Noriyuki Kasahara

University of Miami, كورال غيبلز، فلوريدا, Florida, United States

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Publications (132)592.32 Total impact

  • Source
    Shuji Kubo, Misato Takagi-Kimura, Noriyuki Kasahara
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    ABSTRACT: Anti-angiogenic gene therapy represents a promising strategy for cancer; however, it has rarely been tested in malignant mesothelioma, a highly aggressive tumor associated with asbestos with poor prognosis. In the present study, we investigated whether anti-angiogenic factors such as angiostatin, endostatin and the soluble form of vascular endothelial growth factor receptor 2 (sFlk1) were able to inhibit endothelial cell proliferation via lentivirus-mediated gene transfer into malignant mesothelioma cells in culture. We also assessed whether a dual-agent strategy had greater therapeutic benefit. Human malignant pleural mesothelioma MSTO-211H cells were transduced using lentiviral vectors that individually expressed angiostatin, endostatin and sFlk1 and linked to enhanced green fluorescent protein (EGFP) marker gene expression via an internal ribosome entry site. The lentivirus expressing EGFP alone was used as a control. The resultant cells designated as MSTO-A, MSTO-E, MSTO-F and MSTO-C were confirmed by western blot analysis and fluorescence microscopy to stably express the corresponding proteins. No differences were observed in the in vitro growth rates between any of these cells. However, co-culture of MSTO-A, MSTO-E and MSTO-F showed significant suppression of human umbilical endothelial cell growth in vitro compared with that of MSTO-C. Furthermore, a combination of any two among MSTO-A, MSTO-E and MSTO-F significantly enhanced efficacy. These results suggest that combinatorial anti-angiogenic gene therapy targeting different pathways of endothelial growth factor signaling has the potential for greater therapeutic efficacy than that of a single-agent regimen.
    Oncology Reports 06/2015; DOI:10.3892/or.2015.4058 · 2.19 Impact Factor
  • Cytotherapy 06/2015; 17(6):S51-S52. DOI:10.1016/j.jcyt.2015.03.484 · 3.10 Impact Factor
  • Cytotherapy 06/2015; 17(6):S18. DOI:10.1016/j.jcyt.2015.03.363 · 3.10 Impact Factor
  • Cytotherapy 06/2015; 17(6):S52. DOI:10.1016/j.jcyt.2015.03.486 · 3.10 Impact Factor
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    ABSTRACT: Despite advances in screening, colorectal cancer (CRC) remains the fourth most commonly diagnosed cancer and the second leading cause of cancer death for both men and women in the US. Approximately one half of patients with CRC develop liver metastases (mCRC). The standard treatment for mCRC is 5-fluorouracil (5-FU) based combination chemotherapy. 5-FU combination chemotherapy has extended the median survival of these patients from 6 to >20 months. We are pursuing a unique investigational approach to treat cancer via in situ production of 5-FU. Toca 511 (vocimagene amiretrorepvec), a retroviral replicating vector (RRV), selectively replicates and spreads in malignant cells and encodes an optimized yeast cytosine deaminase (CD) gene. Within infected cells, the CD enzyme is expressed and converts 5-FC (flucytosine, an orally available anti-fungal drug) to the anti-cancer drug 5-FU. Both a direct cytotoxic effect and an extended immunotherapeutic effect have been reported using this approach. Toca 511, in conjunction with subsequent oral extended-release 5-fluorocytosine (Toca FC), is currently under investigation in patients with recurrent high grade glioma. In these studies, Toca 511 is delivered either intratumorally (NCT01156584), by injection into the surgical resection bed (NCT01470794), or intravenously (NCT01985256) in subjects scheduled for subsequent resection. We tested the suitability of the intravenous approach for the treatment of mCRC in a mouse syngeneic liver metastasis model. CT-26-luciferase colon carcinoma cells were delivered via intrasplenic injection producing multiple tumor foci within the liver. Intravenous delivery of RRV resulted in expression of the vector encoded transgene in tumor foci but not in adjacent normal liver tissue. Intravenous delivery of Toca 511 followed by courses of 5-FC resulted in shrinkage or elimination of tumor foci and improved survival in this model of mCRC. The data is supportive of future clinical trials of intravenous Toca 511 followed by cycles of Toca FC in metastatic CRC.
    ASGCT 2015, New Orleans, LA; 05/2015
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    ABSTRACT: The aim of the present study was to enhance the efficiency of leukemia immunotherapy by increasing the antigen‑specific cytotoxic T lymphocyte‑inducing ability of leukemia cells. The leukemic plasmacytoid dendritic cell line PMDC05 containing the HLA‑A02/24 antigen, which was previously established in our laboratory (Laboratory of Hematology and Oncology, Graduate School of Health Sciences, Niigata University, Niigata, Japan), was used in the present study. It exhibited higher expression levels of CD80 following transduction with lentiviruses encoding the CD80 gene. This CD80‑expressing PMDC05 was named PMDC11. In order to establish a more potent antigen‑presenting cell for cellular immunotherapy of tumors or severe infections, PMDC11 cells were transduced with a constitutively active (ca) toll‑like receptor 4 (TLR4) gene using the Tet‑On system (caTLR4‑PMDC11). CD8+ T cells from healthy donors with HLA‑A02 were co‑cultured with mutant WT1 peptide‑pulsed PMDC11, lipopolysaccharide (LPS)‑stimulated PMDC11 or caTLR4‑PMDC11 cells. Interleukin (IL)‑2 (50 IU/ml) and IL‑7 (10 ng/ml) were added on day three of culture. Priming with mutant WT1 peptide‑pulsed PMDC11, LPS‑stimulated PMDC11 or caTLR4‑PMDC11 cells was conducted once per week and two thirds of the IL‑2/IL‑7 containing medium was replenished every 3‑4 days. Immediately prior to the priming with these various PMDC11 cells, the cultured cells were analyzed for the secretion of interferon (IFN)‑γ in addition to the percentage and number of CD8+/WT1 tetramer+ T cells using flow cytometry. caTLR4‑PMDC11 cells were observed to possess greater antigen‑presenting abilities compared with those of PMDC11 or LPS‑stimulated PMDC11 cells in a mixed leukocyte culture. CD8 T cells positive for the WT1 tetramer were generated following 3‑4 weeks of culture and CD8+/WT1 tetramer+ T cells were markedly increased in caTLR4‑PMDC11‑primed CD8+ T cell culture compared with PMDC11 or LPS‑stimulated PMDC11‑primed CD8+ T cell culture. These CD8+ T cells co‑cultured with caTLR4‑PMDC11 cells were demonstrated to secrete IFN‑γ and to be cytotoxic to WT1‑expressing target cells. These data suggested that the antigen‑specific cytotoxic T lymphocyte (CTL)‑inducing ability of PMDC11 was potentiated via transduction of the caTLR4 gene. The present study also suggested that caTLR4‑PMDC11 cells may be applied as potent antigen‑presenting cells for generating antigen‑specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infections.
    Molecular Medicine Reports 04/2015; DOI:10.3892/mmr.2015.3685 · 1.48 Impact Factor
  • Article: LBP12
  • Article: LBP14
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    ABSTRACT: Autologous human induced pluripotent stem cells (hiPSCs) should allow cellular therapeutics without an associated immune response. This concept has been controversial since the original report that syngeneic mouse iPSCs elicited an immune response after transplantation. However, an investigative analysis of any potential acute immune responses in hiPSCs and their derivatives has yet to be conducted. In the present study, we used correlative gene expression analysis of two putative mouse "immunogenicity" genes, ZG16 and HORMAD1, to assay their human homologous expression levels in human pluripotent stem cells and their derivatives. We found that ZG16 expression is heterogeneous across multiple human embryonic stem cell and hiPSC-derived cell types. Additionally, ectopic expression of ZG16 in antigen-presenting cells is insufficient to trigger a detectable response in a peripheral blood mononuclear cell coculture assay. Neither of the previous immunogenicity-associated genes in the mouse currently appears to be relevant in a human context. ©AlphaMed Press.
    STEM CELLS TRANSLATIONAL MEDICINE 01/2015; 4(2). DOI:10.5966/sctm.2014-0117 · 3.60 Impact Factor
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    ABSTRACT: We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.
    Journal of Visualized Experiments 01/2015; DOI:10.3791/52416
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    ABSTRACT: Toca 511 (vocimagene amiretrorepvec), a nonlytic, amphotropic retroviral replicating vector (RRV), encodes and delivers a functionally optimized yeast cytosine deaminase (CD) gene to tumors. In orthotopic glioma models treated with Toca 511 and 5-fluorocytosine (5-FC) the CD enzyme within infected cells converts 5-FC to 5-fluorouracil (5-FU), resulting in tumor killing. Toca 511, delivered locally either by intratumoral injection or by injection into the resection bed, in combination with subsequent oral extended-release 5-FC (Toca FC), is under clinical investigation in patients with recurrent high-grade glioma (HGG). If feasible, intravenous administration of vectors is less invasive, can easily be repeated if desired, and may be applicable to other tumor types. Here, we present preclinical data that support the development of an intravenous administration protocol. First we show that intravenous administration of Toca 511 in a preclinical model did not lead to widespread or uncontrolled replication of the RVV. No, or low, viral DNA was found in the blood and most of the tissues examined 180 days after Toca 511 administration. We also show that RRV administered intravenously leads to efficient infection and spread of the vector carrying the green fluorescent protein (GFP)-encoding gene (Toca GFP) through tumors in both immune-competent and immune-compromised animal models. However, initial vector localization within the tumor appeared to depend on the mode of administration. Long-term survival was observed in immune-competent mice when Toca 511 was administered intravenously or intracranially in combination with 5-FC treatment, and this combination was well tolerated in the preclinical models. Enhanced survival could also be achieved in animals with preexisting immune response to vector, supporting the potential for repeated administration. On the basis of these and other supporting data, a clinical trial investigating intravenous administration of Toca 511 in patients with recurrent HGG is currently open and enrolling.
    Human Gene Therapy 11/2014; 26(2):150127063138006. DOI:10.1089/hum.2014.100 · 3.62 Impact Factor
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    ABSTRACT: Cellular immunity is pivotal in HIV-1 pathogenesis, but hampered by viral sequence diversity. An approach to minimize this diversity is to focus immunity on conserved proteome sequences; therefore we selected four relatively conserved regions (Gag amino acids 148-214 and 250-335, Env 521-606, and Nef 106-148), each created in three mosaics to provide better coverage of M-group HIV-1 sequences. A conserved region vaccine (CRV) delivering genes for these four regions as equal mixtures of three mosaics (each region at a separate injection site) was compared to a whole protein vaccine (WPV) delivering equimolar amounts of genes for whole Gag, Env, and Nef as clade B consensus sequences (separate injection sites). Three rhesus macaques were vaccinated via three DNA primes and a recombinant adenovirus-5 boost (weeks 0, 4, 8, and 24 respectively). Although CRV inserts were about a fifth that of WPV, the CRV generated comparable magnitude blood CD4(+) and CD8(+) T lymphocyte responses against Gag, Env, and Nef. WPV responses preferentially targeted proteome areas outside the selected conserved regions in direct proportion to sequence lengths, indicating similar immunogenicities for the conserved regions versus the outside regions. The CRV yielded conserved region targeting density that was approximately five-fold that of the WPV. A similar pattern was seen in bronchoalveolar lymphocytes, but quadruple the magnitudes in blood. Overall, these findings demonstrated that the selected conserved regions are highly immunogenic, and that anatomically isolated vaccinations with these regions focuses immunodominance compared to full-length protein vaccination. IMPORTANCE HIV-1 sequence diversity is a major barrier limiting the capability of cellular immunity to contain infection and the ability of vaccines to match circulating viral sequences. To date, vaccines tested in humans have delivered whole proteins or genes for whole proteins, and it is unclear whether including only conserved sequences would yield sufficient cellular immunogenicity. We tested a vaccine delivering genes for four small conserved HIV-1 regions compared to a control vaccine with genes for whole Gag, Env, and Nef. Although the conserved regions ranged from 43 to 86 amino acids and comprised less than one fifth of whole Gag/Env/Nef, the vaccines elicited equivalent total magnitudes of both CD4(+) and CD8(+) T lymphocyte responses. These data demonstrate immunogenicity of these small conserved regions, and the potential for a vaccine to steer immunodominance towards conserved epitopes.
    Journal of Virology 11/2014; 89(2). DOI:10.1128/JVI.02370-14 · 4.65 Impact Factor
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    ABSTRACT: A tumor-selective non-lytic retroviral replicating vector (RRV), Toca 511, and an extended-release formulation of 5-fluorocytosine (5-FC), Toca FC, are currently being evaluated in clinical trials in patients with recurrent high-grade glioma (NCT01156584, NCT01470794 and NCT01985256). Tumor-selective propagation of this RRV enables highly efficient transduction of glioma cells with cytosine deaminase (CD), which serves as a prodrug activator for conversion of the anti-fungal prodrug 5-FC to the anti-cancer drug 5-fluorouracil (5-FU) directly within the infected cells. We investigated whether, in addition to its direct cytotoxic effects, 5-FU generated intracellularly by RRV-mediated CD/5-FC prodrug activator gene therapy could also act as a radiosensitizing agent. Efficient transduction by RRV and expression of CD were confirmed in the highly aggressive, radioresistant human glioblastoma cell line U87EGFRvIII and its parental cell line U87MG (U87). RRV-transduced cells showed significant radiosensitization even after transient exposure to 5-FC. This was confirmed both in vitro by a clonogenic colony survival assay and in vivo by bioluminescence imaging analysis. These results provide a convincing rationale for development of tumor-targeted radiosensitization strategies utilizing the tumor-selective replicative capability of RRV, and incorporation of radiation therapy into future clinical trials evaluating Toca 511 and Toca FC in brain tumor patients.Cancer Gene Therapy advance online publication, 10 October 2014; doi:10.1038/cgt.2014.38.
    Cancer Gene Therapy 10/2014; 21(10). DOI:10.1038/cgt.2014.38 · 2.55 Impact Factor
  • Cancer Research 10/2014; 74(19 Supplement):708-708. DOI:10.1158/1538-7445.AM2014-708 · 9.28 Impact Factor
  • Cancer Research 10/2014; 74(19 Supplement):1660-1660. DOI:10.1158/1538-7445.AM2014-1660 · 9.28 Impact Factor
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    ABSTRACT: Toca 511 is a novel retroviral replicating vector, encoding a modified yeast cytosine deaminase, administered to recurrent high grade glioma patients in Phase 1 trials by stereotactic, transcranial injection into the tumor or into the walls of the resection cavity. A key issue, with little published data, is vector biocompatibility with agents likely to be encountered in a neurosurgical setting. We tested biocompatibility of Toca 511 with: delivery devices; MRI contrast agents, including ProHance supporting coinjection for real time MRI-guided intratumoral delivery; hemostatic agents; biofluids (blood and cerebrospinal fluid); potential adjuvants; and a needleless vial adapter that reduces risk of accidental needle sticks. Toca 511 is stable upon thawing at ambient temperature for at least 6 hours, allowing sufficient time for administration, and its viability is not reduced in the presence of: stainless steel and silica-based delivery devices; the potential MRI contrast agent, Feraheme; ProHance at several concentrations; the hemostatic agent SURGIFOAM; blood; cerebrospinal fluid; and the needleless vial adapter. Toca 511 is not compatible with the hemostatic agent SURGICEL or with extended exposures to titanium-based biopsy needles.
    06/2014; 1:14024. DOI:10.1038/mtm.2014.24
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    ABSTRACT: We are developing a retroviral replicating vector (RRV) encoding cytosine deaminase (CD) as an anticancer agent for gliomas. Despite its demonstrated natural selectivity for tumors, and other safety features, such a virus could potentially cause off-target effects by productively infecting healthy tissues. Here, we investigated whether incorporation of a hematopoietic lineage-specific microRNA target sequence in RRVs further restricts replication in hematopoietic lineage-derived human cells in vitro and in murine lymphoid tissues in vivo. One or four copies of a sequence perfectly complementary to the guide strand of microRNA 142 3p were inserted into the 3'UTR of the RRV genome expressing the transgene GFP. Viral spread and GFP expression of these vectors in hematopoietic-lineage cells in vitro and in vivo were measured by qPCR, qRT-PCR and flow cytometry. In hematopoietic lineage-derived human cell lines and primary human stimulated PBMCs, vectors containing 142 3pT sequence showed a remarkable decrease in GFP expression relative to the parental vector, and viral spread was not observed over time. In a syngeneic subcutaneous mouse tumor model, RRV with and without the 142 3pT sequences spread equally well in tumor cells, were strongly repressed in blood, bone marrow and spleen, and generated antiviral immune responses. In an immune-deficient mouse model, RRV with 142 3pT sequences were strongly repressed in blood, bone marrow and spleen compared to unmodified RRV. Tissue-specific MicroRNA-based selective attenuation of retroviral replication can maintain antiviral immunity, and if needed, provide an additional safeguard to this delivery platform for gene therapy applications.
    Human gene therapy 05/2014; 25(8). DOI:10.1089/hum.2012.216 · 3.62 Impact Factor
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    ABSTRACT: Therapeutic gene transfer is currently being evaluated as a potential therapy for inflammatory bowel disease. This study investigates the safety and therapeutic benefit of a locally administered lentiviral vector encoding murine interleukin-10 in altering the onset and relapse of dextran sodium sulfate induced murine colitis. Lentiviral vectors encoding the reporter genes firefly-luciferase and murine interleukin-10 were administered by intrarectal instillation, either once or twice following an ethanol enema to facilitate mucosal uptake, on Days 3 and 20 in Balb/c mice with acute and relapsing colitis induced with dextran sulfate sodium (DSS). DSS colitis was characterized using clinical disease activity, macroscopic, and microscopic scores. Bioluminescence optical imaging analysis was employed to examine mucosal lentiviral vector uptake and transgene expression. Levels of tumor necrosis factor-alpha and interleukin-6 in homogenates of rectal tissue were measured by ELISA. Biodistribution of the lentiviral vector to other organs was evaluated by real time quantitative PCR. Mucosal delivery of lentiviral vector resulted in significant transduction of colorectal mucosa, as shown by bioluminescence imaging analysis. Lentiviral vector-mediated local expression of interleukin-10 resulted in significantly increased levels of this cytokine, as well as reduced levels of tumor necrosis factor-alpha and interleukin-6, and significantly reduced the clinical disease activity, macroscopic, and microscopic scores of DSS colitis. Systemic biodistribution of locally instilled lentiviral vector to other organs was not detected. Topically-delivered lentiviral vectors encoding interleukin-10 safely penetrated local mucosal tissue and had therapeutic benefit in this DSS model of murine colitis.
    BMC Gastroenterology 04/2014; 14(1):68. DOI:10.1186/1471-230X-14-68 · 2.11 Impact Factor
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    ABSTRACT: Despite recent advances in molecular classification, surgery, radiotherapy, and targeted therapies, the clinical outcome of patients with malignant brain tumors remains extremely poor. In this study, we have identified the tetraspan protein epithelial membrane protein-2 (EMP2) as a potential target for glioblastoma (GBM) killing. EMP2 had low or undetectable expression in normal brain, but was highly expressed in GBM as 95% of patients showed some expression of the protein. In GBM cells, EMP2 enhanced tumor growth in vivo in part by upregulating αvβ3 integrin surface expression, activating FAK and Src kinases, and promoting cell migration and invasion. Consistent with these findings, EMP2 expression significantly correlated with activated Src kinase in patient samples and promoted tumor cell invasion using intracranial mouse models. As a proof of principle to determine if EMP2 could serve as a target for therapy, cells were treated using specific anti-EMP2 antibody reagents. These reagents were effective in killing GBM cells in vitro and in reducing tumor load in subcutaneous mouse models. These results support the role of EMP2 in the pathogenesis of GBM and suggest that anti-EMP2 treatment may be a novel therapeutic treatment.
    Journal of Biological Chemistry 03/2014; 289(20). DOI:10.1074/jbc.M113.543728 · 4.60 Impact Factor
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    Molecular Cancer Therapeutics 01/2014; 12(11_Supplement):B225-B225. DOI:10.1158/1535-7163.TARG-13-B225 · 6.11 Impact Factor

Publication Stats

2k Citations
592.32 Total Impact Points

Institutions

  • 2014–2015
    • University of Miami
      • Department of Cell Biology
      كورال غيبلز، فلوريدا, Florida, United States
  • 2001–2014
    • University of California, Los Angeles
      • • Department of Medicine
      • • Department of Molecular and Medical Pharmacology
      • • Department of Chemistry and Biochemistry
      Los Ángeles, California, United States
  • 2006–2013
    • Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center
      • Department of Medicine
      Torrance, California, United States
  • 2009
    • University of Southampton
      Southampton, England, United Kingdom
  • 2008
    • Molecular and Cellular Biology Program
      Seattle, Washington, United States
  • 2003–2007
    • Memorial Sloan-Kettering Cancer Center
      New York City, New York, United States
  • 2001–2006
    • University of Southern California
      • • Keck School of Medicine
      • • Institute for Genetic Medicine
      • • Department of Pathology
      Los Angeles, California, United States