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Date of Award

9-2012

Document Type

Campus Access

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemical Engineering

First Advisor

Neil S. Forbes

Second Advisor

Susan C. Roberts

Third Advisor

Sallie Smith Schneider

Subject Categories

Chemical Engineering

Abstract

To effectively treat cancer, therapeutics must target tumor tissue and be minimally toxic to healthy tissues. Engineered bacteria, such as Salmonella typhimurium and Escherichia coli , are known to target tumors and can be exploited as tumor-specific drug delivery vectors. Incorporating inducibility increases specificity by controlling the timing of drug expression. Recent studies demonstrate that basal expression in commonly used systems is too high for use with strongly toxic molecules, such as bacterial toxins. Pseudomonas aeruginosa Exotoxin A (PEA) and Staphylococcus aureus Alpha-Hemolysin (SAH), are highly cytotoxic and are promising moieties for bacterial cancer therapy. This dissertation presents an improved genetic switch for tighter regulation of toxic molecules and demonstrates the potential effectiveness of these toxins in bacterial cancer therapy.

It has been shown that a clinically relevant trigger, γ-radiation, can induce gene expression using the recApromoter, a component from the bacterial SOS system. Self-regulation of this promoter causes high basal expression and low induction. The ce1a promoter, from another SOS-controlled gene, was hypothesized to exhibit tighter control over gene expression. Testing this hypothesis, a non-pathogenic strain of S. typhimurium, VNP20009, was engineered to express a green fluorescent protein under control of Pce1a . This strain demonstrated low basal expression, with 94% of noninduced bacteria having exhibited an "off" phenotype, equivalent to that of bacterial controls. Exposing VNP20009-ce1aGFP to ultraviolet radiation caused 72% of bacteria to acquire an "on" phenotype, and yielded a 24-fold increase in overall protein production. Induction with 100 Gray of γ-radiation resulted in significant induction, causing a 329% increase in fluorescence in 4 hours. Using this system allows future development of bacterial protein delivery to include highly cytotoxic bacterial toxins.

Hypotheses assessing the utility of PEA and SAH as therapeutic moieties for bacterial therapy were tested, in vitro and in vivo , with bacteria engineered to express PEA or SAH under the arabinose-inducible promoter, PBAD . Toxins secreted into the medium following induction and were subsequently applied to monolayer cultures of MCF7 cells for three days. Treatment with PEA or SAH generated an 82% or 99% decrease in cell viability, respectively. Mice treated with PEA-expressing S. typhimurium experienced a delay in tumor growth. SAH-producing E. coli caused tumor regression in mice, yielding a 76% reduction in tumor volume over 5 days. SAH-delivery corresponded with a 6-fold lower viable tissue fraction within tumors compared to bacterial controls. Overall, these data indicate the therapeutic potential of bacterially-delivered toxins and suggest that their further development, in combination with tightly-controlled gene regulation, will yield a safe, efficacious option in cancer treatment.

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