Therapeutic Cell Engineering: Electrogenetics, Bioelectronic Implant Design, and Rewiring of Intracellular Signaling Pathways Using dCas9
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Date
2020
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Doctoral Thesis
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Abstract
Cell therapies utilize functions of the entire living cell to fight diseases. The first chimeric antigen receptor T-cells are already approved for cancer treatment and many others are being developed. Furthermore, multiple proof-of-concept animal studies showed promising results in the treatment of metabolic diseases using designer cells. To face the challenges of their clinical translation, new purpose-driven methods to regulate the behavior of engineered cells are required.
Electronic devices can collect numerous diagnostic or biometric data. Combining them with therapeutic cells to create hybrid, bioelectronic systems could leverage strengths of both components. To achieve this, it would be essential to create an electrogenetic interface that translates the information collected by electronic devices to a format which engineered cells are able to interpret. CHAPTER I of this thesis presents the first direct interface between therapeutic cells and electronic devices. Electrical stimulation opens L-type voltage gated calcium channels and causes calcium influx, which can be linked to either transgene expression, or fast vesicular secretion. The resulting engineered human cell line Electroβ is capable of rapid insulin secretion in the timescale of minutes, bypassing the transcriptional delay typical for current synthetic systems. A wireless-powered subcutaneous implant encapsulating Electroβ cells can provide real-time control of glycaemia in a type I diabetes mouse model. Such electrogenetic devices offer new opportunities for advanced healthcare in the future.
Reprogramming of cellular behavior typically focuses on achieving an input-specific reaction. Current designs that rely on engineered receptors are limited to single inputs, and often suffer from high leakiness and low fold induction. CHAPTER II of this thesis focuses on a new method of rewiring of endogenous signaling pathways to alternative genomic targets, to upregulate expression of genes important for therapeutic purposes. It introduces Generalized Engineered Activation Regulators (GEARs) that consist of the MS2 bacteriophage coat protein fused to regulatory or transactivation domains. GEARs are driven by catalytically inactive Cas9 (dCas9), and can hijack intracellular signaling dependent on NFAT, NFκB, SMAD2 and Elk1. Because of being pathway-specific, they can integrate and process multiple input signals. GEARs enable a membrane depolarization-induced activation of insulin production in β mimetic cells, interleukin 12 expression in activated immortalized T-cells (Jurkat), interleukin 12 production in response to the immunomodulatory cytokines TGFβ and TNFα in HEK293T cells, as well as a simultaneous activation of two genes. Engineered cells with the ability to reinterpret their behavioral programs have potential for applications in immunotherapy and in the treatment of metabolic diseases.
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Examiner: Fussenegger, Martin
Examiner: Vörös, Janos
Examiner: Dittrich, Petra S.
Examiner: Platt, Randall
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ETH Zurich
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synthetic biology; electrogenetics; Genetic circuit; bioelectronics; therapeutic cell engineering
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03694 - Fussenegger, Martin / Fussenegger, Martin
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