cu-CB-TE1A1P-USL-trastuzumab was cleaved into smaller radioactive fragments by 20 000 IU/mL urokinase treatment in vitro at an efficacy of ∼95%. The probe targeted HER2 in A431 tumors in mice within 24 h post-injection, and approximately two-thirds of the probe in the blood circulation was eliminated via renal clearance of radioactive fragments after three urokinase injections. Therefore, the tumor/blood ratio increased 3.0-fold. Without urokinase injection, the tumor accumulation of 64Cu-CB-TE1A1P-USL-trastuzumab slowly increased, and the blood radioactivity decreased over 72 h. However, the tumor/blood ratios in mice after three urokinase injections were higher at 24 h than those in mice without injections at 72 h. The results indicate that our approach shortened the time required to develop adequate imaging contrast of immuno-PET by >2 days. Therefore, this approach can benefit high-sensitivity imaging under lower radioactive decay conditions and can decrease patient radiation exposure. In addition, it could reduce other adverse effects of radioimmunotherapy.

Tumor recognition of peanut agglutinin-immobilized fluorescent nanospheres in biopsied human tissues.

We are investigating an imaging agent for early detection of colorectal cancer. The agent, named the nanobeacon, is coumarin 6-encapsulated polystyrene nanospheres whose surfaces are covered with poly(N-vinylacetamide) and peanut agglutinin that reduces non-specific interactions with the normal mucosa and exhibits high affinity for terminal sugars of the Thomsen-Friedenreich antigen, which is expressed cancer-specifically on the mucosa, respectively. We expect that cancer can be diagnosed by detecting illumination of intracolonically administered nanobeacon on the mucosal surface. In the present study, biopsied human tissues were used to evaluate the potential use of the nanobeacon in the clinic. Prior to the clinical study, diagnostic capabilities of the nanobeacon for detection of colorectal cancer were validated using 20 production batches whose characteristics were fine-tuned chemically for the purpose. Ex vivo imaging studies on 66 normal and 69 cancer tissues removed from the colons of normal and orthotopic mouse models of human colorectal cancer, respectively, demonstrated that the nanobeacon detected colorectal cancer with excellent capabilities whose rates of true and false positives were 91% and 5%, respectively. In the clinical study, normal and tumor tissues on the large intestinal mucosa were biopsied endoscopically from 11 patients with colorectal tumors. Histological evaluation revealed that 9 patients suffered from cancer and the rest had adenoma. Mean fluorescence intensities of tumor tissues treated with the nanobeacon were significantly higher than those of the corresponding normal tissues. Correlation of magnitude relation of the intensity in individuals was observed in cancer patients with a high probability (89%); however, the probability reduced to 50% in adenoma patients. There was a reasonable likelihood for diagnosis of colorectal cancer by the nanobeacon applied to the mucosa of the large intestine.

Elucidation of the Mechanism of Increased Activity of Immunostimulatory DNA by the Formation of Polypod-like Structure.

PURPOSE: We previously demonstrated that the immunostimulatory activity of CpG DNA is increased by the formation of polypod-like structures. The present study was designed to elucidate the mechanism underlying this increase. METHODS: Tripodna (three pods) and hexapodna (six pods) were prepared. The cellular uptake of Alexa Fluor 488-labeled DNA samples was examined in several cell lines by measuring the MFI of cells. TNF-α release from RAW264.7 cells was measured after addition of polypodna containing CpG motifs. Dissociation of double stranded DNA was evaluated using FRET. RESULTS: Tripodna and hexapodna were efficiently taken up by macrophage-like RAW264.7 cells and dendritic DC2.4 cells, but not by fibroblast or endothelial cell lines. The uptake by RAW264.7 cells was highest for hexapodna, followed by tripodna, dsDNA, and ssDNA. The release of TNF-α from RAW264.7 cells was also highest for hexapodna. The ratio of TNF-α release to cellular uptake was highest for ssDNA, and lowest for dsDNA. Tripodna and hexapodna were more easily dissociated into single strands after cellular uptake than was dsDNA. CONCLUSIONS: The efficient cellular uptake and prompt dissociation into single strands can be directly related to the high immunostimulatory activity of polypod-like structured DNAs containing CpG motifs.

Evaluation of a novel fluorescent nanobeacon for targeted imaging of Thomsen-Friedenreich associated colorectal cancer.

The Thomsen-Friedenreich (TF) antigen represents a prognostic biomarker of colorectal carcinoma. Here, using a nanobeacon, the surface of which was fabricated with peanut agglutinin as TF-binding molecules, we demonstrate that the nanobeacon is able to detect TF antigen in frozen and freshly biopsied polyps using fluorescence microscopy. Our results provide important clues about how to detect aberrant colonic tissues in the most timely fashion. Given the versatile application method for this topical nanobeacon, the protocol used in this work is amenable to clinical colonoscopy. Moreover, the prospects of clinical translation of this technology are evident.

Optimal Arrangement of Four Short DNA Strands for Delivery of Immunostimulatory Nucleic Acids to Immune Cells.

Nanosized DNA assemblies are useful for delivering immunostimulatory cytosine-phosphate-guanine (CpG) DNA to immune cells, but little is known about the optimal structure for such delivery. In this study, we designed three different DNA nanostructures using four 55-mer oligodeoxynucleotides (ODNs), that is, tetrapod-like structured DNA (tetrapodna), tetrahedral DNA (tetrahedron), and tetragonal DNA (tetragon), and compared their potencies. Electrophoresis showed that tetrapodna was obtained with high yield and purity, whereas tetrahedron formed multimers at high ODN concentrations. Atomic force microscopy revealed that all preparations were properly constructed under optimal conditions. The thermal stability of tetrapodna was higher than those of the others. Dynamic light scattering analysis showed that all of the assemblies were about 8 nm in diameter. Upon addition to mouse macrophage-like RAW264.7 cells, tetrahedron was most efficiently taken up by the cells. Then, a CpG DNA, a ligand for toll-like receptor 9, was linked to these DNA nanostructures and added to RAW264.7 cells. CpG tetrahedron induced the largest amount of tumor necrosis factor-α, followed by CpG tetrapodna. Similar results were obtained using human peripheral blood mononuclear cells. Taken together, these results indicate that tetrapodna is the best assembly with the highest yield and high immunostimulatory activity, and tetrahedron can be another useful assembly for cellular delivery if its preparation yield is improved.

Toxicity studies of coumarin 6-encapsulated polystyrene nanospheres conjugated with peanut agglutinin and poly(N-vinylacetamide) as a colonoscopic imaging agent in rats.

We are investigating an imaging agent that detects early-stage primary colorectal cancer on the mucosal surface in real time under colonoscopic observation. The imaging agent, which is named the nanobeacon, is fluorescent nanospheres conjugated with peanut agglutinin and poly(N-vinylacetamide). Its potential use as an imaging tool for colorectal cancer has been thoroughly validated in numerous studies. Here, toxicities of the nanobeacon were assessed in rats. The nanobeacon was prepared according to the synthetic manner which is being established as the Good Manufacturing Practice-guided production. The rat study was performed in accordance with Good Laboratory Practice regulations. No nanobeacon treatment-related toxicity was observed. The no observable adverse effect levels (NOAEL) of the nanobeacon in 7-day consecutive oral administration and single intrarectal administration were estimated to be more than 1000mg/kg/day and 50mg/kg/day, respectively. We concluded that the nanobeacon could be developed as a safe diagnostic agent for colonoscopy applications. FROM THE CLINICAL EDITOR: Colon cancer remains a major cause of death. Early detection can result in early treatment and thus survival. In this article, the authors tested potential systemic toxicity of coumarin 6-encapsulated polystyrene nanospheres conjugated with peanut agglutinin (PNA) and poly(N-vinylacetamide) (PNVA), which had been shown to bind specifically to colonic cancer cells and thus very promising in colonoscopic detection of cancer cells.

Self-assembling DNA dendrimer for effective delivery of immunostimulatory CpG DNA to immune cells.

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.

Injectable, self-gelling, biodegradable, and immunomodulatory DNA hydrogel for antigen delivery.

DNA nanotechnology-based nanosystems and macrosystems have attracted much attention in the biomedical research field. The nature of DNA endows these systems with biodegradable, biocompatible, and immunomodulatory properties. Here, we present an injectable hydrogel system that consists only of chemically synthesized short DNA strands, water, and salts. Several preparations of polypod-like structured DNA, or polypodna, were designed, including tri-, tetra-, penta- and hexapodna, as the building blocks of self-gelling DNA hydrogel. Under physiological conditions, properly designed polypodna preparations formed a hydrogel. The analysis of the modulus data of the hydrogel consisting of two sets of hexapodna preparations showed that this injectable hydrogel was reorganized at a time scale of 0.25s. Then, DNA hydrogel containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides was used to stimulate innate immunity through Toll-like receptor 9, the receptor for CpG DNA. Gel formation significantly increased the activity of immunostimulatory CpG DNA, retarded the clearance after intradermal injection into mice, and increased the immune responses to ovalbumin (OVA) incorporated into the hydrogel as a model antigen. OVA/CpG DNA hydrogel induced much less local or systemic adverse reactions than OVA injected with complete Freund's adjuvant or alum. GpC DNA hydrogel containing no CpG sequences was less effective, indicating the importance of immunomodulation by CpG DNA hydrogel. Thus, we have created an efficient system for sustained delivery of antigens or other bioactive compounds.

Efficient delivery of immunostimulatory DNA to mouse and human immune cells through the construction of polypod-like structured DNA.

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.

Increased immunostimulatory activity of polypod-like structured DNA by ligation of the terminal loop structures.

The immunostimulatory activity of phosphodiester DNA containing unmethylated cytosine-guanine (CpG) dinucleotides can be increased by converting it into branched structures. These structures could be stabilized by ligating the 5'- and 3'-ends to form a closed loop with no terminal ends. To further increase the ability of branched DNA assemblies to induce cytokines, a series of tetrapod-like structured DNA, or tetrapodna, were designed using four 48-base oligodeoxynucleotides (ODNs). All these preparations were designed to have the same sequence except for the nick sites, and all the ODNs of one of the tetrapodna preparations were ligated to obtain circular tetrapodna. The nick site significantly influenced the formation of the structure and melting temperature (Tm), but hardly affected the enzymatic stability of the tetrapodna preparations. Circular tetrapodna exhibited a significantly higher Tm and was more stable in mouse serum than its non-ligated counterparts. The amounts of cytokines released from macrophage-like RAW264.7 cells or dendritic DC2.4 cells after addition of circular tetrapodna were not significantly higher than those after addition of other tetrapodna preparations under conditions when no serum was present. However, when serum was present, circular tetrapodna induced the greatest amount of tumor necrosis factor-α, indicating that circular tetrapodna is effective in inducing cytokines under conditions where DNA-degrading enzymes are present. The cellular association of tetrapodna preparations was almost unaffected by ligation of the terminal ends. These results indicate that circular tetrapodna with no terminal ends is more effective than its non-ligated counterparts in the presence of serum.

Design and development of nanosized DNA assemblies in polypod-like structures as efficient vehicles for immunostimulatory CpG motifs to immune cells.

The immunostimulatory activity of phosphodiester DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides, or CpG motifs, was significantly increased by the formation of Y-, X-, or dendrimer-like multibranched shape. These results suggest the possibility that the activity of CpG DNA is a function of the structural properties of branched DNA assemblies. To elucidate the relationship between them, we have designed and developed nanosized DNA assemblies in polypod-like structures (polypod-like structured DNA, or polypodna for short) using oligodeoxynucleotides (ODNs) containing CpG motifs and investigated their structural and immunological properties. Those assemblies consisting of three (tripodna) to eight (octapodna) ODNs were successfully obtained, but one consisting of 12 ODNs was not when 36-mer ODNs were annealed under physiological sodium chloride concentration. High-speed atomic force microscopy revealed that these assemblies were in polypod-like structures. The apparent size of the products was about 10 nm in diameter, and there was an increasing trend with an increase in ODN length or with the pod number. Circular dichroism spectral data showed that DNA in polypodna preparations were in the B-form. The melting temperature of polypodna decreased with increasing pod number. Each polypodna induced the secretion of tumor necrosis factor-α and interleukin-6 from macrophage-like RAW264.7 cells, with the greatest induction by those with hexa- and octapodna. Increasing the pod number increased the uptake by RAW264.7 cells but reduced the stability in serum. These results indicate that CpG DNA-containing polypodna preparations with six or more pods are a promising nanosized device with biodegradability and high immunostimulatory activity.

Biodegradable CpG DNA hydrogels for sustained delivery of doxorubicin and immunostimulatory signals in tumor-bearing mice.

Immunostimulatory CpG DNA was self-assembled to form DNA hydrogels for use as a sustained delivery system for both intercalated doxorubicin (DXR) and immunostimulatory CpG motifs for cancer treatment. X-shaped DNA (X-DNA) was designed as a building unit, and underwent ligation to form DNA hydrogels. Two types of X-DNA were constructed using four oligodeoxynucleotides each, one containing six potent CpG motifs (CpG X-DNA) and the other with none (CpG-free X-DNA). CpG X-DNA was more effective than its components or the CpG-free counterpart in terms of the production of tumor necrosis factor-α from murine macrophage-like RAW264.7 cells, as well as maturation of the murine dendritic DC2.4 cells. The cytotoxic effects of X-DNA, DXR and their complexes were examined in a co-culture system of colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, and RAW264.7 cells. DXR/CpG X-DNA showed the highest ability to inhibit the proliferation of colon26/Luc cells. DXR was slowly released from CpG DNA hydrogels. Injections of DXR/CpG DNA hydrogels into a subcutaneous colon26 tumor effectively inhibited tumor growth. These results show that CpG DNA hydrogels are an effective sustained system for delivery of immunostimulatory signals to TLR9-positive immune cells and DXR to cancer cells.

Structural and immunostimulatory properties of Y-shaped DNA consisting of phosphodiester and phosphorothioate oligodeoxynucleotides.

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.