Doctor of Philosophy
Ronald, John A.
Scholl, Timothy J.
Introduction: The ability to non-invasively detect cancer cells and other cell types within tumours has many valuable applications for early cancer diagnosis and grading, monitoring disease progression, and assessing treatment response. While technologies for detecting endogenous cancer biomarkers in bodily fluids or through imaging are being rapidly developed, they often face challenges that result in low sensitivity and/or specificity. To address these limitations, an emerging strategy involves building exogenously delivered tools that use reporter genes as synthetic biomarkers. Leveraging synthetic biology, reporter genes can be assembled into cancer-activatable gene circuits which link their expression to the presence of endogenous cancer molecules not readily assayed, generating synthetic biomarkers that serve as unambiguous signals for cancer. In this thesis, we describe the design and evaluation of cancer-activatable synthetic biomarker systems which are encoded upon both gene- and cell-vector platforms. Methods: We first outline the detection and grading of prostate cancer using survivin-driven, tumour-activatable DNA minicircles (MCs) which upon intratumoural injection produce detectable reporter levels in blood related to cancer aggressiveness. We subsequently built a theranostic MC system by combining urinary identification of aggressive prostate lesions with the ability to selectively treat them using a prodrug-activated gene therapy. Finally, we describe an activatable imaging system in T cells which allows immune-cancer cell communication to be visualized through multimodal reporter gene imaging. Results: Using diagnostic tumour-activatable MCs, we discriminated between aggressive and non-aggressive prostate cancers in mice by measuring the differing levels of an exogenous blood or urine reporter protein. Furthermore, we paired lesion detection with a therapeutic MC which specifically attenuated the growth of aggressive prostate lesions. Finally, we engineered T cells with a synthetic notch imaging system which targeted these cells to antigen-expressing tumours, inducing expression of imaging reporter genes in response to antigen binding. Notably, for the first time we observed in vivo antigen-dependent cell-cell communication using magnetic resonance imaging. Conclusion: The studies presented here contribute to the growing repertoire of synthetic biomarker systems, laying a foundation for activatable gene- and cell-based technologies for improved cancer detection, grading, and monitoring.
Summary for Lay Audience
The ability to non-invasively detect cancers in the body is extremely valuable for diagnosing cancers early, predicting prognoses, and monitoring disease progression. While cancers naturally produce detectable signals which indicate their presence, the reliability of these signals is limited. To address these limitations, reporter genes which produce a protein that is not naturally found in humans can be used as a surrogate marker for cancer. Moreover, reporter genes can be incorporated into an “activatable” system which only produces the reporter proteins in cancer cells or in cells that interact with cancers. In this thesis, we built activatable reporter gene systems which function in a highly cancer-specific manner. The first system we designed used tiny deoxyribonucleic acid (DNA) rings called minicircles which temporarily force cancer cells, but not healthy cells, to produce a unique blood or urine reporter. When these minicircles were administered into mice, we reliably distinguished mice with aggressive prostate tumours from mice with benign tumours and healthy mice via a blood or urine test. We then explored minicircles which can be used to delay the growth of aggressive prostate tumours. Finally, we designed a system that visualizes immune cells within a tumour. We were able to engineer T cells which, upon sensing cancer cells with specific markers, produced reporter proteins that can be detected by several imaging technologies, including magnetic resonance imaging. The works presented here contribute to the growing repertoire of reporter gene technologies, laying a foundation for activatable systems to advance current methods of cancer detection and treatment, ultimately helping to improve patient outcomes.
Wang, TianDuo, "Activatable synthetic biomarker systems for gene- and cell-based cancer detection and theranostics" (2022). Electronic Thesis and Dissertation Repository. 8690.
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Available for download on Thursday, July 20, 2023