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Author ORCID Identifier

https://orcid.org/0000-0003-0252-4513

AccessType

Campus-Only Access for Five (5) Years

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemical Engineering

Year Degree Awarded

2022

Month Degree Awarded

May

First Advisor

Neil S. Forbes

Second Advisor

Ashish Kulkarni

Third Advisor

Lisa M. Minter

Subject Categories

Bacteria | Biochemical and Biomolecular Engineering | Biological Engineering | Cancer Biology | Immunotherapy | Molecular, Cellular, and Tissue Engineering | Viruses

Abstract

The greatest opportunity for the treatment of cancer lies with intranuclear protein and localized viral delivery. Conventional macromolecular therapies fail to selectively accumulate in tumors, are membrane impermeable, and inactive in the nucleus. The only clinical viral therapy is approved for intratumoral injection as the virus is cleared prior to colonization. As such, an intranuclear delivery vehicle is needed to overcome the limitations faced by traditional therapies.

Salmonella is a beneficial delivery vehicle for anti-cancer therapies. Non-pathogenic Salmonella colonize and grow within tumors at ratios greater than ten thousand to one over other organs. The highly motile bacteria invade epithelial cells and activate a set of genes to control for intracellular survival. Salmonella have been engineered to lyse intracellularly and deliver protein therapeutics to the cytoplasm of cancerous cells.

This thesis had two purposes: (1) to demonstrate that bacteria can deliver protein and plasmid DNA to the nucleus of cancerous cells for therapeutic effect and (2) increase the safety of therapeutic bacteria by utilizing a failsafe genetic circuit. We hypothesized that bacterially delivered proteins reach the nucleus for a therapeutic effect and that a delivered plasmid can express a virus. To test this, bacteria were engineered to release protein, and the location of said protein was determined using cell-based infection assays. To test the delivery of plasmid, both GFP and a viral genome were placed on a plasmid and delivered to cancer cells. Delivered plasmids resulted in protein expression by the cancer cells. Salmonella delivery of protein and plasmid reach the nucleus for therapeutic effect and protein expression.

To enhance the safety of therapeutic non-pathogenic Salmonella, we hypothesized that the creation of a failsafe genetic circuit would enable the clearance of bacteria following treatment. To test this hypothesis, we engineered a genetic circuit that controls the expression of a vital bacterial gene, aspartate semialdehyde dehydrogenase, under the control of a small molecule inducer, sodium propionate. In the presence of sodium propionate, Salmonella grow and replicate; when sodium propionate is removed, bacteria lyse. These results demonstrates that an engineered genetic circuit can control the growth of Salmonella to ensure bacterial clearance following treatment to prevent subsequent infection.

DOI

https://doi.org/10.7275/28403685

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

Available for download on Sunday, November 13, 2022

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