Design of synthetic promoter-based gene circuits for the surveillance of cellular state transitions
Embargoed until 2025-10-11
Date
2023Type
- Doctoral Thesis
ETH Bibliography
yes
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Abstract
In recent years, synthetic biology and biomolecular computing have joined forces to
create programmable synthetic gene networks resembling naturally occurring
signaling or regulatory pathways. Among them, ‘cell classifiers’ autonomously assess
the presence or absence of multiple endogenous bimolecular inputs, integrate them
according to user-defined logic programs and elicit customizable responses in a cell
state-specific manner. Several in vivo studies have demonstrated that such devices
hold great potential to herald a new era of gene-therapeutic modalities that will
eventually replace the prevalent ‘one-target – one-drug’ paradigm and revolutionize
the treatment of complex multifactorial diseases, such as cancer. However, despite
considerable progress in the design and clinical translation of gene circuits, the
dynamic nature of cellular states has not yet received adequate attention.
Conventional classifiers have primarily been employed for distinguishing between
static ‘state snapshots’ of different cells, e.g., healthy vs. cancerous. Conversely, the
use of multi-input biomolecular computing systems for monitoring and, if necessary,
manipulating dynamic cell state transitions, a process otherwise known as
surveillance, remains unexplored. In this thesis, I describe the rational design and
implementation of a synthetic surveillance gene circuit capable of continuously
monitoring the state of an individual cell and triggering a programmable response only
when the cell transitions to another predefined state. As a prototypical state transition,
I use the epithelial-to-mesenchymal transition (EMT), a reversible and evolutionarily
conserved developmental program with major physiological and pathological
relevance. To recapitulate EMT in vitro, I employ a well-established cell culture model,
wherein A-549 lung adenocarcinoma cells are treated with transforming growth factorß1
(TGF-ß1). Starting from global RNA-sequencing (RNA-seq) analysis, I devise an
unbiased systematic approach for constructing and testing promoter-based state
detectors that utilize endogenous gene expression levels as inputs. Further, I show
that two state detectors with opposing state specificity can readily be combined in
multi-input gene circuits implementing ‘M-state’ AND NOT (‘E-state’) logic. This circuit
architecture significantly enhances targeting precision and, given its high modularity,
facilitates tunable control over output expression strength. Upon lentiviral integration, the surveillance circuit robustly responds to EMT induction with minimal false positive
activation in epithelial cells and maintains stable expression over extended periods of
time. Finally, substituting the fluorescent output with a suicide gene, commonly used
for cancer gene therapy, enables the highly selective killing of cells that undergo EMT.
These findings demonstrate that the circuit’s surveillance capacity can effectively be
translated into a biologically relevant effect, providing the opportunity to purposefully
interfere with cellular behavior. Ultimately, I discuss how the current circuit design
could be further optimized and propose potential future clinical applications for the
EMT surveillance gene circuit developed in this thesis. Show more
Permanent link
https://doi.org/10.3929/ethz-b-000636129Publication status
publishedExternal links
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Publisher
ETH ZurichSubject
synthetic biology; gene circuits; logic gates; state transitions; Epithelial-to-mesenchymal transitionOrganisational unit
03860 - Benenson, Yaakov (ehemalig) / Benenson, Yaakov (former)
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ETH Bibliography
yes
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