scholarly journals Novel Semi-Replicative Retroviral Vector Mediated Double Suicide Gene Transfer Enhances Antitumor Effects in Patient-Derived Glioblastoma Models

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1090 ◽  
Author(s):  
Lee ◽  
Kim ◽  
Lee ◽  
Kang ◽  
Shin ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cytosine Deaminase ◽  
Suicide Gene ◽  
Transduction Efficiency ◽  
Antitumor Effects ◽  
Suicide Genes ◽  
Wide Range ◽  
Double Suicide ◽  
Differentiation And Proliferation

As glioblastomas are mostly localized infiltrative lesions, gene therapy based on the retroviral replicating vector (RRV) system is considered an attractive strategy. Combinations of multiple suicide genes can circumvent the limitations associated with each gene, achieving direct and synergistic cytotoxic effects, along with bystander cell killing. In this study, we constructed a semi-and pseudotyped-RRV (sp-RRV) system harboring two suicide genes—herpes simplex virus type 1 thymidine kinase (TK) and yeast cytosine deaminase (CD)—to verify the dissemination and antitumor efficacy of our sp-RRV system (spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD) in seven patient-derived glioblastoma stem-like cells (GSCs). Flow cytometry and high-content analysis revealed a wide range of transduction efficiency and good correlation between the delivery of therapeutic genes and susceptibility to the prodrugs ganciclovir and 5-fluorocytosine in patient-derived GSCs in vitro. Intra-tumoral delivery of spRRVe-sEF1α-TK/sRRVgp-sEF1α-CD, combined with prodrug treatment, synergistically inhibited cell proliferation and angiogenesis while increasing apoptosis and the depletion of tumor-associated macrophages in orthotopic glioblastoma xenografts. Genomic profiling of patient-derived GSCs revealed that the key genes preventing sp-RRV infection and transmission were associated with cell adhesion, migration, development, differentiation, and proliferation. This is the first report demonstrating that a novel sp-RRV-mediated TK/CD double suicide gene transfer system has high oncolytic power against extremely heterogeneous and treatment-refractory glioblastomas.

scholarly journals Peptide-guided JC polyomavirus-like particles specifically target bladder cancer cells for gene therapy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wei-Hong Lai ◽  
Chiung-Yao Fang ◽  
Ming-Chieh Chou ◽  
Mien-Chun Lin ◽  
Cheng-Huang Shen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bladder Cancer ◽  
Cancer Cells ◽  
Suicide Gene ◽  
Transduction Efficiency ◽  
Ligand Molecule ◽  
Jc Polyomavirus ◽  
Exogenous Dna ◽  
Bladder Cancer Cells

AbstractThe ultimate goal of gene delivery vectors is to establish specific and effective treatments for human diseases. We previously demonstrated that human JC polyomavirus (JCPyV) virus-like particles (VLPs) can package and deliver exogenous DNA into susceptible cells for gene expression. For tissue-specific targeting in this study, JCPyV VLPs were conjugated with a specific peptide for bladder cancer (SPB) that specifically binds to bladder cancer cells. The suicide gene thymidine kinase was packaged and delivered by SPB-conjugated VLPs (VLP-SPBs). Expression of the suicide gene was detected only in human bladder cancer cells and not in lung cancer or neuroblastoma cells susceptible to JCPyV VLP infection in vitro and in vivo, demonstrating the target specificity of VLP-SPBs. The gene transduction efficiency of VLP-SPBs was approximately 100 times greater than that of VLPs without the conjugated peptide. JCPyV VLPs can be specifically guided to target particular cell types when tagged with a ligand molecule that binds to a cell surface marker, thereby improving gene therapy.

scholarly journals Imaging Expression of Cytosine Deaminase-Herpes Virus Thymidine Kinase Fusion Gene (CD/TK) Expression with [124I]FIAU and PET

2002 ◽  
Vol 1 (1) ◽  
pp. 153535002002000 ◽  
Author(s):  
Trevor Hackman ◽  
Michail Doubrovin ◽  
Julius Balatoni ◽  
Tatiana Beresten ◽  
Vladimir Ponomarev ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Thymidine Kinase ◽  
Enzyme Assay ◽  
Fusion Gene ◽  
Cytosine Deaminase ◽  
Prodrug Activation ◽  
Wide Range ◽  
Different Levels

Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CD)herpes simplex virus type 1 thymidine kinase ( HSV1-tk) fusion gene ( CD/TK) with 5-fluorocytosine (5FC), ganciclovir (GCV), and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodo-uracil) and positron emission tomography (PET) for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells). The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.

Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells

1996 ◽  
Vol 124 (1) ◽  
pp. 49-60 ◽  
Author(s):  
J. Kreuzer ◽  
S. Denger ◽  
F. Reifers ◽  
C. Beisel ◽  
K. Haack ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Gene Transfer ◽  
Smooth Muscle Cells ◽  
Cytosine Deaminase ◽  
Muscle Cells

Comparison of Dfferent Suicide Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3753-3753 ◽  
Author(s):  
Virna Marin ◽  
Irene Pizzitola ◽  
Andrea Biondi ◽  
Ettore Biagi ◽  
Martin Pule
Keyword(s):  
T Cells ◽  
High Efficiency ◽  
Viral Vector ◽  
Conflicts Of Interest ◽  
Marker Gene ◽  
Suicide Gene ◽  
Severe Toxicity ◽  
Suicide Genes ◽  
Target Effects

Abstract Abstract 3753 Immunotherapy with TCR or chimeric receptor-genetically modified T cells may result in toxicity related to direct target effects, unanticipated off-target effects or lymphoproliferation due to insertional mutagenesis. Therefore it is required to include a suicide gene in the viral vector. We compared different suicide genes in vitro in Epstein Barr Virus-specific cytotoxic T cells (EBV-CTL). Herpes Simplex Virus Thymidine Kinase (HSV-tk), human inducible Caspase 9 (iCasp9), human CD20 and mutant human thymidylate kinase (mTMPK) genes were cloned in frame with truncated CD34 (dCD34, marker gene), separated by the 2A peptide in a SFG-based vector. We previously reported with the iCasp9-coding construct, a lower transduction efficiency and instable expression of the dCD34 marker gene. We therefore codon-optimized the iCasp9 sequence (iCasp9opt) and repeated the cloning in frame with 2A-dCD34 in the SFG vector. EBV-CTLs could be similarly and efficiently transduced with iCasp9opt-2A-dCD34, HSV-TK-2A-dCD34, mTMPK-2A-dCD34 and CD20-2A-dCD34 (mean % CD34+, 80%, n=5), similarly to the control vector containing dCD34 alone. Expression of the marker gene was stable up to 3 weeks. Expression of the suicide genes was not associated with alterations in the expansion rate, immunophenotype and capacity to kill autologous lymphoblastoid cell lines. Transduced and CD34-selected EBV-CTLs have been tested for their sensibility to the corresponding activator in vitro by evaluating residual CD34+ cells. iCasp9opt- transduced cells were rapidly killed with high efficiency by CID, (mean survival, 11% after 24 hours, and 5% after 7 days; n=7). Gancyclovir treated HSV-tk expressing cells showed similar levels of efficacy only after 3 days and CD20 and mTMPK-transduced cells showed only minimal killing at all time points (mean survival after 7 days,84% and 32% respectively).The same results were obtained by analyzing apoptosis induction through Annexin-7AAD staining. In fact, after 24 hours of incubation with CID, nearly 100% iCasp9opt+ cells were apoptotic, whereas a significant lower % of apoptotic cells was observed with the other suicide genes. Altogether our results suggest that the faster activity of iCasp9 might be advantageous in case of occurring severe toxicity, and, together with its lack of immunogenicity and the absence of side-effects of CID, support the clinical applicability of iCap9-based suicide strategy. Disclosures: No relevant conflicts of interest to declare.

Antitumor effects of cytosine deaminase and thymidine kinase fusion suicide gene under the control of mdr1 promoter in mdr1 positive leukemia cells

2007 ◽  
Vol 48 (8) ◽  
pp. 1600-1609 ◽  
Author(s):  
Xiangling Wang ◽  
Chunyan Ji ◽  
Daoxin Ma ◽  
Jianqiang Zhao ◽  
Ming Hou ◽  
...  
Keyword(s):  
Thymidine Kinase ◽  
Cytosine Deaminase ◽  
Suicide Gene ◽  
Leukemia Cells ◽  
Antitumor Effects ◽  
Mdr1 Promoter

Activation of the CMV-IE Promoter by Hyperthermia In Vitro and In Vivo: Biphasic Heat Induction of Cytosine Deaminase Suicide Gene Expression

2010 ◽  
Vol 46 (2) ◽  
pp. 197-205 ◽  
Author(s):  
Dennis Kobelt ◽  
Jutta Aumann ◽  
Iduna Fichtner ◽  
Ulrike Stein ◽  
Peter M. Schlag ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cytosine Deaminase ◽  
Suicide Gene ◽  
Heat Induction

scholarly journals Retroviral transduction of human progenitor cells: use of granulocyte colony-stimulating factor plus stem cell factor to mobilize progenitor cells in vivo and stimulation by Flt3/Flk-2 ligand in vitro

Blood ◽  
1996 ◽  
Vol 88 (12) ◽  
pp. 4452-4462 ◽  
Author(s):  
NJ Elwood ◽  
H Zogos ◽  
T Willson ◽  
CG Begley
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Fold Increase ◽  
Transduction Efficiency ◽  
Granulocyte Colony Stimulating Factor ◽  
Retroviral Transduction ◽  
Stimulating Factor

The clinical application of gene transfer is hindered by the availability of the multipotential stem cells and the difficulty in obtaining efficient retroviral transduction. To assess potential means by which gene transfer into human hemopoietic stem cells might be enhanced, the retroviral transduction efficiency of human bone marrow cells (BM) or peripheral blood progenitor cells (PBPC) was compared at multiple time points after in vivo administration of granulocyte colony- stimulating factor (G-CSF). This was further compared with the transduction efficiency of cells mobilized with G-CSF plus stem cell factor (SCF) in a cohort of patients randomized to receive either one or two growth factors and with normal BM function. Using the LNL6 retrovirus, retroviral transduction efficiencies of up to 19% were observed for both PBPC and BM (n = 26 patients). There was at least a 100-fold increase in PBPC with G-CSF alone and a further 30-fold increase in the total number of progenitor cells available for retroviral transduction using the combination of SCF plus G-CSF. However, pretreatment of patients with G-CSF with or without SCF did not enhance the retroviral infectability of growth factor-mobilized progenitor cells. The effect of the growth factor, Flk-2/Flt3 ligand (FL), was also examined with respect to retroviral transduction efficiency of human progenitor cells. FL plus IL-3 in vitro increased the retroviral transduction efficiency up to eightfold compared with results observed using other combinations of cytokines tested (P < .001). These findings have clinical implications both for increasing the number of target cells for in vivo gene-marking/gene-therapy studies and improving the efficiency of gene transfer.

Ultrasound targeted gene therapy in a mouse model of prostate cancer: Evaluation of immune response.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 120-120
Author(s):  
Flavia De Carlo ◽  
Litty Thomas ◽  
Rounak Nande ◽  
Olivia Boskovic ◽  
Gailen Marshall ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Gene Transfer ◽  
Cancer Cells ◽  
Transduction Efficiency ◽  
Acquired Immunity ◽  
Prostate Cancer Cells ◽  
Adenoviral Gene Transfer ◽  
Specific Tissue

120 Background: Gene transfer to malignant sites using human adenoviruses (hAd) has been limited because of their immunogenicity. Murine cells often lack some of the receptors needed for hAd infection; therefore, are generally non-permissive for hAd infection and replication, which limits translational studies of adenoviral gene transfer techniques. We developed a gene transfer method, which uses a combination of lipid-encapsulated perfluorocarbon microbubbles (MBs) and ultrasound (US) to shield and deliver hAds to a specific tissue bypassing the requirement of the coxsackie and adenovirus receptor (CAR). Methods: Transduction efficiency and GFP protein expression of hAd.GFP was assessed by flow cytometry and fluorescence microscopy in murine TRAMP-C2 and human DU145 prostate cancer cells. Innate and acquired immunity response was determined by ELISA and CTL assay in C57BL/6 mice bearing TRAMP-C2 syngeneic tumor grafts following injections of MBs-Ad.GFP complexes in the presence or absence of ultrasound. Results: We observed that the murine prostate cancer cells TRAMP-C2 were transduced less efficiently by hAd.GFP than the human DU145 cells. We showed in vitro that the transduction rate was increased significantly in both TRAMP-C2 and DU145 prostate cancer cells when delivering the Ad particles by a combination of MBs and US. Moreover, we observed expression of the GFP transgene in both cell lines at 48 hours and 72 hours. Lack of activation of the innate and acquired immunity was observed in vivo by quantifying IL-6 and TNF-α cytokines, and by assaying neutralizing IgG antibodies and CTLs activity, following intratumoral or intravenous injections of MBs-Ad.GFP complexes in the presence or absence of ultrasound. Conclusions: This study demonstrates the feasibility of using the TRAMP-C2 murine model of prostate adenocarcinoma to translate our ultrasound-mediated MB-Ad delivery system from the bench to the clinic. Our data provides evidence that the TRAMP-C2 prostate cancer graft model is a suitable system to study in immune competent animals the capacity of lipid-encapsulated perfluorocarbon MBs and US, to shield and deliver hAds to a site-specific tissue bypassing the requirement of specific receptors.

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