Ganciclovir Metabolites Research Articles

Improved Suicide Gene Therapy: Lentiviral Gene Transfer of Equine Herpes Virus Type 4 Thymidine Kinase into Target Cells.

Herpes virus type 1 thymidine kinase (HSV1-TK) with ganciclovir (GCV) prodrug treatment is the most widely used approach for suicide gene therapy. This ‘suicide' strategy allows direct reduction of tumors and clearance of donor cells should graft-versus-host disease (GvHD) arise after bone marrow transplantation. Given recent clinical outcomes, this suicide approach may also provide a key safety component for therapeutic gene transfer vectors that integrate. Although suicide gene therapy using HSV1-TK-encoding oncoretroviral vectors has been evaluated in the clinic, the success of this approach has been relatively modest. Reasons for this include: low gene transfer efficacy, reduced expression of the suicide gene, and insufficient conversion of substrate. Our goal is to overcome these limitations by using a novel lentiviral vector (LV) encoding an alternative kinase/prodrug combination. The rational for our innovative suicide gene therapy strategy is two-fold: 1) Lentiviral vectors can efficiently transduce not only dividing cells but also non-dividing cells. 2) Applying a faster viral enzyme like equine herpes virus type 4 thymidine kinase (EHV4-TK) could be advantageous as it has been shown to be kinetically superior to HSV1-TK at GCV phosphorylation. The aim of this study is to evaluate whether LV-mediated gene modification of target cells with EHV4-TK can lead to efficient killing following GCV treatment. We first constructed a LV expression system carrying the wild-type EHV4-TK cDNA with an IRES element followed by a truncated form of human CD19 (hCD19Δ). Human CD19 was chosen as a cell surface marker to allow functional titering of virus and for immuno-enrichment of transduced cells prior to infusion since it is not expressed in the T cell lineage. The truncated form lacks the intracellular domain and therefore does not signal. Use of an IRES element can abrogate some variegated expression seen with vectors having dual promoters. The LV was pseudotyped with VSV-g and concentrated by ultracentrifugation. After one infection, Jurkat cells (human T cell leukemia) showed a more than 80% functional and stable transduction efficiency (MOI = 10). Using hCD19Δ as a selective marker, transduced Jurkat cells were enriched to over 95% positive by immuno-affinity sorting. EHV4-TK-transduced Jurkat cells exhibited increased cell killing in response to GCV treatment (the apoptotic cell indexes with or without GCV were 69.4 ± 1.5 % and 18.8 ± 1.7 %, respectively; n=3). Highly efficient transduction (more than 60%) of primary human T cells was accomplished by a three time exposure to virus over 36 hours at MOI of 20. Next, we found that GCV efficiently killed transduced primary human T cells in a dose dependent manner. We are now comparing the efficiency of GCV conversion by HSV1-TK and EHV4-TK using LV-transduced cells that express the similar protein levels. We are also evaluating intracellular levels of GCV metabolites by HPLC. These results demonstrate that our novel suicide gene therapy strategy has significant potential for many clinical applications.

807. Addition of HU to GCV Induces Bystander Cytotoxicity in Co-Cultures of HSVTK-Expressing and Non-Expressing HeLa Cells

Expression of the herpes simplex virus type I thymidine kinase (HSVTK) gene in tumor cells sensitizes them to the antiviral agent ganciclovir (GCV). Incubation with GCV results not only in cytotoxicity from cells that express the transgene, but neighboring bystander cells as well. Bystander cytotoxicity has been attributed to the transfer of phosphorylated GCV metabolites from HSVTK-expressing to non-expressing cells through protein channels known as gap junctions. We have previously reported good bystander cytotoxicity in the U251 glioblastoma and SW620 colon carcinoma cell lines, which have 84% and 39% gap junction intercellular communication (GJIC), respectively. However, in the HeLa cell line (23% GJIC), no bystander cytotoxicity resulted from incubating a co-culture of HSVTK-expressing and bystander HeLa cells with GCV. Further analysis revealed that lack of bystander cytotoxicity in these cells was not due to an inability to transfer phosphorylated GCV, but resulted from a low level of transfer along with a rapid half-life of GCVTP in bystander cells (3.1 hrs) when compared to the HSVTK-expressing cells (12.3 hrs). We hypothesized that reduction of the endogenous competitor for GCVTP incorporation into DNA, dGTP, would increase levels of GCV incorporation into DNA resulting in the induction of bystander cytotoxicity. To that end, the effect of hydroxyurea (HU), a ribonucleotide reductase inhibitor, on GCV cytotoxicity in cultures of 100% HSVTK-expressing HeLa cells and co-cultures of HSVTK-expressing and bystander HeLa cells was examined. Isobologram analysis of the interaction between GCV and HU demonstrated an additive-antagonistic effect on 100% cultures of HSVTK-expressing HeLa cells, while exhibiting a synergistic effect on the HSVTK-expressing and bystander cells in a co-culture. To determine the mechanism for the synergistic cytotoxicity demonstrated in each of the populations from the co-culture when incubated with GCV and HU, GCVTP and dNTP pools were quantified and the results were compared to 100% cultures of HSVTK-expressing HeLa cells. The results demonstrated a significant increase in the GCVTP:dGTP values in cultures of 100% HSVTK-expressing cells (>2.5 fold) and in co-cultures of HSVTK-expressing cells (>1.4 fold) and bystander cells (>1.8 fold) at 4 hours. However, at 12 hours the GCVTP:dGTP value for 100% cultures of HSVTK-expressing HeLa cells was at control levels (1.3 0.5 fold) while the values for HSVTK-expressing cells (>2.7 fold) and bystander cells (>4.9 fold) in co-culture were still significantly higher. These data suggest that the prolonged increase in the GCVTP:dGTP value in co-cultures incubated with GCV and HU leads to an increase in the levels of GCVTP incorporated into DNA resulting in synergistic cytotoxicity.

Direct evidence for the absence of intercellular trafficking of VP22 fused to GFP or to the herpes simplex virus thymidine kinase.

The treatment of solid tumors by retroviral delivery of the herpes simplex virus thymidine kinase (TK) followed by ganciclovir (GCV) treatment has so far shown only limited success in patients. One major drawback in this approach is the lack of efficient in vivo gene delivery to cancer cells. Although, the transduction of every single tumor cell is not a requirement since the bystander effect (BE) mediated by gap junctions allows the diffusion of the toxic GCV metabolites from TK-expressing cells toward untransduced cells. To render the TK/GCV approach more potent, and independent of the level of gap junctions, we have tested the efficiency of a TK mutant (TK30) fused to VP22, a herpes simplex protein that seems to be capable of intercellular trafficking. We failed to detect an increase in the BE with cells expressing VP22 fused to TK30 versus cells containing TK30 alone, and this result forced us to reinvestigate the trafficking properties of VP22. Using very sensitive Western blot and fluorescence assays, we were not able to detect the spread of VP22 fused either to TK30 or GFP. These results indicate that VP22 cannot be used as a cargo to translocate TK30 or GFP.

Expression of MRP4 Confers Resistance to Ganciclovir and Compromises Bystander Cell Killing

The multidrug resistance protein MRP4, a member of the ATP-binding cassette superfamily, confers resistance to purine-based antiretroviral agents. However, the antiviral agent ganciclovir (GCV) has not been shown to be a substrate of MRP4. GCV is important not only in antiviral therapy, but also in the selective killing of tumor cells modified to express herpes simplex virus thymidine kinase (HSV-TK). We therefore tested the effect of MRP4 on the cytotoxicity of GCV, on the ability of GCV to kill cells genetically modified to express HSV-TK, and on the bystander effect in which unmodified target cells are killed by GCV. Cells overexpressing MRP4 had markedly increased resistance to the cytotoxicity of GCV. Although, expression of recombinant HSV-TK increased the intracellular concentration of GCV nucleotide, cells were rescued by the cytoprotective effect of MRP4. In cells that overexpressed MRP4, intracellular accumulation of GCV metabolites was reduced, efflux of these metabolites was increased, and resistance to bystander killing was increased. Therefore, MRP4 can strongly reduce the susceptibility of HSV-TK-expressing cells to GCV, and its overexpression in adjacent cells protects them from bystander cell death. These findings indicate that a nucleotide transporter, such as MRP4, modulates the cellular response to GCV and thus may influence not only the efficacy of antiviral therapy, but also prodrug-based gene therapy, which is critically dependent upon bystander cell killing.

Connexin-independent ganciclovir-mediated killing conferred on bystander effect-resistant cell lines by a herpes simplex virus-thymidine kinase-expressing colon cell line.

A novel gap junction-independent mechanism for ganciclovir-mediated bystander effect killing by a herpes simplex virus thymidine kinase (HSV-TK)-expressing SW620 human colon tumor cell line has been characterized. The mechanism of the HSV-TK/GCV bystander effect for many tumor cell lines has been demonstrated to be due to connexin gap junction transfer of phosphorylated ganciclovir (GCV) metabolites; however, there may be as yet uncharacterized connexin-independent mechanisms for the effect. To address this, the bystander effect was further evaluated in a panel of cell lines mixed with homologous HSV-TK-expressing cell lines, a SW620.TK cell line, or a high connexin43-expressing PA-317.TK cell line. Of the 10 cell lines tested, 4 were found to be resistant to bystander effect killing by their homologous HSV-TK-expressing cell lines and the PA-317.TK cells, but all of the cell lines were sensitive to GCV killing when mixed with the SW620.TK cells. The SW620.TK cells were then further evaluated for any indication of extracellular GCV metabolite efflux. Culture medium from SW620.TK cells labeled with [(3)H]GCV was evaluated for the presence of GCV nucleotides by ion-exchange column separation and HPLC analysis. The presence of GCV mono-, di-, and triphosphate metabolites in the medium was detected. Inclusion in the medium of inhibitors of extracellular phosphatases and ecto-ATPases increased the proportion of GCV metabolites recovered. These results indicate that phosphorylated GCV metabolites can be effluxed from SW620.TK cells and that some type of cellular uptake mechanism independent of gap junctions exists for nucleotide entry into neighboring cells.

Ganciclovir-mediated cell killing and bystander effect is enhanced in cells with two copies of the herpes simplex virus thymidine kinase gene.

Delivery and expression of the herpes simplex virus thymidine kinase (HSVtk) gene in combination with the prodrug ganciclovir is currently being evaluated for the treatment of many types of cancer. After initial phosphorylation by HSVtk, cellular kinases generate the toxic triphosphate form of ganciclovir (GCV). To further define the role of GCV metabolism in cells expressing HSVtk, two human tumor cell lines, UMSCC29 and T98G, were transduced with HSVtk and screened for insertion of one or two copies of the viral transgene by Southern blot analysis. Both the relative capacities for incorporating labeled GCV and the levels of GCV metabolites were determined for each of the parental cell lines and their derivatives containing either one or two copies of the HSVtk gene. The efficiency of GCV killing and the magnitude of the bystander effect were compared for the single- and double-copy HSVtk cell lines. Consistently, cells that expressed two copies of HSVtk metabolized GCV more efficiently, were more sensitive to GCV, and demonstrated improved bystander killing relative to single-copy HSVtk cells. The implications of these results for future and current therapies employing HSVtk and GCV are discussed.

The bystander effect in the HSVtk/ganciclovir system and its relationship to gap junctional communication.

The bystander effect (BSE) is an interesting and important property of the herpes thymidine kinase/ganciclovir (hTK/GCV) system of gene therapy for cancer. With the BSE, not only are the hTK expressing cells killed upon ganciclovir (GCV) exposure but also neighboring wild-type tumor cells. On testing a large number of tumor cell lines in vitro, a wide range of sensitivity to bystander killing was found. Since transfer of toxic GCV metabolites from hTK-modified to wild-type tumor cells via gap junctions (GJ) seemed to be a likely mechanism of the BSE, we tested GJ function in these various tumors with a dye transfer technique and pharmacological agents known to affect GJ communication. We confirmed that mixtures of tumor cell resistant to the BSE did not show dye transfer from cell to cell while bystander-sensitive tumor cells did. Dieldrin, a drug known to decrease GJ communication, diminished dye transfer and also inhibited the BSE. Forskolin, an upregulator of cAMP did increase GJ, but directly inhibited hTK and therefore its effect on BSE could not be determined. We conclude that these observations further support port the concept that functional GJ play an important role in the BSE and further suggest that pharmacological manipulation of GJ may influence the outcome of cancer therapy with hTK/GCV.

Superior cytostatic activity of the ganciclovir elaidic acid ester due to the prolonged intracellular retention of ganciclovir anabolites in herpes simplex virus type 1 thymidine kinase gene-transfected tumor cells.

Ganciclovir (GCV) and its lipophilic elaidic acid ester prodrug E-GCV were evaluated for their antiherpetic, cytostatic and metabolic properties, E-GCV proved exquisitely inhibitory to the replication of herpes simplex virus type 1 (HSV-1) and HSV-2 in cell cultures (50% effective concentration (EC50): 0.002 microM). It was five- to 10-fold more effective than its parent drug GCV. E-GCV was at least 2000-fold more cytostatic to HSV-1 or HSV-2 thymidine kinase (tk) gene-transfected mammary carcinoma FM3A tk-/HSVtk+ tumor cells than to the corresponding nontransfected tumor cells. The cytostatic activity of E-GCV to the HSVtk gene-transfected tumor cells was far superior to that of GCV. Metabolic studies revealed that both GCV and E-GCV were converted to the mono-, di- and tri-phosphate derivatives of GCV to a markedly higher extent in FM3Atk-/HSV-1 tk+ cells than in wild-type FM3A/0 cells. Strikingly, mono-, di- and tri-phosphate metabolites of GCV were retained for a substantially longer time in E-GCV-treated cells (half-life approximately 50 h) than in GCV-treated cells (half-life approximately 20 h). The longer retention time of the GCV metabolites most likely explains why E-GCV is superior to GCV against herpes simplex virus replication and HSVtk gene-transfected tumor cell proliferation. Taking into account the marked stability of E-GCV in human plasma and its much higher lipophilicity than GCV, E-GCV should be considered as an effective lipophilic prodrug of GCV with a markedly enhanced cytostatic activity in HSVtk gene-transfected tumor cells compared with parental ganciclovir. Its usefulness in the combined gene/chemotherapy of HSVtk gene-transfected tumors should be further pursued.

Stereotactic Injection of Herpes Simplex Thymidine Kinase Vector Producer Cells (PA317-G1Tk1SvNa.7) and Intravenous Ganciclovir for the Treatment of Progressive or Recurrent Primary Supratentorial Pediatric Malignant Brain Tumors. St. Jude Children's Research Hospital, Memphis, Tennessee

This study will evaluate the safety and efficacy of in vivo gene transfer of the herpes simplex thymidine kinase (HSV-Tk1) gene using PA317/G1Tk1SvNa.7 vector producer cells (VPC) in pediatric patients with progressive or recurrent primary supratentorial malignant brain tumors. Insertion of the HSV-Tk1 gene confers a sensitivity to the anti-herpes drug ganciclovir. It has been demonstrated that the direct injection of HSV-Tk vector producer cells into growing tumors in animals can result in their complete destruction with ganciclovir therapy. This selective destruction of growing tumors in situ is thought to result from the transfer of the HSV-Tk gene into the tumor cells and the production of toxic ganciclovir metabolites which result from the interaction of HSV-Tk and ganciclovir. This procedure can result in the cure of some experimental animals with limited systemic toxicity due to selective gene transfer into tumors. This clinical trial will focus on maximizing the relative number of vector producer cells to the tumor mass by stereotactically injecting VPCs into the tumor mass. Children with progressive or recurrent primary supratentorial malignant brain tumor which is accessible to stereotactic injection will be evaluated for the extent and location(s) of their disease before being entered into the study. Fifteen days after stereotactic injection of the tumor mass, ganciclovir will be administered at 5 mg/kg IV b.i.d. for 14 days. Upon completion of the treatment with HSV-Tk1 vector producer cells and ganciclovir, the patient will be followed monthly for the first three months, then every two months for the next twenty-one months, and annually for life thereafter.