ABS 033: Engineering Bacteria to Launch and Control an Oncolytic Virus for Targeted Cancer Therapy

Kailyn R. Grant ¹ , Zakary S. Singer ¹ ² , Jonathan Pabón ¹ , Hsinyen Huang ¹ , William Sun ¹ , Hongsheng Luo ¹ , Ijeoma Obi ¹ , Courtney Coker ¹ , Charles M Rice ² , Tal Danino ¹ ³ ⁴

¹ Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA
² Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA
³ Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA
⁴ Data Science Institute, Columbia University, New York, NY 10027, USA.

Van Wickle (2025) Volume 1, ABS 033

Introduction: Oncolytic viruses and bacteria have shown promise as cancer therapies, yet both face significant challenges. Viral therapies are often neutralized by pre-existing immunity, while bacterial therapies are typically confined to the tumor core, limiting their ability to impact distant tumor cells. To overcome these barriers, CAPPSID (Coordinated Activity of Prokaryote and Picornavirus for Safe Intracellular Delivery) was developed as an engineered system in which Salmonella typhimurium transcribes and delivers the genome of Senecavirus A (SVA) directly into tumor cells, initiating an oncolytic infection. This bacterially encapsulated viral delivery enables the virus to evade circulating antibodies, allowing for viral replication and tumor targeting even in pre-vaccinated hosts. Additionally, SVA was engineered to require a bacterially delivered protease for maturation, ensuring that viral replication remains dependent on bacterial presence, improving safety and control. This was achieved by introducing a cleavage site in the viral polyprotein that depends on Salmonella-delivered Tobacco Etch Virus protease (TEVp) for proper maturation.

Through in vitro invasion assays and in vivo tumor models, CAPPSID efficiently delivers and amplifies SVA, leading to tumor regression in both immunodeficient and immunocompetent models. To regulate viral genome release, SVA delivery was linked to bacterial intracellular sensing, allowing the virus to be transcribed and released only after bacterial invasion of tumors. Notably, systemic administration of CAPPSID resulted in tumor-selective colonization with minimal bacterial presence in healthy tissues, highlighting its potential for clinical translation. These findings validate the ability of engineered bacterial systems to enhance viral delivery and tumor targeting in complex immune environments.
This work represents an engineered cooperation between bacteria and oncolytic viruses, demonstrating how microbial consortia can be programmed to enhance therapeutic efficacy. By integrating bacterial and viral therapies, CAPPSID offers a novel approach for improving tumor targeting, immune evasion, and controlled viral spread, advancing the potential of microbial-based cancer therapeutics.

Methods: We engineered Salmonella typhimurium to deliver Senecavirus A (SVA) RNA into tumor cells using a plasmid-based system under the control of an intracellularly activated promoter. To ensure safety and control, the SVA polyprotein was modified to include a cleavage site dependent on the bacterially expressed Tobacco Etch Virus protease (TEVp), linking viral maturation to bacterial presence. In vitro invasion assays measured bacterial entry and viral RNA release in mouse and human cancer cell lines. In vivo, immunodeficient mice bearing subcutaneous tumors received intra-tumoral CAPPSID treatment. To evaluate immune evasion, CAPPSID was tested intravenously in immunocompetent mice and mice pre-vaccinated against SVA.

Results: By developing a bacterially delivered platform for viral RNA, we show successful launch of a viral infection capable of eradicating tumors, the ability to cloak and deliver viral genomes into tumors in mice with humoral immunity, and that viral spreading controlled in trans by a bacterially donated protease can enhance persistence compared to a replicon alone.

Discussion: This work establishes a multi-layered microbial platform that coordinates S. typhimurium and Senecavirus A (SVA) to overcome key challenges in oncolytic therapy, including immune neutralization and limited tissue penetration. By using bacteria to deliver and control viral replication through protease-dependent maturation, CAPPSID enhances safety, persistence, and tumor targeting. Importantly, this system demonstrates efficacy in both immunodeficient and immunocompetent models, expanding its translational potential. The ability to deliver large viral RNAs and activate replication selectively in tumors marks a significant step forward for bactofection strategies. Future iterations may incorporate additional safeguards to further limit mutational escape and increase therapeutic precision.