Virotherapy with conditionally replicating adenoviruses (CRADs) has been explored for enhanced tumor transduction and amplification of effect. Expression of ...
CANCER-TARGETED GENE THERAPY: ADENOVIRUS AND HERPES VIRUS 648. Transductionally Modified Oncolytic Adenoviruses for Treatment of Advanced Pancreatic Cancer Tommi Pisto,1 Lotta Kangasniemi,1 Tanja Hakkarainen,1 Tuula Kiviluoto,2 Kilian Guse,1 Anna Kanerva,1 Akseli Hemminki.1 1 Cancer Gene Therapy Group, University of Helsinki, Helsinki, Finland; 2Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland. 216 367 new cases of pancreatic cancer are estimated worldwide, with 213 462 subsequent deaths and the incidence is increasing. A reason for the strikingly low survival is the fact that clinical symptoms appear late and the lack of good therapy modalities. Virotherapy with conditionally replicating adenoviruses (CRADs) has been explored for enhanced tumor transduction and amplification of effect. Expression of the coxsackie adenovirus receptor (CAR) has been shown to be expressed variably in many cancer types thus compromising adenoviral transduction. We evaluated infectivity enhancement approaches for pancreatic cancer in vitro using a selection of cell lines (HPAC, Capan-2, HS766T,SW1990, Panc-1) and clinical samples fresh from patients. Capsid modifications utilized were RGD-4C, serotype chimerism (5/3), and polylysine modification of the adenoviral fiber. The RGD4C motif in the fiber knob allows adenovirus binding to integrins, serotype chimerism targets the Ad3 receptor and polylysine modification allows binding to heparin sulphate proteoglycans, thus circumventing potentially lacking or variable expression of CAR. To measure transduction efficacy, cell lines and clinical samples were infected with replication deficient viruses expressing firefly luciferase or b-galactosidase. After 24 hours of infection, cells were lysed and relative light units were measured and compared to wild type capsid containing viruses. The oncolytic effect of CRADs with similiar capsid modifications was tested by measuring mitochondrial activity of the cell lines post infection. Our data showed the benefits of infectivity enhancement for pancreatic cancer. In all cell lines and clinical samples either the pk7 or 5/3 capsid modified viruses showed significantly better transduction efficacy over wild type. Also CRADs with chimeric or polylysine modifications showed superior in vitro oncolytic potency compared to wild type. For in vivo analysis, we utilized a subcutaneous model of pancreatic cancer and comparison of capsid modified CRADs is ongoing. More importantly, we have developed orthotopic models of disseminated pancreatic cancer for comparing candidate CRADs and data will be presented at the meeting.
649. Increased Therapeutic Efficacy of the Prostate-Specific Oncolytic Adenovirus Ad[I/PPTE1A] by Introduction of the Entire E3 Region or the Adenovirus Death Protein Angelika Danielsson,1 Helena Dzojic,1 Berith Nilsson,1 WingShing Cheng,1 Magnus Essand.1 1 Clinical Immunology, Uppsala University, Uppsala, Sweden. Conditionally replicating adenoviruses are developing as a complement to traditional cancer therapies. Ad[I/PPT-E1A] is a virus that replicates exclusively in prostate cells since the expression of E1A is controlled by the tissue specific promoter PPT. The regulatory PPT sequence is composed of a PSA enhancer, a PSMA enhancer and a TARP promoter. The H19 insulator (I) placed in front of PPT protects the regulatory sequence from interfering signals from the adenoviral backbone that otherwise can make the expression unspecific. This virus has now been improved by including only the active part of the H19 insulator and by adding either the entire adenoviral E3 region (Ad[iPPT-E1A, E3]) or just ADP (Ad[iPPTE1A, ADP]). Since proteins expressed by the E3 region play a S250
crucial role in blocking immune responses from the host, these proteins are important for the persistence of the viral infection. ADP, which is located in the E3 region, is important for efficient viral release. These two viruses have been compared to Ad[I/PPTE1A] and results from in vitro studies demonstrate a more efficient cell kill without altered specificity. The Ad[iPPT-E1A, E3] vector has also been evaluated in vivo by intratumoral injection of the virus in subcutaneous LNCaP tumors on nude mice. Treated mice showed tumor suppression compared to the control group. Ad[iPPT-E1A, E3] and Ad[iPPT-E1A, ADP] will be further investigated but so far both of them seem promising for use in the clinic.
650. AU-rich 3’UTR Elements for Increasing the Specificity of Promoter Regulated Adenoviral Gene Therapy Merja Sarkioja,1,2 Minna Eriksson,1,2 Ristimaki Ari,3,4 Kanerva Anna,1,2,5 Hakkarainen Tanja,1,2 Hemminki Akseli.1,2 1 Rational Drug Design, University of Helsinki, Finland; 2 Department of Oncology, Helsinki University Central Hospital, Finland; 3Department of Pathology, Helsinki University Central Hospital, Finland; 4Molecular and Cancer Biology Research Program, University of Helsinki, Finland; 5Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Finland. Backround: Selective expression of genes in cancer cells would improve the safety and efficacy of many cancer gene therapy approaches. Adenoviruses can be targeted on a transductional level with capsid modification, or on a transcriptional level using tumor specific promoters (TSPs) and other regulatory elements. The control of mRNA stability and translation could be useful for restricted gene expression. There are a number of TSPs used in adenoviral gene therapy including the cyclooxygenase 2 (Cox2) promoter. Cox2 is expressed in various cancer tissues but also at inflammatory sites, while it is undetectable in normal healthy tissues. Therefore, it is potentially attractive for cancer gene therapy. However, it is currently unknown if Cox2 expression is induced by adenoviral infection per se, given its role as an inflammatory mediator, which might compromise specificity. The Cox2 promoter is partly regulated post-transcriptionally either by stimulation or inhibition of mRNA degregation by AU-rich elements (AREs) which are situated within 3’-untranslated regions (3’UTR) of mRNA. We sought to determine if adenovirus infection of tumor or normal cells results in Cox2 expression. Further, our hypothesis was that if infection induces Cox2, we might regain selectivity by utilization of AREs. Moreover, as the ARE of interest is controlled by the RAS pathway, which is frequently dysregulated in cancer cells, this control moiety might provide an additional level of tumor selectivity. Methods: 293, FHS173WE (non-malignant) and A549 (cancer) cells were transfected with plasmids containing either the Cox2 or CMV promoter driving luciferase, with or without additional posttranscriptional ARE control. Then, the transfected cells were infected with conditionally replicating adenoviruses (CRAds) Ad5-∆24E3, Ad5/3-∆24, Ad5-∆24RGD or Ad5300wt and Ad5LacZ as control viruses. Selectivity and activity of the promoters and 3’UTR elements in vitro were analysed by lusiferase assay. Analysis will also be done in vivo. Results: Our data indicates that adenoviral infection induces the Cox2 promoter in normal and tumor cells, which might compromise utility of the promoter for tumor specific gene therapy. On the other hand, AREs in the 3’UTR downregulated the expression of Cox2 and CMV driven luciferase in 293-and FHS173WE-cells and upregulated or maintained the luciferase expression in A549 cells. This was able to rescue the tumor selectivity of the Cox2 promoter and provided additional tumor selectivity per se. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
