Conditionally-replicating adenoviruses (CRAds) have generally demonstrated only modest therapeutic efficacy in human clinical trials, in part due to their poor ability to spread throughout a tumor mass. In these studies, I first examined whether inclusion of an intact early region 3 (E3) and the p14 fusion-associated small transmembrane (FAST) protein in a CRAd vector can enhance oncolytic efficacy by improving viral spread. E3 encodes the adenovirus death protein (ADP), which enhances virus progeny release from infected cells, while p14 FAST can allow spread of the virus through cell-cell fusion. I generated viruses with (CRAdRC109) or without (CRAdRC111) an intact E3 region, which encoded the p14 FAST gene between the fiber coding sequence and E4 region of their viral genomes. In the A549 human lung cancer cell line, both CRAdRC109 and CRAdRC111 expressed p14 FAST at very low levels when compared to CRAdFAST, a similar virus that expressed the protein from within the E3 deletion, and thus had a relatively poor ability to mediate cell-cell fusion. Although inclusion of E3/ADP in CRAdRC109 did result in larger plaques and increased virus spread relative to CRAdRC111, neither virus showed improved oncolytic activity relative to CRAdFAST. I subsequently developed CRAdRC116, in which the E3 region of the viral genome was replaced with a bicistronic expression cassette containing the p14 FAST and ADP coding sequences separated by a self-cleaving 2A peptide sequence. This virus co-expressed p14 FAST and ADP and caused extensive cell-cell fusion in A549 cells. However, expression of ADP from CRAdRC116 did not increase cancer cell killing nor virus spread, and thus did not enhance oncolytic efficacy relative to CRAdFAST. These studies suggest that p14 FAST and ADP do not exhibit synergy when co-expressed from a CRAd vector. Future studies should instead focus on combining other methods of improving viral spread in conjunction with expression of ADP or FAST proteins from CRAd.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/38379 |
| Date | 01 November 2018 |
| Creators | Clarkin, Ryan Gregory |
| Contributors | Parks, Robin |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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