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PDF file - 316K, Supplementary Fig. S1: Effective depletion of PDCA-1+ cells in mouse splenocytes. Supplementary Fig. S2: Validation of gene expression by quantitative real-time PCR. Supplementary Fig. S3: Effects of TLR ligands on body weight changes in mice treated with subcutaneous injections of LLC/SeV/GM cells. Supplementary Fig. S4: Increased number of TVS-infiltrating PDCA-1+ cells in mice treated with combined irLLC/SeV/GM cells and imiquimod. Supplementary Fig. S5: Increased serum IFN-a level in mice treated with combined irLLC/SeV/GM cells and imiquimod. Supplementary Fig. S6: Prophylactic tumor vaccination using irLLC/SeV/GM cells induced significant antitumor effects in a syngeneic mouse model. Supplementary Fig. S7: Enhanced expression levels of maturation and costimulatory molecules including CCR7, CD40, MHC class I, and MHC class II in tumor cells-derived GM-CSF-sensitized DCs in TDLNs.

Vaccination with irradiated granulocyte macrophage colony-stimulating factor (GM-CSF)–transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity. However, its effectiveness is limited. We therefore attempted to improve the antitumor effect by identifying little-known key pathways in GM-CSF–sensitized dendritic cells (GM-DC) in tumor-draining lymph nodes (TDLN). We initially confirmed that syngeneic mice subcutaneously injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) remarkably rejected the tumor growth. Using cDNA microarrays, we found that expression levels of type I interferon (IFN)–related genes, predominantly expressed in plasmacytoid DCs (pDC), were significantly upregulated in TDLN-derived GM-DCs and focused on pDCs. Indeed, mouse experiments demonstrated that the effective induction of GM-CSF–induced antitumor immunity observed in immunocompetent mice treated with LLC/SeV/GM cells was significantly attenuated when pDC-depleted or IFNα receptor knockout (IFNAR−/−) mice were used. Importantly, in both LLC and CT26 colon cancer–bearing mice, the combinational use of imiquimod with autologous GVAX therapy overcame the refractoriness to GVAX monotherapy accompanied by tolerability. Mechanistically, mice treated with the combined vaccination displayed increased expression levels of CD86, CD9, and Siglec-H, which correlate with an antitumor phenotype, in pDCs, but decreased the ratio of CD4+CD25+FoxP3+ regulatory T cells in TDLNs. Collectively, these findings indicate that the additional use of imiquimod to activate pDCs with type I IFN production, as a positive regulator of T-cell priming, could enhance the immunologic antitumor effects of GVAX therapy, shedding promising light on the understanding and treatment of GM-CSF–based cancer immunotherapy. Cancer Immunol Res; 2(6); 568–80. ©2014 AACR.

A key challenge in pulmonary gene therapy for cystic fibrosis is to provide long-term correction of the genetic defect. This may be achievable by targeting airway epithelial stem/progenitor cells with an integrating vector. Here, we evaluated the ability of a lentiviral vector, derived from the simian immunodeficiency virus and pseudotyped with F and HN envelope proteins from Sendai virus, to transduce progenitor basal cells of the mouse nasal airways. We first transduced basal cell-enriched cultures ex vivo and confirmed efficient transduction of cytokeratin-5 positive cells. We next asked whether progenitor cells could be transduced in vivo. We evaluated the transduction efficiency in mice pretreated by intranasal administration of polidocanol to expose the progenitor cell layer. Compared to control mice, polidocanol treated mice demonstrated a significant increase in the number of transduced basal cells at 3 and 14 days post vector administration. At 14 days, the epithelium of treated mice contained clusters (4 to 8 adjacent cells) of well differentiated ciliated, as well as basal cells suggesting a clonal expansion. These results indicate that our lentiviral vector can transduce progenitor basal cells in vivo, although transduction required denudation of the surface epithelium prior to vector administration.

Islet transplantation, including β-cells, has proven to be effective for diabetes in many recent studies; however, this treatment strategy requires sufficient organ donors. One attractive approach for the generation of β-cells is to utilize the expansion and differentiation of cells from pancreatic stem cells (PSCs), which are closely associated to the β-cells lineage. In this study, we investigated whether important transcription factors (Pdx-1, Ngn3, NeuroD, and MafA) in islet cells could be efficiently transduced into mouse PSCs (mPSCs) using Sendai virus (SeV) vectors and found that the transduced cells were differentiated into insulin-producing pancreatic β-cells. The mPSCs transduced with single transcription factors using SeV vectors could not express the insulin-2 mRNA. When combinations of two transcription factors were transduced using the SeV vectors, including combinations of Pdx-1 + NeuroD, Pdx-1 + MafA, and NeuroD + MafA, the expression of insulin-2 mRNA was low but could be detected. When combinations of three or more transcription factors were transduced using SeV vectors, the expression of insulin-2 mRNA could be detected. In particular, the transduction of the combination of PDX-1, NeuroD, and MafA produced the most effective for the expression of insulin-2 mRNA out of all of the different combinations examined. These data suggest that the transduction of transcription factors using SeV vectors facilitates mPSC differentiation into insulin-producing cells and showed the possibility of regenerating β-cells by using transduced PSCs.

Genetic manipulations with mammalian cells often require introduction of two or more genes that have to be in trans-configuration. However, conventional gene delivery vectors have several limitations, including a limited cloning capacity and a risk of insertional mutagenesis. In this paper, we describe a novel gene expression system that consists of two differently marked HAC vectors containing unique gene loading sites. One HAC, 21HAC, is stably propagated during cell divisions; therefore, it is suitable for complementation of a gene deficiency. The other HAC, tet-O HAC, can be eliminated, providing a unique opportunity for transient gene expression (e.g., for cell reprogramming). Efficiency and accuracy of a novel bi-HAC vector system have been evaluated after loading of two different transgenes into these HACs. Based on analysis of transgenes expression and HACs stability in the proof of principle experiments, the combination of two HAC vectors may provide a powerful tool toward gene and cell therapy.

Summary The use of induced pluripotent stem cells (iPSCs) as an autologous cell source has shed new light on cell replacement therapy with respect to the treatment of numerous hereditary disorders. We focused on the use of iPSCs for cell-based therapy of haemophilia. We generated iPSCs from mesenchymal stem cells that had been isolated from C57BL/6 mice. The mouse iPSCs were generated through the induction of four transcription factor genes Oct3/4, Klf-4, Sox-2 and c-Myc. The derived iPSCs released functional coagulation factor VIII (FVIII) following transduction with a simian immunodeficiency virus vector. The subcutaneous transplantation of iPSCs expressing FVIII into nude mice resulted in teratoma formation, and significantly increased plasma levels of FVIII. The plasma concentration of FVIII was at levels appropriate for human therapy at 2–4 weeks post transplantation. Our data suggest that iPSCs could be an attractive and prospective autologous cell source for the production of coagulation factor, and that engineered iPSCs expressing coagulation factor might provide a cell-based therapeutic strategy appropriate for haemophilia.

Studies of rare genetic bone disorders are often limited due to unavailability of tissue specimens and the lack of animal models fully replicating phenotypic features. Craniometaphyseal dysplasia (CMD) is a rare monogenic disorder characterized by hyperostosis of craniofacial bones concurrent with abnormal shape of long bones. Mutations for autosomal dominant CMD have been identified in the ANK gene (ANKH). Here we describe a simple and efficient method to reprogram adherent cells cultured from peripheral blood to human induced pluripotent stem cells (hiPSCs) from eight CMD patients and five healthy controls. Peripheral blood mononuclear cells (PBMCs) were separated from 5–7 mL of whole blood by Ficoll gradient, expanded in the presence of cytokines and transduced with Sendai virus (SeV) vectors encoding OCT3/4, SOX2, KLF4, and c-MYC. SeV vector, a cytoplasmic RNA vector, is lost from host cells after propagation for 10–13 passages. These hiPSCs express stem cell markers, have normal karyotypes, and are capable of forming embryoid bodies in vitro as well as teratomas in vivo. Further differentiation of these patient-specific iPSCs into osteoblasts and osteoclasts can provide a useful tool to study the effects CMD mutations on bone, and this approach can be applied for disease modeling of other rare genetic musculoskeletal disorders.

Hemophilia is an X-linked bleeding disorder, and patients with hemophilia are deficient in a biologically active coagulation factor. This study was designed to combine the efficiency of lentiviral vector transduction techniques with murine adipose tissue-derived stem/stromal cells (mADSCs) as a new method to produce secreted human coagulation factor IX (hFIX) and to treat hemophilia B. mADSCs were transduced with simian immunodeficiency virus (SIV)-hFIX lentiviral vector at multiplicities of infection (MOIs) from 1 to 60, and the most effective dose was at an MOI of 10, as determined by hFIX production. hFIX protein secretion persisted over the 28-day experimental period. Cell sheets composed of lentiviral vector-transduced mADSCs were engineered to further enhance the usefulness of these cells for future therapeutic applications in transplantation modalities. These experiments demonstrated that genetically transduced ADSCs may become a valuable cell source for establishing cell-based gene therapies for plasma protein deficiencies, such as hemophilia.

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

Summary. Background: Transplantation of cells overexpressing a target protein represents a viable gene therapeutic approach for treating hemophilia. Here, we focused on the use of autologous mesenchymal stem cells (MSCs) expressing coagulation factor for the treatment of coagulation factor VIII (FVIII) deficiency in mice. Methods and Results: Analysis of luciferase gene constructs driven by different promoters revealed that the plasminogen activator inhibitor-1 (PAI-1) gene promoter coupled with the cytomegalovirus promoter enhancer region was one of the most effective promoters for producing the target protein. MSCs transduced with the simian immunodeficiency virus (SIV) vector containing the FVIII gene driven by the PAI-1 promoter expressed FVIII for several months, and this expression was maintained after multiple mesenchymal lineage differentiation. Although intravenous injection of cell supernatant derived from MSCs transduced with an SIV vector containing the FVIII gene driven by the PAI-1 promoter significantly increased plasma FVIII levels, subcutaneous implantation of the MSCs resulted in a transient and weak increase in plasma FVIII levels in FVIII-deficient mice. Interestingly, intra-articular injection of the transduced MSCs significantly ameliorated the hemarthrosis and hemophilic arthropathy induced by knee joint needle puncture in FVIII-deficient mice. The therapeutic effects of a single intra-articular injection of transduced MSCs to inhibit joint bleeding persisted for at least 8 weeks after administration. Conclusions: MSCs provide a promising autologous cell source for the production of coagulation factor. Intra-articular injection of MSCs expressing coagulation factor may offer an attractive treatment approach for hemophilic arthropathy.

The induced pluripotent stem cell (iPSC) technology enables derivation of patient-specific pluripotent stem cells from adult somatic cells without using an embryonic cell source. Redifferentiation of iPSCs from diabetic patients into pancreatic islets will allow patient-specific disease modeling and autologous cell replacement therapy for failing islets. To date, diabetes-specific iPSCs have been generated from patients with type 1 diabetes using integrating retroviral vectors. However, vector integration into the host genome could compromise the biosafety and differentiation propensities of derived iPSCs. Although various integration-free reprogramming systems have been described, their utility to reprogram somatic cells from patients remains largely undetermined. Here, we used nonintegrating Sendai viral vectors to reprogram cells from patients with type 1 and type 2 diabetes (T2D). Sendai vector infection led to reproducible generation of genomic modification-free iPSCs (SV-iPSCs) from patients with diabetes, including an 85-year-old individual with T2D. SV-iPSCs lost the Sendai viral genome and antigens within 8–12 passages while maintaining pluripotency. Genome-wide transcriptome analysis of SV-iPSCs revealed induction of endogenous pluripotency genes and downregulation of genes involved in the oxidative stress response and the INK4/ARF pathways, including p16INK4a, p15INK4b, and p21CIP1. SV-iPSCs and iPSCs made with integrating lentiviral vectors demonstrated remarkable similarities in global gene expression profiles. Thus, the Sendai vector system facilitates reliable reprogramming of patient cells into transgene-free iPSCs, providing a pluripotent platform for personalized diagnostic and therapeutic approaches for diabetes and diabetes-associated complications.

Cytotoxic T lymphocyte (CTL) responses play a central role in viral suppression in human immunodeficiency virus (HIV) infections. Prophylactic vaccination resulting in effective CTL responses after viral exposure would contribute to HIV control. It is important to know how CTL memory induction by vaccination affects postexposure CTL responses. We previously showed vaccine-based control of a simian immunodeficiency virus (SIV) challenge in a group of Burmese rhesus macaques sharing a major histocompatibility complex class I haplotype. Gag 206-216 and Gag 241-249 epitope-specific CTL responses were responsible for this control. In the present study, we show the impact of individual epitope-specific CTL induction by prophylactic vaccination on postexposure CTL responses. In the acute phase after SIV challenge, dominant Gag 206-216 -specific CTL responses with delayed, naive-derived Gag 241-249 -specific CTL induction were observed in Gag 206-216 epitope-vaccinated animals with prophylactic induction of single Gag 206-216 epitope-specific CTL memory, and vice versa in Gag 241-249 epitope-vaccinated animals with single Gag 241-249 epitope-specific CTL induction. Animals with Gag 206-216 -specific CTL induction by vaccination selected for a Gag 206-216 -specific CTL escape mutation by week 5 and showed significantly less decline of plasma viral loads from week 3 to week 5 than in Gag 241-249 epitope-vaccinated animals without escape mutations. Our results present evidence indicating significant influence of prophylactic vaccination on postexposure CTL immunodominance and cooperation of vaccine antigen-specific and non-vaccine antigen-specific CTL responses, which affects virus control. These findings provide great insights into antigen design for CTL-inducing AIDS vaccines.

After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34 + cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.

Human malignant pleural mesothelioma (HMPM) is highly resistant to conventional therapy, and therefore novel therapies are required. We previously reported that overexpression of the FUSE-binding protein-interacting repressor (FIR), a c-myc transcriptional repressor, induces apoptosis via c-Myc suppression, and is thus a suitable cancer therapy. In the current preclinical trial, a fusion gene deleted non-transmissible Sendai virus vector encoding FIR (SeV/ΔF/FIR) was prepared and its cytotoxic activity against an orthotopic xenograft model of HMPM, in combination with cisplatin, was assessed. SeV/ΔF/FIR and a fusion gene deleted non-transmissible Sendai virus vector encoding green fluorescent protein (SeV/ΔF/GFP) were prepared. The transduction efficiency of these agents in terms of dose-dependent cytotoxicity and/or apoptosis induction was then assessed in a few HMPM cells. Combination therapy with SeV/ΔF/FIR plus cisplatin was evaluated in vitro and in a mouse model. SeV/ΔF/FIR significantly reduced cell viability in three HMPM cell lines but was less effective in non-tumor immortalized mesothelial cells. SeV/ΔF/FIR cytotoxicity was partly due to apoptosis induction via c-Myc suppression. In addition, SeV/ΔF/FIR showed synergistic antitumor effects in combination with cisplatin, as was revealed by isobologram analysis in MSTO-211H. Moreover, combination therapy with SeV/ΔF/FIR plus cisplatin demonstrated significant tumor reduction and improvement in survival rate in an animal model. Combination therapy with SeV/ΔF/FIR plus cisplatin has therapeutic potential against HMPM. SeV/ΔF/FIR plus cisplatin will be an attractive modality against HMPM in the future.

Malignant ascitis (MA) is a highly intractable and immunotherapy-resistant state of advanced gastrointestinal and ovarian cancers. Using a murine model of MA with CT26 colon cancer cells, we here determined that the imbalance between the VEGF-A/vascular permeability factor and its decoy receptor, soluble fms-like tryrosine kinase receptor-1 (sFLT-1), was a major cause of MA resistance to dendritic cell (DC)-based immunotherapy. We found that the ratio of VEGF-A/sFLT-1 was increased not only in murine but also in human MA, and F-gene–deleted recombinant Sendai virus (rSeV/dF)-mediated secretion of human sFLT-1 by DCs augmented not only the activity of DCs themselves, but also dramatically improved the survival of tumor-bearing animals associated with enhanced CTL activity and its infiltration to peritoneal tumors. These findings were not seen in immunodeficient mice, indicating that a VEGF-A/sFLT-1 imbalance is critical for determining the antitumor immune response by DC-vaccination therapy against MA. Mol Cancer Ther; 10(3); 540–9. ©2011 AACR.

Dendritic cell (DC)-based immunotherapy has potential for treating infections and malignant tumors, but the functional capacity of DC must be assessed in detail, especially maturation and Ag-specific CTL priming. Recent reports suggest that DC that are provided with continuous maturation signals in vivo after transfer into patients are required to elicit the full DC functions. We demonstrate in this study that the rSendai virus vector (SeV) is a novel and ideal stimulant, providing DC with a continuous maturation signal via viral RNA synthesis in the cytosol, resulting in full maturation of monocyte-derived DC(s). Both RIG-I–dependent cytokine production and CD4 T cell responses to SeV-derived helper Ags are indispensable for overcoming regulatory T cell suppression to prime melanoma Ag recognized by T cell-1–specific CTL in the regulatory T cell abundant setting. DC stimulated via cytokine receptors, or TLRs, do not show these functional features. Therefore, SeV-infected DC have the potential for DC-directed immunotherapy.

Objective The homeobox B4 ( HoxB4 ) gene promotes expansion of hematopoietic stem cells (HSCs). However, frequent development of leukemia in large animals due to retrovirally transduced HoxB4 gene has been reported. To prevent tumorigenesis, we developed a nonintegrating and nonreplicating Sendai virus vector that did not contain the phosphoprotein gene (SeV/ΔP), which enabled clearance of the vector and transgene shortly after transduction. We tested the SeV/ΔP vector expressing the HoxB4 gene (SeV/ΔP/ HoxB4 ) for the ex vivo expansion of human cord blood CD34 + cells (HSCs) using a sheep in utero transplantation assay. Materials and Methods Human HSCs were ex vivo−expanded by transduction with SeV/ΔP/ HoxB4 vector and transplanted into the abdominal cavity of fetal sheep. The engraftment of human HSCs in the lambs was quantitatively evaluated by hematopoietic colony-forming unit assays. Results After transplantation, the HoxB4 -transduced HSCs contributed to longer-period (up to 20 months) repopulation in sheep, and human hematopoietic progenitors were detected more frequently in the bone marrow of the HoxB4 group as compared with the control untreated group ( p HoxB4 vector was comparable with previously reported retroviral vectors expressing HoxB4. The SeV/ΔP/ HoxB4 vector and the transgene were cleared from the recipient sheep and leukemia was not detected at 20 months post-transplantation. Conclusions The SeV/ΔP vector would be suitable for transient expression of HoxB4 in human CD34 + cells. In addition, the SeV/ΔP vector is free of concern about transgene-related and insertional leukemogenesis and should be safer than retroviral vectors.

Sendai virus (SeV) is an enveloped virus with a non-segmented negative-strand RNA genome. SeV envelope fusion (F) glycoproteins play crucial roles in the viral life cycle in processes such as viral binding, assembly, and budding. In this study, we developed a viable recombinant SeV designated F-EGFP SeV/ΔF, in which the F protein was replaced by an F protein fused to EGFP at the carboxyl terminus. Living infected cells of the recombinant virus were directly visualized by green fluorescence. The addition of EGFP to the F protein maintained the activities of the F protein in terms of intracellular transport to the plasma membrane via the ER and the Golgi apparatus and fusion activity in the infected cells. These results suggest that this fluorescent SeV is a useful tool for studying the viral binding, assembly, and budding mechanisms of F proteins and the SeV life cycle in living infected cells.

We recently demonstrated highly efficient antitumor immunity against dermal tumors of B16F10 murine melanoma with the use of dendritic cells (DCs) activated by replication-competent, as well as nontransmissible-type, recombinant Sendai viruses (rSeV), and proposed a new concept, “immunostimulatory virotherapy,” for cancer immunotherapy. However, there has been little information on the efficacies of thismethod: 1) inmore clinically relevant situations including metastatic diseases, 2) on other tumor types and other animal species, and 3) on the related molecular/cellular mechanisms. In this study, therefore, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV on a rat model of lung metastasis using a highly malignant subline of Dunning R-3327 prostate cancer, AT6.3. rSeV/dF-green fluorescent protein (GFP)-activated bone marrow-derived DCs (rSeV/dF-GFP-DC), consistent with results previously observed in murine DCs. Vaccination of rSeV/dF-GFP-DC was highly effective at preventing lung metastasis after intravenous loading of R-3327 tumor cells, compared with the effects observed with immature DCs or lipopolysaccharide-activated DCs. Interestingly, neither CTL activity nor DC trafficking showed any apparent difference among groups. Notably, rSeV/dF-DCs expressing a dominant-negative mutant of retinoic acid-inducible gene I (RIG-I) (rSeV/dF-RIGIC-DC), an RNA helicase that recognizes the rSeV genome for inducing type I interferons, largely lost the expression of proinflammatory cytokines without any impairment of antitumor activity. These results indicate the essential role of RIG-I-independent signaling on antimetastatic effect induced by rSeV-activated DCs and may provide important insights to DC-based immunotherapy for advanced malignancies.

We have previously shown that recombinant Sendai virus (SeV) vector, derived from murine parainfluenza virus, is one of the most efficient vectors for airway gene transfer. We have also shown that SeV-mediated transfection on second administration, although reduced by 60% when compared with levels achieved after a single dose, is still high because of the efficient transfection achieved by SeV vector in murine airways. Here, we show that these levels further decrease on subsequent doses. In addition, we validated SeV vector repeat administration in a non-natural host model, the sheep. As part of these studies we first assessed viral stability in a Pari LC Plus nebuliser, a polyethylene catheter (PEC) and the Trudell AeroProbe. We also compared the distribution of gene expression after PEC and Trudell AeroProbe administration and quantified virus shedding after sheep transduction. In addition, we show that bronchial brushings and biopsies, collected in anaesthetized sheep, can be used to assess SeV-mediated gene expression over time. Similar to mice, gene expression in sheep was transient and had returned to baseline values by day 14. In conclusion, the SeV vector should be strongly considered for lung-related applications requiring a single administration of the vector even though it might not be suitable for diseases requiring repeat administration.

Sendai virus (SeV) vectors can efficiently introduce foreign genes without toxicity into various organs and are expected to be clinically applicable. We previously compared the transfectional efficiency of SeV and adenovirus (AdV) vectors by assessing the transfer of the green fluorescent protein (GFP) gene to pancreatic stem cells. Although the gene transfer efficiency was similar between these vectors, SeV vector had a lower toxicity in comparison to the AdV vector. In this study, we assessed the gene transfer efficiency of SeV vector in the floating state to pancreatic stem cells. The efficiency of gene transfer was much higher at all time points and at all concentrations in the floating state versus in the adhesion state. In addition, the pancreatic stem cells transfected with SeV in the floating state maintained their differentiation ability. These data suggest that SeV transfection to pancreatic stem cells in the floating state may be useful in gene transfer technology.

BACKGROUND: Carbon-ion beam (CIB) treatment is a powerful tool for controlling primary tumors in the clinical setting. However, to date, few clinical or experimental studies have investigated the effects of CIB treatment on tumor recurrence and antitumor immunity. METHODS: A multiple challenge test was performed using syngenic and nude mouse models of a poorly immunogenic squamous cell carcinoma cell line (SCCVII) after CIB treatment at a clinically available dose (77 kiloelectron volts [keV]/μm) to primary tumors. To further examine changes in antitumor immunity in this model, the authors used dendritic cell (DC)-based immunotherapy. RESULTS: In a syngenic model, CIB treatment itself resulted not only in efficient elimination of the primary tumor but also in a dramatic reduction of tumor formation after secondary tumor challenge at a contralateral site (P < .0001). Conversely, CIB treatment eliminated neither the primary nor the secondary tumor in nude mice. This antitumor effect produced by CIB treatment was enhanced significantly by combining it with DC immunotherapy (P = .0007). Combined CIB and DC treatment induced more intense cytolytic activity than CIB in a chromium-release assay. The third challenge tests, which included challenge with a third-party tumor cell line (FM3A) and effector depletion, revealed that the antitumor effects were the results of tumor-specific, long-lasting antitumor immunity through CD8-positive T lymphocytes. CONCLUSIONS: To the authors' knowledge, this is the first demonstration of strong antitumor immunity induced by CIB treatment in a dermal tumor, and this effect was enhanced by combining it with DC-based immunotherapy. The authors concluded that this combination warrants further investigation as a promising modality for the prevention of tumor recurrence. Cancer 2010. © 2010 American Cancer Society.

Sendai viral vector (SeV) is emerging as a promising vector for gene therapy. However, little information is available regarding the combination of SeV-mediated gene and mesenchymal stem cell (MSC) therapy in dealing with ischemic diseases. In this study, we infected SeV to the MSCs in vitro; and injected MSCs modified with SeV harboring human angiopoietin-1 gene (SeVhAng-1) into the ischemic limb of rats in vivo. We found SeV had high transductive efficiency to the MSCs. Both MSCs and SeVhAng-1-modified MSCs improved the blood flow recovery and increased the capillary density of the ischemic limb, compared with the control. However, in contrast to MSCs, SeVhAng-1-modified MSCs had a better improvement of blood flow recovery in the ischemic limb. We further found the ischemic limb injected with SeVhAng-1-modified MSCs had strong expression of p-Akt, which improved survival of MSCs injected into the ischemic limb. This indicated SeVhAng-1 modification enhanced angiogenetic effect of MSCs by both angiogenesis and cell protection. We conclude that SeVhAng-1-modified MSCs may serve as a more effective tool in dealing with ischemic limb disease.

Emerging evidence indicates that the tight communication between vascular endothelial cells and mural cells using platelet-derived growth factor (PDGF)-BB is essential for capillary stabilization during the angiogenic process. However, little is known about the related regulator that determines PDGF-BB expression. Using murine models of therapeutic neovascularization, we here show that a typical lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, is an essential regulator determining PDGF-BB expression for vascular stabilization via a paracrine mode of action. The blockade of VEGF type 3 receptor (VEGFR3) using neutralizing antibody AFL-4 abrogated FGF-2-mediated limb salvage and blood flow recovery in severely ischemic hindlimb. Interestingly, inhibition of VEGFR3 activity not only diminished lymphangiogenesis, but induced marked dilatation of capillary vessels, showing mural cell dissociation. In these mice, VEGF-C and PDGF-B were upregulated in the later phase after induced ischemia, on day 7, when exogenous FGF-2 expression had already declined, and blockade of VEGFR3 or PDGF-BB activities diminished PDGF-B or VEGF-C expression, respectively. These results clearly indicate that VEGF-C is a critical mediator, not only for lymphangiogenesis, but also for capillary stabilization, the essential molecular mechanism of communication between endothelial cells and mural cells during neovascularization.

Chagas' disease, caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), is intractable showing a high mortality rate, and the development of effective vaccines is much desired. To examine the efficacy of a new mode of recombinant viral vaccine, we constructed two non-transmissible Sendai viruses (rSeV/dF) encoding the full-length parasite antigen amastigote surface protein-2 (ASP2) or ASP2 fused with a mono-ubiquitin on its N-terminus (UASP2). C57BL/6 mice immunized intranasally with rSeV/dF expressing either ASP2 or UASP2 showed significantly suppressed parasitemia and could be protected from lethal T. cruzi challenge. Depletion of CD8(+) T cells around the time of infection with T. cruzi completely abolished this protection, confirming that acquired immunity against the infection of T. cruzi is dependent on CD8(+) T cells. We also demonstrated that the protective immunity correlated with higher secretion of interferon-gamma (IFN-gamma) by spleen cells on in vitro-specific or non-specific stimulation. Increased CTL activity was also confirmed by degranulation or CTL assays. Interestingly, the control virus, rSeV/dF-GFP, induced even a higher IFN-gamma production from spleen cells following non-specific but not specific stimulation in vitro, suggesting that SeV may also be a good adjuvant when used as a vaccine vehicle. Taking together, the current findings indicate that recombinant Sendai virus expressing the ASP2 or UASP2 antigens of T. cruzi are interesting candidates for the development of a new mode of recombinant viral vaccine against Chagas' disease.

We recently demonstrated efficient antitumor immunity against murine tumors using dendritic cells (DCs) activated by recombinant Sendai viruses (rSeVs), and proposed a new concept, “immunostimulatory virotherapy,” for cancer immunotherapy. However, there has been little information on the efficacy of this method in preventing metastatic diseases. In this study, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV (rSeV/dF) using a murine model of lung metastasis. Bolus and i.v. administration of DCs harboring rSeV/dF-expressing GFP without pulsation of tumor Ag (DC-rSeV/dF-GFP) 2 days before tumor inoculation showed efficient prevention against lung metastasis of c1300 neuroblastoma, but not of RM-9 prostatic cancer. We found that the timing of DC therapy was critical for the inhibition of pulmonary metastasis of RM-9, and that the optimal effect of DCs was seen 28 days before tumor inoculation. Interestingly, the antimetastatic effect was sustained for over 3 mo, even when administered DCs were already cleared from the lung and organs related to the immune system. Although NK cell activity had already declined to baseline at the time of tumor inoculation, Ab-mediated depletion studies revealed that CD4+ cells as well as the presence of, but not the activation of, NK cells were crucial to the prevention of lung metastasis. These results are the first demonstration of efficient inhibition of lung metastasis via bolus administration of virally activated DCs that was sustained and NK/CD4+ cell-dependent, and may suggest a potentially new mechanism of DC-based immunotherapy for advanced malignancies.

Efficient gene transfer into stem cells is essential for the basic research and for therapeutic applications in gene-modified regenerative medicine. Adenovirus (AdV) vectors, one of the most commonly used types of vectors, can mediate high, albeit transient, levels of expression of the transgene in pancreatic stem/progenitor cells. However, high multiplicity of infection (MOI) with AdV vectors can result in cellular toxicity. Therefore, AdV vectors have been of limited usefulness in clinical applications. In this study, we investigated the in vitro gene transfer efficiency of Sendai virus (SeV) vectors, a paramyxovirus vector that can efficiently introduce foreign genes without toxicity into several cell types, including pancreatic stem cells. The dose-dependent GFP expression of pancreatic stem cells transfected with SeV vectors after 48 h of culture at 37°C was observed. The transfection of pancreatic stem cells with SeV vectors and AdV vectors results in equal expression of the transgene (GFP expression) in the cells after 48 h of culture at 37°C. Although the transfection of pancreatic stem cells with AdV vectors at high MOIs was cytotoxic, transfection with SeV vectors at high MOIs was rarely cytotoxic. In addition, pancreatic stem cells transfected with SeV maintained their differentiation ability. These data suggest that SeV could provide advantages with respect to safety issues in gene-modified regenerative medicine.

Induced pluripotent stem cells (iPSC) have been generated from somatic cells by introducing reprogramming factors. Integration of foreign genes into the host genome is a technical hurdle for the clinical application. Here, we show that Sendai virus (SeV), an RNA virus and carries no risk of altering host genome, is an efficient solution for generating safe iPSC. Sendai-viral human iPSC expressed pluripotency genes, showed demethylation characteristic of reprogrammed cells. SeV-derived transgenes were decreased during cell division. Moreover, viruses were able to be easily removed by antibody-mediated negative selection utilizing cell surface marker HN that is expressed on SeV-infected cells. Viral-free iPSC differentiated to mature cells of the three embryonic germ layers in vivo and in vitro including beating cardiomyocytes, neurons, bone and pancreatic cells. Our data demonstrated that highly-efficient, non-integrating SeV-based vector system provides a critical solution for reprogramming somatic cells and will accelerate the clinical application.(Communicated by Kumao TOYOSHIMA, M.J.A.)

We previously reported the development of a prototype 'oncolytic Sendai virus (SeV) vector' formed by introducing two major genomic modifications to the original SeV, namely deletion of the matrix (M) gene to avoid budding of secondary viral particles and manipulation of the trypsin-dependent cleavage site of the fusion (F) gene to generate protease-specific sequences. As a result, the 'oncolytic SeV' that was susceptible to matrix metalloproteinases (MMPs) was shown to selectively kill MMP-expressing tumors through syncytium formation in vitro and in vivo. However, its efficacy has been relatively limited because of the requirement of higher expression of MMPs and smaller populations of MMP-expressing tumors. To overcome these limitations, we have designed an optimized and dramatically powerful oncolytic SeV vector. Truncation of 14-amino acid residues of the cytoplasmic domain of F protein resulted in dramatic enhancement of cell-killing activities of oncolytic SeV, and the combination with replacement of the trypsin cleavage site with the new urokinase type plasminogen activator (uPA)-sensitive sequence (SGRS) led a variety of human tumors, including prostate (PC-3), renal (CAKI-I), pancreatic (BxPC3) and lung (PC14) cancers, to extensive death through massive cell-to-cell spreading without significant dissemination to the surrounding noncancerous tissue in vivo. These results indicate a dramatic improvement of antitumor activity; therefore, extensive utility of the newly designed uPA-targeted oncolytic SeV has significant potential for treating patients bearing urokinase-expressing cancers in clinical settings.

Photoreceptor apoptosis is a critical process of retinal degeneration in retinitis pigmentosa (RP), a group of retinal degenerative diseases that result from rod and cone photoreceptor cell death and represent a major cause of adult blindness. We previously demonstrated the efficient prevention of photoreceptor apoptosis by intraocular gene transfer of pigment epithelium-derived factor (PEDF) in animal models of RP; however, the underlying mechanism of the neuroprotective activity of PEDF remains elusive. In this study, we show that an apoptosis-inducing factor (AIF)-related pathway is an essential target of PEDF-mediated neuroprotection. PEDF rescued serum starvation-induced apoptosis, which is mediated by AIF but not by caspases, of R28 cells derived from the rat retina by preventing translocation of AIF into the nucleus. Nuclear translocation of AIF was also observed in the apoptotic photoreceptors of Royal College of Surgeons rats, a well-known animal model of RP that carries a mutation of the Mertk gene. Lentivirus-mediated retinal gene transfer of PEDF prevented the nuclear translocation of AIF in vivo, resulting in the inhibition of the apoptotic loss of their photoreceptors in association with up-regulated Bcl-2 expression, which mediates the mitochondrial release of AIF. These findings clearly demonstrate that AIF is an essential executioner of photoreceptor apoptosis in inherited retinal degeneration and provide a therapeutic rationale for PEDF-mediated neuroprotective gene therapy for individuals with RP.

Sendai virus (SeV) vectors can introduce foreign genes efficiently and stably into primate embryonic stem (ES) cells. For the application of these cells, the control of transgene expression is important. Cynomolgus ES cells transduced with a SeV vector expressing the green fluorescent protein (GFP) gene were propagated in Knockout serum replacement (KSR)-supplemented medium, used widely for the serum-free culture of ES cells, and growth and transgene expression were evaluated. The SeV vector-mediated GFP expression was suppressed in the KSR-supplemented medium, although it was stable in regular fetal bovine serum (FBS)-supplemented medium. Propagation in the KSR-supplemented medium eventually resulted in a complete suppression of GFP expression and eradication of the SeV genome. The inhibitory effect of KSR on the transduction was attributable to the positive selection of untransduced ES cells in addition to the removal of the SeV vector from transduced cells. KSR also reduced the efficiency of the transduction. SeV vector-mediated transgene expression in ES cells was suppressed in the KSR-supplemented medium. Although the suppression is limited in specified cells such as ES cells, these findings will help elucidate how to control transgene expression.

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.

A current promising AIDS vaccine strategy is to elicit CD8(+) cytotoxic T lymphocyte (CTL) responses that broadly recognize highly-diversified HIVs. In our previous vaccine trial eliciting simian immunodeficiency virus (SIV) mac239 Gag-specific CTL responses, a group of Burmese rhesus macaques possessing a major histocompatibility complex haplotype 90-120-Ia have shown vaccine-based viral control against a homologous SIVmac239 challenge. Vaccine-induced Gag(206-216) epitope-specific CTL responses exerted strong selective pressure on the virus in this control. Here, we have evaluated in vivo efficacy of vaccine-induced Gag(206-216)-specific CTL responses in two 90-120-Ia-positive macaques against challenge with a heterologous SIVsmE543-3 that has the same Gag(206-216) epitope sequence with SIVmac239. Despite efficient Gag(206-216)-specific CTL induction by vaccination, both vaccinees failed to control SIVsmE543-3 replication and neither of them showed mutations within the Gag(206-216) epitope. Further analysis indicated that Gag(206-216)-specific CTLs failed to show responses against SIVsmE543-3 infection due to a change from aspartate to glutamate at Gag residue 205 immediately preceding the amino terminus of Gag(206-216) epitope. Our results suggest that even vaccine-induced CTL efficacy can be abrogated by a single amino acid change in viral epitope flanking region, underlining the influence of viral epitope flanking sequences on CTL-based AIDS vaccine efficacy.

Background Recombinant Sendai virus vectors (rSeV) constitute a new class of cytoplasmic RNA vectors that have shown efficient gene transfer in various organs, including retinal tissue; however, the related immune responses remain to be overcome in view of clinical applications. We recently developed a novel rSeV from which all envelope-related genes were deleted (rSeV/dFdMdHN) and, in the present study, assess host immune responses following retinal gene transfer. Methods rSeV/dFdMdHN or conventional F-gene deleted rSeV (rSeV/dF) was injected into subretinal space of adult Wistar rats or C57BL/6 mice. The transgene expression and histopathological findings were assessed at various time points. Immunological assessments, including the expression of proinflammatory cytokines, natural killer (NK)-cell activity, as well as SeV-specific cytotoxic T lymphocytes (CTLs) and antibodies, were performed following vector injection. Results rSeV/dFdMdHN showed high gene transfer efficiency into the retinal pigment epithelium at an equivalent level to that seen with rSeV/dF. In the early phase, the upregulation of proinflammatory cytokines, local inflammatory cell infiltration and tissue damage that were all prominently seen in rSeV/dF injection were dramatically diminished using rSeV/dFdMdHN. NK cell activity was also decreased, indicating a reduction of the innate immune response. In the later phase, on the other hand, CTL activity and anti-SeV antibodies were similarly induced, even using rSeV/dFdMdHN, and resulted in transient transgene expression in both vector types. Conclusions Deletion of envelope-related genes of rSeV dramatically reduces the vector-induced retinal damage and may extend the utility for ocular gene transfer; however, further studies regulating the acquired immune response are required to achieve long-term transgene expression of rSeV. Copyright © 2007 John Wiley & Sons, Ltd.

Sendai virus (SeV) vector has been shown to efficiently transduce airway epithelial cells. As a precursor to the potential use of this vector for cystic fibrosis (CF) gene therapy, the correct maturation of the SeV vector-derived CF transmembrane conductance regulator (CFTR) protein was examined using biochemical and functional analyses. We constructed a recombinant SeV vector, based on the fusion (F) gene-deleted non-transmissible SeV vector, carrying the GFP-CFTR gene in which the N terminus of CFTR was fused to green fluorescence protein (GFP). This vector was recovered and propagated to high titers in the packaging cell line. Western blotting using an anti-GFP antibody detected both the fully glycosylated (mature) and the core-glycosylated (immature) proteins, indicating that SeV vector-derived GFP-CFTR was similar to endogenous CFTR. We also confirmed the functional channel activity of GFP-CFTR in an iodide efflux assay. The efficient expression of GFP-CFTR, and its apical surface localization, were observed in both MDCK cells in vitro, and in the nasal epithelium of mice in vivo. We concluded that recombinant SeV vector, a cytoplasmically maintained RNA vector, is able to direct production of a correctly localized, mature form of CFTR, suggesting the value of this vector for studies of CF gene therapy.

Recent clinical studies revealed 'proof of concept' of gene therapy targeting hematopoietic stem cells (HSCs) to treat hematopoietic disorders. However, vector integration-related adverse events of retroviral vectors have slowed progress in this field. As an initial step to overcoming this hurdle, we examined the potential of an improved cytoplasmic RNA vector, temperature-sensitive mutant non-transmissible recombinant Sendai virus (ts-rSeV/dF), for gene transfer to murine HSCs and progenitors. Both conventional vector and ts-rSeV/dF-GFP showed efficient gene transfer to T-lymphocyte-depleted syngeneic bone marrow cells (BMCs) (>85%), but only BMCs treated with ts-rSeV/dF-GFP but not with conventional vector efficiently repopulated in the recipient mice, associated with multilineage differentiation in vitro and in vivo. To our knowledge, this is the first demonstration of the in vivo reconstruction of hematopoietic series by cytoplasmically gene transferred BMCs, that warrants further investigation to realize this strategy in clinical settings.

We demonstrated previously that the additive-type recombinant Sendai virus (rSeV) is highly efficient for use in pulmonary gene transfer; however, rSeV exhibits inflammatory responses. To overcome this problem, we tested newly developed non-transmissible constructs, namely, temperature-sensitive F-deleted vector, rSeV/dF (ts-rSeV/dF) and a rSeV with all the envelope-related genes deleted (rSeV/dFdMdHN), for pulmonary gene transfer in neonatal mice, by assessing their toxicity and immune responses. The gene expression in the lungs of neonatal ICR mice peaked on day 2, then gradually decreased until almost disappearing at 14 days after infection in all constructs. Loss of body weight and mortality rate, however, were dramatically improved in mice treated with SeV/dFdMdHN (mortality=0%, n=41) and ts-rSeV/dF (24.2%, n=33) compared with additive rSeV (70.7%, n=58). Although the deletion of envelope-related genes of SeV had a small impact on the production of antibody and cytotoxic T-lymphocyte activity in both adults and neonates, a dramatic reduction was found in the events related to innate responses, including the production of proinflammatory cytokines, particularly in the case of neonates. These results indicate that pulmonary gene transfer using SeV/dFdMdHN warrants further investigation for its possible use in developing safer therapeutics for neonatal lung diseases, including cystic fibrosis.