Herpesvirus Thymidine Kinase Gene Research Articles

THYMIDINE KINASE AS A CAUSATIVE FACTOR FOR TYPE 1 HERPES SIMPLEX VIRUS RESISTANCE AGAINST ACYCLOVIR

ABSTRACTBackground: Herpes Simplex Virus (HSV) infection demonstrates a high prevalence in the world. Acyclovir, one of guanine synthetic analogues, is commonly used to treat infections caused by HSV. HSV resistance against acyclovir may occur, especially in immunocompromised and immunocompetent patients, as the consequence of viral mutations. Thymidine kinase (TK) is an HSV tegument protein which plays an important role in HSV-1 resistance against acyclovir. Purpose: The purpose of this article is to review the mechanisms of TK mutation that cause HSV-1 resistance against acyclovir. Review: Acyclovir involves three stages of viral thymidine kinase phosphorylation to form acyclovir triphosphate. It prevents HSV replication by acting as a competitive inhibitor of viral DNA polymerase and a chain terminator in viral DNA synthesis. Resistance is associated with viral TK mutation that is encoded by UL23 gene. Long-term use of acyclovir may promote thymidine kinase mutation in immunocompromised and immunocompetent patients via three mechanisms, namely absolute insufficiency in TK activity (TK-negative), depletion in TK synthesis, and inability in TK phosphorylation which consequently hinders the phosphorylation of acyclovir. Herpes TK gene contains a series of cytosine and guanosine, that are important for the function and the mutation of HSV by producing incomplete or fewer enzymes as the result of nucleotide addition or elimination in homopolymer process. Conclusion: HSV-1 resistance against acyclovir is evolved from TK mutations, in the form of TK-negative, TK low-producing, and TK altered mutants, that are unable to phosphorylate TK and accordingly disrupt acyclovir phosphorylation to convert acyclovir triphosphate. Keyword : Acyclovir, Herpes simplex virus, Resistance, Thymidine kinase

Investigation of a Cyprinid Herpesvirus 1 Disease Episode in a Group of Pond-Reared Koi.

Fifteen adult koi (a variant of Common Carp Cyprinus carpio) simultaneously developed white cutaneous proliferations affecting up to 30% of their bodies. The onset of these lesions (in September 2014) was associated with their return to a remodeled backyard water garden after temporarily being maintained in a plastic swimming pool. A single water temperature taken during the outbreak read 21°C on November 17, 2014. The water garden had no extrinsic heat source, with average ambient temperatures ranging from 9.4 to 26.4°C during the outbreak (September 2014-January 2015). Representative skin biopsies were obtained from two fish; the histologic features included severe epidermal hyperplasia, dysplasia, keratinocyte apoptosis, decreased and haphazardly distributed goblet cells with the absence of club cells, keratinocyte hydropic degeneration, and moderate infiltration by lymphocytes and eosinophilic granular cells. Ultrastructural findings included intranuclear nonenveloped hexagonal nucleocapsids and abundant cytoplasmic-enveloped virions morphologically consistent with the Alloherpesviridae family. Polymerase chain reaction was performed on formalin-fixed, paraffin-embedded shavings from the two biopsied koi targeting the thymidine kinase gene of cyprinid herpesvirus 1 (CyHV-1). Together with the aforementioned findings, these results are consistent with an outbreak of CyHV-1 in a population of adult koi.

Role of Homology and DNA Double‐Strand Breaks in Initiation of Intrachromosomal Recombination in Mammalian Cells

As part of our continuing investigations into the mechanisms of intrachromosomal recombination in mammalian cells, we stably transfected thymidine kinase deficient mouse fibroblasts with a DNA construct, pKM1, harboring a herpes thymidine kinase (tk) gene rendered nonfunctional by insertion of an oligonucleotide containing the recognition site for endonuclease I‐SceI. We refer to this latter tk sequence as the “recipient.” The construct also contained a closely linked truncated “donor” tk sequence. The recipient and donor were positioned as direct repeats with respect to transcriptional orientation. The donor could potentially restore function to the recipient gene via recombination with the recipient, and such events could be provoked by induction of a double‐strand break (DSB) at the I‐SceI site in the recipient. Such DSB repair events were recoverable by selection for tk‐positive clones. The donor contained 33 mismatches relative to the recipient. The mismatches were clustered, forming a localized segment of DNA sequence displaying about 20% divergence relative to the recipient, and the mismatched segment was surrounded by regions of high homology. When the donor was aligned with the recipient in pKM1, the DSB site in the recipient aligned opposite the mismatched segment, allowing us to potentially capture recombinational repair events initiating between diverged sequences. Our earlier work demonstrated that mammalian cells effectively avoid recombination between 20% diverged sequences. In work presented here, we asked whether flanking regions of high homology would enable genetic exchange between highly diverged sequences or, instead, would rejection of exchange between diverged sequences remain unchanged. We found that by surrounding mismatches with high homology, suppression of recombination between diverged sequences was overcome. Strikingly, we recovered a high frequency of gene conversion tracts positioned entirely within the mismatched sequences. We infer that such events were enabled by homologous pairing interactions between sequences surrounding the site of strand invasion. Our results suggest that the search for high homology prior to recombination is not mediated by an invading DNA terminus and may actually be independent of DSB formation. Further, the homology that enabled the recovered recombination events was located at a discrete distance from the actual invading DNA terminus. In an extension of our studies, we designed two additional constructs, pCW1 and pCW2. These constructs were similar to pKM1 except the recipient sequence in each was disrupted by an 8bp XhoI linker insertion and each construct contained a I‐SceI recognition site positioned within vector sequences at a location outside of either the recipient or donor sequence. Cells transfected with constructs pCW1 and pCW2 are being used to further explore whether a chromosomal DSB can induce and/or influence the nature of genetic exchanges occurring at a discrete distance from the DSB when the genetic exchanges themselves do not directly repair the DSB. Progress addressing this latter issue is presented.Support or Funding InformationThis work was supported by grant MCB 1157416 from the National Science Foundation to ASW and BCW.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High homology is not required at the site of strand invasion during recombinational double-strand break repair in mammalian chromosomes

We investigated the impact of sequence divergence on DNA double-strand break (DSB) repair occurring via recombination in cultured thymidine kinase deficient mouse fibroblasts. We stably transfected cells with a DNA construct harboring a herpes thymidine kinase (tk) gene (the “recipient”) rendered nonfunctional by insertion of an oligonucleotide containing the recognition site for endonuclease I-SceI. The construct also contained a closely linked truncated “donor” tk sequence. The donor could potentially restore function to the recipient gene via recombination provoked by induction of a DSB at the I-SceI site in the recipient. Repair events were recoverable by selection for tk-positive clones. The donor contained 33 mismatches relative to the recipient. The mismatches were clustered, forming a localized segment of DNA sequence displaying about 20% divergence relative to the recipient, and the mismatched segment was surrounded by regions of high homology. When the donor was aligned with the recipient, the DSB site in the recipient aligned opposite the mismatched segment, allowing us to potentially capture recombinational repair events initiating between diverged sequences. Previous work demonstrated that mammalian cells effectively avoid recombination between 20% diverged sequences. In the current study we asked whether flanking regions of high homology would enable genetic exchange between highly diverged sequences or, instead, would rejection of exchange between diverged sequences remain unchanged. We found that by surrounding mismatches with high homology, suppression of recombination between diverged sequences was overcome. Strikingly, we recovered a high frequency of gene conversion tracts positioned entirely within the mismatched sequences. We infer that such events were enabled by homologous pairing interactions between sequences surrounding the site of strand invasion. Our results suggest a search for high homology prior to recombination that is not mediated by an invading DNA terminus.

Polymer functionalized gold nanoparticles as nonviral gene delivery reagents.

In the present study, we investigated the ability of polyethylene glycol (PEG) functionalized gold nanoparticles to function as nonviral vectors in the transfection of different cell lines, comparing them with commercial lipoplexes. Positively-charged gold nanoparticles were synthesized using polyethylenimine (PEI) as a reducing and stabilizer agent and its cytotoxicity was reduced by its functionalization with PEG. We bound the nanoparticles to three plasmids with different sizes (4-40 kpb). Vector internalization was evaluated by confocal and electronic microscopy. Its transfection efficacy was studied by fluorescence microscopy and flow cytometry. The application of the resulting vector in gene therapy was evaluated indirectly using ganciclovir in HeLa cells transfected to express the herpes virus thymidine kinase. An appropriate ratio between the nitrogen from the PEI and the phosphorous from the phosphate groups of the DNA, together with a reduced size and an elevated electrokinetic potential, are responsible for an increased nanoparticle internalization and enhanced protein expression when carrying plasmids of up to 40 kbp (plasmid size close to the limit of the DNA-carrying capacity of viral vectors). Compared to a commercial transfection reagent, an equal or even higher expression of reporter genes (on HeLa and Hek293t) and a suicide effect on HeLa cells transfected with the herpes virus thymidine kinase gene were observed when using this novel nanoparticulated vector. Nonviral vectors based on gold nanoparticles covalently coupled with PEG and PEI can be used as efficient transfection reagents showing expression levels that are the same or greater than those obtained with commercially available lipoplexes.

Abstract B84: Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition

Abstract Early clinical trial data show that blockade of PD-1 signaling leads to significant anticancer responses in a subset of patients in certain cancer types. While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics in brain tumors has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune checkpoint inhibitors block the body's natural checkpoint response, the combination of these two approaches offers a potentially advantageous interaction. One of the molecular underpinnings of T-cell exhaustion is the expression of Programmed Death-1 (PD-1) on T-cells that recognizes its ligand PD-L1. AdV-TK is an immunostimulatory virus-based approach, known as Gene-Mediated Cytotoxic Immunotherapy (GMCI), that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene (TK) followed by administration of an anti-herpetic prodrug (ganciclovir GCV) and recently showed encouraging results in a Phase II trial in glioblastoma (Wheeler et al., 2016). The immunological component results from the delivery vehicle being a virus, the mode of cell death, through both necrosis and apoptosis, and the pro-immunogenic properties of the TK protein. We confirm that this approach induces glioblastoma cell death and a consistent anti-tumor immune stimulation. Not surprisingly, however, this immune stimulation also leads to increase in cell surface of immune checkpoint inhibitory ligands on tumor cells, including PD-L1, detected by flow cytometry and immunohistochemistry. We show that GMCI induces a type-I interferon response, and using IFN decoy we demonstrated that the release of IFNβ in vitro is at least partially responsible for autocrine/paracrine PD-L1 up-regulation both in human and mouse glioblastoma cell lines. In vivo studies using an intracranial GL261 model showed high numbers of long term survivors in the GMCI/PD-1 combination (11/14), compared with GMCI (6/16), anti-PD-1 (5/12) and untreated (0/11). In addition, long term survival mice were no longer able to form tumors after rechallenge indicating the establishment of anti-tumor immunity. Finally, tumor infiltrating lymphocytes after GMCI showed an increase in CD8+, CD8+/GranzymeB+, and IFNγ+ cells suggestive of cytotoxic T-cell activation. However, there was also a significant increase in CD4+, CD4+/FoxP3+, and IL-10 indicating a significant infiltration by Tregs, releasing immunosuppressive cytokines. Additionally, there was a significant increase in PD-1+ /TIM3+ T-cells, indicative of an immunosuppressive microenvironment. Overall, our data show that GMCI/anti-PD-1 combinatorial therapy is effective in a syngeneic tumor model, and strongly support clinical trials of GMCI/checkpoint inhibitor combinations in glioblastoma patients. Citation Format: Maria Carmela Speranza, Franz Ricklefs, Carmela Passaro, Sarah R. Klein, Kazue Kasai, Johanna Kaufmann, Hiroshi Nakashima, Bronisz Agnieszka, Estuardo Aguilar-Cordova, Brian W. Guzik, Gordon J. Freeman, David A. Reardon, Patrick Wen, E. Antonio Chiocca, Sean E. Lawler. Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B84.

Abstract A42: Alternate pseudotypes overcome receptor interference and enable combination suicide gene therapy with retroviral replicating vectors

Abstract Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. An amphotropic murine leukemia virus (MLV)-based RRV encoding the yeast cytosine deaminase (CD) prodrug activator gene, designated Toca 511 (vocimagene amiretrorepvec), is now being investigated in combination with Toca FC (extended-release 5-FC) in multi-center Phase IIB / III clinical trials for patients with recurrent high-grade glioma (clinicaltrials.gov: NCT02414165). RRV-mediated prodrug activator gene therapy represents the ultimate form of “intracellular” chemotherapy, generated selectively and directly from within the infected cancer cells themselves, without incurring systemic toxicity. Moreover, preclinical data support subsequent activation of the immune system selectively against the cancer. We further propose that combination with additional therapies may be desirable to optimize treatment outcomes. Here we first evaluated two different RRVs derived from MLV and gibbon ape leukemia virus (GALV), and expressing either the same fluorescent reporter gene (MLV-GFP and GALV-GFP, respectively), or different reporter genes (MLV-GFP vs. GALV-RFP; MLV-RFP vs. GALV-GFP), in different types of human cancer cells. Individually, RRVs expressing the same reporter gene efficiently infected human glioma, prostate cancer, and ovarian cancer cells, and showed efficient replication and spread in culture. When marked with different fluorescent reporter genes, it was found that MLV-GFP can spread in cells that had been pre-transduced with GALV-RFP, but not in MLV-RFP pre-transduced cells. Similarly, GALV-GFP can spread in MLV-RFP pre-transduced cells, but not in GALV-RFP pre-transduced cells. This mutually exclusive infection pattern is likely due to the phenomenon of receptor interference, which occurs when vectors derived from the same viral strain compete for binding to cell surface receptors, resulting in superinfection resistance. Notably, however, replication and spread of either RRV in culture was not affected by pre-transduction with RRV derived from a different strain, indicating that this approach could overcome receptor interference. In order to investigate the effect of combined prodrug-dependent cell killing in vitro, cells were then transduced with MLV- or GALV-based RRV expressing the yeast cytosine deaminase suicide gene (MLV-CD, GALV-CD), which converts the prodrug 5-fluorocytosine (5-FC) into the active drug 5-fluorouracil (5-FU), or with RRV expressing the Herpes thymidine kinase gene (MLV-TK, GALV-TK) which converts Ganciclovir (GCV) to GCV-monophosphate, either individually or in combination. In vitro cytocidal effects obtained by combining different prodrug activator genes were significantly greater when these genes were delivered with RRV derived from two different strains. These data indicate the potential utility of using RRV pseudotyped with envelopes derived from different viral strains to overcome receptor interference leading to superinfection resistance, and achieve effective combined prodrug activator gene therapy. Citation Format: James Grosso, Sara Collins, Akihito Inagaki, Suzanne Matsuura, Brian Slomovitz, Tan Ince, Noriyuki Kasahara. Alternate pseudotypes overcome receptor interference and enable combination suicide gene therapy with retroviral replicating vectors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A42.

EXTH-23. PRECLINICAL ANALYSIS OF COMBINATORIAL GLIOBLASTOMA THERAPY WITH THE PRODRUG-MEDIATED GENE THERAPY VECTOR AdV-tk AND IMMUNE CHECKPOINT INHIBITION

While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune-checkpoint inhibitors block tumor-induced T-cell exhaustion, the combination of these two approaches offers a potentially synergistic interaction. One of the molecular underpinnings of T-cell exhaustion is the expression of Programmed Death-1 (PD1) on T-cells that recognizes its ligand PD-L1. AdV-TK is an immunostimulatory virus-based approach, known as Gene-Mediated Cytotoxic Immunotherapy (GMCI), that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene(TK) followed by administration of an anti-herpetic prodrug(ganciclovir-GCV) and recently showed encouraging results in a Phase II trial in glioblastoma(Wheeler et al.,2016). To provide a rationale for this therapeutic combination we investigated PD-L1 expression during GMCI therapy in human and mouse glioma cells in vitro and found that there was a consistent increase in cell surface PD-L1 levels. Interestingly, this was not associated with an increase of mRNA or protein. We also show that GMCI induces a type-I interferon response, and that the release of IFNβ is at least partially responsible for autocrine/paracrine PD-L1 up-regulation. In vivo studies using an intracranial GL261 model showed high levels of long term survivors in the GMCI/PD1 combination(11/14), compared with GMCI(6/16), anti-PD1(5/12) and controls(0/11). In addition, tumor infiltrating lymphocytes after GMCI showed an increase in CD8+, CD8+/GranzymeB+, and CD8+/IFNγ+/TNFα+cells suggestive of cytotoxic T-cell activation. However, there was also a significant increase in CD4+, CD4+/FoxP3+, and IL-10 indicating a significant infiltration by Tregs, releasing immunosuppressive cytokines. Additionally, there was a significant increase in PD1+/TIM3+ T-cells, indicative of an immunosuppressive microenvironment. Overall, our data show that GMCI/anti-PD1 combinatorial therapy is effective in a syngeneic tumor model, and strongly support clinical trials of GMCI/checkpoint inhibitor combinations in glioblastoma patients.

Abstract A075: Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition in GBM therapy

Abstract While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune-checkpoint inhibitors block tumor-induced T-cell exhaustion, the combination of these two approaches offers a potentially synergistic interaction. One of the molecular underpinnings of T-cell exhaustion is the expression of Programmed Death-1 (PD1) on T-cells that recognizes its ligand PD-L1. AdV-tk is an immunostimulatory virus-based approach, known as Gene-Mediated Cytotoxic Immunotherapy (GMCI), that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene(TK) followed by administration of an anti-herpetic prodrug(ganciclovir-GCV) and recently showed encouraging results in a Phase II trial in glioblastoma(Wheeler et al.,2016). To provide a rationale for this therapeutic combination we investigated PD-L1 expression during GMCI therapy in human and mouse glioma cells in vitro and found that there was a consistent increase in cell surface PD-L1 levels. Interestingly, this was not associated with an increase of mRNA or protein. We also show that GMCI induces a type-I interferon response, and that the release of IFNβ is at least partially responsible for autocrine/paracrine PD-L1 up-regulation. In vivo studies using an intracranial GL261 model showed high levels of long term survivors in the GMCI/PD1 combination (11/14), compared with GMCI (6/16), anti-PD1 (5/12) and controls (0/11). In addition, tumor infiltrating lymphocytes after GMCI showed an increase in CD8+, CD8+/GranzymeB+, and CD8+/IFNγ+/TNFα+cells suggestive of cytotoxic T-cell activation. However, there was also a significant increase in CD4+, CD4+/FoxP3+, and IL-10 indicating a significant infiltration by Tregs, releasing immunosuppressive cytokines. Additionally, there was a significant increase in PD1+/TIM3+ T-cells, indicative of an immunosuppressive microenvironment. Overall, our data show that GMCI/anti-PD1 combinatorial therapy is effective in a syngeneic tumor model, and strongly support clinical trials of GMCI/checkpoint inhibitor combinations in glioblastoma patients. Citation Format: Maria Carmela Speranza, Kazue Kasai, Franz Ricklefs, Sarah R. Klein, Carmela Passaro, Hiroshi Nakashima, Johanna Kaufmann, Agnieszka Bronisz, Estuardo Aguilar-Cordova, Brian W. Guzik, Gordon J. Freeman, David A. Reardon, Patrick Wen, E. Antonio Chiocca, Sean E. Lawler. Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition in GBM therapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A075.

Abstract 2349: Evaluation of the combination of the prodrug-mediated gene therapy vector AdV-tk and immune checkpoint inhibitor for glioblastoma treatment in a syngeneic mouse model

Abstract There has been a recent surge of interest in cancer immunotherapies due to newly FDA approved immunologic treatments based on targeting immune checkpoint inhibition pathways. Despite the dramatic responses in animal models and early clinical human trials, particularly in melanoma, durable clinical responses are observed only in approximately 30% of patients, therefore combinatorial immune therapeutic approaches may be needed to improve outcomes further. While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics in brain tumors has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune checkpoint inhibitors block the body's natural checkpoint (ICI) response, the combination of these two approaches offers a potentially advantageous interaction. AdV-tk is an immunostimulatory virus-based approach that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene (AdV-tk) followed by administration of an anti-herpetic prodrug (ganciclovir - GCV). This approach has shown benefit in clinical trials of glioblastoma among other tumor types. The immunological component results from the delivery vehicle being a virus, the mode of cell death, through both necrosis and apoptosis, and the pro-immunogenic properties of the thymidine kinase protein (TK). This approach has consistently demonstrated anti-tumor immune stimulation and an increased intra-tumoral CD8+ T-cell infiltrate. Not surprisingly, however, this immune stimulation also leads to increased expression of immune checkpoint inhibitory ligands on tumor cells, including PD-L1. Our initial in vitro studies showed that the immune checkpoint ligand PD-L1 is expressed across a panel of human and mouse conventional and stem-like glioblastoma-derived cells (GSCs). After AdV-tk infection and GCV treatment PD-L1 is consistently up-regulated both at protein and mRNA levels, while no changes were detected after AdV-tk alone. In vivo experiments in immunocompetent mouse models with GL261 glioma cell lines demonstrate a significant improvement in survival when we combine AdV-tk+GCV and anti-PD-1 antibodies. This data suggests that combination of immune checkpoint blockade with AdV-tk treatment should be explored in glioblastoma clinical trials. Citation Format: Maria Carmela Speranza, Kazue Kasai, Johanna Kaufmann, Estuardo Aguilar-Cordova, Brian W. Guzik, E. Antonio Chiocca, Sean Lawler. Evaluation of the combination of the prodrug-mediated gene therapy vector AdV-tk and immune checkpoint inhibitor for glioblastoma treatment in a syngeneic mouse model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2349.

Bovine herpesvirus type 5 in semen samples from bulls in Iran.

Bovine herpesvirus 5 (BoHV-5) is an important pathogen of the central nervous system and has already been described in the genital tract of cattle and in semen. This virus is responsible for sporadic epizootics of fatal meningoencephalitis of calves. The objective of the present study was the identification and characterization of BoHV-5 in semen samples from bulls for the first time in Iran. DNA was extracted from bull semen samples, and the glycoprotein D (gD) gene of BoHV-5 and also the thymidine kinase (tK) gene of bovine herpesvirus 1 (BoHV-1) were amplified by PCR assay. The results showed a high prevalence of BoHV-5 (73.2 %) and BoHV-1 (25.89 %) in Iranian bull semen samples. In addition, in order to identify and compare BoHV-5 isolated from Iranian bulls with other isolates from all over the world, the gD gene of this virus was cloned and sequenced. A BLAST search showed that the sequence of the gD gene of BoHV-5 from Iran was 99 % identical to other sequences in the GenBank database. The present study indicated that semen samples are important transmission sources of BoHV-5 virus in Iranian bulls.

Live and Let Die: A New Suicide Gene Therapy Moves to the Clinic

The concept of suicide gene therapy dates from the beginning of the field of gene therapy and was one of the first clinical applications. Initially described as an anticancer therapy, it quickly found a specific application in the practice of hematopoietic stem cell transplantation. A recent publication by Di Stasi et al. presents the first clinical data on a relatively new suicide gene system that targets caspase 9–mediated apoptosis in an inducible fashion.1 Specifically, five children undergoing haplo-identical stem cell transplantation for leukemia received donor T lymphocytes that had been genetically engineered with the inducible caspase 9 (iCasp9) gene. Four of the five patients developed graft-vs.-host disease (GVHD) caused by the donor lymphocytes that was quickly reversed by induction of the iCasp9 suicide gene. These results further validate the findings of others that suicide gene therapy for the control of GVHD is a valuable clinical procedure and, in the broader context, may have implications for a variety of other gene therapy applications in which a suicide safety switch may be useful.

Persistent anti-tumor effects via recombinant adeno-associated virus encoding herpes thymidine kinase gene monitored by PET-imaging

Despite the well-documented advantages of the recombinant adeno-associated virus (rAAV) as a gene delivery vehicle, including its non-pathogenic and long-term therapeutic gene expression, there have been very limited studies on its potential for producing persistent anti-tumor effects, particularly in vivo. To address this issue, we constructed rAAV vectors encoding herpes simplex virus 1-thymidine kinase (HSV-TK) or its mutant form (sc39TK) as therapeutic genes, and GFP as a control gene. Effective rAAV-mediated gene delivery was readily observed in human cancer cells using immunocytochemistry and Western blotting. Cell survival analysis following prodrug ganciclovir treatment implied that both preferential and superior cytotoxicity was achieved by rAAV-sc39TK introduction. Persistent anti-tumor effects in vivo were investigated in Balb/c nude mice bearing human cancer cells treated with either rAAV-sc39TK or -GFP. Severe tumor growth inhibition was clearly observed only in the case of sc39TK with ganciclovir treatment. Non-invasive micro-PET imaging using 18F-FHBG directly correlated with persistent anti-tumor effects by sc39TK. Therefore, the present study provides evidence that rAAV-mediated persistent therapeutic gene expression can occur, resulting in long-term anti-tumor activities and that these events can be readily monitored using micro-PET imaging.

Parabiosis model does not show presence of circulating osteoprogenitor cells

The goal of this study was to determine the presence of osteoprogenitor cells in the peripheral blood. Experiments were conducted with a parabiosis model in which osteoblast specific transgenic mice (Col2.3GFP or hOC-GFP) were surgically joined with a transgenic mouse where herpes virus thymidine kinase gene is under the control of the collagen alpha1 promoter (Col2.3DeltaTK). This method permits conditional ablation of osteoblasts by ganciclovir (GCV) treatment. In parabionts treated with GCV for 15 days or 1.5-2 months, GFP (hOC-GFP or Col2.3GFP) expression was not detected in histological preparations or in marrow stromal cell cultures from the Col2.3DeltaTK parabiont. Finally, Col2.3GFP/Col2.3DeltaTK pairs were treated with GCV for 15 days and allowed to recover from GCV for 3 months. Again there was a failure to detect Col2.3GFP expressing cells in the Col2.3DeltaTK parabiont. These observations, at least within the limits of this model system, allow the conclusion that osteoprogenitor cells do not readily circulate.

Detection and quantitation of gallid herpesvirus 1 in avian samples by 5′ Taq nuclease assay utilizing Minor Groove Binder technology

A 5′ Taq nuclease assay utilizing Minor Groove Binder technology and targeting the thymidine kinase gene of gallid herpesvirus 1 (GaHV-1) was designed and optimized for use in diagnosing avian infectious laryngotracheitis. The assay was specific for GaHV-1 in that it did not react with other avian viral or bacterial pathogens. The detection limit was 1.0×10−2 median tissue culture infectious dose per reaction or 90 target copies per reaction. Fifteen out of 41 diagnostic samples from sick birds reacted in the assay, five of which produced a typical alphaherpesvirus cytopathic effect (CPE) on chicken kidney (CK) cells. Sequencing, using amplicons generated by a polymerase chain reaction with primers flanking the 5′ Taq nuclease amplicon, confirmed the presence of GaHV-1 in six samples (two producing alphaherpesvirus CPE on CK cells, three not producing alphaherpesvirus CPE, and one that was not inoculated onto CK cells). Tracheal swabs taken from 18 healthy broilers did not react in the assay. The ability of the assay to determine viral load in samples was demonstrated. Overall the assay is suitable for the rapid diagnosis of infectious laryngotracheitis.

Phase I Study of Noninvasive Imaging of Adenovirus-mediated Gene Expression in the Human Prostate

To monitor noninvasively potentially therapeutic adenoviruses for cancer, we have developed a methodology based on the sodium iodide symporter (NIS). Men with clinically localized prostate cancer were administered an intraprostatic injection of a replication-competent adenovirus, Ad5-yCD/utTK(SR39)rep-hNIS, armed with two suicide genes and the NIS gene. NIS gene expression (GE) was imaged noninvasively by uptake of Na(99 m)TcO(4) in infected cells using single photon emission-computed tomography (SPECT). The investigational therapy was safe with 98% of the adverse events being grade 1 or 2. GE was detected in the prostate in seven of nine (78%) patients at 1 x 10(12) virus particles (vp) but not at 1 x 10(11) vp. Volume and total amount of GE was quantified by SPECT. Following injection of 1 x 10(12) vp in 1 cm(3), GE volume (GEV) increased to a mean of 6.6 cm(3), representing, on average, 18% of the total prostate volume. GEV and intensity peaked 1-2 days after the adenovirus injection and was detectable in the prostate up to 7 days. Whole-body imaging demonstrated intraprostatic gene expression, and there was no evidence of extraprostatic dissemination of the adenovirus by SPECT imaging. The results demonstrate that noninvasive imaging of adenovirus-mediated gene therapy in humans is feasible and safe.

Radiographic and immunologic responses to adjuvant immunotherapy for malignant gliomas

2039 Background: The invasive nature of malignant gliomas leads to a high rate of recurrence. Recruitment of the immune system to eradicate residual tumor may provide a significant outcome improvement. AdV-tk, an adenoviral vector containing the Herpes virus thymidine-kinase gene (GliAtak, Advantagene) plus prodrug has shown promise as an immuno-stimulatory adjuvant to standard therapies. The mechanisms involve tumor cytotoxicity combined with local elicitation of danger signals, recruitment and activation of antigen presenting cells and stimulation of systemic antitumor T- cell immunity. Methods: Patients with newly diagnosed malignant glioma received one of three dose levels of AdV-tk (3×1010, 1×1011 and 3×1011 vector particles) delivered to tumor cells via tumor bed injection followed by valacyclovir 2gm TID×14d. Radiation began within 7 days and temozolomide was allowed after prodrug completion. Results: The study enrolled 3 patients in each of the first two dose levels and 6 in the third. Tumors were classified as WHO grade IV (GBM) in 10 patients and anaplastic astrocytoma in two. Possibly related toxicity included transient elevation of liver transaminase, creatinine and fever. There were no significant related delays or interruptions in radiation or temozolomide. Five patients are alive 12–21 months after treatment. Significant intratumoral T-cell and macrophage infiltrate was seen in two patients who underwent re-resection at 30 and 36 weeks, suggesting a treatment induced antitumor response. One GBM patient, with persistent enhancement and FLAIR signal resolving slowly over 9 months is progression-free >20 months after treatment. Three patients have persistent enhancement and FLAIR signals but no progression for >12 months. Radiation and immune effects may confound interpretation of conventional imaging. Perfusion MRI was used in two of these three patients in an attempt to differentiate tumor progression from treatment effects. Conclusions: AdV-tk can be safely combined with surgery and radio-chemotherapy in patients with newly diagnosed malignant gliomas. Novel imaging techniques may help differentiate tumor progression from inflammation after radio-immunotherapy. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Advantagene Advantagene

The 3.6 kb DNA fragment from the rat Col1a1 gene promoter drives the expression of genes in both osteoblast and osteoclast lineage cells

The type I collagen promoter has been used to develop transgenic constructs that are able to mark different stages of osteoblastic differentiation. The pOBCol3.6 promoter is active in early mesenchymal progenitors, including preosteoblasts and osteoblasts, while the pOBCol2.3 promoter is more restricted, showing expression in mature osteoblasts and osteocytes. Transgenic mouse lines have been created that express various GFP reporters under the control of both promoters. These transgenic mice permit the tracking of osteoblastic lineage progression in vitro. They also represent a system to test lineage progression in vivo after the transplantation of progenitors. A parabiosis system was used in which pOBCol3.6GFP transgenic mice were surgically joined with mice bearing a Col2.3ΔTK transgene. The Col2.3ΔTK transgenic mouse bears a herpes thymidine kinase gene driven by the pOBCol2.3 promoter, and upon treatment with gancyclovir (GCV) displays extensive destruction of the bone lining cells. After a common circulation was established, parabiotic pairs were treated with GCV for 15 days. Histological analysis of their bones showed the clear presence of GFP positive cells in the Col2.3ΔTK parabionts, around trabecular bone and on the endosteal and periosteal surfaces. Stromal cell cultures from these Col2.3ΔTK parabionts did not display mineralized colonies coexpressing GFP. In contrast, scattered GFP positive clusters that contained large cells with morphology similar to osteoclast like cells (OCLs) were observed. These cells were also TRAP positive. They were readily detected in Col2.3ΔTK mice treated with GCV and transplanted with purified hematopoietic stem cells (HSCs) isolated from pOBCol3.6GFP mice. OCLs were also generated in vitro from osteoclast progenitor cells obtained from pOBCol3.6GFP mice that were defined by the B220− CD3− CD11b− c-fms+ phenotype. Molecular analysis showed that OCLs did not express type I collagen indicating that the Col3.6 promoter contains elements that are active during osteoclastogenesis and are not strictly related to collagen transcription. In summary, we demonstrate that pOBCol3.6 unexpectedly directs the expression of transgenes in the osteoclast lineage and this effect must be considered when utilizing this promoter to study of mesenchymal progenitor cells.

Five year follow up of a phase II study of cytotoxic immunotherapy combined with radiation in newly diagnosed prostate cancer

4635 Background: In the U.S. there are about 70,000 annual prostate cancer recurrences. The purpose of this study is to evaluate a product to decrease incidence of recurrence. This study is based on objective clinical responses in Phase I studies with AdV-tk (ProstAtak™, Advantagene, Inc) as monotherapy in recurrent disease and preclinical data demonstrating synergy between AdV-tk and radiation. AdV-tk is an adenoviral vector expressing the herpes thymidine kinase gene delivered to the prostate via TRUS-guided injection followed by 14 days of oral prodrug. The mechanisms of function involve direct tumor cytotoxicity, local elicitation of danger signals, recruitment and activation of antigen presenting cells and stimulation of systemic anti-tumor T-cell immunity. Method: AdV-tk was evaluated in combination with radiation in 66 newly diagnosed patients: 33 low risk (Arm A, PSA <10, Gleason <7, and T1c-T2a) and 33 intermediate-high risk (Arm B, PSA ≥10, Gleason ≥7, or T2b-T3). Arm A received two treatments with AdV-tk, immediately before and 14 days into radiation. Arm B received an additional treatment at initiation of androgen deprivation therapy. Results: Two surrogate and one definitive end-point were evaluated. Frequency of patients in Arm A with PSA nadir ≤0.2 ng/ml was 71% vs 56% in a control group of concurrent patients without AdV-tk. The two-year pathologic complete response (pCR) rate by sextant biopsy was 90% in Arm A and 94% in Arm B, compared to an expected range of 70–73%. Freedom from failure (FFF) after 60 month median follow up is 100% for Arm A and 90% for Arm B (95% for intermediate, 75% for high risk) vs best reported results of 79–90% for low risk and 48–79% for intermediate-high risk patients. The three failures in Arm B occurred within months after treatment leading to a Kaplan-Meier curve that plateaus at 90% beyond year 3. This is notably different than previous reports in which the curves continue to drop beyond year 5. Conclusion: These results suggest that AdV-tk combined with radiation therapy may significantly reduce the recurrence rate in patients with prostate cancer, particularly in patients with intermediate-high risk disease. A randomized controlled trial is warranted. [Table: see text]

Recurrent herpes simplex virus hepatitis after liver retransplantation despite acyclovir therapy

Herpes virus hepatitis (HSV) represents a form of acute necrotizing hepatitis, which most frequently develops in immunocompromised patients. Therapeutic options include high-dose intravenous acyclovir and liver transplantation. We report the first case of recurrent HSV hepatitis after liver retransplantation, which occurred despite continuous administration of high-dose intravenous antiviral therapy. Because explant histology pointed to initial therapy response, we thought that the reason for recurrence might be due to acyclovir resistance. Most acyclovir resistance is caused by inactivating mutations in the herpes virus thymidine kinase gene. HSV infection was detected by histology and proofed by immunohistochemistry. PCR amplification of the herpes virus thymidine kinase gene was performed on histology specimens to demonstrate the course of viral infection in liver tissue. Genotypic resistance testing of the herpes virus was performed by sequencing the thymidine kinase amplicon. In serial biopsy, HSV-DNA sequences were only detectable when histology revealed herpes hepatitis. Whereas the primary explant exhibited the wild-type thymidine kinase gene, a biopsy of the second graft one month after retransplantation, which showed recurrent herpes virus hepatitis, had a single base insertion within a homopolymeric cytosine stretch. This mutation causes a frame shift leading to a premature stop codon and results in a known acyclovir-resistant herpes strain. In conclusion, we believe that testing for acyclovir-resistant herpes strains should be considered in high-risk patients in whom viral clearance is not achieved serologically to prevent fatal recurrence of disease by using antiviral drugs such as inhibitors of HSV-DNA polymerase or viral helicase primase inhibitors.