Comparison of HSV-1 Thymidine Kinase-Dependent

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Comparison of HSV-1 thymidine kinase-dependent and -independent inhibition of replication-competent adenoviral vectors by a panel of drugs Oliver Wildner, Dennis Hoffmann, Christian Jogler, and Klaus U¨ berla Ruhr-Universita¨t Bochum, Abteilung fu¨r Molekulare und Medizinische Virologie, Bldg. MA, Rm. 6/40, D-44801 Bochum, Germany.

Replication-competent adenoviral vectors hold the promise to be more efficient gene delivery vehicles than their replication- deficient counterparts, but they are also associated with a higher risk for adverse effects, especially in light of the fact that there is no established effective therapy for serious, disseminated adenovirus infection. To assess whether the therapeutic options to inhibit adenoviral replication can be enhanced by expressing a suicide gene, we examined the antiadenoviral effects of 15 drugs against wild-type adenovirus type 5 (Ad5) and an Ad5-based replication-competent vector expressing herpes simplex virus-1 thymidine kinase (HSV-tk) (Ad.OW34) using a real-time polymerase chain reaction -based assay and flow cytometry. Ad5 and Ad.OW34 were highly susceptible to the fluorinated pyrimidine analogs 5-fluoro-20-deoxyuridine (FUdR), 5-fluorouridine (FUR), and trifluorothymidine (TFT), with a mean 50% inhibitory concentration (IC50) ranging from 0.12 to 0.32 mM. The mean IC50 of ribavirin and cidofovir (CDV) for Ad5, the most frequently used drugs to treat adenovirus disease, was 6.87 and 3.19 mM, 0 respectively. In contrast to Ad5, the Ad.OW34 vector was susceptible to (E)-5-(2-bromovinyl)-2 -deoxyuridine (BVdU, IC50 0.09 mM), ganciclovir (GCV, IC50 0.19 mM), and acyclovir (ACV, IC50 32.04 mM). Additionally, we demonstrated in an animal model that Ad.OW34 vector replication can be inhibited significantly by GCV, CDV, and TFT by 35.2, 7.7, and 3.7-fold, respectively, compared to untreated animals. The observed antiadenoviral effects were primarily not through cell killing, since the in vitro 50% cytotoxic concentrations (CC50) were more than 1000 times higher than the antiadenoviral IC50 of the drugs examined, even in cells stably expressing HSV-tk. Since for HSV-tk-dependent inhibition of adenoviral vectors, stability of HSV-tk expression is crucial, we examined Ad.OW34 vector stability, by passaging the vector 10 times serially in the presence of 10 mM GCV. The HSV-tk/GCV system neither changed the susceptibility of Ad.OW34 to GCV significantly nor detectable vector rearrangements occurred, suggesting that the system might be suitable as a fail-safe mechanism to stop adenoviral vector replication. Cancer Gene Therapy (2003) 10, 791–802. doi:10.1038/sj.cgt.7700638 Keywords: replication-competent adenoviral vector; nucleoside analogs; antiadenoviral activity; herpes simplex virus-1 thymidine kinase

ntil recently, for safety, crippled, replication-defec- shown encouraging antineoplastic activity.44 However, Utive viruses have been used as vectors for cancer gene due to their ability to replicate and spread from the initial therapy. Consequently, most clinical trials have shown site of infection, replication-competent vectors are also minimal gene transfer at best and few tumor responses.1–3 associated with a higher risk for adverse effects than their Even if at first only a small fraction of tumor cells has replication-deficient counterparts. been infected, replication-competent vectors have the For safety and improved therapeutic index of replica- potential to overcome poor transduction efficiency since tion-competent adenoviral vectors, it has been attempted vector replication leads to in situ amplification of the viral to restrict vector replication to tumors. In contrast to inoculum and spread throughout the tumor by subse- initial findings by Bischoff et al,5 deletion of the quent cycles of infection and lysis of neighboring tumor adenovirus E1B-55 K gene does not restrict virus replica- cells. First controlled clinical trials with an oncolytic tion to p53-dysfunctional cells.6–16 Other approaches adenovirus in combination with chemotherapy have involved the transcriptional targeting of adenovirus E1A genes, which are pivotal for adenoviral replication.17–19 However, targeting of vector replication by this approach Received March 17, 2003. might be poor for two reasons. First, promoter fidelity Address correspondence and reprint requests to: Ruhr-Universita¨t Bochum, Abteilung fu¨r Molekulare und Medizinische Virologie, Bldg. might be abolished by the upstream adenoviral packaging MA, Rm. 6/40, D-44801 Bochum, Germany. sequence that contains two enhancer elements of the E1A E-mail: [email protected] promoter, which are active in most cell types tested and Inhibition of adenovirus vector replication O Wildner et al 792 essential for adenoviral encapsidation.20–23 Second, al- drugs (E)-5-(2-bromovinyl)-20-deoxyuridine (BVdU), tri- ready small amounts of E1A gene products are sufficient fluorothymidine (TFT), and pyrimidine dideoxynucleo- to initiate adenoviral replication.24 In addition, E1A in- side analogs, inhibitors of HIV reverse transcriptase frame deletion mutants have been reported that support inhibitors. Also, we determined the antiviral effects of viral growth in tumor cells but not in normal cells.25 the acyclic nucleoside analogs acyclovir (ACV) and GCV However, in vivo verification of these adenoviral vectors’ that are used for the therapy of herpesvirus infections. We tumor specificity is hampered by the lack of a permissive also examined the antiadenoviral effects of foscarnet non-human host since human adenoviruses replicate only (PFA), a clinically used pyrophosphate analog that in human cells. inhibits not only all human herpesviruses but also HIV. Since their initial description in the 1950’s, adeno- Furthermore, we analyzed the effects of RBV, a nucleo- viruses have been known as a cause of common childhood side analog of guanosine shown to be active against a respiratory illnesses.26–28 In immunocompetent patients, broad spectrum of DNA and RNA viruses, and the most of these infections are asymptomatic, mild, or self- acyclic nucleoside phosphonate CDV that has activity limited. In the immunocompromised host, however, against virtually all DNA viruses. adenovirus infections can cause severe localized disease In addition, we analyzed the Ad.OW34 vector for including pneumonitis, colitis, hemorrhagic cystitis, he- stability of HSV-tk expression and examined in vivo the patitis, nephritis, encephalitis, or disseminated disease effects of GCV, CDV, and TFT on Ad.OW34 vector with multiorgan failure.29–32 Disseminated adenovirus replication. disease has been reported in patients with AIDS or malignancies29,33–35 and in bone marrow or solid organ transplant recipients32,36–39 with increasing frequency. Results The overall incidence of adenoviral disease in immunocompromised patients ranges from 10 to 20%.29,31,37,40 A qPCR-based assay was established to examine the Case fatality rates as high as 50–80% have been effects of 15 potential antiviral compounds (structural described.29,32,40,41 So far, experience is greatest with formulae shown in Fig 1) on the replication of wild-type parenteral administration of the broad-spectrum antiviral Ad5 and Ad. OW34 vector, an Ad5-based (E1+, nE3) ribonucleoside ribavirin (RBV),41 which has documented replication-competent virus expressing HSV-tk (genetic activity against adenovirus in vitro.42 In some cases structures of the vectors used in this study are shown in therapy was successful,43–48 but many patients died Fig 2). despite RBV treatment.49–56 Furthermore, cidofovir (CDV), an acyclic nucleoside phosphonate that has activity against virtually all DNA viruses including In vitro antiviral potency of drugs adenovirus,57,58 has been evaluated for disseminated The in vitro antiviral activity of the evaluated drugs is adenovirus disease with limited success.59–61 Reports of summarized in Table 1. Their potency was not signifi- successful alternative strategies are less common and cantly influenced whether HeLa, Panc-1, or A375 cells include an apparent beneficial effect of donor leukocyte were used for the assay. The following data were obtained infusion,53,54 intravenous immunoglobulin,62 and ganci- in at least three independent experiments on HeLa cells. clovir(GCV) treatment.63,64 The most effective inhibitors of Ad5 replication were the In light of the limited and nonspecific therapeutic fluorinated pyrimidine nucleoside analogs 5-fluoro-20- options for the treatment of wild-type adenovirus infec- deoxyuridine (FUdR) with an inhibitory concentration 65 tions and recent concerns about adenovirus-mediated 50% (IC50) of 0.1370.06 mM, 5-fluorouridine (FUR) with 66,67 gene transfer, we examined whether the therapeutic IC50 of 0.2070.09 mM, and TFT with IC50 of options to inhibit adenoviral replication can be enhanced 0.3270.08 mM. Inhibition of adenoviral replication by by expressing herpes simplex virus-1 thymidine kinase TFT was significantly different compared to FUdR or (HSV-tk). Hence, we analyzed the antiviral activity of 15 FUR (Po.05), whereas there was no significant difference potential antiviral drugs against a wild-type human between FUdR and FUR (P4.05). adenovirus serotype 5 (Ad5)-based replication-competent The IC50 of CDV and RBV were 3.1971.03 and vector expressing HSV-tk (Ad.OW34). To evaluate the 6.8771.89 mM, respectively. In our in vitro assay, the effects attributable to the expression of HSV-tk, we also antiadenoviral efficacy of CDV was significantly greater examined the effects of the drugs on wild-type Ad5 than that of RBV (Pr.03). However, FUdR, FUR, and replication, using a quantitative polymerase chain reac- TFT were more potent than CDV (Po.001). In addition, tion (qPCR)-based assay. Furthermore, we determined 30-deoxy-30-fluorothymidine (FLT) had an effect on Ad5 cytotoxicity of the drugs in vitro and in vivo in order to replication with an IC50 of 3.6570.71 mM. assess whether the observed antiadenoviral effects are just All drugs with antiadenoviral activity inhibited the due to cell killing. replication of the HSV-tk-expressing Ad.OW34 vector to Among the evaluated substances were 10 pyrimidine the same extent as wild-type Ad5. However, the anti- nucleoside analogs, which encompass a diverse group of herpes drugs BVdU, GCV, and ACV inhibited Ad.OW34 drugs that have been employed in the treatment of diverse vector replication strongly, but not Ad.5 replication in afflictions, including neoplastic diseases, psoriasis, infec- concentrations up to 100 mM. The IC50 of BVdU was tions caused by fungi and viruses, including the antiherpes 0.0970.02 mM and that one of GCV was 0.1970.04 mM;

Cancer Gene Therapy Inhibition of adenovirus vector replication O Wildner et al 793 Pyrimidine Nucleoside Analog X Y Z

5-Fluoro-2’-deoxyuridine (FUdR) F H OH 5-Fluoruridine (FUR) F OH OH Trifluorothymidine (TFT) CF3 H OH (E)-5-(2-Bromovinyl)-2’-deoxyuridine (BVdU) CH=CHBr H OH

3’-Deoxy-3’-fluorothymidine (FLT) CH3 H F 2’,3’-Dideoxy-3’-fluorouridine (F-ddU) H H F

3’-Azido-3’-deoxythymidine (AZT) CH3 H N3 3’-Azido-2’,3’-dideoxyuridine (AzdU) H H N3 2’,3’-Dideoxyuridine (ddU) H H H

Figure 1 Chemical structures of drugs evaluated in this study.

E1A/B E3 significantly less than BVdU and GCV (Po.001). The Ad5 IC50 of ACV was 32.0477.42 mM and the calculated IC90 0 20 40 60 80 100 was 256.1743.25 mM. The drugs 30-azido-20,30-dideoxyur- Map Units 0 0 0 0 ∆E3 idine (AzdU), 3 -azido-3 -deoxythymidine (AZT), 2 ,3 - dideoxy-30-fluorouridine (F-ddU), 20,30-dideoxyuridine

IRES E1B 19 & 55-kDa (ddU), 5,6-dihydrothymidine (DHT), and PFA had no HSV-tk effect on the replication of either Ad5 or Ad.OW34 vector E1A BGH pA CMVp in concentrations up to 100 mM. Ad.OW34

GFP In vitro cytotoxicity of antiviral compounds

Ad.OW94 To determine whether the observed in vitro antiadenoviral effects of the compounds are due to inhibition of viral Figure 2 Schematic organization of viral vectors. In both recombi- DNA replication or due to cytotoxic effects, melanoma nant vectors, E1A is transcriptionally coupled by an internal ribosome cells stably expressing HSV-tk (A375 STK) were incu- entry site (IRES) to the gene encoding HSV-tk or GFP, respectively. bated with test compounds for 36 hours at 371C. The 50% Both vectors are Ad5-based, E3-deleted, and the transgene is driven cytotoxic concentrations (CC50) were determined with the by the human CMV-IE promoter and terminated by the bovine growth CellTiter-Glot Luminescent Cell Viability Assay (Pro- hormone polyadenylation site (BHG pA). mega, Madison, WI). The rank order of the cytotoxicity of the compounds was TFT4FUdR, FUR4RBV4GCV4BVdU, FLT4ACV4CDV with a the IC90 was 0.7370.11 and 1.1170.16 mM, respectively. CC50 ranging from 2 to 20 mM, more than 1000-fold The IC50 and IC90 of BVdU and GCV were not significantly different (P4.05). Also, no significant above the IC50. difference was found between FUdR, FUR and BVdU and GCV (P4.05). In addition, there was no statistically Flow cytometric analysis of selected compounds on significant difference in the antiadenoviral activity of adenoviral replication GCV and TFT (P4.005) in contrast to BVdU and TFT To determine whether the observed in vitro antiadenoviral (Pr.005). ACV inhibited Ad.OW34 vector replication effects of the compounds are not only due to inhibition of

Cancer Gene Therapy Inhibition of adenovirus vector replication O Wildner et al 794 Table 1 Comparative in vitro activity of antiviral agents against wild-type adenovirus and Ad.OW34 vector replication on HeLa cells.

Ad5 Ad.OW34

Drug IC50 (mM) IC50 (mM) IC90 (mM)

Acyclovir (ACV) >100 32.0477.42 256.1743.25 Ganciclovir (GCV) >100 0.1970.04 1.1170.16 Ribavirin (RBV) 6.8771.89 8.6472.38 81.0475.05 Cidofovir (CDV) 3.1971.03 2.0970.36 45.3778.33 Foscarnet (PFA) >100 >100 >100 5-Fluoro-20-deoxyuridine (FUdR) 0.1370.06 0.1270.04 0.7770.11 5-Fluorouridine (FUR) 0.2070.09 0.1970.03 1.0370.11 Trifluorothymidine (TFT) 0.3270.08 0.2970.04 3.0270.39 (E)-5-(2-Bromovinyl)-20-deoxyuridine (BVdU) >100 0.0970.02 0.7370.11 5,6-Dihydrothymidine (DHT) >100 >100 >100 30-Deoxy-30-fluorothymidine (FLT) 3.6570.71 3.3770.77 177.2714.86 20,30-Dideoxy-30-fluorouridine (F-ddU) >100 >100 >100 30-Azido-30-deoxythymidine (AZT) >100 >100 >100 30-Azido-20,30-dideoxyuridine (AzdU) >100 >100 >100 20,30-Dideoxyuridine (ddU) >100 >100 >100

Experiments have been performed at least three times and data presented as mean inhibitory concentration 50% (IC50) or 90% (IC90)7SEM.

cells 72 hours after infection with Ad.OW94 vector alone Ad.OW94 resulted in 92% GFP-positive cells and in the presence of control untreated GCV GCV, RBV, CDV, TFT, and BVdU in 47, 81, 64, 63, and 200 2 / 0% 411 / 92% 80 / 47% 42% GFP-positive cells, respectively. The corresponding mean cell fluorescence intensity of untreated Ad.OW94 infected cells was 411 and in the presence of GCV, RBV, CDV, TFT and BVdU 80, 320, 204, 153, and 56, 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 respectively. A375 STK cells not infected with Ad.OW94 RBV CDV vector had a mean fluorescence of 2. Dead cells were 200 320 / 81% 204 / 64% excluded from analysis by propidium iodine staining. Consequently, test compounds not only inhibited viral DNA replication but also spread of the virus. Counts Flow cytometric analysis of A375 STK cells 72 hours 0 100 101 102 103 104 100 101 102 103 104 after transduction with a replication-defective adenovirus TFT BVdU expressing GFP with and without 25 mM antiviral 200 153 / 63% 56 / 42% compounds did not result in a changed GFP expression (data not shown).

0 In vivo inhibition of Ad.OW34 replication by GCV, 100 101 102 103 104 100 101 102 103 104 CDV, and TFT FL1 - GFP A375 cells were infected in vitro with Ad.OW34, mixed at Figure 3 Examination of antiadenoviral effects of selected com- a ratio of 1:10,000 with uninfected A375 cells, and pounds by flow cytometric analysis. HSV-tk-expressing melanoma injected subcutaneously into nude mice. Treatment of cells (A375 STK) were analyzed 72 hours after infection with the nine mice each with GCV, CDV, or TFT (25 mg/kg twice replication-competent adenoviral vector expressing GFP (Ad.OW94) a day (b.i.d.)) was initiated 24 hours after cell injection. in the absence (untreated) and presence of 25 mM antiviral Five tumor-bearing mice were left untreated as a control. compounds, as indicated. Dead cells were excluded by propidium As shown in Figure 4, on day 3, 2.43 Â 10676.19 Â 105 iodine staining. Analysis was carried out in triplicate and representa- Ad.OW34 vector copies/mg tumor DNA were detected by tive histograms are shown. The mean fluorescence intensities of all qPCR in the control group, and 6.92 Â 10478.82 Â 103 in living cells and the percentages of GFP-positive cells are indicated. GCV, 3.15 Â 10576.15 Â 104 in CDV, and 6.50 Â 10577.69 Â 104 in TFT-treated animals. Statistical viral DNA replication but also inhibition of viral spread, data analysis by employing Tukey’s multiple comparison A375 STK cells were infected at a multiplicity of infection test revealed that treatment of animals with GCV, CDV, (MOI) of 5 with the green fluorescent protein (GFP)- or TFT significantly inhibited Ad.OW34 vector replica- expressing replication-competent Ad.OW94 vector, alone tion compared to untreated mice (Pr.001), and that and in the presence of 25 mM antiviral compounds. As GCV or CDV inhibited Ad.OW34 vector replication shown in Figure 3, flow cytometric analysis of A375 STK more effectively than TFT (Pr.014). Furthermore, GCV-

Cancer Gene Therapy Inhibition of adenovirus vector replication O Wildner et al 795 10 7 bearing A375 STK tumors were treated with GCV, CDV, or TFT (25 mg/kg i.p. b.i.d. for 3 days). As a control, one group of animals received GCV (150 mg/kg i.p. b.i.d. for 3 days) at doses used for cytoreductive suicide gene therapy. 6 10 As shown in Figure 5, animals that received test compounds at doses used for antiviral therapy in this study showed only a slight increase of Annexin V 5 µg TU-DNA 10 expression. In addition, antiviral treatment did not significantly increase the number of propidium iodine- positive cells, showing dead cells, when compared to Ad.OW34 copy number/ untreated cells (data not shown). However, at higher dose 4 10 levels of GCV, Annexin V expression of A375 STK tumor ted CV cells was increased. a TFT e G CDV untr Figure 4 In vivo antiadenoviral effects of GCV, CDV, and TFT on the In vitro Ad.OW34 vector stability replication of an HSV-tk-expressing vector (Ad.OW34). A375 cells To examine Ad.OW34 vector stability and functional infected with Ad.OW34 were injected subcutaneously into nude HSV-tk expression, the vector was passaged serially 10 mice. Nine animals each were treated with GCV, CDV, or TFT times in HeLa cells without GCV and in the presence of (25 mg/kg, b.i.d. i.p.) for 3 days consecutively. Five untreated 10 mM GCV. As shown in Figure 6, gel electrophoretic animals served as control. Each mark represents an individual tumor. The horizontal line depicts the median copy number/mg tumor separation of vector DNA after the first and 10th passage DNA. In all groups, there was no adenoviral DNA detectable in the with and without GCV digested with the restriction liver. a MMHind III EcoR I Bgl II control GCV 25 GCV 150 200 p10 p10+ 0.9% 3.4% 31.3% p1 kb

0 5 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 4 CDV TFT

200 3 Counts 1.1% 3.0% 2 1.6 0 100 101 102 103 104 100 101 102 103 104 FL1-H FL1-H FL1 - Annexin V-FITC 1 Figure 5 In vivo apoptosis detection of A375 STK tumors treated with antiviral compounds by flow cytometric analysis of Annexin V expression. To determine whether the in vivo antiviral effect of the test compounds are due to cytotoxic effects, two animals each bearing subcutaneous A375 STK tumors received twice daily 25 mg/ kg or for control purpose 150 mg/kg ganciclovir (GCV 25 and GCV b HSV-tk 150, respectively). Other treatment groups received twice daily 25 mg/kg CDV or TFT for 3 days consecutively. Two untreated animals served as control. The percentages of Annexin V-positive 5000 10000 15000 20000 25000 30000 35000 cells are indicated. bp

Bgl II 4626 5586 6180 5178 4647 1547 2151 1268 1201 1625 treated animals had significantly less vector copies per mg 1497 EcoR I 26842 5950 1916 tumor DNA than those injected with CDV (Po.001). 1070 Hind III 2631 2672 3437 5322 4597 8010 3012 2937 2081 There was no detectable adenoviral DNA in the liver, 1004 even of animals that received no antiadenoviral treatment Figure 6 Examination of Ad.OW34 vector stability. (a) For this, we (detection level 10 Ad5 copies per mg genomic DNA). passaged the vector 10 times serially without GCV (p10) and in the presence of 10 mM GCV (p10+). Vector after passage one (p1) In vivo tumor apoptosis detection served as control. DNA of vector preparations was digested with Hind III, EcoR I and Bgl II and separated by agarose gel To determine whether the observed in vivo antiadenoviral electrophoresis. (b) Position of the expression cassette, consisting effects of the compounds are due to inhibition of viral of HSV-tk, IRES, and E1 ORFs; predicted fragment sizes of vector DNA replication or due to cytotoxic effects, animals DNA digested with restriction endonucleases are indicated.

Cancer Gene Therapy Inhibition of adenovirus vector replication O Wildner et al 796 endonucleases Hind III, EcoR I, and Bgl II did not reveal (A375 STK) to assess the HSV-tk dependent drug effects. any fragment pattern differences indicative of alterations As the mean fluorescence intensity as well as the in the viral genome. percentage of GFP-positive cells decreased when com- In addition, GCV-sensitivity of Ad.OW34 vector did pared to untreated cells, we were able to demonstrate, not change significantly after 10 serial passages in HeLa that not only the viral replication as demonstrated by cells in the absence and presence of 10 mM GCV when qPCR but also the spread of the vector was inhibited by compared side-by-side to parental Ad.OW34 vector by the drugs tested. qPCR-based antiviral assay (data not shown). Further- In contrast to previous studies, we also analyzed the more, sequence analysis of the HSV-tk gene of four vector antiadenoviral effect of GCV, CDV, and TFT on clones did not reveal mutations (data not shown). Ad.OW34 vector replication in vivo. Because injection of a replication-defective adenovirus expressing GFP in established subcutaneous A375 tumor resulted in a tumor transduction of 3.1, 4.8, 7.6, 7.4, and 13.5%, we Discussion mixed Ad.OW34-infected and uninfected cells that are subsequently injected into animals to eliminate We and others combined viral oncolysis with prodrug unpredictable tumor transduction as an additional vari- suicide gene therapy in an attempt to enhance the overall able in the determination of the antiadenoviral drug antineoplastic efficacy of virotherapy. The intrinsic effect in vivo. We are aware that this model does not oncolytic effects of E1B-55K-deleted adenoviruses could represent the clinical setting; however, the main objective be significantly enhanced in several solid xenograft tumor of this study was to determine HSV-tk-dependent and - models when prodrug (GCV alone or in combination with independent inhibition of adenoviral replication by a 5-fluorocytosine (5-FC)) was withheld until maximum panel of drugs. 68–70 vector replication and gene expression occurred. A further limitation of our model is due to the However, GCV administration was not beneficial to the adenoviral host-specificity and the lack of a permissive intrinsic oncolytic activity of an adenovirus (Ad.OW34) animal model for human adenoviruses. Consequently, with a much more robust replication than the E1B-55K- vector replication and spread is restricted to the human RC deleted vector Ad.TK , expressing the entire adenovirus xenograft. As we were not able to detect vector sequences E1 region as well as HSV-tk under conditions that favor in the liver by qPCR, even of untreated animals, we were 14 active viral replication and spread, probably because the not able to demonstrate in vivo inhibition of viral spread potential HSV-tk/GCV-mediated enhancement of the to other organs by the compounds tested. intrinsic viral oncolytic activity was balanced out by the In our study, agents effective against wild-type Ad5 virostatic effects of GCV. were nucleoside analogs that did not need to be Since apparent clinical success in the treatment of phosphorylated by viral enzymes to exert their antiviral severe adenovirus disease is limited to a few case reports, effects. Their antiviral activity was independent of the cell we examined in this study HSV-tk-dependent and- lines tested and inhibited wild-type Ad5 and Ad.OW34 independent inhibition of a replication-competent adeno- vector replication to the same degree. The following six viral vector by a panel of 15 potential antiviral compounds had activity against wild-type Ad5 replication compounds to determine whether the therapeutic options of which only the first two drugs are approved for to inhibit adenoviral replication could be enhanced by the systemic intravenous administration in humans. RBV expression of a suicide gene. demonstrated a moderate antiadenoviral effect, confirm- 42 To screen antiadenoviral agents, previous studies used ing previous reports. The high calculated IC90 of either plaque reduction, fluorescent focus reduction B250 mM might explain the fact that reported clearance assays,59,60,71 or MTT-based cytotoxicity assay.58,72 We of adenovirus infection occurred most frequently in developed a real-time qPCR-based assay to determine the patients with adenovirus-associated hemorrhagic cystitis effects of the examined drugs on the adenoviral replica- due to high bladder concentrations of RBV.43,44,53,74,75 tion in vitro and in vivo. The advantages of qPCR are Intravenous RBV administration is associated with rapid testing time, broad dynamic range, high specificity, anemia due to intravascular hemolysis and bone mar- accuracy, and high sensitivity allowing the detection of row-suppressive effects.76 The antiadenoviral effects of moderate drug effects in vitro and in vivo. Furthermore, CDV, which is approved for the treatment of CMV this assay allows the analysis of drug effects on a single retinitis in AIDS patients, have been described previously viral replication cycle. Despite DNA concentrations not for several serotypes including Ad5.60,61,77,78 However, necessarily correlating with viable virus, similar IC50 the IC50 value in our system was about 10 times lower values have been obtained for Ad.OW34 by plaque than previously reported.78 The major adverse effect reduction assay and qPCR technique.14 observed thus far for CDV is nephrotoxicity.79 In the In addition to the qPCR-based technique, we analyzed clinical setting, the very long intracellular half-life by flow cytometry the antiadenoviral effects of selected (448 hours) of CDV metabolites is advantageous since drugs on the spread of the GFP-expressing Ad.OW94 it allows infrequent dosing (i.e. every week or every 2 vector. Since HSV-tk/GCV-mediated cytotoxic effects weeks).77 are independent of the HSV-tk expression level,73 we With the exception of the investigational HIV-1 reverse used a stable HSV-tk-expressing A375 melanoma cell line transcriptase inhibitor FLT, which had moderate anti-

Cancer Gene Therapy Inhibition of adenovirus vector replication O Wildner et al 797 adenoviral activity, none of the other tested pyrimidine 1000 times higher than the antiadenoviral IC50 of the dideoxynucleoside analogs showed any significant effects drugs examined, even in cells stably expressing HSV-tk. on the replication of the Ad5 or Ad.OW34 vector. Furthermore, there was only a slight increased apoptosis Adenovirus replication was highly susceptible to all detectable in the in vivo studies. three evaluated fluorinated pyrimidine analogs FUdR, Virus replication was considerably more affected than FUR, and TFT. FUR and FUdR are derivatives in the that of cells, most likely since the activated drugs exert intracellular activation pathways of the chemotherapeutic their toxicity only in the S-phase and the replication-cycle agent 5-fluorouracil (5-FU).80,81 5-FU and FUdR have of viruses is much shorter than that of cells. Furthermore, previously been shown to inhibit adenoviral replica- the tumor-suppressor p53 allows cells that have DNA tion.82,83 The third substance in this group, TFT, is damage to arrest in the G1-phase of the cell cycle, approved in the United States for the topical treatment of permitting repair. If DNA repair is inadequate, p53 can epithelial keratitis caused by HSV-1 and -2. TFT has been elicit programmed cell death through apoptosis. reported to have in vitro activity against some strains of In order to achieve cytoreductive effects using the HSV- adenovirus (13, 19, and weakly 8).71 In our in vitro assay, tk/GCV system, much higher dose levels are required than the effects of the three fluorinated pyrimidine analogs on for obtaining antiviral effects. In order to enhance the Ad5 replication were significantly greater than that of antitumor efficacy of the HSV-tk/GCV system, we are RBV or CDV. Although the in vitro antiviral activity of currently conducting a phase I study of intralesional TFT was similar to that of GCV, it was less effective in administration of an HSV-tk expressing adenovirus vivo than CDV, probably due to rapid metabolic vector in combination with escalating doses of GCV in degradation in the liver. patients with cutaneous metastatic malignant melano- To elucidate whether the adenoviral replication could ma.88 be inhibited more effectively by an HSV-tk-dependent Taken together, the highest effects on Ad5 replication mechanism, we evaluated ACV, GCV, and BVdU for of the drugs tested that exerted their antiviral effects their effects on the replication of Ad.OW34 vector. In independent of viral thymidine kinase was seen with the contrast to the other examined compounds, the antiviral fluorinated pyrimidine analogs FUdR, FUR, and TFT. activity and selectivity of these drugs is based on their However, due to their mode of action, these drugs are specific activation by herpesvirus-encoded kinases, that highly toxic in humans with a narrow margin of safety. Of convert these nucleoside analogs into their monopho- the drugs tested that did not require viral activation, the sphate metabolites and BVdU in its mono- and dipho- highest specific antiviral activity was seen with CDV. sphate form.84 The drugs are then phosphorylated to their However, inhibition of Ad.OW34 vector replication was triphosphate form by cellular kinases, which are compe- significantly greater with GCV and BVdU, both requiring titive inhibitors of the viral DNA polymerase. In addition, specific activation by herpesvirus-encoded kinases. As, incorporation of these nucleoside analogs into DNA currently, specific treatment options for severe adenovirus during elongation leads to premature DNA chain disease are limited by the lack of an established effective termination.85 As the drugs are specifically activated by therapy, the incorporation of a fail-safe mechanism, like viral thymidine kinases, none of the drugs had an effect the HSV-tk/GCV or cytosine deaminase/5-FC (which on the replication of Ad5 at the concentrations tested, converts the relatively nontoxic antifungal agent 5-FC confirming the high specificity for HSV-1 thymidine into 5-FU) system, to stop adenoviral vector replication kinase. might be warranted, also in light of the fact that CDV- In this study, we were able to demonstrate directly that resistant Ad5 isolates have been isolated in vitro.78 GCV is a strong inhibitor of Ad.OW34 vector replication not only in vitro but also in vivo. These results also explain why the time point when GCV is given after intratumoral injection of the replication-competent vector expressing Materials and methods HSV-tk is crucial for its oncolytic effect.70 In vitro and in vivo the effect of GCV on Ad.OW34 vector replication Cell lines and drugs was significantly greater than that of CDV. The in vitro The human cervix carcinoma cell line HeLa (CCL-2) and effect of ACV on Ad.OW34 vector replication was more the human melanoma cell line A375 (CRL-1619) were than 100-fold lower compared to GCV. purchased from the American Type Culture Collection Furthermore, Ad.OW34 vector replication was highly (Manassas, VA). The human pancreatic cancer cell line susceptible to BVdU. However, there was no statistically Panc-1 (CRL-1469) was provided by Irmgard Schwarte- significant difference in the antiviral activity when Waldhoff (Knappschaftskrankenhaus, Bochum, Ger- compared to GCV. In contrast to DHT, which had no many). The human embryonic kidney cell line 29389 was antiadenoviral activity, BVdU has a 2(E)-bromovinyl obtained from Microbix Biosystems, Inc. (Toronto, entity that is crucial for the selective activity against HSV- Ontario, Canada). A375 STK90 were generated by 1 and VZV.86 BVdU is currently licensed only in transduction of the parental cell lines with supernatant Germany for VZV therapy.87 In contrast to ACV and from PA317-STK cells20 and subsequent G418 (Invitro- GCV, BVdU has a good oral bioavailability. gen/Gibco, Karlsruhe, Germany, 1 mg/ml for 3 weeks) The observed antiadenoviral effects were primarily not selection followed by subcloning. Cell lines were propa- through cell killing, since the in vitro CC50 were more than gated in D-10, consisting of Dulbecco’s modified Eagle

Cancer Gene Therapy Inhibition of adenovirus vector replication O Wildner et al 798 medium (DMEM) with high glucose, supplemented with Adenovirus qPCR assay 10% heat-inactivated fetal bovine serum and 50 mg/ml 97 A set of TaqMan PCR primers and probe were designed gentamycin. Tissue culture medium and supplements were to quantitate vector sequences using the Beacon Designer purchased from Invitrogen/Gibco (Karlsruhe, Germany). software (version 2; PREMIER Biosoft, Palo Alto, CA). Cells were maintained in the logarithmic phase of growth In our assay, we used a probe 50-(FAM)ACCTCTGAT- at 371C in a humidified atmosphere of 95% air and 5% TAAACATGGCGCCATCC(TAMRA)-30 that annealed CO . 2 within the Ad5 E4orf6 region, flanked by the upstream 50- CDV was kindly provided by Norbert W Bischofber- GTAATTCACCACCTCCCGGTA-30 and downstream ger, Gilead Sciences (Foster City, CA). GCV was 50-GGCTCTCCACTGTCATTGTTC-30 primers. The purchased from FHoffmann-La Roche (Grenzach- fluorescence intensity in each PCR capillary throughout Wyhlen, Germany). ACV, RBV, TFT, FUdR, FUR, the entire amplification procedure was monitored using a FLT, BVdU, AzdU, AZT, F-ddU, ddU, DHT, phospho- Light Cycler II (Roche Diagnostics, Mannheim, Ger- noformic acid (foscarnet, PFA) were purchased from many). Sigma-Aldrich (Munich, Germany). The dynamic linearity of the assay was in the range of 5.101 to 6.106 copies of adenoviral DNA. The coefficient of variation for the qPCR, determined as the standard Construction and production of adenoviral vectors deviation divided by the mean slopes of the standard curves times 100, was found to be 1.8%. The detection The Ad.OW34 and Ad.GFP vectors used in this study are level was determined to be 10 adenovirus DNA copies per described in detail elsewhere.14,15 Briefly, beginning with a mg of human genomic DNA. first-generation adenovirus early region 1 (E1)- and adenovirus early region 3 (E3)-deleted adenoviral vector, we generated the replication-competent adenoviral vector qPCR-based anti-adenoviral assay Ad.OW34, which harbors in the E1 region an HSV-tk- IRES91,92 — Ad5 E1 expression cassette driven by the HeLa, Panc-1, and A375 cells were used for evaluation of human cytomegalovirus immediate-early (CMV-IE) pro- antiadenoviral compound efficacy against wild-type Ad5 and Ad.OW34 vector. Cells were seeded at a density of moter flanked upstream by the Ad5 packaging sequence 5 and downstream by the Ad5 pIX. Ad.OW34 was 10 cells/well in six-well plates. After 12 hours, serial 10- generated by homologous recombination of pAd.CMV- fold dilutions of each antiviral agent, ranging from 0.01 to TK E1 and pBHG1093 in 293 cells, as described 100 mM, were prepared in D-10 culture medium with previously.94 The replication-competent adenoviral re- added Ad5 or Ad.OW34 vector resulting in an MOI of 5. porter gene vector Ad.OW94 was generated by substitut- Culture medium from the subconfluent cell monolayers ing the HSV-tk gene of the Ad.OW34 vector with the was replaced with drug dilutions plus virus. The last well cDNA encoding enhanced GFP from the plasmid of each six-well plate was incubated only with virus and pGreenLantern-1 (Life Technologies, Grand Island, served as an untreated control. After 36 hours, cells were NY). In the replication-deficient adenoviral vector harvested with cell scrapers and centrifuged. After Ad.GFP, the E1 region was replaced by an expression decantation of the culture medium, cells were snap-frozen cassette consisting of GFP (pGreenLantern-1, Life and stored at À801C until DNA extraction using the Technologies), driven by the human CMV-IE promoter DNeasy 96 Tissue Kit (QIAGEN, Hilden, Germany). in parallel to the transcriptional orientation of the Virus DNA copies were normalized to genomic DNA adenovirus E1 gene products, and terminated by the concentration. The 50 and 90% antiviral inhibitory dose bovine growth hormone polyadenylation site. The refer- (IC50 and IC90, respectively) was defined as the concen- ence strain VR-5 (ATCC, Manassas, VA) was used as tration that achieved 50 or 90% inhibition of adenoviral wild-type adenovirus type 5 (Ad5). replication in the qPCR assay, respectively. The replication-competent viruses Ad.OW34 Ad.OW94, and Ad5 were propagated in HeLa cells; the replication-defective Ad.GFP vector was amplified in 293 Adenovirus vector stability assay cells. All viruses were purified by two rounds of CsCl A confluent HeLa cell monolayer was inoculated with density centrifugation,94 dialyzed (Slide-A-Lyzer, Pierce, Ad.OW34 vector at a MOI of 0.1. After adsorption for Rockford, IL) against 1500 ml of PBS with 1 mM MgCl2 2–3 hours at 371C in a 5% carbon dioxide–water vapor and 10% glycerol four times (1 hour each) at 41C, and atmosphere, the inoculum was aspirated and the cells stored at À801C until use. The concentrations of all were washed with PBS and replenished with medium viruses were determined by measuring absorbency at containing 10 mM GCV. At appropriate time intervals, the 260 nm,95 and the infectious titer was determined by cells were examined for progressive viral cytopathic effect plaque assay on 293 cells.96 In all preparations, the ratio (CPE). Medium and cells were collected at peak CPE (B8 of infectious to noninfectious virus particles was approxi- day post infection) and stored at À701C until required for mately 1:80. The Ad.OW34 virus preparation was either further passage, or determination of the IC50. After functionally characterized for HSV-tk expression by 10 times serial passage, GCV sensitivity of passaged [3H]GCV incorporation and for vector replication by vector and parental stock Ad.OW34 vector were deter- lysis of HeLa cell monolayers. mined side-by-side in the qPCR-based antiviral assay.

Cancer Gene Therapy Inhibition of adenovirus vector replication O Wildner et al 799 Animal studies enzymatically digested with collagenase, hyaluronidase, and deoxyribonuclease as described previously.100 For This study was approved by the local authorities and was apoptosis and dead cell detection, Annexin V-FITC conducted in compliance with the Guide for the Care and staining (Annexin V-FITC apoptosis detection kit I, BD Use of Laboratory Animals (NIH Pub. No. 85-23). Biosciences, Heidelberg, Germany) in conjunction with Female athymic nude (NMRI nu/nu) mice, 6–8 weeks the vital dye propidium iodine and subsequent flow old, were obtained from the Central Animal Facility of cytometric analysis has been used according to the the University Essen (Essen, Germany). Animals were manufacturer’s instructions. maintained under pathogen-free conditions and fed ad libitum with standard nude mouse food and water. A375 cells were infected with the Ad.OW34 vector at an Flow cytometric analysis of GFP expression MOI of 5, resulting in a transduction of about 50% of the To visualize the antiviral effects of GCV, RBV, CDV, cells, as previously shown.88 After 12 hours, cells were TFT, and BVdU on adenoviral replication, subconfluent harvested and extensively washed. Afterwards, infected monolayers of A375 STK cells were infected at an MOI of cells were mixed at a ratio of 1:1 Â 104 with uninfected 5 with the replication-competent GFP-expressing vector A375 cells. A total of 50 million viable A375 cells in 100 ml Ad.OW94 in the presence and absence of 25 mM test of serum-free DMEM with 10% Matrigel (Becton compounds. As a control, cells were also transduced with Dickinson, Heidelberg, Germany) were injected subcuta- a replication-defective adenoviral vector carrying GFP.101 neously into the right flank of a nude mouse. For After 72 hours, 2 Â 104 viable cells impermeable to determination of drug-mediated in vivo cytotoxicity, nude propidium iodine were acquired and analyzed with a mice received in the same manner 5 Â 107 A375 STK cells FACSCalibur flow cytometer (BD Biosciences Immuno- subcutaneously into the right flank. The animals were cytometry Systems, San Jose, CA). randomly assigned to treatment groups. Five animals were left untreated and nine animals each received Analytical and statistical methods intraperitoneal administration of GCV, CDV, or TFT The curve fitting software TableCurve 2D version 4 (SPSS (25 mg/kg) in 1 ml of saline twice daily for 3 consecutive Inc., Chicago, IL) was used for calculation of IC and days beginning 24 hours after tumor cell injection. 50 IC , respectively. After logarithmic data transformation, For collection of tumor and liver samples, animals were 90 one-way analysis of variance with multiple comparison euthanized with CO and specimens were harvested 2 procedure (Tukey’s test) was employed to compare immediately with separate sets of sterile, DNA-free treatments for significant differences, using the software instruments. All samples were snap-frozen at the time of STATISTICA 5.5 for Windows (StatSoft, Tulsa, OK). collection and stored in cryotubes at 801C until analysis. À Data are presented throughout this study as mean7SEM. The total cellular DNA was isolated from about 30–40 mg tissue with the DNeasy 96 Tissue Kit (QIAGEN, Hilden, Germany). Abbreviations E1, E3, Adenovirus early region 1 and 3, Respectively; In vitro cytotoxicity assay CMV-IE, Cytomegalovirus immediate-early; HSV-tk, We examined the compounds for cytotoxic effects with the Herpes simplex virus-1 thymidine kinase; GFP, Green CellTiter-Glot Luminescent Cell Viability Assay (Prome- fluorescent protein; Ad5, Wild-type human adenovirus ga, Madison, WI) according to the manufacturer’s instruc- serotype 5; MOI, multiplicity of infection; PFU, plaque tions. This assay determines the number of viable cells in forming unit; qPCR, quantitative polymerase chain culture based on quantitation of ATP, which indicates the reaction. presence of metabolically active cells. ATP-based cell viability assays are more sensitive than other methods.98,99 A375 cells were seeded into 96-well tissue culture plates at a Acknowledgments density of 104 cells/well. 24 hours later, cells in logarithmic phase of growth were incubated with a two-fold dilution We are grateful to Norbert W Bischofberger, Gilead series of the test compounds, ranging in concentration from Sciences (Foster City, CA) for providing cidofovir. We 10 mM to 4.8 mM for 36 hours at 371C. thank Vera Siegmund for cloning and sequence analysis of the HSV-tk gene and her critical review of the manuscript. Furthermore, we are thankful to Klaus Sure In vivo cytotoxicity assay for his support to establish the qPCR and Cathrin S To determine whether in vivo antiviral effects of the test Walter for her critical review of this manuscript and compounds are due to cytotoxic effects, two animals each stimulating discussions. This work was supported by bearing subcutaneous A375 STK tumors received twice grants from Deutsche Forschungsgemeinschaft, Dr. daily 25 mg/kg or for control purpose 150 mg/kg GCV. Mildred Scheel Stiftung fu¨ r Krebsforschung, and For- Other treatment groups received twice daily 25 mg/kg schungsfo¨ rderung Ruhr-Universita¨ t Bochum Medizi- CDV or TFT for 3 days consecutively. At the end of the nischen Fakulta¨ t (FoRUM) to OW. DH was supported treatment period, animals were euthanized and tumors by Sophia & Fritz Heinemann Stiftung and CJ by were removed without overlying skin. Tumors were Konrad-Adenauer-Stiftung.

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