Improving the Oncolytic Potency of Reovirus as a Breast Cancer Treatment through Genetic Modifications
Creators: Cristi Munoz, Francisca
Publisher: name: University of Alberta Library
Year: 2023

<p>Breast cancer is the most diagnosed cancer in women with the second highest mortality rate. Even though current therapies have improved the probability of survival of the disease, there are still many challenges to overcome including the formation of metastases. Oncolytic viruses present a promising approach by selectively targeting cancer cells without destroying healthy normal cells. <br>
Reovirus is a nonpathogenic intestinal virus, and it is considered an oncolytic virus due to its selective replication in cancer cells. However, results from clinical trials using reovirus as monotherapy suggest that wild type reovirus (T3wt) would benefit from an increased potency. To enhance its efficacy, we hypothesized that genetic modifications could enable reovirus to infect tumor cells more effectively, despite its natural adaptation to the intestinal environment. Accordingly, I explored several murine models to study the anti-cancer abilities of T3wt and the reovirus mutants. My findings showed that the murine HER2+ breast tumor model (using TUBO cells) was the most suitable model to observe the oncolytic activity of T3wt and the mutants. Additionally, I created reovirus mutants harboring point mutations or a combination of those mutations and assessed their oncolytic potency in vitro using plaque size as an indicator of virus fitness in tumor cells. Subsequently, I selected some of the mutants that formed larger plaques for further investigation. <br>
Among the generated mutants, a particularly noteworthy variant, named Super Virus 5 (SV5), formed the largest plaques in breast cancer cells. Subsequent evaluation in the TUBO model revealed that mice treated with SV5 exhibited the most substantial tumor reduction and longest overall survival, not only relative to T3wt but also to a single-mutated virus. These results emphasize the enhanced oncolytic potency achieved through the combination of mutations.<br>
Contrary to our initial hypothesis, our findings revealed that SV5 did not exhibit faster replication than T3wt in cultured cells. However, it displayed a remarkable capacity for extended spread compared to T3wt, primarily attributed to its reduced attachment to cancer cells. These observations highlight the significance of increased virus spread as a crucial mechanism for improving oncolytic reovirus activity. <br>
In addition to SV5, we conducted a comprehensive characterization of two mutants, each containing a different mutation in the cell binding protein σ1, that formed larger plaques than T3wt in breast cancer cells. To elucidate the underlying mechanisms behind their increased oncolytic activity, we studied the characteristics of the mutants at various steps in the virus replication cycle. Surprisingly, despite the mutants&#39; reduced binding efficiency to cellular sialic acid, they demonstrated a higher possibility for establishing a successful infection compared to T3wt when their binding to cells was equalized. Furthermore, our research expanded our understanding of the sialic acid binding domain within the σ1 protein.<br>
Lastly, our study focused on investigating the reciprocal influence between the breast tumor microenvironment and reovirus infectivity. Previously, the Shmulevitz lab revealed that breast tumor proteases possess the ability to cleave the reovirus binding protein 1, thereby reducing the virus&#39;s binding capacity and infectivity. In my thesis, I show that the combination of a mutation that impedes the cleavage of 1 with another pro-oncolytic mutation resulted in a remarkable increase in reovirus oncolytic activity in the TUBO model. Additionally, we explored the impact of reovirus on the tumor microenvironment by employing a novel small-biopsy technique. The biopsies confirmed the presence of reovirus within the tumor and revealed alterations in the expression of reovirus-induced cellular cytokines. Further investigation is necessary to fully comprehend the potential and limitations of this innovative technique.<br>
In summary, my thesis work has revealed the significance of introducing multiple genetic modifications to enhance the oncolytic activity of reovirus. By targeting increased virus spread and resistance to tumor proteases, we successfully engineered oncolytic viruses with superior potency against breast cancer. Moreover, my thesis has contributed to our understanding of reovirus biology. Notably, we gained insights into the crucial role of the outer capsid reovirus proteins in in vitro oncolysis. Additionally, we demonstrated that a farther virus spread could be achieved through reduced binding to cells, a previously unreported advantage for reovirus or any virus in the context of oncolytic therapy.</p>