Hemagglutinating Virus Of Japan Envelope Vector Research Articles

A Novel Cellular Imaging Method Using Hemagglutinating Virus of Japan-Envelope (HVJ-E) Vector and Magnetic Particle Imaging.

The purpose of this study was to develop a novel cellular imaging method using the hemagglutinating virus of Japan-envelope (HVJ-E) vector and magnetic particle imaging (MPI). First, we determined the concentration of magnetic nanoparticles (MNPs) suitable for encapsulation into the HVJ-E vector (HVJ-MNPs). Colon-26 cells were labeled with HVJ-MNPs, MNPs conjugated with protamine (Pro-MNPs) or MNPs alone (Res-MNPs), and their labeling efficiencies were evaluated. Second, HVJ-MNPs, Pro-MNPs or Res-MNPs were injected directly into the tumors of tumorbearing mice and the MPI images were obtained using our MPI scanner. The temporal change of the MNPs in the tumor was quantitatively evaluated by calculating the average MPI value. In addition, the microstructures of the resected tumor tissues were observed using a transmission electron microscope (TEM). The amount of iron encapsulated into HVJ-E and the encapsulation efficiency, saturated and decreased linearly with increasing amount of added iron, respectively. The labeling efficiency of HVJ-MNPs was significantly higher than those of Res-MNPs and Pro-MNPs. In animal studies, the average MPI value in the HVJ-MNP group remained almost constant up to 14 days, whereas those in the Res-MNP and Pro-MNP groups significantly decreased at 1 day or later, compared with that at 1 hour after the injection of the agents. In the TEM studies, earlier uptake of HVJ-MNPs in the cytoplasm was observed compared with Res-MNPs and Pro-MNPs. Our results suggest that the present method is useful for cellular imaging and tracking, and that HVJ-E is effective in internalizing MNPs into cells, during cellular imaging using MPI.

Inhibition of Neointima Hyperplasia, Inflammation, and Reactive Oxygen Species in Balloon-Injured Arteries by HVJ Envelope Vector-Mediated Delivery of Superoxide Dismutase Gene

Extracellular superoxide dismutase (EC-SOD) has been implicated in regulation of vascular function but its underlying molecular mechanism is largely unknown. These two-step experiments investigate whether hemagglutinating virus of Japan envelope (HVJ-E) vector-mediated EC-SOD gene delivery might protect against neointima formation, vascular inflammation, and reactive oxygen species (ROS) generation, and also explore cell growth signaling pathways. The first in-vitro experiment was performed to assess the transfection efficacy and safety of HVJ-E compared to lipofectamine®. Results revealed that HVJ-E has higher transfection efficiency and lower cytotoxicity than those of lipofectamine®. Another in-vivo study initially used balloon denudation to rat carotid artery, then delivered EC-SOD cDNA through the vector of HVJ-E. Arterial section with H&E staining from the animals 14 days after balloon injury showed a significant reduction of intima-to-media area ratio in EC-SOD transfected arteries when compared with control (empty vector-transfected arteries) (p < 0.05). Arterial tissue with EC-SOD gene delivery also exhibited lower levels of ROS, as assessed by fluorescent microphotography with dihydroethidium staining. Quantitative RT-PCR revealed that EC-SOD gene delivery significantly diminished mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (p < 0.05 in all comparisons). An immunoblotting assay from vascular smooth muscle cell (VSMC) cultures showed that the EC-SOD transfected group attenuated the activation of MEK1/2, ERK1/2, and Akt signaling significantly. In conclusion, EC-SOD overexpression by HVJ-E vector inhibits neointima hyperplasia, inflammation, and ROS level triggered by balloon injury. The modulation of cell growth-signaling pathways by EC-SOD in VSMCs might play an important role in these inhibitory effects.

Recent advances and developments in the antitumor effect of the HVJ envelope vector on malignant melanoma: from the bench to clinical application

Inactivated Sendai virus particles (hemagglutinating virus of Japan envelope; HVJ-E) are considered to be safe and efficient non-viral vectors used for drug delivery, since they can incorporate DNA, RNA, proteins and drugs. We have recently found that HVJ-E has a novel antitumor immune effect using a colon cancer model. HVJ-E has also been shown to have both direct and immune-mediated indirect actions against malignancy. Intratumoral injection of an inactivated HVJ-E solution significantly reduced the tumor volume and prevented spontaneous lung metastasis, leading to an increased overall survival in C57/BL6 mice transplanted with B16/BL6 mouse melanoma cells, and even in immunodeficient mice transplanted with Mewo human melanoma cells. No severe adverse effects including laboratory data abnormalities or anaphylactic reactions were observed. The comprehensive mechanism(s) underlying the immunological effects of HVJ-E appear to include not only enhanced effector T cell- and/or natural killer (NK) cell-mediated immunity, but also rescue from regulatory T cell (Treg)-mediated immunosuppression, presumably through the interleukin-6 secretion from dendritic cells stimulated by HVJ-E. Since a protocol for a clinical study of HVJ-E in malignant melanoma was approved in 2009 by the ethics committee of Osaka University and of the Medical Center for Translational Research in Osaka University Hospital, a phase I/IIa study for advanced malignant melanoma patients was just started. In this review, we show several favorable results regarding the antitumor effects of HVJ-E and describe the novel mechanism underlying this tumor immune response. Since we are conducting a phase I/IIa clinical trial using HVJ-E in advanced melanoma patients on the basis of preclinical results, detailed clinical information and immune-monitoring data are also introduced. The development of new therapeutic modalities for advanced melanoma patients is urgently needed, and we hope that HVJ-E may provide one such treatment.

In vitroinvestigation of efficient photodynamic therapy using a nonviral vector; hemagglutinating virus of Japan envelope

Photodynamic therapy (PDT) is a photochemical modality approved for cancer treatment. PDT has demonstrated efficacy in early stage lung cancer and esophageal cancer. The accumulation of photosensitizers in cancer cells is necessary to enhance the therapeutic benefits of PDT; however, photosensitizers have low uptake efficiency. To overcome this limitation, a drug delivery system, such as the hemagglutinating virus of Japan envelope (HVJ-E) vector, is required. In this study, the combination of PDT and HVJ-E was investigated for enhancing the efficacy of PDT. The photosensitizers that were evaluated included 5-aminolaevulinic acid (5-ALA), protoporphyrin IX (PPIX), and HVJ-PPIX. The uptake of the photosensitizers as increased twenty-fold with the addition of HVJ-E. The cytotoxicity of conventional 5-ALA was enhanced by the addition of HVJ-E vector. In conclusion, HVJ-E vector improved the uptake of photosensitizers and the PDT effect.

Gene Silencing of MK2 in Hard-to-Transfect Human U937 Cells

Genetic modifications used to answer biological questions in cultured cells are widely used in basic research. Common transfection methods are based on viral components causing cell activation or chemical modifications of small interfering RNA and cytotoxic reagents. Here, we report a rapid and efficient approach to transfect hard-to-transfect human U937 cells via the HVJ Envelope vector system, independent from special transfection media or immobilization of cells. This protocol provides a convenient means of knocking down MAPK-activated kinase 2 in hard-to-transfect cells to study inflammation, cell adhesion, and migration characteristics.

In vitro investigation of efficient photodynamic therapy using a nonviral vector; hemagglutinating virus of Japan envelope

Photodynamic therapy (PDT) is a photochemical modality approved for cancer treatment. PDT has demonstrated efficacy in early stage lung cancer and esophageal cancer. The accumulation of photosensitizers in cancer cells is necessary to enhance the therapeutic benefits of PDT; however, photosensitizers have low uptake efficiency. To overcome this limitation, a drug delivery system, such as the hemagglutinating virus of Japan envelope (HVJ-E) vector, is required. In this study, the combination of PDT and HVJ-E was investigated for enhancing the efficacy of PDT. The photosensitizers that were evaluated included 5-aminolaevulinic acid (5-ALA), protoporphyrin IX (PPIX), and HVJ-PPIX. The uptake of the photosensitizers as increased twenty-fold with the addition of HVJ-E. The cytotoxicity of conventional 5-ALA was enhanced by the addition of HVJ-E vector. In conclusion, HVJ-E vector improved the uptake of photosensitizers and the PDT effect.

Development of Liposomes and Pseudovirions with Fusion Activity for Efficient Gene Delivery

For efficient gene delivery, chimeric vectors combining non-viral vectors with viral components have been developed. In particular, increasing attention has been paid to viral fusion activity. HVJ (hemagglutinating virus of Japan; Sendai virus) fuses with the cell membrane at neutral pH, and HN and F, fusion proteins of the virus, contribute to the cell fusion. For fusion-mediated gene transfer, DNA-loaded liposomes were fused with UV-inactivated HVJ to form the fusion liposome, HVJ-liposome. Fusion-mediated delivery protects the molecules incorporated in the liposome from degradation in endosomes and lysosomes before reaching the cytoplasm. Reconstituted pseudovirions of fusion-competent viruses such as HVJ and influenza virus have been also developed by a detergent-lysis and-removal method. A more direct and practical approach is the conversion of fusion-competent virions to non-viral gene delivery particles. Based on this concept, the HVJ envelope vector was developed using inactivated particles of HVJ and has been utilized for gene therapy experiments and functional screening for therapeutic genes. A tissue-targeting HVJ envelope vector was also constructed.

HVJ Liposomes and HVJ Envelope Vectors

This protocol describes techniques for construction of fusion-mediated vectors based on inactivated HVJ (hemagglutinating virus of Japan; Sendai virus). HVJ liposomes are constructed by fusing liposomes containing DNA with inactivated HVJ. The HVJ envelope vector, a more simplified vector, incorporates DNA into inactivated HVJ particles without liposomes. Both vectors have many advantages. They can be used to introduce proteins, peptides, oligonucleotides (including antisense oligonucleotides, decoy oligonucleotides, and ribozymes), and short interfering RNA (siRNA), as well as plasmid DNA, into cultured cells in vitro and into organs in vivo. Fusion-mediated delivery avoids the degradation of therapeutic molecules before reaching the cytoplasm. Finally, repeated injection of the vector in vivo is not inhibited and even enhances the effects of the delivered molecules. These vectors have been used in many gene therapy experiments in animal models to address problems such as liver cirrhosis, hearing impairment, ischemic brain damage, peripheral arterial diseases, and cancers. This protocol describes methods for the preparation of HVJ liposomes and of HVJ envelope vectors and their use in delivery of plasmid DNA into various cells and tissues.

A non-replicating oncolytic vector as a novel therapeutic tool against cancer

Cancers are still difficult targets despite recent advances in cancer therapy. Due to the heterogeneity of cancer, a single-treatment modality is insufficient for the complete elimination of cancer cells. Therapeutic strategies from various aspects are needed. Gene therapy has been expected to bring a breakthrough to cancer therapy, but it has not yet been successful. Gene therapy also should be combined with other treatments to enhance multiple therapeutic pathways. In this view, gene delivery vector itself should be equipped with intrinsic anti-cancer activities. HVJ (hemagglutinating virus of Japan; Sendai virus) envelope vector (HVJ-E) was developed to deliver therapeutic molecules. HVJ-E itself possessed anti-tumor activities such as the generation of anti-tumor immunities and the induction of cancer-selective apoptosis. In addition to the intrinsic anti-tumor activities, therapeutic molecules incorporated into HVJ-E enabled to achieve multi-modal therapeutic strategies in cancer treatment. Tumor-targeting HVJ-E was also developed. Thus, HVJ-E will be a novel promising tool for cancer treatment.

Immunogene therapy using immunomodulating HVJ-E vector augments anti-tumor effects in murine malignant glioma

The hemagglutinating virus of Japan envelope (HVJ-E) vector derived from inactivated replication-defective Sendai virus enhances anti-tumor immunity through activation of effector T cells and natural killer (NK) cells and inhibition of regulatory T cells (Tregs). Interleukin (IL)-2 enhances T cell proliferation and activates T cells and NK cells. However, recent studies have revealed that the application of IL-2 also has immune suppressive effects through expansion of Tregs. Here, we investigated the efficacy of IL-2 gene therapy using immunomodulating HVJ-E vector in murine malignant glioma models. A single intratumoral injection of HVJ-E containing pVAX-mIL-2 significantly suppressed tumor growth of intracranial gliomas, resulting in prolonged survival. Furthermore, HVJ-E, following intracavitary administration, delivered genes into post-operative residual tumor cells. Consequently, prolonged survival resulted from a single intracavitary administration of HVJ-E containing pVAX-mIL-2 following tumor removal. IL-2 gene therapy delivered via the HVJ-E vector significantly inhibited the expansion of Tregs in tumors compared to IL-2 gene transfer using retroviral vector and resulted in marked infiltration of CD4(+) and CD8(+) T cells into tumors. Through inhibition of Treg-mediated immunosuppression, HVJ-E enhanced effector T cell-mediated anti-tumor immunity induced by IL-2. This combination of an immunomodulating vector and immunostimulating cytokine gene shows promise as an attractive, novel immunogene therapy for malignant glioma.

HVJ envelope vector, a versatile delivery system: Its development, application, and perspectives

An efficient and minimally invasive vector system is the “bottle neck” of both gene transfer and drug delivery. Numerous viral and non-viral (synthetic) delivery systems have been developed and improved. Hemagglutinating virus of Japan (HVJ, Sendai virus) envelope vector is a novel and unique system which combined the advantages of viral and non-viral vectors with the following features and advantages: (1) safe and easy as a “non-viral” transfection reagent; (2) delivery of various molecules including plasmid DNA, siRNA, protein, antisense oligonucleotide; (3) wide usability from in vitro to in vivo. In this review, the development, application, and perspectives of the HVJ envelope vector will be discussed.

Efficient delivery of antibody into living cells using a novel HVJ envelope vector system

Introduction of antibodies which retain their function into cells using a simple and easy method would be very useful for study of the intracellular events in living cells. In this study, we developed a new method for intracellular delivery of antibody. It includes a combination of a novel IgG-capturing protein and hemagglutinating virus of Japan envelope (HVJ-E), an inactivated Sendai virus particle which can deliver a variety of molecules into mammalian cells via membrane-fusing activity. The IgG-capturing protein, which was molecularly designed to have two functions, was prepared as a fusion protein (ZZ–NP) of ZZ-dimer derived from an immunoglobulin-binding domain of protein A and nucleocapsid protein (NP), a part of the structural protein of HVJ. ZZ–NP was efficiently incorporated into the HVJ-E particle by treatment with detergent, and enhanced the incorporation of IgG. Moreover, fluorescence immunostaining revealed that the incorporated antibody was very efficiently introduced into living cells while retaining its function, i.e. anti-NPC (nuclear pore complex) monoclonal antibody was selectively located around cell nuclei. These findings suggest that this method is useful for intracellular delivery of antibody and for analysis of biological function of sub-cellular molecules in living cells.

Development of a transferrin receptor-targeting HVJ-E vector

The development of more effective cancer treatments is anticipated. Tumor-targeted drug delivery is an important strategy in cancer therapy. We have developed an HVJ (hemagglutinating virus of Japan; Sendai virus) envelope (HVJ-E) vector using inactivated Sendai virus. The HVJ-E vector has been observed to target a number of cell lines since its hemagglutinin-neuraminidase (HN) protein recognizes the sialic acids of host cells. Thus, to reduce non-specific binding of the HVJ-E vector, we eliminated HN protein using HN-specific short interfering RNA (siRNA). Then, to further increase its tumor-targeting ability, we constructed HN-depleted HVJ containing the F-transferrin chimeric protein. The modified vectors containing Q-dots demonstrated 32-fold greater tumor-targeting efficiency than wild-type HVJ-E.

A new therapy for highly effective tumor eradication using HVJ-E combined with chemotherapy

BackgroundInactivated HVJ (hemagglutinating virus of Japan; Sendai virus) particles (HVJ envelope vector; HVJ-E can incorporate and deliver plasmid DNA, siRNA, antibody and peptide and anti-cancer drugs to cells both in vitro and in vivo. We attempted to eradicate tumors derived from mouse colon cancer cells, CT26, by combining bleomycin (BLM)-incorporated HVJ-E (HVJ-E/BLM) with cisplatin (CDDP) administration.MethodsCT-26 tumor mass was intradermally established in Balb/c mice. HVJ-E/BLM was directly injected into the tumor mass with or without intraperitoneal administration of CDDP. The anti-tumor effect was evaluated by measuring tumor size and cytotoxic T cell activity against CT26. Re-challenge of tumor cells to treated mice was performed 10 days or 8 months after the initial tumor inoculation.ResultsWe found that three intratumoral injections of HVJ-E/BLM along with a single intraperitoneal administration of CDDP eradicated CT26 tumors with more than 75% efficiency. When tumor cells were intradermally re-injected on day 10 after the initial tumor inoculation, tumors on both sides disappeared in most of the mice that received the combination therapy of HVJ-E/BLM and CDDP. Eight months after the initial tumor eradication, surviving mice were re-challenged with CT26 cells. The re-challenged tumors were rejected in all of the surviving mice treated with the combination therapy. Cytotoxic T lymphocytes specific for CT26 were generated in these surviving mice.ConclusionCombination therapy consisting of HVJ-E and chemotherapy completely eradicated the tumor, and generated anti-tumor immunity. The combination therapy could therefore be a promising new strategy for cancer therapy.

New potential therapy for orthotopic bladder carcinoma by combining HVJ envelope with doxorubicin

To establish a new therapeutic method to treat bladder carcinoma, we investigated the therapeutic potential of doxorubicin hydrochloride (DXR) combined with hemagglutinating virus of Japan-envelope vector (HVJ-E) in an orthotropic mouse bladder cancer model. DXR and/or HVJ-E were instilled into the bladder after implantation of MB49 cells. Antitumor effects of combination therapy were evaluated by histological analysis of the bladder on day 14 after tumor implantation. The survival rate of MB49-disseminated mice was examined for 60 days after single or double administration of DXR alone or DXR/HVJ-E. The surviving mice were re-challenged with intravesical injection of MB49 cells, and the bladder was observed after 3 weeks. Combined intravesical instillation of HVJ-E and DXR resulted in a significantly higher rate of tumor-free mice (11/21) compared with mice treated using DXR alone (3/19, P<0.05). Median survival was >60 days for intravesical instillation of HVJ-E and DXR, compared with the 29 days for DXR instillation alone (P<0.05). After combination therapy, surviving mice formed no tumors in the bladder following intravesical re-instillation of MB49. HVJ-E increased antitumor effects in combination with chemotherapeutic agent (DXR). Antitumor immunity appeared to be enhanced using HVJ-E.

SOX6 Suppresses Cyclin D1 Promoter Activity by Interacting with β-Catenin and Histone Deacetylase 1, and Its Down-regulation Induces Pancreatic β-Cell Proliferation

Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from beta-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with beta-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of beta-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withbeta-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.

Preparation and Evaluation of Gene-transfected Cultured Skin as a Novel Drug Delivery System for Severely Burned Skin

The purpose of this study is to prepare and evaluate gene-transfected cultured skin to establish a dermal patch consisting of cultured skin as a new and novel delivery system for severely burned skin. Plasmid DNA encoding the green fluorescent protein (GFP) gene was used as a model gene and transfected to rat and human cultured dermis models (CDMs) using the hemagglutinating virus of Japan envelope vector (HVJ-E) to prepare gene-transfected CDM and evaluate GFP expression in the CDM. Two kinds of transfection methods were evaluated. In pre-transfection, the gene was first transfected into fibroblasts and then CDM was prepared using these gene-transfected cells. In post-transfection, the gene was transfected directly into CDM. GFP expression was observed in both the pre- and post-transfected CDMs. The post-transfection method showed higher GFP expression in the CDM than pre-transfection, although no statistically significant difference was observed. The cell viability of these transfected CDMs was also examined with MTT assay. Slight decrease in viability was observed in these transfected CDMs. These methods could be useful in preparing gene-transfected cultured skins with low cell damage. Gene transfection to cultured skin may produce several dermal patches that release potent endogenous bioactive peptides.

Rapid transport of plasmid DNA into the nucleolus via actin depolymerization using the HVJ envelope vector

Although nuclear transport of therapeutic genes is an essential requirement of human gene therapy, factors required for nuclear entry of DNA remain to be elucidated. Non-viral vector systems have led to numerous improvements in the efficiency of delivery of exogenous DNA into cells. However, nuclear transport of plasmid is difficult to achieve. We examined nuclear translocation efficiency of Cy3-labeled plasmid DNA (Cy3-pDNA) delivered by the hemagglutinating virus of Japan envelope (HVJ-E) vector, Lipofectamine or microinjection. We also examined the effect of actin depolymerization on nuclear transport of Cy3-pDNA. Cy3-pDNA reached the nucleus, particularly in the nucleolus, in 30 min after fusion-mediated delivery using the HVJ-E vector, while the DNA was retained in the cytoplasm during the observed period after the delivery by cationic liposomes. HVJ-E treatment transiently depolymerized actin filaments, and acceleration of nucleolar entry of microinjected DNA was achieved when treated with either empty HVJ-E or cytochalasin D, an inhibitor of actin depolymerization, prior to microinjection. These results suggest that plasmid DNA can be transported rapidly from the cytoplasm to the nucleolus when actin filaments are depolymerized. Thus, the HVJ-E vector can accelerate the transport of DNA to the nucleolus by actin depolymerization.

Needleless intranasal administration of HVJ-E containing allergen attenuates experimental allergic rhinitis

Allergic rhinitis (AR) is one of the most common chronic diseases. Although current medications are highly effective in controlling its symptoms, they do not reverse the allergen-specific hypersensitivities that underlie the disease. Immunoglobulin E is a key mediator of AR, and preventing its production is clinically important. In this study, we developed an efficient needleless intranasal protein delivery system using the hemagglutinating virus of Japan envelope vector (HVJ-E). Intranasal delivery of ovalbumin (OVA) once a week for 3 weeks using this system enhanced OVA-induced interferon-gamma production by murine splenocytes. This treatment also attenuated the OVA-induced release interleukin-4 (IL-4) and IL-5 from splenocytes and the production of plasma OVA-specific immunoglobulin E in OVA-sensitive AR model mice. Thus, allergen-containing HVJ-E may be useful for noninvasive treatment of AR.

Development of Magnetic Force-Assisted New Gene Transfer System Using Biopolymer-Coated Ferromagnetic Nanoparticles

Development of a simple method for converting the lipid envelope of an inactivated virus to a gene transfer vector was achieved a couple years ago in the medical school of Osaka University. Hemagglutinating virus of Japan (HVJ; Sendai virus) envelope (HVJ-E) vector was constructed by incorporating plasmid DNA into inactivated HVJ particles. This HVJ envelope vector introduced plasmid DNA efficiently and rapidly into various cell lines, including cancer cells and several types of primary cell culture. In the present study, efficiency of gene transfer was found to be greatly enhanced by application of a magnetic field. Therefore, we developed a new type of magnet for magnetically enhancing and targeting gene transfection system by using vectors associated with ferromagnetic particles coated with positively/negatively charged biopolymers, which can help to enhance and target gene delivery with higher efficiency. For the transfection experiment in vitro, the HVJ-E vector was mixed with ferromagnetic particles coated with biopolymer and this mixture was added to cultured cells which were set up under the permanent magnet. The effect of the dose of the ferromagnetic particles on the transfection efficiency was discussed. In order to clarify the effect of magnetic field gradient on the accumulation possibility of the magnetic particles and the accuracy of the targeted site in the blood vessels, calculation of the applied magnetic force for the ferromagnetic particles inside the blood vessel was also performed