922. Improvement of Anti-Tumor Activity by Gene Amplification with a Replicative, Non-Disseminative Adenovirus
Authors: Cara J. Lau, Denis Bourbeau, Jairo Jaime, Josephine Nalbantoglu, Bernard Massie
Journal: Molecular Therapy (2006)

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Gene therapy is a promising approach for cancer treatment; however efficacy of current vectors remains insufficient. In this study, we aimed to improve the success of suicide gene transfer by utilizing a replication competent vector that increases transgene copy number, improves gene expression, and yet does not generate disseminating adenoviral vector particles. One form of suicide gene transfer utilizes an adenoviral vector (AdV) expressing bacterial cytosine deaminase (CDA) to render infected cells susceptible to a prodrug, 5- fluorocytosine. This prodrug is transformed into the highly toxic 5-fluorouracil (5-FU) by CDA in infected cells. 5-FU can then be localized in the tumour area and can also diffuse to neighboring uninfected tumour cells. To increase the potency of CDA, we generated viral constructs that express a fusion protein containing CDA and bacterial uracil phosphoribosyltransferase (CU fusion protein), which is an enzyme that phosphorylates 5-FU to its most cytotoxic form. The replication competent platform we chose is a mutant AdV that is deleted in the protease gene (Ad|[Delta]|PS), which is incapable of producing infectious viral particles in the absence of the protease activity. There is no risk of viral shedding as a result, satisfying safety concerns. Moreover, the ability of Ad|[Delta]|PS to undergo a single round of replication within the host cell will lead to increased expression of the therapeutic gene, resulting in high- level gene transfer. Our results indicate the replication competent and protease-deleted AdV expressing CU (Ad|[Delta]|PSL41) is 45|[times]| more efficacious than its replication deficient counterpart (Ad|[Delta]|PSCU) in tumour cell killing in vitro. In bystander assays, Ad|[Delta]|PSL41 was >20-fold better than Ad|[Delta]|PSCU. In a 3-D spheroid model, 25-fold fewer particles of Ad|[Delta]|PSL41 were required to produce a similar therapeutic outcome when compared to first generation AdV. In vivo evaluation was carried out after intracerebral implantation of mice with U87LacZ human glioma cells or GL261 murine glioma cells. 10-20 days later, the mice were infected intratumourally with virus, and the next day were treated for five days with 500mg/kg of 5-FC injected i.p. twice daily. After 40d, control athymic mice implanted with U87LacZ and treated with 5-FC only (no virus) had a tumour size of 99.4 |[plusmn]| 10.9 mm3, whereas Ad|[Delta]|PSL41 treated mice had a diminished tumour size of 44.5 |[plusmn]| 22.2 mm3 demonstrating that Ad|[Delta]|PSL41 causes tumour regression when compared to control (p<0.05). In survival studies, mice were treated twice with virus where the second treatment occurred 21 days after the first treatment. Athymic mice treated with Ad|[Delta]|PSL41 had a longer median survival of 117d, while Ad|[Delta]|PSCU treated animals had a median survival of 76d (p<0.0001). In the syngeneic model, where B6 mice were implanted with GL261, the effect was more pronounced; Ad|[Delta]|PSCU treated mice had a median survival of 23d, whereas Ad|[Delta]|PSL41 treated animals have yet to reach a median survival after 200d (p = 0.0009). In conclusion, Ad|[Delta]|PSL41 produces a much more potent tumouricidal effect than its replication deficient counterpart, Ad|[Delta]|PSCU.