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Basic Science

issue-Targeted In Vivo Gene Transferoupled with Histone Deacetylase Inhibitorepsipeptide (FK228) Enhances Adenoviral

nfection in Rat Renal Cancer Allograftodel Systems

inoru Kobayashi, Takashi Okada, Takashi Murakami, Keiya Ozawa, Eiji Kobayashi,nd Tatsuo Morita

BJECTIVES Although the adenoviral vector represents an efficient delivery system, hepatotropic accumula-tion often has detrimental effects on adenoviral vector-mediated cancer therapy. To overcomethis disadvantage, we performed in vivo local gene transfer, in combination with the histonedeacetylase inhibitor, depsipeptide (FK228), in a rat renal cancer model.

ETHODS Renal cancer cells induced by ferric nitrilotriacetate in ACI rats were used in this study.Adenoviral vectors containing luciferase cDNA were introduced into the tumor-burdenedkidney by way of a catheter placed in the renal artery. Subcutaneous tumors were treated byherpes simplex virus thymidine kinase cDNA followed by intraperitoneal ganciclovir. The levelsof Coxsackie-adenovirus receptor in various tissue were determined by quantitative reversetranscriptase-polymerase chain reaction. Depsipeptide (1 mg/kg) was intravenously administered24 hours before adenoviral vector transduction.

ESULTS The catheter-based adenoviral vector delivery enabled strong gene transduction of the tumor-burdened kidney. Moreover, depsipeptide treatment before adenoviral vector injection signifi-cantly improved transgene expression at tumor sites. Quantitative reverse transcriptase-poly-merase chain reaction analysis showed that depsipeptide increased the expression levels of theCoxsackie-adenovirus receptor in the renal tumor (13-fold), but not in other normal tissues.Furthermore, the use of herpes simplex virus thymidine kinase cDNA-expressing adenoviralvector followed by ganciclovir markedly inhibited the established tumor growth in combinationwith depsipeptide compared with herpes simplex virus thymidine kinase cDNA alone.

ONCLUSIONS The tissue-targeted in vivo gene transfer coupled with depsipeptide significantly enhancedadenoviral infection at tumor sites. Sensitization of tumor cells with depsipeptide can improvethe efficacy of adenoviral vector-mediated suicide gene therapy. Thus, application of depsipep-tide could be one of the beneficial adjunct for adenoviral vector-mediated cancer gene

therapy. UROLOGY 70: 1230–1236, 2007. © 2007 Elsevier Inc.

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enal cell carcinoma (RCC) is a potentially mortalkidney disease. Surgical resection represents thetherapeutic mainstay for localized RCC. How-

ver, one third of patients with RCC present with me-astasis, and one half of patients treated for a localizedrimary tumor subsequently develop metastatic disease.1

rom the Department of Urology, and Divisions of Genetic Therapeutics and Organeplacement Research, Center for Molecular Medicine, Jichi Medical University,ochigi, Japan; and Department of Molecular Therapy, National Institute of Neuro-

cience, National Center of Neurology and Psychology, Tokyo, JapanReprint requests: Minoru Kobayashi, M.D., Department of Urology, Jichi Medicalniversity, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 Japan. E-mail:

oinoruk@jichi.ac.jpSubmitted: May 16, 2007, accepted (with revisions): September 14, 2007

230 © 2007 Elsevier Inc.All Rights Reserved

n particular, primary tumors that cannot be surgicallyemoved (eg, solitary kidney, renal insufficiency, or bi-ateral tumors) are the most complicated forms of RCC.ntitumor cytokines such as interferon-alpha and inter-

eukin-2 have been widely used, only to lead to limitedherapeutic benefits.2 Thus, a more tumor-selective tar-eting strategy is required for clinical RCC therapy.

Recent advances in cancer therapy have involved these of the adenoviral vector, which has been widelypplied in clinical cancer therapy for its high transduc-ion efficiency. Because systemic adenoviral vector injec-ion results in preferential accumulation in the liver

wing to the hepatocytic tropism3 and can induce unde-

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irable immune responses to viral proteins,4 adenoviralector-mediated cancer therapy has been limited to thentratumor injection approach. However, this approach isot available if the tumor is located in inaccessible sitesr if the tumor has spread extensively in the target organ.herefore, it is necessary to use a more selective tissue-

argeting strategy for adenoviral vector delivery.Improving the efficiency of gene transduction will help

o reduce adenoviral vector-mediated toxicity by decreas-ng the required quantity of viral vectors. It has beenhown that histone deacetylase inhibitors (HDACis) caneactivate virally transduced genes5 and modulate theirxpression at transcriptional levels.6 The novel class ofistone deacetylase inhibitors, depsipeptide (FK228,R901228), which was originally developed as an anti-ancer agent, has been shown to augment adenoviralector-mediated transgene expression in various tumorell lines in vitro.7–9 However, little is known about then vivo effect of depsipeptide on enhancing gene expres-ion10–12 or the adenoviral vector distribution defininghe tissues in vivo, where the vectors spread.

We have demonstrated that tissue-targeted local generansfer using the catheter-based technique overcomeshe adenoviral hepatocytic tropism in a rat orthotopicCC model. Moreover, depsipeptide-mediated sensitiza-

ion increases the efficiency of adenoviral vector-medi-ted in vivo gene transfer in a tumor-selective manner.he mechanism for increasing transgene expression in

he tumor was also evaluated in relation to Coxsackie-denovirus receptor (CAR) expression; the CAR servess a high-affinity receptor for adenoviral vectors anddenovirus type 5.13

ATERIAL AND METHODS

nimals, Cells, and Reagentsale inbred ACI rats (6 to 8 weeks old) were purchased fromLEA Japan (Tokyo, Japan). All experiments were performed

n accordance with the Jichi Medical University Guide foraboratory Animals. Rat renal cancer cells had been previouslystablished from an ACI rat exposed to ferric nitrilotriacetate14

nd were maintained in vivo in the subcutaneous space of ACIats every 2 weeks. Depsipeptide was obtained from Fujisawaharmaceutical (Osaka, Japan).

denovirus Vectorse constructed a replication-deficient adenoviral vectors (se-

otype 5 with E1/E3 deletion) containing firefly luciferaseDNA (AVC2-luc) or herpes simplex virus thymidine kinaseDNA (AVC2-tk) driven under cytomegalovirus promoter,sing the in vitro ligation technique with DNA protein com-lex.15 The adenoviral vectors were purified by sequential CsCltep gradients and isopycnic centrifugation and then titered byetermination of the median tissue culture infection dose. TheacZ (beta-galactosidase)-expressing adenoviral vector AxCA-acZ, driven by chicken beta-actin promoter and cytomegalo-irus enhancer, was donated by Dr. Xiao-Kang Li (Departmentf Regeneration Surgery, National Research Institute for Child

ealth and Development, Tokyo, Japan).16 a

ROLOGY 70 (6), 2007

n Vivo Luminescent Imaging and 5-Bromo-4-hloro-3-Iodolyl-�-D-Galactopyranoside Staining

n vivo luciferase imaging was conducted with the noninvasiveioimaging system IVISTM (Xenogen, Alameda, Calif). Oneinute after an intravenous injection of D-luciferin (30 mg/kg)

potassium salt, Biosynth, Postfach, Switzerland), the signalntensity of the emitted photons from the luciferase-expressingissue were measured with 1 minute of integration time. Toisualize the LacZ expression, frozen sections were stained withhromogenic substrate 5-bromo-4-chloro-3-iodolyl-�-D-galac-opyranoside using a previously described procedure.17

umor Models and Suicide Gene Therapyor the subcutaneous tumor model, harvested tumor cells (4 �05) were injected into the abdominal subcutaneous space ofhe rats. The tumor volume was evaluated using the followingquation: tumor volume � (length in millimeters) � (width inillimeters)2/2. For the orthotopic tumor model of the kidney,

he cells (1 � 106) were suspended in 0.1 mL phosphate-uffered saline and inoculated into the left renal subcapsularpace. An obvious subcapsular tumor was established about 1eek later. For suicide gene therapy, AVC2-tk (1.5 � 109 pfu)as injected into the subcutaneous tumor, followed by contin-ous intraperitoneal infusion of ganciclovir (GCV) (240 mg/g/day) for 7 consecutive days using a miniosmotic pump (Al-et, Palo Alto, Calif) according to the manufacturer’snstructions. Tumor growth was monitored every other day withumor volume measurements.

atheter-Based In Vivo Gene Transferatheter-based gene transfer was performed using a previouslyescribed method18 on the iliac artery and a 2F catheter (Solo-ath, Solomon Scientific, Plymouth Meeting, Pa) was passed

hrough to the abdominal aorta just distal to the origin of theeft renal artery. After the aorta was clamped just proximal tohe left renal artery, 2 mL of saline was injected by way of theatheter to wash out blood in the tissue, followed by 1 mL ofhosphate-buffered saline containing adenoviral vector (5 �08 pfu of AVC2-luc or 1 � 109 pfu of AxCA-LacZ). The leftenal artery and vein were clamped for 10 minutes to allowissue contact with the injected adenoviral vector.

uantitative Real-Time Reverse Transcriptase-olymerase Chain Reactionotal RNA was extracted from rat tissues using RNA-Bee

eagent (Cosmo Bio, Tokyo, Japan). Total RNA (1.5 �g) wassed for first-strand synthesis of cDNA using the SuperScriptreamplification System (Invitrogen, Carlsbad, Calif) and am-lified using QuantiTect SYBR Green polymerase chain reac-ion (PCR) (Qiagen, Valencia, Calif). The following PCRrimers were used: CAR forward, 5=-CTGATCAGTGTAT-CTAC GACTAGATGTT-3=; CAR reverse, 5=-CAAG-AGCGTCCCTATGACA-3=; glyceraldehyde-3-phosphateehydrogenase (GAPDH) forward, 5=-GACAAC TTTG-CATCGTGGA-3=; and GAPDH reverse, 5=-ATGCAGG-ATGATGTTCTGG-3=. The PCR product was analyzed us-

ng the ABI PRISM 7900 HT Sequence Detection SystemApplied Biosystems, Foster City, Calif). Quantitative valuesere obtained from the threshold cycle (Ct) number that indi-ated exponential amplification of the PCR product. The rel-

tive CAR expression level was determined as the N-fold

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ifference relative to GAPDH according to the following for-ula: N � 2corrected �Ct (GAPDH � CAR). The Ct value was

etermined by subtracting the average CAR Ct value from thatf GAPDH. Each experiment was performed two to three timesith similar results.

tatistical Analysisvalues were obtained through Student’s t test or the Mann-hitney U test using the StatView software (Abacus Concepts,

erkeley, Calif). Data are shown as the mean � standardeviation. Differences between groups were considered signifi-ant at P �0.05.

ESULTS

n Vivo Adenoviral Vector Distribution Afterystemic Injectiono examine the distribution of systemically injected ad-noviral vector in vivo, luciferase-expressing adenoviralectors (5 � 108 pfu) were injected intravenously. Vectoristribution was monitored by in vivo luciferase imagingFig. 1A). The vector-derived photons accumulated ex-lusively in the spleen and liver at day 2 after adenoviralector injection, and these organs were substantially lu-iferase positive in the ex vivo analysis (Fig. 1B).

n Vivo Gene Transfer in Tumor-Burdenedidneys of Ratsecause systemic adenoviral vector injection did not

esult in substantial transgene expression at the tumorites, it was necessary to use a more selective strategy forumor-specific targeting. In our previous work, we dem-nstrated a catheter-based in vivo gene delivery methodhat potentially allows for kidney-selective adenoviralector transfer.18 We, therefore, applied this catheter-ased technique to target the orthotopic RCC tumor inhe rat model. The catheter-based injection of luciferase-xpressing adenoviral vector showed strong transgenexpression in the kidney (Fig. 1C), in which rat RCCells were transplanted beneath the renal capsule (Fig.D). Moreover, to determine whether catheter-based ad-noviral vector gene transfer is an effective technique forumor targeting, beta-galactosidase-expressing adenoviralectors (1 � 109 pfu) were administered into the leftidney of the rats using a catheter, and the tissue wasnalyzed using an 5-bromo-4-chloro-3-iodolyl-�-D-galac-opyranoside staining procedure. As shown in Figure 1E,eta-galactosidase expression was dominant in the tumorrea and around the boundary of the renal parenchyma.hus, these results suggest that catheter-based adenoviralector gene delivery might be an effective technique forumor targeting in the kidney.

ffect of Depsipeptide Pretreatment on Adenoviralector-Mediated Tumor Targetingecent evidence has shown that transcriptional modula-

ion using HDACis can augment adenoviral vector-me- G

232

iated gene expression in various tumor cells in vitro.herefore, we hypothesized that the use of an HDACiight improve the efficiency of adenoviral vector-medi-

ted tumor destruction in vivo. To address whether theDACi depsipeptide enhances adenoviral vector-de-

ived transgene expression in tumors, AVC2-luc (5 �08 pfu) was injected directly into subcutaneous tumors4 hours after intravenous injection of depsipeptide1 mg/kg). This dosage and timing of depsipeptide wasreliminary determined to be optimal to show maximalransgene expression in the subcutaneous tumor modeldata not shown). Depsipeptide pretreatment signifi-antly enhanced luciferase gene expression in the tumorompared with the mock treatment, and substantial pho-ons were observed from day 1 to day 5 after adenoviralector transduction (Fig. 2A). A similar enhancement ofene expression was also observed in the orthotopicallyransplanted RCC tumor subjected to local AVC2-lucransfer using the catheter-based method (Fig. 2B). Al-hough adenoviral vector-derived photons in the liverere observed faintly even when using the catheter-based

argeting procedure, depsipeptide treatment did not re-ult in an additional increase of adenoviral vector-de-ived photons in the liver or any other normal tissuesdata not shown). Thus, these findings suggest that dep-ipeptide modulates transgene expression in a tumor-elective manner.

pregulation of Adenoviral Vector Receptor CAR inumors by Depsipeptides shown in recent studies,7,8,11,12 we investigatedhether in vivo exposure of RCC cells with depsipeptidean increase CAR expression levels, which consequentlynvolves enhancement of adenoviral vector transductionn our model systems. CAR mRNA expression in variousissues and subcutaneous tumor was examined after 24ours with or without depsipeptide injection (1 mg/kg).epsipeptide treatment strikingly enhanced CAR ex-ression in the tumor (13-fold increase relative to un-reated tumor) but had no such effect on normal, healthyissues (Fig. 2C). These results explain that depsipeptideensitizes RCC tumors for adenoviral vector-mediatedene therapy partly through an increase in CAR levels.

se of Depsipeptide Enhances Tumor-Selectiveuicide Gene Therapy in Rat Modelinally, we investigated whether sensitization of theCC tumor with depsipeptide promoted herpes simplexirus thymidine kinase cDNA-mediated tumor transduc-ion. Herpes simplex virus thymidine kinase cDNA-ex-ressing adenoviral vectors (AVC2-tk, 1.5 � 109 pfu)ere injected into an established subcutaneous tumorith or without depsipeptide treatment (1 mg/kg) 48ours before intraperitoneal GCV injection. As shown inigure 2D, the use of depsipeptide before adenoviralector-mediated suicide therapy with AVC2-tk and

CV significantly decreased the tumor size compared

UROLOGY 70 (6), 2007

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igure 1. (A) In vivo expression profile in adenoviral vector-mediated gene transfer. Optical bioluminescent imaging ofdenoviral vector-mediated gene transfer. AVC2-luc (5 � 108 pfu) injected intravenously and luciferase images taken at dayafter transduction. Substantial photons detected in spleen and liver. (B) Ex vivo analysis of luciferase expression from

arious tissues of ACI rat. Tissues isolated at day 1 or day 2 after systemic AVC2-luc injection. Luciferase expressionetected exclusively in spleen and liver. Dotted line represents background level (in tissues of control rats). (C) Successfulatheter-based in vivo adenoviral vector delivery into orthotopic renal tumor of rats. Representative image of luciferaseransduction after injection of AVC2-luc (5 � 108 pfu). Luciferase images obtained at day 2 after adenoviral vectorransduction. Red arrow indicates left kidney. Inset shows ex vivo luciferase analysis of isolated tumor-burdened kidney.hotons detected macroscopically in subcapsular area of kidney. (D) Representative histologic section of establishedubcapsular tumor of kidney. Dotted line indicates border of parenchyma. Hematoxylin-eosin stain, original magnification20. (E) Expression pattern of LacZ after catheter-based adenoviral vector transfer. Beta-galactosidase expressionvaluated by 5-bromo-4-chloro-3-iodolyl-�-D-galactopyranoside staining of tumor-burdened kidney of rats (blue area). LacZ-ositive cells predominantly stained blue in subcapsular tumor area. Dotted line indicates border of parenchyma. Original

agnification �40.

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ith vehicle or herpes simplex virus thymidine kinaseDNA/GCV therapy alone. These results, therefore,ave demonstrated that sensitization of RCC tumor cellsith depsipeptide increases the efficacy of adenoviralector-mediated suicide gene therapy.

OMMENTt is well known that most intravenously infused adeno-iruses accumulate in reticuloendothelial organs such ashe liver and spleen.19 Luciferase-based in vivo imagingas demonstrated an exclusive real-time and quantitativedenoviral vector-mediated transgene expression in thesergans (Fig. 1A). This observation suggests that an effi-ient tumor-targeting method is required for adenoviralector-mediated gene therapy.

In the present study, we have demonstrated a selectiveissue-targeting method using the catheter-based generansfer technique. This transduction efficiency was aug-ented by the use of depsipeptide in the rat RCC tumorodel. Several studies have reported decreased CAR

evels in human RCC cell lines,7,8,12,20 suggesting poordenoviral vector infection of the tumor. The most re-ent reports have demonstrated that depsipeptide in-reased CAR expression in tumors at the translationevel, as well as the transcription level, in xenograftodel systems.11,12 Intriguingly, transcriptional modula-

ion of CAR expression in normal tissues was almostlways considerably less sensitive than tumor cells toepsipeptide (Fig. 2C), suggesting this effect might alsoe specific to malignant cells. Therefore, catheter-basedissue targeting in combination with the HDACi, dep-ipeptide, might be an effective strategy for adenoviralector-mediated cancer gene therapy.We also addressed whether augmentation of adenovi-

al vector-mediated gene transduction using depsipeptideould lead to enhanced tumor destruction in a rat model.ubcutaneous RCC tumor growth was significantly in-ibited by intratumor adenoviral vector-thymidine ki-ase injection followed by GCV treatment. This antitu-or effect was significantly enhanced after pretreatmentith depsipeptide. These data strongly support the view

hat sensitization of RCC tumor with depsipeptide can

™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™igure 2. Effect of depsipeptide on transgene expression an subcutaneous tumor. AVC2-luc (2.5 � 108 pfu) injectenjected intravenously 24 hours before adenoviral vector trawo independent experiments with similar results shown.uciferase expression in orthotopic tumor of kidney. AVC2-atheter. Depsipeptide injected intravenously 24 hours befne of two independent experiments with similar resultspregulation of CAR mRNA with depsipeptide treatment. CAeal-time quantitative reverse transcriptase-PCR 24 hoursisted of 4 rats. *P �0.05, Mann-Whitney U test. (D) EnhVC2-tk (1.5 � 109 pfu) injected into established subcutanCV (240 mg/kg/day) for 7 days with or without pretreatmen

njection). *P �0.05, **P �0.01, Mann-Whitney U test. Each e

ROLOGY 70 (6), 2007

mprove the efficacy of adenoviral vector-mediated sui-ide gene therapy.

Evidence has shown that bystander effects for tumorilling are abrogated in T-cell-deficient animals, evenith adenoviral vector-mediated suicide gene therapy,21

uggesting that host cellular immune responses are nec-ssary for tumor destruction. With this viewpoint, ourllograft tumor model in an immune-competent animalight be advantageous to study the exact efficacy and

afety of viral vectors for cancer gene therapy. BecauseDACi can activate components of death receptor path-ays, including FasL and TRAIL in tumor cells,22,23 these of depsipeptide could result in tumor cells becomingulnerable to T-cell attack.The clinical use of depsipeptide when administered

lone showed only partial responses in patients with solidancers.24,25 However, moving the focus onto the mod-lation of tumor factors, targeting HDACis could providereat benefits, particularly for selective gene therapygainst cancer.

This approach has some shortcomings that should beonsidered in the future. First, the safety of intra-arterialdenoviral vector delivery has not yet been established.his approach has been in disfavor, since the case of

reatment-related death in which an adenoviral vectoras injected into the hepatic artery.26 This could haveccurred in a specific patient treated for insufficient liverunction itself, because a later preclinical study demon-trated that hepatic arterial injection of adenoviral vectoraused no liver dysfunction.27 In the latter study, thenimals given intra-arterial adenoviral vector injectionid not show any bodyweight loss or sickness during thebservation period. Nevertheless, the safety should beully addressed with any method of adenoviral vectorelivery. Second, although depsipeptide does result in anncrease in CAR expression, it is unknown whether thencreased CAR expression is the major mechanism forhe enhanced transgene efficiency. Depsipeptide mightossibly amplify a variety of other gene expressions fromhe viral genome itself, which could independently en-ance transgene efficiency. Using adenoviral vectors re-argeted to a CAR-independent pathway could deal with

™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™AR mRNA expression. (A) Enhanced luciferase expressionrectly into subcutaneous tumor. Depsipeptide (1 mg/kg)ction. Each experimental group consisted of 5 rats. One of0.05, **P �0.01, Mann-Whitney U test. (B) Enhanced

5 � 108 pfu) administered through left renal artery usingransduction. Each experimental group consisted of 4 rats.wn.*P �0.05, Mann-Whitney U test. (C) Tumor-selectiveRNA expression was examined in various rat tissues usingexposure to depsipeptide. Each experimental group con-ment of suicide gene therapy by depsipeptide treatment.

s tumor, followed by continuous intraperitoneal infusion ofh depsipeptide (1 mg/kg, 24 hours before adenoviral vector

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his issue.20 Third, it might be worthwhile to study otherotential histone deacetylase inhibitors (eg, trichostatin, sodium phenyl butyrate, and CHAP1), which were

eported to increase CAR expression10,28 compared withepsipeptide. Finally, we could not show the efficacy ofdenoviral vector-mediated suicide gene therapy in therthotopic model because of a lack of an objective mon-toring tool of tumor growth. However, most other stud-es have also had the same limitation. Enhanced trans-ene expression by depsipeptide in the subcutaneousodel could be reproduced clearly in the orthotopicodel in this study. We therefore expect that sensitiza-

ion of tumor cells with depsipeptide, coupled with site-pecific adenoviral vector administration, would alsoncrease the efficacy of suicide gene therapy in the ortho-opic model. The use of in vivo magnetic resonancemaging29 would be feasible to address this issue.

ONCLUSIONSissue-targeted in vivo gene transfer, coupled with dep-

ipeptide, significantly enhanced adenoviral vector-me-iated gene transduction into tumor sites. The catheter-ased gene delivery mimics the clinical scenario exactly;herefore, the combinatorial use of depsipeptide might be

useful adjunct to adenoviral vector-mediated suicideene therapy against human RCC.

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