Keynote Presentation: CRISPR-Enabled Directed Evolution of Enhanced Oncolytic Viruses for Cancer Gene Therapy

Gene therapy has experienced an increasing number of successful human clinical trials, leading to numerous FDA-approved therapies based on recombinant viruses for rare disease, cancer, and other targets.  Oncolytic viruses, for example, are therapeutics engineered to replicate within and induce immune responses against tumor cells.  However, vectors such as vaccinia face a number of barriers and shortcomings that preclude their extension to most human diseases, including limited delivery efficiency to target cells, limited spread within tumors, and poor spread from primary to secondary tumors. These barriers are not surprising, since the parent viruses upon which vectors are based were not evolved by nature for our convenience to use as human therapeutics.  Unfortunately, for most applications, there is insufficient mechanistic knowledge of underlying virus structure-function relationships to empower rational design improvements.

As an alternative, for over two decades we have been implementing directed evolution – the iterative genetic diversification of the viral genome and functional selection for desired properties – to engineer highly optimized, next generation viral variants for efficient and targeted delivery to any cell or tissue target.  To overcome inherent challenges in genetically engineering large DNA genomes, we developed EvolvR (Halperin et al., Nature, 2018; Hurtado et al., Nature Comm., 2025), a CRISPR-guided genetic diversifier that we have harnessed to create large libraries of vaccinia variants.  These have been selected for the capacity to better deliver to and spread between tumor cells in vitro and in mouse models, results that promise to further enhance the efficacy of oncolytic viruses for treating cancer..
 

Learning Objectives

1. Discuss the potential of directed evolution for enhancing gene therapy

2. Explain how engineered vaccinia can serve as a cancer therapy

3.