Triple Gene-Deleted Oncolytic Herpes Simplex Virus Vector Double-Armed with Interleukin 18 and Soluble B7-1 Constructed by Bacterial Artificial Chromosome–Mediated System

Harvard University, Cambridge, Massachusetts, United States
Cancer Research (Impact Factor: 9.33). 01/2006; 65(23):10663-8. DOI: 10.1158/0008-5472.CAN-05-2534
Source: PubMed


Conditionally replicating herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Certain antitumor functions may be added to oncolytic activities of recombinant HSV-1 vectors by inserting transgenes into the viral genome. Because conventional homologous recombination techniques had required time-consuming processes to create "armed" oncolytic HSV-1 vectors, we established an innovative construction system using bacterial artificial chromosome and two recombinase systems (Cre/loxP and FLPe/FRT). Using G47Delta, a safe and efficacious oncolytic HSV-1 with triple gene mutations, as the backbone, this system allowed a rapid generation of multiple vectors with desired transgenes inserted in the deleted ICP6 locus. Four oncolytic HSV-1 vectors, expressing murine interleukin 18 (mIL-18), soluble murine B7-1 [B7-1-immunoglobulin (B7-1-Ig)], both, or none, were created simultaneously within 3 months. In vitro, all newly created recombinant vectors exhibited virus yields and cytopathic effects similar to the parental G47Delta. In two immunocompetent mouse tumor models, TRAMP-C2 prostate cancer and Neuro2a neuroblastoma, the vector expressing both mIL-18 and B7-1-Ig showed a significant enhancement of antitumor efficacy via T-cell-mediated immune responses. The results show that "arming" with multiple transgenes can improve the efficacy of oncolytic HSV-1 vectors. The use of our system may facilitate the development and testing of various armed oncolytic HSV-1 vectors.

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    • "G207) or similar to G207 with an additional deletion in ICP47 locus (e.g., G47D). Oncolytic HSV-1 can also be armed with the prodrug converting gene (Yamada et al, 2012), immunomodulatory molecules (Fukuhara et al, 2005), immune stimulant gene (Liu et al, 2003) or antiangiogenesis agents (Yoo et al, 2012) to augment the efficacy of the viruses beyond their oncolytic abilities. The first oncolytic virus that contains an active gene to enter clinical trial is the oncolytic HSV-1, OncoVex GM-CSF . "
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    ABSTRACT: Background: Emerging studies have shown the potential benefit of arming oncolytic viruses with therapeutic genes. However, most of these therapeutic genes are placed under the regulation of ubiquitous viral promoters. Our goal is to generate a safer yet potent oncolytic herpes simplex virus type-1 (HSV-1) for cancer therapy. Methods: Using bacterial artificial chromosome (BAC) recombineering, a cell cycle-regulatable luciferase transgene cassette was replaced with the infected cell protein 6 (ICP6) coding region (encoded for UL39 or large subunit of ribonucleotide reductase) of the HSV-1 genome. These recombinant viruses, YE-PC8, were further tested for its proliferation-dependent luciferase gene expression. Results: The ability of YE-PC8 to confer proliferation-dependent transgene expression was demonstrated by injecting similar amount of viruses into the tumour-bearing region of the brain and the contralateral normal brain parenchyma of the same mouse. The results showed enhanced levels of luciferase activities in the tumour region but not in the normal brain parenchyma. Similar findings were observed in YE-PC8-infected short-term human brain patient-derived glioma cells compared with normal human astrocytes. intratumoural injection of YE-PC8 viruses resulted in 77% and 80% of tumour regression in human glioma and human hepatocellular carcinoma xenografts, respectively. Conclusion: YE-PC8 viruses confer tumour selectivity in proliferating cells and may be developed further as a feasible approach to treat human cancers.
    Full-text · Article · Nov 2013 · British Journal of Cancer
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    • "These biologic therapeutics demonstrate widespread tumor tropism [33]. We and others have shown neural tumors are sensitive to oncolytic Herpes simplex virus (oHSV) mutants and further hypothesized that these viruses may effectively destroy drug-resistant neuroblastoma tumor initiating cells [34], [35], [36], [37], [38], [39]. Herein we identified and isolated populations of human neuroblastoma cells that expressed CD133 and grew as clonal spheres. "
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    ABSTRACT: Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers. Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice. These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.
    Full-text · Article · Feb 2009 · PLoS ONE
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    • "Somewhat similar recombinase systems have recently been reported that describe the construction of recombinant herpesvirus BACs with inserts into ICP6 rather than UL41 [27,28]. In these systems, the Cre/lox recombinase system was used to insert a transgene into the ICP6 gene of HSV, the same site that was engineered to harbor the BAC replication sequences. "
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    ABSTRACT: Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.
    Full-text · Article · Feb 2007 · BMC Biotechnology
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