Tailored oncolytic Adenovirus-encoded RNAi Delivery: Improvement Strategies (TARDIS)

This thesis explores the development of improved oncolytic adenovirus therapies for cancer treatment through the integration of RNA interference (RNAi)-based gene silencing strategies. Oncolytic virotherapy is an emerging therapeutic approach that employs naturally occurring or genetically engineered viruses to selectively infect, replicate within, and destroy cancer cells, while sparing healthy tissue. In addition to direct tumor cell lysis, oncolytic viruses can stimulate anti-tumor immune responses by promoting immune recognition of malignant cells. Among the different viral platforms available, adenoviruses offer several advantages, including a well-characterized genome, efficient infection of human cells, ease of genetic manipulation, scalable production, and favorable safety profiles. Furthermore, oncolytic adenoviruses have shown the ability to convert immunologically “cold” tumors into “hot” tumors by enhancing immune-cell infiltration within the tumor microenvironment. The work presented in this thesis focuses on combining oncolytic adenoviruses with RNAi technology to improve therapeutic specificity and efficacy. RNAi is a conserved cellular mechanism, in which small RNA molecules mediate sequence-specific degradation of targeted messenger RNA, thereby suppressing target gene expression. Incorporating this mechanism into oncolytic adenoviruses provides opportunities to selectively silence genes involved in tumor growth, immune evasion, or resistance to virotherapy. Chapter 2 introduces the rationale for integrating RNAi into oncolytic adenovirus design and discusses strategies for selecting relevant therapeutic target genes. Chapter 3 investigates whether an oncolytic adenovirus can be made more potent by silencing synoviolin, an endogenous inhibitor of adenoviral replication in cancer cells, resulting in a modest increase in cancer cell killing. In Chapter 4, different RNAi expression cassette formats were compared to establish a platform for adenoviral delivery of RNAi molecules. Among the tested formats, the primary microRNA (pri-miRNA)-based design showed the most consistent and stable expression in infected cancer cells. Chapter 5 applies this platform to melanoma and focuses on identifying genes involved in establishing an immunosuppressive tumor microenvironment. Functional RNAi screens identified several candidate genes that may enhance anti-tumor immune responses when silenced using oncolytic adenoviruses. Chapter 6 expands the translational aspect of this work by investigating approaches for monitoring adenoviral replication through liquid biopsy-based strategies. In particular, the feasibility of using virus-associated RNA as a marker of active adenoviral replication was explored to improve distinction between replicating virus and residual viral material following treatment administration. Together, the studies presented in this thesis provide new strategies for improving oncolytic adenovirus therapy through RNAi-mediated gene regulation and support the further development of RNAi-armed virotherapy for cancer treatment.