Hoechst-Modification on Oligodeoxynucleotides for Efficient Transport to the Cell Nucleus and Gene Regulation.

Various artificial oligodeoxynucleotides (ODNs) that contribute to gene regulation have been developed and their diversity and multifunctionality have been demonstrated. However, few artificial ODNs are actively transported to the cell nucleus, despite the fact that gene regulation also takes place in both the cell nucleus and the cytoplasm. In this study, to prepare ODNs with the ability to accumulate in the cell nucleus, we introduced Hoechst molecules into ODNs that act as carriers of functional molecules to the cell nucleus (Hoe-ODNs). We synthesized Hoe-ODNs and confirmed that they bound strongly to DNA duplexes. When single-stranded Hoe-ODNs or double-stranded ODNs consisting of Hoe-ODNs and its complementary strand were administered into living cells, both ODNs were efficiently accumulated in the cell nucleus. In addition, antisense ODNs, which were tethered with Hoechst unit, were delivered into the cell nucleus and efficiently suppressed the expression of their target RNA. Thus, we constructed a delivery system that enables the transport of ODNs into cell nucleus.

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.

Quantitative Model Analysis and Simulation of Pharmacokinetics and Metastasis-Associated Lung Adenocarcinoma 1 RNA Knockdown Effect After Systemic Administration of Cholesterol-Conjugated DNA/RNA Heteroduplex Oligonucleotide Crossing Blood-Brain Barrier of Mice.

The cholesterol-conjugated heteroduplex oligonucleotide (Chol-HDO) is a double-stranded complex; it comprises an antisense oligonucleotide (ASO) and its complementary strand with a cholesterol ligand. Chol-HDO is a powerful tool for achieving target RNA knockdown in the brains of mice after systemic injection. Here, a quantitative model analysis was conducted to characterize the relationship between the pharmacokinetics (PK) and pharmacodynamics (PD), non-coding RNA metastasis-associated lung adenocarcinoma 1 (Malat1) RNA, of Chol-HDO, in a time-dependent manner. The established PK model could describe regional differences in the observed brain concentration-time profiles. Incorporating the PD model enabled the unique knockdown profiles in the brain to be explained in terms of the time delay after single dosing and enhancement following repeated dosing. Moreover, sensitivity analysis of PK exposure/persistency, target RNA turnover, and knockdown potency identified key factors for the efficient and sustained target RNA knockdown in the brain. The simulation of an adequate dosing regimen quantitatively supported the benefit of Chol-HDO in terms of achieving a suitable dosing interval. This was achieved via sufficient and sustained brain exposure and subsequent strong and sustained target RNA knockdown in the brain, even after systemic injection. The present study provides new insights into drug discoveries and development strategies for HDO in patients with neurogenic disorders. SIGNIFICANCE STATEMENT: The quantitative model analysis presented here characterized the PK/PD relationship of Chol-HDO, enabled its simulation under various conditions or assumptions, and identified key factors for efficient and sustained RNA knockdown, such as PK exposure and persistency. Chol-HDO appears to be an efficient drug delivery system for the systemic administration of desired drugs to brain targets.

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.

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.

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.

Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply.

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3 d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

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.

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.

Incorporation of Gelatin Microspheres into HepG2 Human Hepatocyte Spheroids for Functional Improvement through Improved Oxygen Supply to Spheroid Core.

The multicellular spheroid three-dimensional cell culture system can be used as a formulation for cell-based therapy. However, the viability and functions of the cells in the core region of the spheroid tend to decrease because of limited oxygen supply. In this study, we incorporated gelatin microspheres (GMS) into HepG2 human hepatocyte spheroids to allow oxygen to reach the spheroid core. GMS with an approximate diameter of 37 µm were fabricated by water-in-oil emulsification followed by freeze drying. GMS-containing HepG2 spheroids (GMS/HepG2 spheroids) were prepared by incubation of the cells with GMS at various mixing ratios in agarose gel-based microwells. Increasing the GMS ratio increased the diameter of the spheroids, and few spheroids formed with excess GMS. HepG2 cells in the GMS/HepG2 spheroids were more oxygenated than those in the GMS-free spheroids. GMS incorporation increased the viability of HepG2 cells in the spheroids and increased the CYP1A1 activity of the cells to metabolize 7-ethoxyresorufin, although mRNA expression of the CYP1A1 gene was hardly affected by GMS incorporation. These results indicate that incorporating GMS into HepG2 spheroids improves the hypoxic microenvironment in the spheroids and increases cell viability and CYP1A1 metabolic activity.

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.

Development of RNA/DNA Hydrogel Targeting Toll-Like Receptor 7/8 for Sustained RNA Release and Potent Immune Activation.

Guanosine- and uridine-rich single-stranded RNA (GU-rich RNA) is an agonist of Toll-like receptor (TLR) 7 and TLR8 and induces strong immune responses. A nanostructured GU-rich RNA/DNA assembly prepared using DNA nanotechnology can be used as an adjuvant capable of improving the biological stability of RNA and promoting efficient RNA delivery to target immune cells. To achieve a sustained supply of GU-rich RNA to immune cells, we developed a GU-rich RNA/DNA hydrogel (RDgel) using nanostructured GU-rich RNA/DNA assembly, from which GU-rich RNA can be released in a sustained manner. A hexapod-like GU-rich RNA/DNA nanostructure, or hexapodRD6, was designed using a 20-mer phosphorothioate-stabilized GU-rich RNA and six phosphodiester DNAs. Two sets of hexapodRD6 were mixed to obtain RDgel. Under serum-containing conditions, GU-rich RNA was gradually released from the RDgel. Fluorescently labeled GU-rich RNA was efficiently taken up by DC2.4 murine dendritic cells and induced a high level of tumor necrosis factor-α release from these cells when it was incorporated into RDgel. These results indicate that the RDgel constructed using DNA nanotechnology can be a useful adjuvant in cancer therapy with sustained RNA release and high immunostimulatory activity.

Effects of Localization of Antigen Proteins in Antigen-Loaded Exosomes on Efficiency of Antigen Presentation.

Exosomes are considered to be vehicles of antigen delivery. The localization of antigen proteins, i.e., whether they lie on the outer surface or inner surface of exosomes, might affect antigen presentation after exosomes are taken up by antigen-presenting cells; however, little is known about the importance of this phenomenon. In this study, lactadherin (LA) and group-specific antigen (Gag), exosome-tropic proteins that had previously been shown to cause the localization of luciferase to the outer surface and inner surface of exosomes, respectively [ Takahashi , Y. ; J. Biotechnol. 2013 ; Charoenviriyakul , C. ; Mol. Pharm. 2018 ], were used to examine the importance of the localization of antigen proteins in antigen presentation. Human embryonic kidney cells 293 (HEK293) were selected as exosomes producing cells. First, green fluorescent protein (GFP) was used to trace intracellular behavior of antigen proteins after uptake by murine dendritic DC2.4 cells. GFP-derived fluorescence signals were detected in cells only when GFP-inner-loaded (Gag-GFP) exosomes were added to them. Then, ovalbumin (OVA) was used as a model antigen protein, and OVA-loaded exosomes were added to bone marrow-derived dendritic cells. OVA-inner-loaded (Gag-OVA) exosomes showed enhanced class I antigen presentation capacity as compared with OVA-outer-loaded (OVA-LA) exosomes. Using PKH-labeled exosomes, it was found that the localization of OVA had very little effect on the cellular uptake of exosomes. These results indicate that the loading of antigen proteins inside exosomes helps in efficient antigen presentation.

Rapid Regulation of Human Mesenchymal Stem Cell Proliferation Using Inducible Caspase-9 Suicide Gene for Safe Cell-Based Therapy.

The regulation of transplanted cell proliferation and function is important to achieve safe cell-based therapies. We previously reported that the proliferation and function of transplanted cells, which expressed the herpes simplex virus thymidine kinase (HSVtk) suicide gene, could be controlled by ganciclovir (GCV) administration. However, there are some concerns regarding the use of GCV. It is reported that the inducible caspase-9 (iC9) gene, a human caspase-9-derived genetically engineered suicide gene, rapidly induces cell apoptosis in the presence of apoptosis inducers, such as AP20187. In this study, we used a combination of the iC9 gene and AP20187 to achieve rapid regulation of transplanted cell proliferation. Cells from the human mesenchymal stem cell line UE7T-13 were transfected with the iC9 gene to obtain UE7T-13/iC9 cells. AP20187 significantly reduced the number of UE7T-13/iC9 cells within 24 h in a concentration-dependent manner. This reduction was much faster than the reduction of HSVtk-expressing UE7T-13 cells induced by GCV addition. Subcutaneous AP20187 administration rapidly reduced the luminescence signal from NanoLuc luciferase (Nluc)-expressing UE7T-13/iC9 cells transplanted into mice. These results indicate that the combined use of the iC9 gene and AP20187 is effective in rapidly regulating transplanted cell proliferation.

Click Chemistry as a Tool for Cell Engineering and Drug Delivery.

Click chemistry has great potential for use in binding between nucleic acids, lipids, proteins, and other molecules, and has been used in many research fields because of its beneficial characteristics, including high yield, high specificity, and simplicity. The recent development of copper-free and less cytotoxic click chemistry reactions has allowed for the application of click chemistry to the field of medicine. Moreover, metabolic glycoengineering allows for the direct modification of living cells with substrates for click chemistry either in vitro or in vivo. As such, click chemistry has become a powerful tool for cell transplantation and drug delivery. In this review, we describe some applications of click chemistry for cell engineering in cell transplantation and for drug delivery in the diagnosis and treatment of diseases.

Role of Extracellular Vesicle Surface Proteins in the Pharmacokinetics of Extracellular Vesicles.

Extracellular vesicles (EVs) are small membrane vesicles secreted from cells and have great potential as drug delivery carriers. Surface proteins on EV membranes might play roles in pharmacokinetics. One method which can be used to study the role of surface membrane of EV is to modify the inner space of EV. In the present study, we constructed a plasmid DNA expressing a fusion protein of Gag protein derived from Moloney murine leukemia virus (Gag) and Gaussia luciferase (gLuc) (Gag-gLuc) to modify the inner space of EVs. EVs were collected from B16BL6 melanoma cells, transfected with the plasmid, and isolated by a differential ultracentrifugation method. Gag-gLuc EVs were negatively charged globular vesicles with a diameter of approximately 100 nm. gLuc labeling of the Gag-gLuc EVs was stable in serum. gLuc activity of Gag-gLuc EVs was minimally decreased by proteinase K (ProK) treatment, indicating that gLuc was modified in the inner space of EV. Then, to evaluate the effect of the surface proteins of EVs on their pharmacokinetics, Gag-gLuc EVs treated with ProK were intravenously administered to mice. Volume of distribution (Vd) was significantly smaller for treated EVs than untreated EVs. Moreover, integrin α6ß1, an integrin known to be involved in lung targeting, was degraded after ProK treatment. The ProK treatment significantly reduced the lung distribution of EVs after intravenous injection. These results indicate that the surface proteins of EVs such as integrin α6ß1 play some roles in pharmacokinetics in terms of reducing Vd and their distribution to the lung.

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

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

Accelerated growth of B16BL6 tumor in mice through efficient uptake of their own exosomes by B16BL6 cells.

Exosomes are extracellular vesicles released by various cell types and play roles in cell-cell communication. Several studies indicate that cancer cell-derived exosomes play important pathophysiological roles in tumor progression. Biodistribution of cancer cell-derived exosomes in tumor tissue is an important factor for determining their role in tumor proliferation; however, limited studies have assessed the biodistribution of exosomes in tumor tissues. In the present study, we examined the effect of cancer-cell derived exosomes on tumor growth by analyzing their biodistribution. Murine melanoma B16BL6-derived exosomes increased the proliferation and inhibited the apoptosis of B16BL6 cells, which was associated with an increase and decrease in the levels of proliferation- and apoptosis-related proteins, respectively. GW4869-induced inhibition of exosome secretion decreased the proliferation of B16BL6 cells, and treatment of GW4869-treated cells with B16BL6-derived exosomes restored their proliferation. Next, we treated B16BL6 tumors in mice with B16BL6-derived exosomes and examined the biodistribution and cellular uptake of these exosomes. After the intratumoral injection of radiolabeled B16BL6-derived exosomes, most radioactivity was detected within the tumor tissues of mice. Fractionation of cells present in the tumor tissue showed that fluorescently labeled exosomes were mainly taken up by B16BL6 cells. Moreover, intratumoral injection of B16BL6-derived exosomes promoted tumor growth, whereas intratumoral injection of GW4869 suppressed tumor growth. These results indicate that B16BL6 cells secrete and take up their own exosomes to induce their proliferation and inhibit their apoptosis, which promotes tumor progression.

Enhanced Class I Tumor Antigen Presentation via Cytosolic Delivery of Exosomal Cargos by Tumor-Cell-Derived Exosomes Displaying a pH-Sensitive Fusogenic Peptide.

Tumor-cell-derived exosomes contain endogenous tumor antigens and can be used as a potential cancer vaccine without requiring identification of the tumor-specific antigen. To elicit an effective antitumor effect, efficient tumor antigen presentation by MHC class I molecules on dendritic cells (DC) is desirable. Because DC endocytose exosomes, an endosomal escape mechanism is required for efficient MHC class I presentation of exosomal tumor antigens. In the present study, efficient cytosolic delivery of exosomal tumor antigens was performed using genetically engineered tumor-cell-derived exosomes and pH-sensitive fusogenic GALA peptide. Murine melanoma B16BL6 cells were transfected with a plasmid vector encoding a streptavidin (SAV; a protein that binds to biotin with high affinity)-lactadherin (LA; an exosome-tropic protein) fusion protein to obtain SAV-LA-modified exosomes (SAV-exo). SAV-exo was mixed with biotinylated GALA to obtain GALA-modified exosomes (GALA-exo). Fluorescent microscopic observation using fluorescent-labeled GALA showed that the exosomes were modified with GALA. GALA-exo exerted a membrane-lytic activity under acidic conditions and efficiently delivered exosomal cargos to the cytosol. Moreover, DC treated with GALA-exo showed enhanced tumor antigen presentation capacity by MHC class I molecules. Thus, genetically engineered GALA-exo are effective in controlling the intracellular traffic of tumor-cell-derived exosomes and for enhancing tumor antigen presentation capacity.

Amelioration of Experimental Autoimmune Encephalomyelitis in Mice by Interferon-Beta Gene Therapy, Using a Long-Term Expression Plasmid Vector.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Repeated injections of the interferon-ß (IFN-ß) protein are required for relapse prevention therapy in patients with MS. IFN-ß gene transfer can be an alternative treatment that continuously supplies IFN-ß protein to the patient without requiring repeated injections. In a previous study, we constructed a novel long-term IFN-ß-expressing plasmid vector (pMx-IFN-ß). In the present study, we examined whether gene transfer of pMx-IFN-ß could be effective for the treatment of MS in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Seven days after injection of the EAE-inducing peptide, the EAE mice received hydrodynamic injections pMx-IFN-ß. The severity of EAE symptoms in the pMx-IFN-ß-treated mice was significantly lower for 1 month than that observed in the untreated mice. An evaluation of blood-brain barrier (BBB) function, using Evans Blue, showed that injection of pMx-IFN-ß suppressed the BBB disruptions normally observed in EAE mice, while BBB disruptions remained evident in the untreated EAE mice. Histological analysis showed fewer invasive inflammatory cells in the spinal cords of the pMx-IFN-ß-treated mice than in the spinal cords of the other mice. Serum interferon gamma protein (IFN-γ) concentrations in the pMx-IFN-ß-treated mice were significantly lower than that in the untreated mice, indicating that IFN-ß gene transfer suppressed the production of IFN-γ from pathogenic T cells. These results indicate that IFN-ß transgene expression by single administration of the pMx-IFN-ß can be an effective long-term treatment for MS.