299. In Vivo Luciferase Imaging for Analysis of Oncolytic Adenoviruses for Renal Cell Cancer
Authors: Kilian Guse, Tuuli Ranki, Tanja Hakkarainen, Martti Ala- Opas, Tommi Pisto, Merja Särkioja, Maria Rajecki, Anna Kanerva, Akseli Hemminki
Journal: Molecular Therapy (2006)

Introduction:Renal cell cancer (RCC) is a common disease, which lacks effective treatments, when metastatic. Adenoviruses (Ads) are well characterized oncolytic agents with good safety data and promising initial efficacy. However, a major determinant of the oncolytic potency of Ad is the capacity for entering target cells. Unfortunately, recent data suggests that expression of the coxsackie-adenovirus receptor (CAR) is often low in many tumors. Therefore, oncolytic potency can be improved by utilization of non-CAR receptors such as 5/3 serotype chimerism, an integrin binding arginine-glycine- aspartic acid (RGD) moiety in the HI-loop of the fiber knob and fiber modification with polylysine (pK) motifs, which allows binding to heparan sulphate glycans.We hypothesized that Ads featuring these capsid modifications would show enhanced transduction of RCC cells and that the respective oncolytic Ads would display increased oncolytic potency in vitro and enhanced antitumor effect in vivo.Moreover, to facilitate determination of virus replication in vivo,we sought to develop a system for non-invasive, repeatable measurements of virion replication in live mice.Such a system would reduce the number of required test animals and provide information about in vivo virus replication kinetics.Methods:RCC cell lines and clinical tumor samples were infected with replication deficient,marker gene expressing Ads featuring 5/3, RGD and/or pK modifications.Oncolytic Ads with the same capsids were constructed,featuring a 24bp deletion in the E1A region,rendering the viruses unable to bind Rb and therefore able to replicate preferentially in cancer cells.These viruses were tested in in vitro cell killing assays as well as in vivo,in a xenograft RCC mouse model,where conditionally replicating oncolytic adenoviruses (CRAds) were co-injected with an E1 deleted,luciferase expressing Ad which replicates when present in the same cell as the replication competent virus. Therefore,luciferase expression is linked to replication kinetics of the CRAd and can be analyzed by repeated non-invasive imaging.This provides longitudinal data in individual mice and alleviates the need for killing animals for analysis of produced virions.Results/Conclusion:Capsid modified viruses showed increased transduction and oncolysis of RCC substrates in comparison to Ad5 based agents. In vivo, RGD, pK and 5/3 modified Ads exhibited significantly higher antitumor effects than wild type Ad. Therefore, capsid modified Ads are promising agents for the treatment of RCC.Good correlation between virus replication and imaging results was seen, suggesting that the proposed system of co-injecting a CRAd with a replication deficient, luciferase expressing Ad could be used as a non-invasive surrogate for analysis of virus replication in vivo.Currently,we are setting up orthotopic models for disseminated renal cancer, which will then be utilized for in vivo comparison of CRAds. Introduction: Renal cell cancer (RCC) is a common disease, which lacks effective treatments, when metastatic. Adenoviruses (Ads) are well characterized oncolytic agents with good safety data and promising initial efficacy. However, a major determinant of the oncolytic potency of Ad is the capacity for entering target cells. Unfortunately, recent data suggests that expression of the coxsackie-adenovirus receptor (CAR) is often low in many tumors. Therefore, oncolytic potency can be improved by utilization of non-CAR receptors such as 5/3 serotype chimerism, an integrin binding arginine-glycine- aspartic acid (RGD) moiety in the HI-loop of the fiber knob and fiber modification with polylysine (pK) motifs, which allows binding to heparan sulphate glycans. We hypothesized that Ads featuring these capsid modifications would show enhanced transduction of RCC cells and that the respective oncolytic Ads would display increased oncolytic potency in vitro and enhanced antitumor effect in vivo. Moreover, to facilitate determination of virus replication in vivo,we sought to develop a system for non-invasive, repeatable measurements of virion replication in live mice.Such a system would reduce the number of required test animals and provide information about in vivo virus replication kinetics. Methods: RCC cell lines and clinical tumor samples were infected with replication deficient,marker gene expressing Ads featuring 5/3, RGD and/or pK modifications.Oncolytic Ads with the same capsids were constructed,featuring a 24bp deletion in the E1A region,rendering the viruses unable to bind Rb and therefore able to replicate preferentially in cancer cells.These viruses were tested in in vitro cell killing assays as well as in vivo,in a xenograft RCC mouse model,where conditionally replicating oncolytic adenoviruses (CRAds) were co-injected with an E1 deleted,luciferase expressing Ad which replicates when present in the same cell as the replication competent virus. Therefore,luciferase expression is linked to replication kinetics of the CRAd and can be analyzed by repeated non-invasive imaging.This provides longitudinal data in individual mice and alleviates the need for killing animals for analysis of produced virions. Results/Conclusion: Capsid modified viruses showed increased transduction and oncolysis of RCC substrates in comparison to Ad5 based agents. In vivo, RGD, pK and 5/3 modified Ads exhibited significantly higher antitumor effects than wild type Ad. Therefore, capsid modified Ads are promising agents for the treatment of RCC. Good correlation between virus replication and imaging results was seen, suggesting that the proposed system of co-injecting a CRAd with a replication deficient, luciferase expressing Ad could be used as a non-invasive surrogate for analysis of virus replication in vivo. Currently,we are setting up orthotopic models for disseminated renal cancer, which will then be utilized for in vivo comparison of CRAds.