Hemagglutinating Virus Of Japan Envelope Vector Research Articles

Targeted chemotherapy against intraperitoneally disseminated colon carcinoma using a cationized gelatin–conjugated HVJ envelope vector

The hemagglutinating virus of Japan envelope (HVJ-E; Sendai virus) vector derived from inactivated HVJ particles can be used to deliver DNA, proteins, and drugs into cells both in vitro and in vivo. HVJ-E is capable of delivering bleomycin, an anticancer drug, to various cancer cell lines, thereby producing 300-fold greater cytotoxicity than administration of bleomycin alone. In a mouse model of peritoneally disseminated colon cancer, we injected HVJ-E containing the luciferase gene into the peritoneum. Unexpectedly, luciferase gene expression was not observed within the tumor deposits or any organs. However, when combined with cationized gelatin (CG), CG-HVJ-E produced a high level of luciferase gene expression primarily within the tumor deposits. Forty-eight hours after introducing colon cancer cells into the peritoneum of experimental mice, CG-HVJ-E with or without bleomycin was injected into the abdominal cavity. Following six injections of bleomycin-incorporated CG-HVJ-E, complete responses were observed in 40% of the mice examined. All of the mice that received either empty CG-HVJ-E or bleomycin alone died within 40 days of having cancer cells introduced into the peritoneum. When the mice with complete responses were rechallenged with colon cancer cells from the same cell line, no tumors developed. Thus, CG-HVJ-E may suppress peritoneal dissemination of cancer.

Development of High-Throughput Functional Screening of Therapeutic Genes, Using a Hemagglutinating Virus of Japan Envelope Vector

Isolation of effective therapeutic genes is critical for the advancement of gene therapy for various diseases, including vascular diseases and cancers. The goal of the present study was to screen a human cDNA library, using a hemagglutinating virus of Japan envelope (HVJ-E) vector, to isolate candidate genes with potent therapeutic potential. The advantages of a high-throughput functional screening system based on the HVJ-E vector include (1) rapid preparation of the vector containing the DNA library, (2) effective fusion-mediated transfer of the plasmids to various cells with minimal toxicity, and (3) easy cloning of candidate genes by transformation of Escherichia coli. These advantages resulted in a lower probability of damage to isolated clones and in minimization of the time needed to screen for candidate genes. Screening of a human heart library for candidate genes to regulate endothelial cell growth identified three growth-stimulating genes, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and c-fos promoter activity, the products of which were more potent than vascular endothelial growth factor. Similarly, two growth-inhibiting genes were identified, the effects of which were similar to angiostatin. Overall, this novel system will help advance our understanding of cell biology and promote the utility of human gene therapy.

873. Development of High Throughput Functional Screening of Cardiovascular Therapeutic Genes Using the HVJ-E Vector

Isolation of effective therapeutic genes to control angiogenesis is critical for the advancement of gene therapy for various diseases, including cardiovascular diseases and cancer. The goal of the present study is to screen a human cDNA library using Hemagglutinating Virus of Japan envelope (HVJ-E) vector to isolate candidate genes to regulate endothelial cell growth with potent therapeutic potential. cDNA library were randomly infused into HVJ-E vector, which were then transferred to Human Aortic Endothelial Cells (HAEC) that were seeded in 96-well plates. Then, the MTS assay revealed variable HAEC proliferation among individual wells. To characterize the candidate genes responsible for inducing or reducing endothelial cell proliferation, DNA was extracted from the cell population showing the highest or lowest degree of proliferation and was then transformed directly into E. coli. Use of this system allowed isolation of candidate genes rapidly, because HVJ-E vector does not require preparation of a viral library or packaging cell constructions. In addition, use of this vector allows easy cloning of candidate genes by transformation of E. Coli (12|[ndash]|16 hours). These advantages resulted in a lower probability of damage to isolated clones and in minimization of the time needed to screen for candidate genes.

Magnetic resonance imaging using hemagglutinating virus of Japan-envelope vector successfully detects localization of intra-cardially administered microglia in normal mouse brain

Although the therapeutic use of microglia has received some attention for the treatment of brain diseases, few non-invasive techniques exist for monitoring the cells after administration. Here, we present a technique using magnetic resonance imaging (MRI) to track microglia injected intra-cardially. We labeled microglia expressing enhanced green fluorescent protein with superparamagnetic iron oxide (Resovist) using the hemagglutinating virus of Japan-envelope vector. We injected labeled microglia into the left ventricle of the heart of mice. After monitoring exogenously administered microglia in the mouse brain in vivo using T 2*-weighted MRI at a magnetic field of 7 T, we compared the MR images with histochemical localization of exogenous microglia in vitro. MRI revealed clear signal changes attributable to Resovist-containing microglia in the mouse brain. Histochemistry demonstrated the presence of exogenous microglia in the brain at the same locations shown by MRI. This study demonstrates the usefulness of MRI for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia/macrophages as therapeutic tools for brain disease.

Liver-directed gene therapy of diabetic rats using an HVJ-E vector containing EBV plasmids expressing insulin and GLUT 2 transporter

Insulin gene therapy in clinical medicine is currently hampered by the inability to regulate insulin secretion in a physiological manner, the inefficiency with which the gene is delivered, and the short duration of gene expression. To address these issues, we injected the liver of streptozotocin-induced diabetic rats with hemagglutinating virus of Japan-envelope (HVJ-E) vectors containing Epstein-Barr virus (EBV) plasmids encoding the genes for insulin and the GLUT 2 transporter. Efficient delivery of the genes was achieved with the HVJ-E vector, and the use of the EBV replicon vector led to prolonged hepatic gene expression. Blood glucose levels were normalized for at least 3 weeks as a result of the gene therapy. Cotransfection of GLUT 2 with insulin permitted the diabetic rats to regulate their blood glucose levels upon exogenous glucose loading in a physiologically appropriate manner and improved postprandial glucose levels. Moreover, cotransfection with insulin and GLUT 2 genes led to in vitro glucose-stimulated insulin secretion that involved the closure of K(ATP) channels. The present study represents a new way to efficiently deliver insulin gene in vivo that is regulated by ambient glucose level with prolonged gene expression. This may provide a basis to overcome limitations of insulin gene therapy in humans.

Development of novel method of non-viral efficient gene transfer into neonatal cardiac myocytes

To establish new treatment for cardiovascular disease, the development of safe and highly efficient vectors is necessary. Especially, non-viral vectors are considered to be ideal for human gene therapy, since recent adverse events with retroviral or adenoviral vectors have highlighted the issue of safety. Although we previously reported safety and high efficiency of HVJ-liposome method, we have modified the envelope of HVJ (Sendai virus). In this novel non-viral vector, the envelope of HVJ alone was utilized as a carrier to deliver proteins, genes and oligodeoxynucleotides (ODN). Thus, we optimized the transfection efficiency of HVJ-envelope vector into neonatal cardiac myocytes in this study, since cardiac myocytes is one of the most difficult cells to be transfected. HVJ-envelope, obtained after complete destruction of HVJ genome, containing FITC-labeled ODN or luciferase plasmid was incubated with cardiac myocytes. In addition, the concentration of protamine sulfate was modified (0–700 μg/ml) to increase transfection efficacy. Without HVJ-envelope vector, few cells showed fluorescence, whereas most cells demonstrated fluorescence with HVJ-envelope vector. Consistent with the high transfection efficiency of ODN, high luciferase activity was also detected using HVJ-envelope vector. Moreover, the transfection efficiency varied according to the concentration of protamine sulfate. No obvious cytotoxicity was observed in cells transfected with HVJ-envelope vector. The present study demonstrated the development of a highly efficient novel non-viral vector for cardiac myocytes, suggesting that further development may provide a new useful tool for research and clinical gene therapy in the field of cardiovascular disease.

Magnetic nanoparticles with surface modification enhanced gene delivery of HVJ-E vector

To enter the realm of human gene therapy, a novel drug delivery system is required for efficient delivery of small molecules with high safety for clinical usage. We have developed a unique vector “HVJ-E (hemagglutinating virus of Japan-envelope)” that can rapidly transfer plasmid DNA, oligonucleotide, and protein into cells by cell-fusion. In this study, we associated HVJ-E with magnetic nanoparticles, which can potentially enhance its transfection efficiency in the presence of a magnetic force. Magnetic nanoparticles, such as maghemite, with an average size of 29 nm, can be regulated by a magnetic force and basically consist of oxidized Fe which is commonly used as a supplement for the treatment of anemia. A mixture of magnetite particles with protamine sulfate, which gives a cationic surface charge on the maghemite particles, significantly enhanced the transfection efficiency in an in vitro cell culture system based on HVJ-E technology, resulting in a reduction in the required titer of HVJ. Addition of magnetic nanoparticles would enhance the association of HVJ-E with the cell membrane with a magnetic force. However, maghemite particles surface-coated with heparin, but not protamine sulfate, enhanced the transfection efficiency in the analysis of direct injection into the mouse liver in an in vivo model. The size and surface chemistry of magnetic particles could be tailored accordingly to meet specific demands of physical and biological characteristics. Overall, magnetic nanoparticles with different surface modifications can enhance HVJ-E-based gene transfer by modification of the size or charge, which could potentially help to overcome fundamental limitations to gene therapy in vivo.

Radiation-induced apoptosis in peritoneal resident macrophages of C3H mice: Selective involvement of superoxide anion, but not other reactive oxygen species

Remarkably, apoptosis was induced by gamma-ray-irradiation in peritoneal resident macrophages (PRM) of C3H mice, but not other strains of mice. The mechanism of this strain-specific apoptosis induction was studied. Apoptosis in PRM was detected microscopically. Various radical scavengers were examined to identify the critical radicals involved in apoptosis induction. Intracellular peroxide levels were measured with a redox-sensitive dye, 2’,7′-dichlorofluorescin diacetate (DCFH). Superoxide dismutase or catalase was introduced into the cells using commercially available Hemagglutinating Virus of Japan (HVJ) envelope vector kit. The enzyme activity of superoxide dismutase was also measured. Radiation-induced apoptosis in C3H mouse PRM was significantly suppressed by treatment with a pharmacological scavenger of superoxide anion, Tiron, but not with other radical scavengers. Intracellular peroxide levels were not elevated by irradiation at doses high enough to induce apoptosis maximally. Radiation-induced apoptosis in C3H mouse PRM was markedly suppressed by superoxide dismutase introduced into the cells using the HVJ envelope vector, but not catalase. The enzyme activity of superoxide dismutase in C3H mouse PRM was comparable with that in B6 mouse PRM. It was concluded that superoxide played the major role in radiation-induced apoptosis in the C3H mouse PRM and that cellular responses downstream or unrelated to superoxide might be responsible for the strain difference in radiation-induced apoptosis of mouse PRM.

216. Enhancement of Gene Transfection Both In Vitro and In Vivo Using Polymer Conjugated HVJ-Envelope Vector

Vector development is critical for the advancement of human gene therapy. However, the use of viral vectors raises many safety concerns and most non-viral methods are less efficient for gene transfer. One of the breakthroughs in vector technology is the combination of the vector with various polymers. HVJ (hemagglutinating virus of Japan) envelope vector (HVJ-E) has been developed as a versatile gene transfer vector. In this study, we combined HVJ-E with cationized gelatin(CG) to make it a more powerful tool and assessed its transfection efficiency in vitro and in vivo. CG is one of the most suitable polymers for the complex formation with HVJ-E because of the positive charge and biocompatibility. As shown in Fig. 1, we combined it with HVJ-E and used it in vitro and in vivo experiments. In addition, we investigated the mechanism of the gene transfer by means of the inhibition of fusion or endocytosis. The combination of both protamine sulfate(PS) and CG with HVJ-E, referred to as PS-CG-HVJ-E, further enhanced the in vitro transfection efficiency. In CT26 cells, the luciferase gene expression of PS-CG-HVJ-E was approximately 10 times higher than that of the combination of PS with HVJ-E or the combination of CG with HVJ-E, referred to as PS-HVJ-E or CG-HVJ-E, respectively. In case of in vivo study, we tried intravenous administration at first. In conclusion, the luciferase gene expression in liver mediated by intravenous administration of CG-HVJ-E was much higher than the luciferase gene expression mediated by PS-HVJ-E or PS-CG-HVJ-E and approximately 100 times higher than that mediated by HVJ-E alone. Next, we tried intraperitoneal administration of CG-HVJ-E containing luciferase gene to the peritoneal disseminated colon tumor model mice. As shown in Fig. 2, the luciferase gene expression was specifically detected at tumor nodules. Thus, cationized gelatin-conjugated HVJ-E enhanced gene transfection efficiency both in vitro and in vivo. These results suggest that the conjugation with low molecular weight cationized gelatin may increase in vivo transfection efficiency of various recombinant viral vectors such as retrovirus, herpes virus and lentivirus vector.

452. The Enhancement of Anticancer Effect of Chemotherapy by the Delivery of Rad51 siRNA

Top of pageAbstract Chemotherapy is one of the effective therapies to cancer, however, cancers gradually develop machineries to resist to chemotherapy. To overcome the problem of drug resistance, we tested the combination of short interfering RNA (siRNA) against Rad51, related to the homologous recombination repair, with cis-diamminedichloroplatinum (II) (CDDP; cisplatin). Moreover, to deliver siRNA into cancer cells effectively, we have developed the HVJ (hemagglutinating virus of Japan, Sendai virus) envelope vector which has the high membrane fusion ability. When synthetic Rad51 siRNA was delivered to HeLa cells using HVJ envelope vector, no Rad51 transcripts were detected on day 2, and Rad51 protein completely disappeared for 4 days after siRNA transfer. When HeLa cells were incubated with 0.02 ug/ml CDDP for 3 hours after siRNA transfer, the number of colonies decreased to approximately 10% of that with scrambled siRNA. The sensitivity to CDDP was enhanced in various human cancer cells, but not in normal human fibroblasts. The synthetic Rad51 siRNA was also introduced into subcutaneous tumor mass of HeLa cells in SCID mice with or without intraperitoneal injection of CDDP, and tumor growth was monitored. When Rad51 siRNA was delivered into tumors using HVJ envelope vector, the delivery efficiency was approximately 50%, and the Rad51 transcript level was reduced to approximately 25%. Rad51 siRNA combined with CDDP significantly inhibited the tumor growth when compared to siRNA or CDDP alone. Rad51 siRNA could enhance the sensitivity to CDDP in cancer cells both in vitro and in vivo. Rad51 siRNA also enhanced the sensitivity of cancer cells to bleomycin. Our results suggest that the combination of CDDP and Rad51 siRNA will be an effective anticancer protocol.

Rad51 siRNA delivered by HVJ envelope vector enhances the anti‐cancer effect of cisplatin

Every cancer therapy appears to be transiently effective for cancer regression, but cancers gradually transform to be resistant to the therapy. Cancers also develop machineries to resist chemotherapy. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing a target protein by specifically digesting its mRNA. Suppression of the machineries using siRNA may enhance the sensitivity to chemotherapy in cancers when combined with an effective delivery system. To enhance the anti-cancer effect of chemotherapy, we transferred siRNA against Rad51 into various human cancer cells using the HVJ (hemagglutinating virus of Japan, Sendai virus) envelope vector in the presence or absence of cis-diamminedichloroplatinum(II) (CDDP, cisplatin). The inhibition of cell growth was assessed by a modified MTT assay, counting cell number, or fluorescence-activated cell sorting (FACS) analysis after Annexin V labeling. The synthetic Rad51 siRNA was also introduced into subcutaneous tumor masses of HeLa cells in SCID mice with or without intraperitoneal injection of CDDP, and tumor growth was monitored. When synthetic Rad51 siRNA was delivered into HeLa cells using the HVJ envelope vector, no Rad51 transcripts were detected on day 2, and Rad51 protein completely disappeared for 4 days after siRNA transfer. When HeLa cells were incubated with 0.02 microg/ml CDDP for 3 h after siRNA transfer, the number of colonies decreased to approximately 10% of that with scrambled siRNA. The sensitivity to CDDP was enhanced in various human cancer cells, but not in normal human fibroblasts. When Rad51 siRNA was delivered into tumors using the HVJ envelope vector, the Rad51 transcript level was reduced to approximately 25%. Rad51 siRNA combined with CDDP significantly inhibited tumor growth when compared to siRNA or CDDP alone. Rad51 siRNA could enhance the sensitivity to CDDP in cancer cells both in vitro and in vivo. Our results suggest that the combination of CDDP and Rad51 siRNA will be an effective anti-cancer protocol.

Biocompatible polymer enhances the in vitro and in vivo transfection efficiency of HVJ envelope vector

Vector development is critical for the advancement of human gene therapy. However, the use of viral vectors raises many safety concerns and most non-viral methods are less efficient for gene transfer. One of the breakthroughs in vector technology is the combination of the vector with various polymers. HVJ (hemagglutinating virus of Japan) envelope vector (HVJ-E) has been developed as a versatile gene transfer vector. In this study, we combined HVJ-E with cationized gelatin to make it a more powerful tool and assessed its transfection efficiency in vitro and in vivo. In addition, we investigated the mechanism of the gene transfer by means of the inhibition of fusion or endocytosis. The combination of both protamine sulfate and cationized gelatin with HVJ-E, referred to as PS-CG-HVJ-E, further enhanced the in vitro transfection efficiency. In CT26 cells, the luciferase gene expression of PS-CG-HVJ-E was approximately 10 times higher than that of the combination of protamine sulfate with HVJ-E or the combination of cationized gelatin with HVJ-E, referred to as PS-HVJ-E or CG-HVJ-E, respectively. Furthermore, the luciferase gene expression in liver mediated by intravenous administration of CG-HVJ-E was much higher than the luciferase gene expression mediated by PS-HVJ-E or PS-CG-HVJ-E and approximately 100 times higher than that mediated by HVJ-E alone. Cationized gelatin-conjugated HVJ-E enhanced gene transfection efficiency both in vitro and in vivo. These results suggest that low molecular weight cationized gelatin may be appropriate for complex formation with various envelope viruses, such as retrovirus, herpes virus and HIV.

Direct in vivo protein transduction into a specific restricted brain area in rats

Attempts at protein transduction into specific restricted brain areas have remained unsuccessful. We attempted targeted, direct in vivo protein transduction by microinjecting β-galactosidase (β-gal) with hemagglutinating virus of Japan envelope (HVJ-E) vector into the rat nucleus tractus solitarius (NTS). The medulla oblongata including the NTS was removed 6 h post-injection and cryostat sections were histochemically stained to detect β-gal enzymatic activity. β-gal-positive cells were present in these sections as was β-gal activity determined by colorimetric analysis. β-gal-positive cells were not present in the rats microinjected only β-gal protein without HVJ-E vector. Our findings suggest that direct in vivo protein transduction into specific restricted brain areas is possible. The type of targeted delivery system we present may have wide applications in the administration of therapeutic proteins to the central nervous system.

Local OPG Gene Transfer to Periodontal Tissue Inhibits Orthodontic Tooth Movement

Previously, we discovered that RANKL expression is induced in compressed periodontal ligament cells, and that this promotes osteoclastogenesis on the compression side in orthodontic tooth movement. We hypothesized that local OPG gene transfer to the periodontium would neutralize the RANKL activity induced by mechanical compressive force, thereby inhibiting osteoclastogenesis and diminishing tooth movement. The upper first molars of six-week-old male Wistar rats were moved palatally by means of a fixed-orthodontic wire. A mouse OPG expression plasmid [pcDNA3.1(+)-mOPG] was constructed, and the production of functional OPG protein was confirmed in vitro. The inactivated HVJ envelope vector containing pcDNA3.1(+)-mOPG or PBS was injected periodically into the palatal periodontal tissue of upper first molars. When this local OPG gene transfer was performed, OPG production was induced, and osteoclastogenesis was inhibited. Local OPG gene transfer significantly diminished tooth movement. In this study, we report that OPG gene transfer to periodontal tissue inhibited RANKL-mediated osteoclastogenesis and inhibited experimental tooth movement.

Noncytotoxic ribonuclease, RNase T1, induces tumor cell death via hemagglutinating virus of Japan envelope vector.

Several ribonucleases, including onconase and alpha-sarcin, are known to be toxic to tumor cells. On the other hand, although its structure is related to that of alpha-sarcin, RNase T1 is noncytotoxic because of its inability to internalize into tumor cells. In this study, we internalized RNase T1 into human tumor cells via a novel gene transfer reagent, hemagglutinating virus of Japan (HVJ) envelope vector, which resulted in cell death. This cytotoxicity was drastically increased by pretreatment of HVJ envelope vector with protamine sulfate, and was stronger than that of onconase, which is in phase III human clinical trials as a nonmutagenic cancer chemotherapeutic agent. Furthermore, internalized RNase T1 induced apoptotic cell death programs. Because its cytotoxicity is unfortunately not specific to tumor cells, it cannot at present be developed as an anticancer drug. However, we believe that RNase T1 incorporated in HVJ envelope vector will be a unique anticancer drug if HVJ envelope vector can be targeted to tumor cells.

The HVJ-envelope as an innovative vector system for cardiovascular disease.

Recently promising results of gene therapy clinical trials have been reported for treatment of peripheral vascular and cardiovascular diseases using various angiogenic growth factors and other therapeutic genes. Viral vector and non-viral vector systems were employed in preclinical studies and clinical trials. Adenoviral vector and naked plasmid have been used most in the clinical studies. HVJ (hemagglutinating virus of Japan or Sendai virus)-liposome vector, a hybrid non-viral vector system with fusion of inactivated HVJ virus particle and liposome, has developed and demonstrated high transfection efficiency in preclinical studies of many different disease models, including a wide range of cardiovascular disease models. However, some limitations exist in the HVJ-liposome technology, especially in the scalability of its production. Recently an innovative vector technology, HVJ envelope (HVJ-E) has been developed as a non-viral vector, consisting of HVJ envelope without its viral genome, which is eliminated by a combination of inactivation and purification steps. HVJ-E is able to enclose various molecule entities, including DNA, oligonucleotides, proteins, as single or multiple therapeutic remedies. The therapeutic molecule-included HVJ-E vector can transfect various cell types in animals and humans with high efficiency. In this review, vector technology for cardiovascular disease and the biology of HVJ-E vector technology is discussed.

228. A Novel Therapeutic Strategy To Treat Brain Ischemia: Over-Expression of Hepatocyte Growth Factor Gene Reduced Ischemic Injury without Cerebral Edema in Rat Model

Top of pageAbstract Cerebral occlusive disease caused by atherosclerosis of the cerebral arteries or Moyamoya disease often causes global ischemia of the brain. Although such a condition leads to not only cerebral ischemic events, but also neuropathological changes including dementia, an effective treatment to improve brain ischemic injury has not yet been established. It is known that ischemic stroke induces active angiogenesis, particularly in the ischemic penumbra, which correlates with longer survival in humans. However, the natural course of angiogenesis is not sufficient to compensate for the hypoperfusion state. Recently, a novel therapeutic strategy using genes of angiogenic growth factors and neurotrophic factors has been proposed for the treatment of cerebrovascular disease. In the present study, we examined the effects of hepatocyte growth factor (HGF) in a rat middle cerebral artery occlusion model. First, human HGF gene was transferred into the brain via the cisterna magna using HVJ-envelope vector. The concentration of human HGF and rat HGF in CSF were measured by ELISA at 5 and 12 days after gene transfer. On day 5, as expected, human HGF could be detected in the CSF of rats transfected with human HGF vector, while human HGF protein could not be detected in control rats. The increase in human HGF protein in the CSF continued up to 12 days after transfection. Consistently, up-regulation of c-met was observed in the cerebral cortex as well as the brain stem and cerebellum. Next, the middle cerebral artery was occluded 5 days after HGF gene transfer. Over-expression of the HGF gene resulted in a significant decrease in the infarcted brain area after 24 hours of ischemia, whereas control group showed large infarction (48 ± 3.0 % vs 72 ± 5.9 %, P<0.05). Consistently, the neurological deficit was attenuated in rats transfected with HGF gene (score = 1.0 ± 0.2 in HGF group and 1.4 ± 0.2 in control group, P<0.01). Numerous TUNEL-positive cells were observed in rats transfected with control vector, while a significant decrease in TUNEL positive cells was detected in rats transfected with HGF gene. Stimulation of angiogenesis without destruction of the blood-brain barrier was also detected in rats transfected with the HGF gene as compared to control group (the vessel area = 152.7 ± 6.9 mm2/mm2 vs 95.8 ± 7.7 mm2/mm2, p<0.01). Of importance, no exacerbation of cerebral edema was observed in rats transfected with the HGF gene (water content = 75.9 ± 0.01% in HGF group vs 77.9 ± 0.01% in control group, p<0.01). Overall, HGF gene transfer using HVJ-envelope vector reduced ischemic injury without exacerbation of cerebral edema or BBB leakage. Although further studies on the safety are necessary, gene therapy using HGF may provide new therapeutic options for the treatment of cerebral ischemia. Continuous development of systems involving vectors, promoters or alternative routes of administration may help to achieve human gene therapy for cerebrovascular disease in the future.

Transduction of protein into rat brainstem using the Hemagglutinating virus of Japan-envelope vector in vivo

Transduction of protein into rat brainstem using the Hemagglutinating virus of Japan-envelope vector in vivo

New vector innovation for drug delivery: development of fusigenic non-viral particles.

Efficient and minimally invasive drug delivery systems have been developed to treat intractable human diseases. One approach has been the development of chimeric vector systems combining at least two different vector systems. Based on this concept, chimeric drug delivery systems that combine viral and non-viral features have been developed. Fusigenic non-viral particles have been constructed by conferring viral fusion proteins onto non-viral vectors. HVJ (hemagglutinating virus of Japan; Sendai virus)-liposomes were constructed by the combination of DNA-loaded liposomes with a fusigenic envelope derived from HVJ (hemagglutinating virus of Japan, Sendai virus). Reconstituted HVJ-liposomes were also developed by the insertion of isolated fusion proteins of HVJ into DNA-loaded liposomes. Recently, the technology has been developed to incorporate macromolecules directly into inactivated HVJ particles without liposomes. The resulting HVJ envelope vector introduced plasmid DNA, efficiently and rapidly into both cultured cells in vitro and organs in vivo. Furthermore, proteins, synthetic oligonucleotides and drugs have also been effectively introduced into cells using the HVJ envelope vector. The HVJ envelope vector will be a promising tool for both ex vivo and in vivo gene therapy experiments.

HVJ-envelope vector for gene transfer into central nervous system

To overcome some problems of virus vectors, we developed a novel non-viral vector system, the HVJ-envelope vector (HVJ-E). In this study, we investigated the feasibility of gene transfer into the CNS using the HVJ-E both in vitro and in vivo. Using the Venus reporter gene, fluorescence could be detected in cultured rat cerebral cortex neurons and glial cells. In vivo, the reporter gene (Venus) was successfully transfected into the rat brain by direct injection into the thalamus, intraventricular injection, or intrathecal injection, without inducing immunological change. When the vector was injected after transient occlusion of the middle cerebral artery, fluorescence due to EGFP gene or luciferase activity could be detected only in the injured hemisphere. Finally, luciferase activity was markedly enhanced by the addition of 50 U/ml heparin ( P<0.01). Development of efficient HVJ-E for gene transfer into the CNS will be useful for research and clinical gene therapy.