620. An Efficient and Safe Herpes Simplex Virus Type 1 Amplicon Vector for Transcriptionally Targeted Therapy of Human Hepatocellular Carcinomas

620. An Efficient and Safe Herpes Simplex Virus Type 1 Amplicon Vector for Transcriptionally Targeted Therapy of Human Hepatocellular Carcinomas

618. A Novel Chromogranin-A Promoter-Driven Oncolytic Adenovirus for Midgut Carcinoid Therapy Justyna Leja, I Helena Dzojic, I Elisabet Gustafson,' K...

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618. A Novel Chromogranin-A Promoter-Driven Oncolytic Adenovirus for Midgut Carcinoid Therapy

Justyna Leja, I Helena Dzojic, I Elisabet Gustafson,' Kjell Oberg,' Valeria Giandomcnico,' Magnus Essand.' 'Clinical lmmunology: Rudbeck Laboratory, Uppsala University. Uppsala. Sweden; JSI/rgicalSciences. University Hospital. Uppsala , Sweden; 'Endocrine Oncology. University Hospital. Uppsala, Sweden. Replica tion-selective oncolytic adenoviruses arc emerging anticancer agents. We have constructed Ad[CgA- E IA] where the chromogranin A (CgA) promoter controls expression of the adenoviral EIA gene. To our knowledge Ad[CgA-EIA] is the first oncolytic virus to specifically target neuroendocrine tumors. We found that Ad[CgA-E IA] selectively replicates in and kills cells where the CgA promoter is acti ve, including carcinoid and neuroblastoma cells. By means of in vivo bioluminescence we showed thatAd[CgA-E IA] is able to suppress fast growing human carcinoid BON tumors in nude mice. Most carcinoid patients have disseminated disease at the time ofdiagnosis with metastases most frequently detected in mesentery and liver, In order to bc able to treat carcinoid liver metastases with Ad[CgA-E IA] it is important that normal hcpatocytes do not support virus replication to minimize hepatotoxicity. We found that CgA is highly expressed in microdissected cells from carcinoid metastases while it is not expressed in normal hepatocytes. Therefore, Ad [CgAE IA] is an interesting agent for treatment of liver metastases in conjunction with standard therapy for these malignancies. :t :t :t :t :t W W W iii W

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to RCC cell lines, clinical samples and orthotopic murine models of metastatic RCC. In addition, kidney vascular targeting moieties HTTHREP. HITSLLS and APASLYN were placed into the fiber knob of AdSII9p and studied for utility in RCC. Six different human RCC cell lines (ACI-IN, Sv7tert, Caki-2, 786-0, 769-1' and SK-UT-1B) were infected. AdSII9p was at Icast as effective asAdS in genc delivery to all RCC cell lines, Together with the lack of liver transduction, these data suggest the potential for a substantial gain in the ratio of gene delivery to tumor versus liver following systemic administration. Vascular targeting moieties l-rn'HREPand HITSLLS showed modest utility in ACHN but not other cell lines in vitro. In vivo experiments with orthotopic models is ongoing and clinical samples are being analyzed. Full data will be presented at the meeting, In summary. AdSII9 is promising for gcnc delivery to RCC.

620. An Efficient and Safe Herpes Simplex Virus Type 1 Amplicon Vector for Transcriptionally Targeted Therapy of Human Hepatocellular Carcinomas Kian Chuan Sia,' Lv Miao ,' Grace Y. Wang,' Ivy A. W. Ho,1 I-I. Huynh,' Kam M. I-Iui,' Paula Y. P. Lam.' 'Cellular and Molecular Research, National Cancer Centre, Singapore. Singapore .

Our previous studies have shown that transgene expression could, indeed, be targeted to specifically to proliferating cells whcn cell cycle transcriptional regulatory elements are incorporated into herpes simplex virus type I (IISV-I) amplicon backbone vectors. In this study, we demonstrate that transcriptional regulation could be rendered specific to human hepatocellular carcinoma cells (I-ICC) by inserting the chimeric gene, GaI4/NF- YA, under the regulation ofthe I-ICC-specifie, hybrid promoter. The hybrid promoter which consists of four copies ofthe ApoE enhancer clement inserted upstream ofthe hAATpromoter induced an elevated level oftranscription compare to other liver-specific promoters (AFP and Alb, respectively) tested in both HCC and non-IICC cells (Fig. l a),

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619. Ad5/19p for Targeting of Renal Cancer and Detargeting the Liver Iulia Diaconu,' Laura Denby,' Gerd Bauerschrnitz,' Kilian Guse .' Anna Kanerva,' Joao D. Dias,l Andrew H. Baker.' Akseki Hemminki.' 'Cancer Gene Therapy. Molecular Cancer Biology Program & Haartman lnstitute, University ofHelsinki. and Department of Oncology. Helsinki University Central Hospital, Helsinki, Finland; 2B1-IFGCRC, University ofGlasgow, Glasgow, Scotland, United Kingdom.


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Despite advances in cancer research and therapeutics, patients with advanced renal clear cell carcinoma (RCC) cannot usually be cured. The aim of this study is to utilize novel targeting moieties for adenoviral gene therapy of RCC. Our previous work suggests that utilization of the Ad 19p fiber in the context of AdS (AdS/19p: AdS capsid with 19p fiber) may be useful for targeting structures of the rat kidney and kid ney vasculature. Further, reduced transduction of rat livers in vivo and human cells of hepatic origin (1-lepG2) was observed for AdSII9p compared to control AdS/S . Therefore, we sought to investigate the utility of AdSII9p for gene delivery Molecular Therapy Volume 15• .surrk-mt"m t, ~ br 2()()7

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Luciferase reporter activities in proliferating I-ICC cells (HuH-7 and PLC/PRF/S) were subsequently observed to be significantly higher than the corresponding GI-arrested cells (Fig. Ibi), as confinned with elevated endogenous cyclin A protein expression (Fig. Ibii). In contrast, background level with no significant difference between the proliferating and corresponding G.-arrested cells was observed in non-I-ICC cells (Gli36 and l-IeLa). As a consequence, the enhancement of tissue-specific expression in the context of GaI4/NF-YA fusion proteins enabled the real-time visualization of S237

the luciferase reporter activities mediated by these viral vectors (Fig. 2), which is to our best knowledge, the first report.

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In our current study, we explore the possibility ofwhether multiple tandem repeats of the CDE/CHR elements could further tighten the cell cycle regulation in the context of the newly generated veetor against HCC. To evaluate the efficacies of these vectors, the prodrug yeast cytosine deaminase therapeutic gene will be fused in frame with the open reading frames of luciferase gene and the 2A sequence. The cytosine deaminease gene is capable of converting 5-fluorocytosine into the known antitumor drug 5-fluorouracil, which is already used as a common chemotherapy treatment for both primary human liver cancers and metastasis colon cancers in clinical setting. Together with the relatively high infectivity in primary HCC eclls, we believe that the new hybrid vectors could provide alternatives to the design ofsafe and efficient systemic gene therapeutic strategies for human HCC.

621. Fluorescently Tagged Conditionally Replicative Adenovirus Via Modification with Protein IX Red Fluorescent Protein Fusion

Ana Nedeljkovic-Kurepa, 1 Heike Creutz,' Mariam A. StoffKhalili,' Yoshinobu Odaka,' Jagat Poduturri,' Xiao L. Li,1 Angel A. Rivera,' David T. Curiel.' J. Michael Mathis.' 'Cellular Biology and Anatomy, Gene Therapy Program, LSU Health Sciences Center; Shreveport, LA; ]Division ofHuman Gene Therapy, Departments 0/Medicine, Obstetrics and Gynecology, Pathology, and Surgery; and the Gene Therapy Center; University ofAlabama at Birmingham, Birmingham, AL; 'Department a/Obstetrics and Gynecology. University 0/ Duesseldorf Medical Center; Duesseldorf, Germany. Using conditionally replicative adenoviruses (CRAds) represent a therapeutic method to achieve efficient tumor cell oncolysis and mitigate the limitations of tumor infection. Ideally, cancer specific replication ofCRAds results in viral mediated oncolysis of infected tumor tissues and release of the virus progeny, capable of further propagating in surrounding tumor cells but not in those of norma l tissues. A number of characteristics of the Adenovirus type 5 (Ad5) make it an optimal gene therapy/virotherapy vector suitable for a wide array of cancer therapeutic approaches. Despite these advantages, overall efficacy ofAd5-based cancer gene therapy/vitotherapy approaches remains limited by sub-optimal vector delivery efficiency in cancer tissues and non-specific delivery to normal tissues. The ability ofCRAds and other replicative agents to eradicate tumors critically depends on two key functions, efficient replication and spread within the tumor. A monitoring system that can capture quantitatively and dynamically the replication and spread of these oncolytic viruses would be a useful assessment tool for developing these agents as well as for their application in patients. The labeling system should also reflect the replication, lysis, and dispersion efficiency of the virus. Recent work by several groups has defined the C-terminus of the minor capsid protein piX as a locus capable ofpresenting incorporated ligands on the virus surface. In this study, S238

two CRAd vectors labeled with the fluorescent capsid fusion protein IX-red fluorescent protein (pIX-EGFP) were developed. Expression of the fluorescent fusion-protein label in infected cells could be detected. The labeled virions could be visualized by fluorescence microscopy and quantified by flow cytornetry, These results were was applicable to the tracking ofCRAd infection, as well as localizing the distribution of the vector in tissues . Expression of pIX-RFP could be exploited to detect the replication and spread of CRAds. These results indicate that pIX can serve as a platform for incorporation of heterologous proteins in the context of a replicating adenovirus. It is believed that capsid-labeled CRAds have utility for vectordevelopment studies and for monitoring CRAd-based oncolytic adenovirus replication.

622. Fiber Region-Modified Adenoviruses Produced Anti-Tumor Effects to CAR-Low Expressing Tumors

Masatoshi Tagawa.l-' Kiyoko Kawamura,' Guangyu Ma,I,2A Quanhai Li,I'~ Osamu Shimozato,' Ayako Sato,' Taketo Suzuki,' Nobuo Suzuki,' Hideaki Shimada,':' Takenori Ochiai.' :' IDivision 0/Pathology, Chiba Cancer Center Research Institute, Chiba, Japan; 2Departmen( a/Environmental Biochemistry, Graduate School ofMedicine, Chiba University, Chiba, Japan; JDepartment 0/ Frontier Surgery, Graduate School ofMedicine, Chiba University, Chiba, Japan; "Center 0/Excellence Program in the 21st Century, Graduate School ofMedicine, Chlba University, Chiba, Japan. Infectivity of adenoviruses type 5 (Ad5) to cells is primarily mediated by the interaction between their fibers-knob regions and the coxsackievirus and adenovirus receptor (CAR) on target cells. Downregulated CAR expression, often found in human tumors, hampered Ad5-mediated gene transfer. Ad II andAd 35, belonging to a subtype B group, use CD46 molecules as their cellular receptors; accordingly, chimerieAd5 whose fiber structure was substituted with that of the type II or 35 (Ad5/11 or Ad5135) could infect human cells in a different manner from Ad5. We found that Ad5135 infected human tumors better than Ad5 and Ad5/11 and that infectivity of Ad5/35 to these cells was correlated with that ofAd5/11. Infection of human hepatoma cells with measles virus, whose cellular receptor is CD46, down regulated the CD46 expression and decreased subsequent infectivity ofAd5/35. Infection ofAd5 suppressed subsequent gene transfer by Ad5 but not by Ad5/11 or Ad5/35 , and infection of Ad5/35 downregulated following transduction by Ad5/35 and Ad5/11 and to lesser extent by Ad5. These data collectively showed that combinatory usc ofAd 5 and the chimeric Ad enables dual gene transfer into target cells. We then prepared recombinantAd5 in which the E IA expression was regulated by an exogenous promoter such as a regulatory region of the midkine gene and the fiber structure was replaced by that of Ad35. The fiber-modified oncolytic Ad showed the similar cytotoxic activities as prototype Ad5 to tumors with CAR-high expression and their replication in CAR-high tumors remained the same as the oncolytic Ad5. The fiber-modified Ad however produced greater anti-tumor effects than thc Ad5 to tumors with CAR-low expression, suggesting a possible usc of ehimcric Ad for eanccr therapy.

Molecular Therapy Yofume 15. Supplement I, .\by 2007 Copyright © '111C AmericanSocietyo f Gene TIICr.lpr