Engineering Large DNA Viruses for Enhanced Oncolytic Virotherapy

Abstract

Oncolytic viruses (OVs) offer a promising approach to cancer immunotherapy, but challenges in safety, efficacy, and engineering complex platforms remain. This thesis addresses these hurdles with novel tools and strategies. We designed an array of chemically inducible systems, controlled by FDA-approved small molecules, that grant unprecedented precision over both viral replication and transgene expression. We demonstrate the application of these chemically inducible systems for modulating viral replication dynamics, providing a potential strategy to mitigate cytokine release syndrome (CRS) associated with cytokine delivery, and regulating the activity of fusogenic proteins within the tumor microenvironment (TME). This allows for a more nuanced optimization of therapeutic outcomes. We illustrate the combined power of these systems by engineering a virus with multiple control levels for replication and expression of the potent immune modulator IL-12. Furthermore, we introduce an innovative antibiotic-based selection method that significantly improves the efficiency of OV engineering. This approach facilitates rapid platform discovery through random mutagenesis and demonstrates its utility in the parallelized incorporation of 20 different cytokines in two distinct viral backbones, enabling the assessment of their in vivo efficacy. This work advances the development of next-generation OVs, designed for precise control, potent immune activation, and enhanced therapeutic efficacy in cancer immunotherapy.