Retrovector Encoding a Green Fluorescent Protein–Herpes Simplex Virus Thymidine Kinase Fusion Protein Serves as a Versatile Suicide/Reporter for Cell and Gene Therapy Applications

Paquin, André; Jaalouk, Diana E.; Galipeau, Jacques

doi:10.1089/104303401450924

Cited by 39 publications

(34 citation statements)

References 33 publications

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“…One hundred percent of eGFP-expressing cells were positive for HSV by immunohistochemistry. Of the fluorescence-activated cell sorted cells that did not express eGFP, none were positive for HSV by immunohistochemistry, except for Ͻ1% of cells infected at a MOI of 1 at 24 h. This finding supports the argument that eGFP is an accurate marker of HSV infection (45,46). This was also confirmed in an in vivo model, in which we administered NV1066 i.p.…”

Section: Mechanism Of Induction Of Apoptosissupporting

confidence: 82%

Infection with Oncolytic Herpes Simplex Virus-1 Induces Apoptosis in Neighboring Human Cancer Cells

Stanziale

Petrowsky

Adusumilli

et al. 2004

Clinical Cancer Research

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Purpose:The antitumor efficacy of a herpes simplex virus (HSV)-1 oncolytic virus depends on the cytotoxic effect of the virus, but also on viral replication and spread within the tumor. Apoptosis is considered a defense mechanism of infected cells that minimizes the spread of viral progeny by limiting cellular production of virus. We sought to determine whether oncolytic HSV-1 infection induces apoptosis in neighboring, uninfected cells and whether manipulation of apoptosis can increase viral replication and cytotoxicity.Experimental Design: NV1066 is an oncolytic HSV-1 mutant that contains the marker gene for enhanced green fluorescent protein. OCUM human gastric cancer cells were infected with NV1066 in vitro and inspected for apoptosis by Hoechst and terminal deoxynucleotidyltransferase-mediated nick end labeling staining and for infection by expression of green fluorescence.Results: A significant increase in apoptosis was seen in cells infected by NV1066. More interestingly, a significant percentage (10%) of uninfected cells also proceeded to apoptosis. After NV1066 infection, cells were also treated with N-acetylcysteine (NAC), an inhibitor of apoptosis. By day 4 after infection, 2.7؋ more NV1066 was produced in cells exposed to NAC than in those not exposed to NV1066 (P ‫؍‬ 0.04). NAC also increased tumor kill when administered with virus.Conclusions: These data suggest that NV1066 induces apoptosis in uninfected cocultured cells, potentially hindering propagation of viral progeny and concomitant tumor kill. Inhibition of apoptosis may improve the efficacy of oncolytic HSV-1 therapy.

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Section: Mechanism Of Induction Of Apoptosissupporting

confidence: 82%

Infection with Oncolytic Herpes Simplex Virus-1 Induces Apoptosis in Neighboring Human Cancer Cells

Stanziale

Petrowsky

Adusumilli

et al. 2004

Clinical Cancer Research

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“…The retrovirus used in this study is a MMLVderived retrovirus vector produced by the 293-GPG packaging cell line (39). This cell line, a generous gift from J. Galipeau (Lady Davis Institute for Medical Research, Montreal, QC, Canada), generates a VSV-G-pseudotyped retrovirus vector encoding a fusion protein between the herpes simplex virus thymidine kinase (TK) protein and the green fluorescent protein (48). Suspension-adapted cell cultures were maintained in calcium-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and tetracycline (1 g/ml; Fisher Scientific, Nepean, ON, Canada).…”

Section: Methodsmentioning

confidence: 99%

Identification of Host Proteins Associated with Retroviral Vector Particles by Proteomic Analysis of Highly Purified Vector Preparations

Segura

Garnier

Falco

et al. 2008

J Virol

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The Moloney murine leukemia virus (MMLV) belongs to the Retroviridae family of enveloped viruses, which is known to acquire minute amounts of host cellular proteins both on the surface and inside the virion. Despite the extensive use of retroviral vectors in experimental and clinical applications, the repertoire of host proteins incorporated into MMLV vector particles remains unexplored. We report here the identification of host proteins from highly purified retroviral vector preparations obtained by rate-zonal ultracentrifugation. Viral proteins were fractionated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-gel tryptic digested, and subjected to liquid chromatography/tandem mass spectrometry analysis. Immunogold electron microscopy studies confirmed the presence of several host membrane proteins exposed at the vector surface. These studies led to the identification of 27 host proteins on MMLV vector particles derived from 293 HEK cells, including 5 proteins previously described as part of wild-type MMLV. Nineteen host proteins identified corresponded to intracellular proteins. A total of eight host membrane proteins were identified, including cell adhesion proteins integrin ␤1 (fibronectin receptor subunit beta) and HMFG-E8, tetraspanins CD81 and CD9, and late endosomal markers CD63 and Lamp-2. Identification of membrane proteins on the retroviral surface is particularly attractive, since they can serve as anchoring sites for the insertion of tags for targeting or purification purposes. The implications of our findings for retrovirusmediated gene therapy are discussed.

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“…NV1066 carries such a marker gene, a constitutively expressed transgene for EGFP, the protein product of which is identifiable 4-6 hours following viral entry into cells. While green fluorescent protein and its derivative proteins have been used extensively as reporters and/or markers in numerous biologic studies (60)(61)(62)(63)(64)(65), their use to determine infection and spread of replication-competent viral vectors in vivo is an emerging technology. Previous studies have demonstrated that the fluorescent signal from cells constitutively expressing GFP correlates strongly with cell number in vitro (66).…”

Section: Discussionmentioning

confidence: 99%

Minimally invasive localization of oncolytic herpes simplex viral therapy of metastatic pleural cancer

et al. 2005

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Purpose-Herpes simplex virus-one (HSV-1) oncolytic therapy and gene therapy are promising treatment modalities against cancer. NV1066, one such HSV-1 virus carries a marker gene for enhanced green fluorescent protein (EGFP). The purpose of this study was to determine whether NV1066 is cytotoxic to lung cancer and whether EGFP is a detectable marker of viral infection in vitro and in vivo. We further investigated whether EGFP expression in infected cells can be used to localize the virus and to identify small metastatic tumor foci (< 1 mm.) in vivo by means of minimally invasive endoscopic systems equipped with fluorescent filters.Experimental Design-In A549 human lung cancer cells, in vitro viral replication was determined by plaque assay, cell kill by LDH release assay, and EGFP expression by flow cytometry. In vivo, A549 cells were injected into the pleural cavity of athymic mice. Mice were treated with intrapleural injection of NV1066 or saline and examined for EGFP expression in tumor deposits using a stereomicroscope or a fluorescent thoracoscopic system. Results-NV1066 replicated in, expressed EGFP in infected cells and killed tumor cells in vitro.In vivo, treatment with intrapleural NV1066 decreased pleural disease burden, as measured by chest wall nodule counts and organ weights. EGFP was easily visualized in tumor deposits, including microscopic foci, by fluorescent thoracoscopy.Conclusions-NV1066 has significant oncolytic activity against a human NSCLC cell line and is effective in limiting the progression of metastatic disease in an in vivo orthotopic model. By incorporating fluorescent filters into endoscopic systems, a minimally-invasive means for diagnosing small metastatic pleural deposits and localization of viral therapy for thoracic malignancies may be developed using the EGFP marker gene inserted in oncolytic herpes simplex viruses.

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Retrovector Encoding a Green Fluorescent Protein–Herpes Simplex Virus Thymidine Kinase Fusion Protein Serves as a Versatile Suicide/Reporter for Cell and Gene Therapy Applications

Cited by 39 publications

References 33 publications

Infection with Oncolytic Herpes Simplex Virus-1 Induces Apoptosis in Neighboring Human Cancer Cells

Infection with Oncolytic Herpes Simplex Virus-1 Induces Apoptosis in Neighboring Human Cancer Cells

Identification of Host Proteins Associated with Retroviral Vector Particles by Proteomic Analysis of Highly Purified Vector Preparations

Minimally invasive localization of oncolytic herpes simplex viral therapy of metastatic pleural cancer

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