Engineered Adenovirus for Selective Replication in Tumors and Druggable Control of Virus Progression
- Author(s): Partlo, William
- Advisor(s): O'Shea, Clodagh C
- et al.
The oncolytic Adenovirus has shown promise as a cancer treatment and is under development in numerous laboratories. Two important requirements of an oncolytic Adenovirus are safety and effectivity. These requirements translate to selectivity against replication in normal cells and potent replication in tumor cells. Often, potency is sacrificed for selectivity, resulting in limited clinical effectivity. The work described in this dissertation attempts to engineer an oncolytic Adenovirus with selectivity based on an arbitrary transcription factor while maintaining wildtype or near-wildtype replication kinetics in the targeted cell type. The example transcription factor used in this work is p53. A significant percentage of all tumors have been found to be p53-null, while all normal cells are p53-positive. An oncolytic Adenovirus based on the p53 status would thus be a powerful clinical tool.
Development of a robust method for assessing Adenovirus replication kinetics is described. This method employs a genetic modification to the Adenovirus genome such that each infected cell is forced to express a fluorophore. This fluorophore expression is monitored over time to create an exponential growth curve as the virus passes through its initial lifecycle and produces secondary and tertiary infections. With this fluorescence-based viral kinetics assay, the replication kinetics of any Adenovirus type infecting any cell type can be quantified.
A selectively replicating Adenovirus was created by placing a single, critical Adenovirus ORF under control of the Two Step Transcriptional Activation (TSTA) system. For a positively regulated virus, the TSTA system is used with a selective, but weak promoter driving expression of the Tet-On transcriptional activation factor and the Tet-Response Element (TRE) promoter driving expression of the chosen Adenovirus ORF. For a negatively-regulated virus, Tet-On is replaced with the TetR transcriptional repressor while the TRE promoter is replaced with the CMV-Tet-O promoter, subject to repression by TetR.
Negative selectivity based on transcriptional activity of p53 is demonstrated with approximately 100X differential between p53+/+ and p53-/- cell lines.