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Receptors, Small Moleculesand Physical Cues - ControllingCellular Behavior with SyntheticBiology
Doctoral Thesis
Author(s):Strittmatter, Tobias
Publication date:2021
Permanent link:https://doi.org/10.3929/ethz-b-000493914
Rights / license:In Copyright - Non-Commercial Use Permitted
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DISS. ETH NO. 27358
RECEPTORS, SMALL MOLECULES AND PHYSICAL CUES - CONTROLLING CELLULAR BEHAVIOR WITH
SYNTHETIC BIOLOGY
A thesis submitted to attain the degree of
DOCTOR OF SCIENCES of ETH ZURICH
(Dr. sc. ETH Zurich)
presented by
TOBIAS STRITTMATTER
Dipl. Biochem., Eberhard Karls Universität Tübingen
born on 20.08.1987
citizen of Germany
accepted on the recommendation of
Prof. Dr. Martin Fussenegger
Prof. Dr. Randall Platt
Prof. Dr. Ivan Martin
Prof. Dr. Renato Paro
2021
Tobias Strittmatter i
Table of Contents:
Abstract ........................................................................................................................................................ 1
Zusammenfassung ........................................................................................................................................ 3
Introduction .................................................................................................................................................. 5 Concepts of Synthetic Biology ........................................................................................................................... 5 The Synthetic Biology Toolbox ........................................................................................................................... 6 Applications of Synthetic Biology in Biotechnology ........................................................................................... 9 Synthetic Biology-Enabled (Future) Medicine ................................................................................................. 10 Contributions of this work ............................................................................................................................... 13
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events15 Author Contributions ....................................................................................................................................... 15 Contributions to each figure ............................................................................................................................ 15 Author Affiliations ........................................................................................................................................... 15 Abstract ........................................................................................................................................................... 17 Introduction ..................................................................................................................................................... 19 Results ............................................................................................................................................................. 23
General receptor design strategy ............................................................................................................... 23 DARPin selection ........................................................................................................................................ 25 Designing AMBERs in homodimeric, heterodimeric and tandem configurations ...................................... 26 AMBER screening setup ............................................................................................................................. 27 Control experiments show no unspecific induction ................................................................................... 28 AMBER screening ....................................................................................................................................... 28 Characterization of candidate AMBERs ...................................................................................................... 31 Generating monomeric tandem AMBERs .................................................................................................. 32 Linking fibrinogen activation to induction .................................................................................................. 34 D-dimer- and fragment E-mediated activity ............................................................................................... 36 Generating mammalian designer cells for the detection of coagulation ................................................... 39 Preventing coagulation with hirudin-producing designer cells .................................................................. 41
Discussion ........................................................................................................................................................ 43 Acknowledgements ......................................................................................................................................... 45 Materials & Methods ...................................................................................................................................... 47
Chemicals and proteins .............................................................................................................................. 47 Selection and screening of DARPin binders specific for FDP ...................................................................... 47 Sequence analysis ...................................................................................................................................... 48 Plasmid preparation ................................................................................................................................... 48 SDS-PAGE and Coomassie staining ............................................................................................................. 48 Cell culture ................................................................................................................................................. 49 Transfections and stable cell line generation ............................................................................................. 49 Reporter Assays .......................................................................................................................................... 50 Size-exclusion chromatography of reconstituted plasma .......................................................................... 51 Mass spectrometry ..................................................................................................................................... 51 tPA activity measurements ........................................................................................................................ 51 Statistics ..................................................................................................................................................... 51
Tobias Strittmatter ii
Supplementary Information ............................................................................................................................ 53 Table S1: Plasmids used in this work .......................................................................................................... 53 Table S2: Plasmids transfected in each experiment ................................................................................... 61
Supplementary Figures .................................................................................................................................... 69 References ....................................................................................................................................................... 93
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells ............... 99 Author Contributions ....................................................................................................................................... 99 Contributions to each figure ............................................................................................................................ 99 Author Affiliations ........................................................................................................................................... 99 Abstract ......................................................................................................................................................... 101 Introduction ................................................................................................................................................... 103 Results ........................................................................................................................................................... 107
Design of an L-glucose-responsive gene circuit ........................................................................................ 107 Testing of metabolic enzymes .................................................................................................................. 110 Functionality of ON- vs OFF-switch .......................................................................................................... 112 L-Glucose-induced rituximab production ................................................................................................. 112
Discussion ...................................................................................................................................................... 119 Acknowledgements ....................................................................................................................................... 121 Materials & Methods .................................................................................................................................... 123
Cell culture ............................................................................................................................................... 123 Plasmid generation and transfection ....................................................................................................... 123 Generation of stably expressing cell lines ................................................................................................ 124 Induction with L-glucose .......................................................................................................................... 124 Assessment of reporter gene expression ................................................................................................. 125 Measurement of rituximab production .................................................................................................... 125 Analysis of glycosylation patterns ............................................................................................................ 126 Statistical Analysis .................................................................................................................................... 127
Supplementary Information .......................................................................................................................... 129 Table S1: Plasmid Information ................................................................................................................. 129
References ..................................................................................................................................................... 133
Chapter 3: Controlling gene expression through mechanical cues ............................................................... 137 Author Contributions ..................................................................................................................................... 137 Contributions to each figure .......................................................................................................................... 137 Author Affiliations ......................................................................................................................................... 137 Abstract ......................................................................................................................................................... 139 Introduction ................................................................................................................................................... 141 Results ........................................................................................................................................................... 143
Endogenous and engineered depolarization activated gene switches .................................................... 143 Amplifiers of calcium response ................................................................................................................ 144 Population-based induction of shear stress through turbulent flow ....................................................... 148
Discussion ...................................................................................................................................................... 155 Materials & Methods .................................................................................................................................... 157
Cell culture ............................................................................................................................................... 157 Plasmid generation and transfection ....................................................................................................... 157
Tobias Strittmatter iii
Stimulation of shear stress using the piston device ................................................................................. 157 Generation of stable cell lines .................................................................................................................. 158 Assessment of reporter gene expression ................................................................................................. 158 Statistical Analysis .................................................................................................................................... 159
Supplementary Information .......................................................................................................................... 161 Table S1: Plasmid Information ................................................................................................................. 161 Table S2: Plasmids transfected in each experiment ................................................................................. 167
References ..................................................................................................................................................... 171
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation .................................................................................................................................... 177
Author Contributions ..................................................................................................................................... 177 Contributions to each figure .......................................................................................................................... 177 Author Affiliations ......................................................................................................................................... 177 Abstract ......................................................................................................................................................... 179 Introduction ................................................................................................................................................... 181 Results ........................................................................................................................................................... 185 Discussion ...................................................................................................................................................... 191 Materials and Methods ................................................................................................................................. 193
Plasmid preparation ................................................................................................................................. 193 Cell culture ............................................................................................................................................... 193 Directed differentiation of hiPSCs ............................................................................................................ 194 Mechano-regulation of Wnt/β-catenin signaling ..................................................................................... 195 Analytical assays ....................................................................................................................................... 195 Gene expression profiling by RT-qPCR ..................................................................................................... 195 Statistical analyses .................................................................................................................................... 196
Supplementary Information .......................................................................................................................... 197 Table S1: Plasmid Information ................................................................................................................. 197 Table S2: List of primers for qRT-PCR ....................................................................................................... 199
Supplementary Figures .................................................................................................................................. 200 References ..................................................................................................................................................... 203
Discussion .................................................................................................................................................. 207 New receptors to expand the range of targetable disease ........................................................................... 207 Controlling cellular behavior with small molecules ....................................................................................... 210 Mechanical cues to control gene expression and lineage commitment ........................................................ 212 Developments and future applications of mammalian synthetic biology ..................................................... 214
Conclusion ................................................................................................................................................. 219
Additional References ................................................................................................................................ 221
Acknowledgements .................................................................................................................................... 233
Curriculum Vitae ................................................................................................. Error! Bookmark not defined.
Abstract
Tobias Strittmatter 1/240
Abstract Synthetic biology applies an engineering mindset to the classical field of biology in
order to facilitate the design of new proteins and cellular systems. Synthetic biology-inspired
solutions and products are already a part of modern-day biotechnology and medicine.
Drawing from genetic components from all kingdoms of life, engineers are able to design
sophisticated genetic circuits that respond to various physical and chemical inputs
One key motivation of synthetic biological engineering is the creation of tools to
control cellular behavior typically through genetic circuits. However, the generation of gene
circuits that respond to extracellular soluble ligands remains limited by the availability of
suitable receptors. Although there is a vast number of endogenous receptors at hand, some
proteins, especially disease markers, lack a native sensor module. To tackle this problem our
group designed a novel receptor platform based on the established erythropoietin receptor
scaffold that can accommodate various binding domains to detect small molecules as well as
larger proteins. We took this idea further by employing a high-throughput selection for the
generation of designed ankyrin repeat proteins (DARPins) to detect an even broader range of
ligands in a streamlined fashion as described in chapter 1. We tested this pipeline by creating
receptors that can detect sub-pathological concentrations of fibrin degradation products
arising from e.g., thrombotic events.
In order to detect a ligand without known receptor it is not necessarily the receptor
that needs to be engineered – instead, the ligand itself can be modified to enable its
detection. We show in chapter 2 how we designed an inducible gene switch that responds to
the inert-to-human small molecule L-glucose. Here, we harnessed a metabolic process found
in the soil bacterium Paraccocus species 43P to convert L-glucose into the active ligand of the
bacterial transcriptional regulator LgnR. By fusing transcriptional modulator domains to LgnR,
we could control gene expression from a synthetic promoter in an L-glucose responsive
manner to enable production of the model biopharmaceutical rituximab.
Physical stimuli like mechanical cues are omnipresent in our surroundings and aid cells
to make informed decisions on propagation, tissue organization and cell fate. In chapter 3 we
present results from our work on implementing a mechanically triggered gene switch in
mammalian cells. With the help of a custom-made shear stress induction device we screened
a small library of genetic circuits, transcription factors as well as accessory channels and
Abstract
Tobias Strittmatter 2/240
receptors for their potential to improve shear stress induced calcium responses. This
approach allowed us to identify key components that sensitize cells to mechanical cues to
enable the exogenous control of gene expression.
In chapter 4 we used the same device to study the effects of shear stress on lineage
commitment of human induced pluripotent stem cells (hiPSCs) into the three germ layers. We
were able to show that commitment to the ectoderm and mesoderm lineages was sensitive
to shear stress through modulation of the Wnt/β-catenin signaling pathway while endoderm
was not affected. More detailed analysis revealed that the effect is reversible and can be used
to modulate gene expression levels and lineage commitment even in further matured cells of
the mesoderm lineage.
Zusammenfassung
Tobias Strittmatter 3/240
Zusammenfassung Synthetische Biologie wendet die Denkweise der Ingenieurswissenschaften auf das Gebiet der
klassischen Biologie an, um die Entwicklung von neuen Proteinen und Zellsystemen zu
vereinfachen. Durch synthetische Biologie inspirierte Lösungsansätze und Produkte sind
bereits Teil moderner Biotechnologie und Medizin. Durch die Verwendung von genetischen
Komponenten verschiedenster Spezies können aufwändige genetische Schaltkreise
konstruiert werden, die auf unterschiedliche physikalische und chemische Eingaben
reagieren. Eines der wichtigsten Ziele der Synthetischen Biologie ist das Entwickeln von
Werkzeugen, um das Zellverhalten mittels genetischer Schaltkreise zu kontrollieren. Die
Entwicklung solcher Schaltkreise, die auf extrazelluläre Liganden reagieren können, wird
durch die Verfügbarkeit geeigneter Rezeptoren begrenzt. Obwohl es eine grosse Zahl
endogener Rezeptoren gibt, fehlt zu einigen Proteinen, speziell zu krankheitsrelevanten, ein
natürliches Sensormodul. Um dieses Problem zu lösen, hatte unsere Gruppe bereits eine
neuartige Rezeptorplattform etabliert die auf dem Gerüst des Erythropoietinrezeptors
basiert. Diese Plattform erlaubt es mit Hilfe verschiedener austauschbarer Bindungsdomänen
gleichermassen kleine Moleküle wie auch grosse Proteine zu erkennen. Wie in Kapitel 1
beschrieben entwickelten wir die Idee weiter indem wir eine Hochdurchsatz-
Selektionsmethode zur Generierung von künstlichen repetitiven Ankyrinproteinen (designed
ankyrin repeat proteins; DARPins) verwendeten, um die Bandbreite an möglichen Liganden
auf effiziente Art und Weise noch zu vergrössern. Wir testeten unsere Pipeline durch die
Konstruktion von Rezeptoren, welche Abbauprodukte des Fibrins, die in der Folge von
thrombotischen Ereignissen auftreten, bereits vor dem Erreichen des pathologischen
Grenzwerts erkennen können.
Wie wir in Kapitel zwei darlegen, muss man jedoch nicht immer den Rezeptor
verändern, um einen Liganden erkennen zu können, der keinen bekannten Rezeptor hat,
sondern kann auch den Liganden an den Rezeptor anpassen. Hierzu verwendeten wir einen
metabolischen Prozess aus dem Bodenbakterium Paraccocus species 43P, um den für
Menschen inerten Stoff L-Glucose in einen aktiven Liganden für das bakterielle
Regulatorprotein LgnR umzuwandeln. Indem wir LgnR mit Domänen zur Modulation der
Transkription verbanden, konnten wir die Expression eines synthetischen Promoters L-
Zusammenfassung
Tobias Strittmatter 4/240
Glucose-abhängig kontrollieren, um die Produktion von Rituximab als Modell für ein
biopharmazeutisches Produkt, zu steuern.
Physikalische Reize wie zum Beispiel mechanische Signale sind omnipräsent in unserer
Umgebung und helfen Zellen informierte Entscheidungen bezüglich Zellteilung,
Gewebeorganisation und Zellentwicklung zu treffen. In Kapitel drei präsentieren wir die
Ergebnisse unserer Arbeit zur Umsetzung eines mechanisch aktivierten genetischen Schalters
in Säugerzellen. Mit Hilfe eines speziell angepassten Scherstressgenerators waren wir in der
Lage, einen Pool an genetischen Schaltkreisen, Transkriptionsfaktoren sowie zusätzlichen
Ionenkanälen und Rezeptoren auf ihren Nutzen zur Verbesserung der Signalqualität hin zu
untersuchen.
In Kapitel vier verwendeten wir denselben Scherstressgenerator, um Auswirkungen
von Scherstress auf das Differenzierungsverhalten von menschlichen induzierten
Stammzellen (human pluripotent stem cells; hiPSCs) in die drei Keimblätter zu studieren. Wir
konnten zeigen, dass die Festlegung auf Ektoderm und Mesoderm durch Scherstress
beeinflusst wird, wohingegen die Entwicklung hin zu Endoderm nicht betroffen ist. In einer
detaillierteren Untersuchung fanden wir heraus, dass der Effekt abhängig vom
Induktionszeitpunkt sogar die Entwicklung von Zellen des lateralen Mesoderms oder des
paraxialen Mesoderms begünstigt.
Introduction
Tobias Strittmatter 5/240
Introduction This work aims to expand on the available tools of synthetic biology to control cellular
behavior by engineered receptors, conversion of small-molecule inducers through metabolic
pathways as well as through mechanical cues. This introduction provides an overview of the
underlying design principles of synthetic biology-inspired engineering citing examples of how
synthetic biology is used in industrial biotechnology as well as current and future applications
in medicine.
References included in this introduction, as well as the closing discussion and
conclusion sections can be found at after the conclusion at the end of this work.
Concepts of Synthetic Biology
Synthetic biology applies concepts of classical engineering to the field of biology by
describing functional biological entities as independent modules. By recombining these
modules, synthetic biologists create novel functions in RNAs (Auslander et al. 2014; Auslander
et al. 2016), proteins ( Scheller et al. 2018; Chassin et al. 2019) and whole cells (Gitzinger et
al. 2012).
The modular approach on biological engineering draws from discoveries in protein
biochemistry that established protein domains as independently folding entities with distinct
functions. Domains that are of particular interest for synthetic biology applications are
signaling components as well as metabolically active domains. Bacterial signaling pathways
can generally be very simple, compared to sophisticated interdependent phosphorylation
cascades that can be found in mammalian systems. Instead, prototypic bacterial gene circuits
are based on induced binding or dissociation of transcriptional regulators to the target
sequence on the DNA. Therefore, chimeric transcription factors comprising fusion proteins of
bacterial regulators and mammalian transactivator domains have become a mainstay in
biological research and bioengineering. The most prominent implementation of such a
chimeric transcription factor is the tetracycline transactivator (tTA) (Gossen et al. 1992),
which still is the current gold standard for orthogonal gene expression systems.
The Synthetic Biology Toolbox
Tobias Strittmatter 6/240
The Synthetic Biology Toolbox
The modules that are used for synthetic biology-inspired engineering are often
referred to as the synthetic biology toolbox. This toolbox comprises items from all levels of
cellular organization in the form of protein domains, DNA sequences and RNA elements which
can be rationally assembled to generate new functions. For example, structural
rearrangements of protein domains allow for the creation of novel protein scaffolds used in
binder development for biopharmaceutical products. Cell-based applications, in turns, often
need signaling functions that are not naturally available but that can be engineered by
recombining existing modules to form novel regulatory gene networks.
Figure I: The mammalian synthetic biology toolbox consists of receptors, signal mediators
(NFAT1c) and promoters from all kingdoms of life that enable triggering or rewiring of
endogenous as well as orthogonal signaling pathways.
Such gene networks can be designed on the basis of native or engineered receptors
and channels residing in the plasma membrane that integrate the signal transduction events
through endogenous or orthogonal signaling cascades (figure I). There is a multitude of native
receptors to be found in all kingdoms of life. However, it can prove to be challenging to
integrate transmembrane proteins from bacteria, fungi or plants into mammalian systems
due to their specific requirements on the local membrane environment. Nevertheless,
synthetic biology toolbox
RE RERE promoter GOI
receptors, e.g.- catenines- channels- RTKs- GPCRs- TCRs
signal mediators- signaling cascades- synthet ic transcript ion factors- dCas9
promoter- endogenous- synthet ic
Introduction
Tobias Strittmatter 7/240
mammalian cells are already equipped with specific receptors that can sense a diverse set of
inputs, including temperature (Bai et al. 2019; Zhang et al. 2018), ion concentrations (Yoast
et al. 2020), membrane potential (Helton et al. 2005; Kerstein et al. 2013) or mechanical stress
(Nonomura et al. 2018; Saotome et al. 2018). Additionally, the largest class of cellular sensors,
the family of G-protein-coupled receptors (GPCRs) contains members that respond to small
molecules (Muller et al. 2017) as well as light (Mansouri et al. 2019).
Artificial membrane receptors can be designed with customizable in- and outputs by
equipping existing membrane receptor scaffolds with new binding moieties to trigger
endogenous (Scheller et al. 2018; Kuwana et al. 1987) or orthogonal signaling cascades
(Schwarz et al. 2017). Native receptors are heavily evolved to excel at their respective task in
cell signaling. The sometimes sophisticated, fine-tuned conformational changes that lead to
receptor activation hamper attempts to engineer new functionalities into the existing
receptor scaffold. The class of dimerization dependent receptors like the erythropoietin
receptor (EpoR) however employs a rather simple mechanism of activation. EpoR is expressed
as a dimer of two identical receptor chains. The receptor is activated by binding of its ligand
erythropoietin to the extracellular domains of each receptor chain, which induces
dimerization and a conformational change that translates into a rotation of the intracellular
signaling domains towards each other. Through this turning motion, the associated Janus
kinases (JAKs) move into close proximity leading to mutual phosphorylation of the kinases as
well as of the signaling domains of the receptor chains which triggers downstream STAT
signaling (Watowich, 2011). Because of this comparably simple mechanism of activation,
EpoR has been the target for engineering artificial signaling pathways already.
In a recent study, we were able to demonstrate a generalized extracellular molecule
sensor (GEMS) platform for programming cellular behavior based on EpoR (Scheller et al.
2018). This platform allows for the modular addition and switching of extracellular ligand-
binding domains as well as intracellular signal transduction domains enabling the detection
of a selection of diverse soluble ligands ranging from small molecules to protein cancer
markers that could be used to trigger different signaling cascades.
Native GPCRs signal through binding of small G-proteins that are specific for each
pathway and receptor. In an attempt to alter the signaling from a given GPCR, chimeric G-
proteins have been created that still bind to the respective GPCR but trigger an alternative
pathway (Coward et al. 1999). GPCRs were also engineered through the more cumbersome
The Synthetic Biology Toolbox
Tobias Strittmatter 8/240
method of directed evolution to alter their binding specificity to form receptors activated
solely by synthetic ligands (RASSLs) (Redfern et al. 1999; Armbruster et al. 2007; Conklin et al.
2008).
A more flexible rewiring of endogenous signaling cascades via a modular approach
was described recently (Krawczyk et al. 2020). Here, a nuclease deficient Cas9 was targeted
to endogenous promoters on the genome via specific guide RNAs (gRNAs). Activation of gene
expression was mediated by the interaction of a bacteriophage MS2 coat protein (MCP)
moiety with an MS2-binding RNA loop in the gRNA (figure I). By fusing the MCP to a pathway-
specific regulatory protein, this system can be used to either activate specific pathways or
rewire input from one pathway to the expression of an unrelated target. In addition to this
modular rewiring of endogenous signaling pathways, dCas9 can also be engineered to
respond to light, temperature changes as well as small molecules, constituting a highly flexible
architecture for the design of artificial transcription factors (Richter et al. 2017).
Alternative approaches tackle detection of small molecules from a different angle by
avoiding membrane receptors and rely solely on cytosolic modules instead. To this end,
different approaches have been employed, including light-sensitive dimerization-dependent
gene switches (Levskaya et al. 2009), small molecule-dependent gene switches based on
bacterial regulators (Gitzinger et al. 2009; Wang et al. 2015) or endogenous temperature-
sensitive promoter elements (Miller et al. 2018).
Besides controlling the expression of a given gene, the resulting transcript can also be
engineered to tune mRNA concentrations (Auslander et al. 2016), localize the expression to
predefined regions of the cell (Berkovits et al. 2015), allow for the interaction with specific
RNA-binding proteins like L7Ae (Auslander et al. 2014) or even to sense temperature
(Giuliodori et al. 2010).
On another note, the modular architecture of proteins can not only be exploited for
the design of epitope-binding proteins like designed ankyrin repeat proteins (DARPins)
(Schilling et al. 2014) and antibodies (Liu et al. 2017) but also allows to directly control protein
concentrations inside the cell by addition of destabilizing domains, so-called degrons, that
trigger proteasomal degradation (Chassin et al. 2019).
Cells themselves can be regarded as the biggest available module in synthetic biology
as they can also serve as signaling entities within higher-order cellular systems. Such systems
employ communication between different cell populations via the common metabolite
Introduction
Tobias Strittmatter 9/240
tryptophan (Bacchus et al. 2012), small molecules dopamine and histamine (Auslander et al.
2018) or by direct surface contact through the modular syn-notch receptor platform (Morsut
et al. 2016). Especially the syn-notch platform is nowadays considered a promising tool to
alter and study tissue generation in genetically modified stem cells. With the help of cell-cell
contact-mediated signaling, researchers aim to recapitulate the in vivo situation where cells
receive multiple inputs from their neighbors within a specific developmental niche.
Applications of Synthetic Biology in Biotechnology
Synthetic biology is present on many levels in biotechnological production guiding
protein design as well as genetic modifications of the production cell line. For example,
engineering of antibodies is feasible through their modular structure that enables the
generation of truncated versions comprising only domains essential for epitope binding (Liu
et al. 2017). Fusion of different binding domains for the simultaneous targeting of multiple
epitopes (Cuesta et al. 2010) or addition of T-cell recruiting affinity domains (Lejeune et al.
2020) are further examples of synthetic biology-inspired engineering of biopharmaceuticals.
With the rise of gene therapy, adeno associated viruses (AAVs) are getting broader
attention as safe DNA delivery vehicles in vivo. By engineering capsid proteins of the AAV hull,
scientists are now able to tune the specificity of AAVs for several tissues and cell types (Li et
al. 2020). Novel holistic screening approaches covering all steps from DNA modification, AAV
production and assessment of tissue specificity in live animals are poised to speed up this
process even more, enabling the design of targeted next-generation designer drugs for gene
therapy (Weinmann et al. 2020).
Inducible gene expression is commonly used in microbial fermentations for small to
large scale production of peptides and proteins in lab and industry settings (Huang et al.
2014). Large scale production can be divided into two phases: (i) the growth phase where the
cells are grown to a critical density before (ii) proliferation is limited and the cells enter a
stationary phase. Induced protein production offers a way to time biopharmaceutical
production to the second phase to reduce stress on the culture during the growth phase.
Thereby excess production of harmful metabolites which would lead to a decrease in product
quality can be avoided. In mammalian systems those issues have been tackled by traditional
process optimization rather than genetic engineering. However, inducible biopharmaceutical
Synthetic Biology-Enabled (Future) Medicine
Tobias Strittmatter 10/240
production is slowly making its way into mammalian bioreactors for the production of hard-
to-express proteins that show sophisticated post-translational modifications (Kallunki et al.
2019).
Besides the integration of genetic circuits to respond to exogenous triggers for timed
expression of a transgene - which is still in its infancy for mammalian systems in industrial
applications - researchers have early on tried to improve the productivity of the host cell
through means of genetic engineering. To this end, expression of metabolic enzymes is either
reduced or shut down to limit excess production of metabolites that pose a danger to the
host cell or the product in order to improve overall productivity of the cell line. A prime
example of metabolic engineering is the ongoing effort to reduce overflow metabolism and
lactate production in Chinese hamster ovary (CHO) cells by genetically tuning the activity of
glucose metabolism (Kim et al. 2007; Toussaint er al, 2016; Zhou et al. 2011). Specific post-
translational modifications in the cell can be achieved by ectopic expression of additional
enzymes to either expand the range of products that can be expressed or to reduce unwanted
potentially immunogenic modifications instead (Amann et al. 2019).
A complementary route for increased cellular productivity is offered by protocols to
increase the copy number of the transgene in the genome of stable cell lines. Increased copy
number can be achieved by selection protocols employing glutamine synthetase (GS)-
mediated gene amplification (Fan et al. 2012; Yu et al. 2018). Here, GS-deficient cells can be
adapted for higher expression of a GS-coupled transgene by adding increasing concentrations
of the GS-inhibitor methionine sulfoximine (MSX).
Synthetic Biology-Enabled (Future) Medicine
Treatments based on small molecules and antibodies are still the backbone of
modern-day medicine. However, with the approval of engineered T-cells equipped with
chimeric antigen receptors (CAR-T cells), which were developed already in the last century
(Kuwana et al. 1987), the field of cellular therapies saw its first application outside clinical
trials in 2017. In light of these advances, the application of cellular therapies in patients
becomes more likely. There are already multiple potential treatments based on human
designer cells for the treatment of psoriasis (Schukur et al. 2015) or gouty arthritis (Kemmer
et al. 2010). Equipped with specific receptors to sense disease-related markers and
Introduction
Tobias Strittmatter 11/240
metabolites in patients’ blood, engineered human cells can take over lost body functions to
counteract disease symptoms and potentially restore the quality of life for people with e.g.
diabetes (Scheller et al. 2019; Teixeira et al. 2019) (figure II).
Figure II: Therapeutic designer cells allow the detection of disease markers, integrate the
input and respond in a predefined manner. Amongst others, therapeutic designer cells have
been described that sense and respond to elevated blood glucose or fibrin degradation
products.
Inspired by electrical engineering, synthetic biology recombines interdependent
signaling pathways and transcription factors for the implementation of logic gates in living
cells (Auslander et al. 2018). By doing so, a cell can integrate multiple inputs to cater to the
more complex environment of potential disease niches. For example, logic AND gates based
on gene circuits as well as natural signal-integrating cytokine receptors for the simultaneous
detection of two inflammatory blood markers have been demonstrated to be useful to fight
auto-inflammatory diseases (Schukur et al. 2015; Chassin et al. 2017).
In the new field of electrogenetics, the connection between electrical engineering and
synthetic biology is taken to a new level. A one-way electrical interface was developed to
trigger a prosthetic gene network inside a cell implant for the remote-controlled release of
insulin in diabetic mice (Krawczyk et al. 2020). Besides electricity, other physical stimuli can
be used as well as there are applications for gene switches triggered by light (Mansouri et al.
2019), temperature (Bai et al. 2019; Miller et al. 2018; Vilaboa et al. 2005) and even
ultrasound (Y. Pan et al. 2018).
In conclusion there are currently numerous therapeutic applications in the making
that are directly drawing from synthetic biology-inspired engineering. The designer cells used
disease markers- blood glucose- coagulation products- ...
designer cell therapeutic output- insulin- fibrinolyt ics- ...
detect process respond
Synthetic Biology-Enabled (Future) Medicine
Tobias Strittmatter 12/240
in these therapies are envisioned to enable autonomous treatment of disease in patients and
– once implanted – would require little or no intervention (Kitada et al. 2018).
Introduction
Tobias Strittmatter 13/240
Contributions of this work
The work presented here aims to improve our understanding of mammalian synthetic
biology by adding new modules to the synthetic biology toolbox to control cellular behavior.
Chapter 1 comprises a protocol for the acceleration of receptor development against targets
without a known natural receptor that is based on the well-characterized erythropoietin
receptor. We also describe how an inaccessible small molecule can be harnessed for induction
of gene expression by employing a metabolic process to convert it into an active ligand of a
bacteria-derived artificial transcription factor (chapter 2).
We furthermore present our work on how mechanical cues can potentially be used to
control gene expression in a non-invasive, ligand-free manner in chapters 3 and 4. We show
how mechanical stimuli can alter calcium signaling and downstream gene expression (chapter
3) as well as how shear stress can change morphogen signaling in induced pluripotent stem
cells (iPSCs) to modulate lineage commitment (chapter 4).
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 15/240
Chapter 1: Programmable DARPin-based Receptors for the Detection
and Treatment of Thrombotic Events
Chapter 1 describes a work in progress that has not yet been submitted for publication.
Author Contributions
Tobias Strittmatter1, Leo Scheller2 and Martin Fussenegger1,6 designed the project together
with Jonas V. Schaefer3 and Andreas Plueckthun4. Tobias Strittmatter1, Pascal Stuecheli1,
Patrick C. Freitag3, Jonas V. Schaefer4, Thomas Reinberg3 and Dimitrios Tsakiris5 performed
experiments and analyzed the results. Leo Scheller2 performed initial experiments that are
not covered in this manuscript and provided plasmids.
Contributions to each figure
Tobias Strittmatter1: figures 1a-c, 2, 3, 4a-e, S1-6, S8-14
Jonas V. Schaefer3, Thomas Reinberg4 and Andreas Plueckthun4: figures 1d, S3
Patrick C. Freitag4: prepared binders for figure S7
Dimitrios Tsakiris5: figure 4f
Jens Sobek4: performed SPR measurements in figure S7
Steven Schmitt1: assisted with HPLC in figure S11
Ulrike Lanner6 as well as Alexander Schmidt6: performed mass spectrometry for figure S11
Author Affiliations 1Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland. 2Laboratory of Protein Design & Immunoengineering, École Polytechnique Fédérale de
Lausanne | EPFL, Lausanne, Switzerland 3Present Address: Novartis Institutes for BioMedical Research (NIBR), Basel, Switzerland 4University of Zurich, Zürich, Switzerland 5Diagnostic Hematology, University Hospital Basel, Basel, Switzerland 6University of Basel, Faculty of Science, Basel, Switzerland.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 17/240
Abstract Sophisticated design strategies have been developed for engineering mammalian receptors
with different intracellular signaling capabilities. However, cellular therapies remain
constrained by the limited availability of sensors for the relevant disease states and markers,
because detection of new markers currently relies on pre-existing binders. To address the
need for an integrated target-to-receptor approach, we present here a pipeline for the
generation of a customizable advanced modular bispecific extracellular receptor (AMBER)
that combines our generalized extracellular molecule sensor (GEMS) system with a high-
throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of
concept, we chose human fibrin degradation products (FDPs) as markers with high clinical
relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19
DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers in
combination with a known scFv binder. Tandem receptors consisting of combinations of the
validated DARPins are also functional. We demonstrate an application of these AMBER
receptors by constructing designer cell lines that detect pathological concentrations of FDPs
and respond with the production of a therapeutic anti-thrombotic protein.
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Introduction
Cell-based therapies are still hampered by the limited availability of cellular receptors
for the detection of clinical disease markers. To address this issue, various types of receptors
have been employed to change cellular behavior (Scheller and Fussenegger 2019). These
include well-known chimeric antigen receptors (CARs) that rely on endogenous signaling of T
cells to drive T-cell responses to tumors. Other chimeric receptors that consist of fusions of
extracellular receptor domains and intracellular signaling domains of native receptors have
been engineered to reroute cellular responses (Mohammed et al. 2017). In addition, synthetic
dimerization-dependent receptors can drive transgene expression by employing bacterial
transcription factors (Schwarz et al. 2017). However, all of these receptor systems require
careful fine-tuning for each new ligand, and the ligand space is limited by the characteristics
of the existing binding proteins.
The recently developed generalized extracellular molecular sensor (GEMS) platform
allows for more robust switching of extracellular binding domains as well as intracellular
signaling domains (Scheller et al. 2018). GEMS receptors consist of an extracellular target-
binding moiety linked to the erythropoietin receptor (EpoR) extracellular domain. Activated
receptors amplify the incoming signal through signal transduction domains, such as the
intracellular domain of interleukin receptor 6B, which activates the signal transducer and
activator of transcription 3 (STAT3) pathway and triggers expression of a protein of interest
from STAT3-responsive promoters (fig. 1a). However, changing the specificity of the receptor
still relies on the availability of a suitable target-binding domain.
Most engineered receptors in mammalian synthetic biology use well-characterized
protein-protein interactions of native receptor-ligand interactions or single chain variable
fragments (scFv). However, the ligand space for native receptor-ligand interactions is severely
limited, and scFvs are typically derived from antibodies (AB) that are generated by laborious
immunizations of animals. Besides Fabs or scFvs obtained by classic AB selection combined
with protein engineering (Helma et al. 2015), there are various other kinds of protein scaffolds
that can be modified to change their binding properties. Some of these scaffolds have been
used in high-throughput screenings and for selecting binders to non-native targets. Examples
include fibronectin III (FN3)-derived monobodies (Karatan et al. 2004), αRep proteins
containing HEAT-like repeats (Guellouz et al. 2013), camelid-derived VHH domains, termed
nanobodies (Skerra et al. 1988), and designed ankyrin repeat proteins (DARPins) (Binz et al.
Introduction
Tobias Strittmatter 20/240
2004; Schilling et al. 2014). While monobodies and αRep are generated by phage-display
(Guellouz et al. 2013) and yeast-surface display (Uchanski et al. 2019), respectively, DARPins
(Binz et al. 2004; Dreier et al. 2012) and nanobodies (Bencurova et al. 2015) have been shown
to work robustly with cell-free ribosome display selection systems that enable the screening
of bigger libraries and can be combined with error-prone PCR to increase sequence variation.
DARPins are small proteins of 14 kDa that preferentially bind to three-dimensional features
of target proteins (Pluckthun 2015). The DARPin technology enables the production of high-
affinity binders in an efficient manner. However, screening efforts generally aim to select just
a few high-affinity binders for a given target, and may discard potentially valuable binders.
In the context of synthetic dimerization-dependent receptors, selection of multiple
binders in parallel tackles an intrinsic bottleneck, i.e., that at least two independent binding
modules are required for a given target. Therefore, we aimed to combine the GEMS platform
with the DARPin screening technology to generate a customizable advanced modular
bispecific extracellular receptor (AMBER). To demonstrate the feasibility of this strategy, we
decided to develop and characterize the AMBER system by focusing first on cellular sensors
of human fibrin degradation products (FDPs), which are used as a clinical marker to exclude
pathologic coagulation events (Adam et al. 2009) and are also a candidate prognostic marker
for the severity of viral infections (Yao et al. 2020).
In the physiological context, blood coagulation is necessary to prevent hemorrhages
and the intrusion of pathogens during injury by forming stable clots consisting of fibrin fibrils.
Fibrin formation from its precursor, fibrinogen, is mediated by thrombin, which is activated
via the proteolytic blood coagulation cascade (Smith et al. 2015). Excessive coagulation can
happen due to stalling of blood flow (e.g., in the case of atrial fibrillation (Watson et al. 2009)),
reaction of the immune system to foreign material (Xu et al. 2014) (e.g., artificial heart valves
(Iung et al. 2014), stents (Luscher et al. 2007)), hereditary disorders (e.g. hyperactive factor V
Leiden (Dahlback et al. 1993; Bertina et al. 1994)), or cancer (Dipasco et al. 2011). Pathological
blood coagulation events can cause serious disability and even death (Benjamin et al. 2019),
so a cell-therapy approach employing autonomous detection of FDPs and delivery of a
therapeutic response directly in patients at risk could help to improve patient outcomes and
reduce mortality.
Therefore, we set out to demonstrate the translational potential of the AMBER system
by engineering mammalian cells equipped with AMBERs to detect pathological coagulation
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 21/240
events and to respond by triggering the secretion of a therapeutic protein, either
tenecteplase (TNK), which is an engineered version of the fibrinolytic protein tissue
plasminogen activator (tPA), or the anticoagulant hirudin, which naturally is found in leeches.
To slow down or inhibit blood coagulation, either direct (e.g. with hirudin (Sohn et al.
2001)) or indirect inhibition (e.g. with heparin (Baglin et al. 2006)) of thrombin can be
employed. However, even very recently developed drugs are constitutively active and hence
increase the risk of bleeding, or can lead to induced thrombocytopenia (Bonaca et al. 2020;
Blackmer et al. 2009). An integrated cellular system able to measure efficacy and toxicity in
parallel could improve the lead times of innovative anticoagulant therapies. We envision that
the presented DARPins and the receptors developed here will be useful for the benchmarking
of existing and novel blood-thinning agents, as well as inspiring the development of next-
generation designer cells to reduce the deleterious effects of thrombotic events.
The AMBER pipeline provides streamlined and predictable methodology for the
generation of new cellular receptors for soluble targets of choice. We believe this technology
will facilitate research on cellular signaling as well as the development of future cellular
therapies to treat diseases for which no relevant cellular receptors are known. Accordingly,
this study aims to expand the available repertoire of receptors against soluble targets by
introducing a new type of binder as well as to improve reliability and predictability of receptor
design as a whole. To this end a high-throughput DARPin-selection protocol was included to
open up new epitope spaces and streamline receptor development. In a proof-of-concept
experiment functionality of the resulting receptors for use in therapeutic settings was
assessed by targeting fibrin degradation products (FDPs) as clinical markers for thrombosis.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 23/240
Results
General receptor design strategy
The AMBER system employs the GEMS receptor architecture. We focused here on the
interleukin 6 signal transduction domain (IL6ST) as the intracellular domain. IL6ST signals
through the JAK/STAT pathway via signal transducer and activator of transcription 3 (STAT3),
and hence a synthetic reporter construct equipped with STAT3 response elements (RE) was
used to record STAT3 signaling (fig. 1a). Specificity of the sensor system is ensured by
introducing a previously described mutation (F93A) (Middleton et al. 1996) into the
extracellular domain of the EpoR scaffold to decrease native erythropoietin sensitivity.
We use this receptor scaffold in combination with an established high-throughput
DARPin selection method. This leads us to a four-step pipeline for the generation of functional
AMBERs (fig. 1b). Briefly, DARPins are first selected by ribosome display and then their
sequences are classified by means of phylogenetic comparison and pairwise amino acid
alignments. Distinct DARPins are fused to the receptor scaffold and tested for induction in a
homodimeric or heterodimeric receptor setup. Finally, a more compact tandem receptor
design may be applied to validated DARPin binders. Each step is described in detail below for
the case of xFDP receptors as an example.
Due to the dimeric nature of the AMBER platform, successful activation of the
receptors relies on binding of both receptor chains to their target. This requirement implies a
limitation concerning the distance between the epitopes. Based on the crystal structure of
activated EpoR (Livnah et al. 1996) we estimated the optimal theoretical distance between
the attachment sites of the C-termini of the two binders to the extracellular domains to be
less than 8 nm. Identical epitopes may be located along the whole 45 nm length of the dimeric
fibrinogen molecule (Kollman et al. 2009; Brown et al. 2000), providing a range of differently
spaced epitope pairs that should offer a good chance to find working receptors. However, the
large size of the molecule also reduces the likelihood that two epitopes would lie in close
proximity. These considerations led us to select cross-linked fibrin degradation products
(xFDPs) as the preferred target for demonstrating the feasibility of our receptor-building
pipeline; a choice that is supported by their established clinical relevance.
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Tobias Strittmatter 24/240
Figure 1: a) Schematic illustration of the final coagulation-responsive cellular sensor system.
A sensor on the cell surface detects fibrin-derived fibrin degradation products (FDP) (grey
hexagons) via engineered extracellular binding domains (orange). Activation of generalized
extracellular molecular sensor (GEMS)-based advanced modular bispecific extracellular
receptor (AMBER) (green) triggers the signal transducer and activator of transcription 3
(STAT3; blue) transcription factor pathway, which is rerouted towards expression of a
reporter or a therapeutic output (tenecteplase, TNK or hirudin, Hir). b) A four-step pipeline
for the generation of functional AMBERs. First, binders (designed ankyrin repeat proteins;
DARPins) are generated by ribosome display and the genetic information of candidate
binders is analyzed. Next, genetically distinct binders are used to build AMBERs, which are
tested in cellular assays in homodimeric or heterodimeric configuration. After the
identification of functional binders, they can be used to build more compact tandem
a) The AMBER architecture
d) DARPin screening against xFPDs
b) The AMBER pipeline
c) Ribosome display for DARPin generation
ribosome
dissolvecomplex
reverse transcription
in vitrotranslation
translation stopsat last codon
mRNA library
nascentpeptide
stalled ribosome
reporter
STAT3
AMBER
inducer
binder
scaffold
signaling
hSAxF
DPs0
1
2
3
4
5H
TFR
ratio
relat
ive
to S
trept
avid
in [A
U]
1.45
118 = 31 %160 = 42 %
1.15
binder selection
(ribosome display)
sequence analysis
homodimeric
AMBERs
heterodimeric
AMBERs
tandem AMBERs
of validated hits
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 25/240
receptors with similar specificity. c) Overview of ribosome-display-based DARPin selection
to generate novel binding domains. An optimized DARPin library containing a mixture of
N3C-DARPins with randomized and non-randomized N- and C-caps is transcribed to mRNA,
which is translated in vitro. Since the mRNA lacks a stop codon, the ribosome is stalled on
the mRNA with the nascent peptide still attached upon completion of translation. The fully
folded DARPin can now bind to FDPs coupled to streptavidin-coated beads via their
biotinylated lysine residues. FDP-DARPin-ribosome-mRNA complexes are washed and
subsequently dissolved. The resulting mRNA is reverse-transcribed to yield an enriched
plasmid library, which is subjected to three additional rounds of the same selection cycle. d)
Results from high-throughput homogeneous time-resolved fluorescence assay (HTRF) to
assess binding of DARPins to human serum albumin (hSA) or cross-linked fibrin degradation
products (xFDPs) depicted as the ratio of maximum fluorescence of the sample versus the
streptavidin control. Streptavidin is used to couple biotinylated target protein to beads
during the selection run. DARPins are labeled via a FLAG-tag with an antibody coupled to a
fluorescent acceptor dye (d2) while xFDPs are labeled in a similar fashion via their biotin tag
with streptavidin carrying a Tb FRET donor. Binding of a DARPin to FDP protein hence results
in an increase of FRET efficiency (increased signal-to-noise ratio). AU: arbitrary units.
DARPin selection
To identify a suitable target for FDP binder screening we assessed the composition of
commercially available xFDPs enriched in D-dimers by means of SDS-PAGE. We confirmed
that the major component is D-dimers (fig. S1), accompanied with other degradation
products, most likely fragment E and fragment D of fibrin, as well as DD-E complexes, which
serve to broaden the available epitope space. xFDPs are protein complexes ranging from 120
kDa (D-E complex) to 220 kDa (DD-E complex) in size, and may include larger unprocessed
fragments of several hundred kDa. Those complexes have large surface areas that are likely
to present numerous epitopes. We biotinylated these xFDPs and applied them to an in vitro
ribosome display-based screening system for the generation of DARPins (fig. 1c). Briefly, an
optimized plasmid DARPin library comprising N3C-DARPins with randomized and non-
randomized N-terminal and C-terminal cap structures was transcribed into mRNA, and the
obtained mRNA was translated using isolated ribosomes. However, because the mRNA lacks
a stop codon, translation is stalled at the last codon, since the ribosome can neither progress
Results
Tobias Strittmatter 26/240
nor complete the translation. Consequently, the mRNA is attached through the ribosome to
the corresponding nascent peptide. This peptide undergoes folding into a functional DARPin
that can bind to immobilized FDPs coupled to streptavidin-coated beads through biotinylated
lysine residues. The resulting FDP-DARPin-ribosome-mRNA complexes are purified and
disassembled to extract the enriched mRNA. The selected mRNA is used in a subsequent cycle
to further increase the affinities of DARPins in the pool. We employed four rounds of selection
under the same conditions.
We selected 380 DARPins through ribosome display and tested them for binding to
the target in a homogeneous time-resolved fluorescence (HTRF)-based assay (fig. 1d). We
excluded unspecific binding to human serum albumin (hSA), used as a stabilizing agent in the
xFDP preparation.
Of the 380 DARPins, 160 candidates (42 %) gave a signal sufficiently above background
in the HTRF assay (fold induction > 1.15). Of these, 118 (31 %) were considered good binders
(fold induction > 1.45). Next, DNA of a subset of 32 DARPins (8 %) was sequenced, leading to
the exclusion of 13 duplicated sequences. This left a set of 19 genetically unique DARPins (5
%). Phylogenetic analysis (fig. S2a) showed some of the 19 binders to have a high degree of
sequence similarity (e.g. DARPins A6, A7 and B4), which was confirmed by sequence
alignment (fig. S2b) and pairwise comparison of the hits (fig. S2c); DARPins with high similarity
would potentially bind to the same epitope. All 19 binders were further characterized by size-
exclusion chromatography (SEC) analysis to check for the presence of aggregates, which were
observed in four of the 19 DARPins, leaving 15 (4 %) DARPins with monomeric characteristics
(fig. S3). Nevertheless, we used all 19 binders in subsequent receptor designs, since we
hypothesized that constraints introduced by receptor fusion might affect dimerization in the
final receptor constructs.
Designing AMBERs in homodimeric, heterodimeric and tandem configurations
Different receptor configurations were explored to find the best setup to sense FDPs
and also to help us deduce some general guidelines for targeting other proteins of interest
(fig. 2a). A homodimeric receptor configuration in which all receptors are equipped with the
same binding module is the smallest and most straight-forward receptor architecture (fig. 2a,
i). However, homodimeric receptors rely on the presence of two epitopes in close proximity,
so that functional receptors require dimeric or symmetric ligands.
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Tobias Strittmatter 27/240
Heterodimeric receptors are pairwise combinations of receptors with different
binding moieties (fig. 2a, ii), and hence two independent receptor chains always need to be
expressed. Pairwise combinations grow exponentially with the number of available binders
(following the equation for pairwise combinations N of two out of n elements: N = 0.5 n2 - 0.5
n). Assuming a unique epitope for each receptor, this would greatly increase the likelihood of
finding active receptors, i.e. receptors that allow the binders to come into close proximity
upon ligand binding.
Tandem receptors consist of two binders fused in sequence to the N-terminus of the
receptor scaffold; this results in an increase of avidity and hence a possibly higher sensitivity
(fig. 2a, iii). Because the tandem approach involves only one receptor chain to build receptors
with two binding sites, all receptors presented on the cell surface are of the same
architecture, which increases the effective concentration. Tandem receptors also reduce the
size of the genetic construct (2.5-2.9 kb for the receptor alone) allowing it to be used in size-
restricted vectors, such as vectors derived from adeno-associated viruses (AAVs) (Grieger et
al. 2005). The drawback to building functional tandem receptors is the even greater
combinatorial space, which grows to the power of four for each binder involved (following
the equation for N pairwise combination of two out of n elements in pairs: N = 0.5 x2 - 0.5 x
with x = 0.5 n2-0.5 n, N = 0.125 n4 - 0.25 n3 - 0.125 n2 + 0.25 n).
For the AMBER platform, we therefore decided to screen homodimeric or
heterodimeric receptors prior to confirming the functionality of tandem receptors built with
the validated binders.
AMBER screening setup
To benchmark new FDP-DARPins as well as to explore potential benefits of cross-
platform combinations of binders, we used a previously described single-chain variable
fragment (scFv) targeting human D-dimers (Laroche et al. 1991). We equipped the AMBER
platform with both the anti-D-Dimer-scFv (scFv-AMBER) and the new repertoire of 19 FDP-
binding DARPins. This yielded a small library of 20 receptors, each bearing a single binding
moiety on the extracellular domain. HEK293T cells were co-transfected with plasmids each
encoding a single receptor under the control of a constitutive synthetic promoter derived
from human cytomegalovirus (hCMV) (PhCMV-receptor-pA), along with a plasmid encoding a
constitutively expressed STAT3-transcription factor (PhCMV-STAT3-pA) and a secreted human
Results
Tobias Strittmatter 28/240
placental alkaline phosphatase (SEAP) reporter plasmid controlled by two STAT3 response
elements (RE) and a minimal version of the hCMV promoter (SEAP, RE2-PhCMVmin-SEAP-pA).
Control experiments show no unspecific induction
Insensitivity of the receptors to erythropoietin (EPO) was confirmed by measuring
EPO-mediated activation of the receptor scaffold (fig. S4a). A previously reported construct
based on an scFv responding exclusively to the non-toxic industrial dye reactive red (RR120)
(Scheller et al. 2018) was used as a negative control for testing potential receptor-
independent effects on reporter activity (fig. S4b). We assessed the impact of purified human
xFDPs on productivity and on the viability of HEK293T cells by measuring changes in
constitutive SEAP expression (fig. S4c) and reductive capacity via resazurin, which is reduced
by healthy mitochondria and is an indicator of metabolically active cells (fig. S4d). We found
xFDPs to be non-toxic at concentrations up to 10 µg/mL. We also tested for background
effects of human plasma and found that concentrations of up to 2 % (v/v) had little effect on
constitutive SEAP expression (fig. S4e) or viability (fig. S4f).
AMBER screening
We finally screened all 210 possible pairwise combinations of the available receptors
(including all 20 receptors as homodimers) for inducibility by 0.5 µg/mL xFDPs as well as by
0.5 % reconstituted plasma in cell culture (fig. S5a-d), and calculated the fold inductions
relative to the uninduced samples (fig. 2b). We divided the responses in two categories: weak
(fold induction >2) and strong (fold induction >5).
Induction with xFDPs yielded four receptors (1.9 %) in the weak and 3 (1.4 %) in the
strong category. Notably, all receptors responding strongly to xFDPs were heterodimers co-
expressed with the scFv receptor.
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Tobias Strittmatter 29/240
Figure 2: a) Engineered receptors based on the GEMS platform bind to fibrin degradation
products, including fragment E (blue), fragment D (green) and cross-linked D-dimers (green,
indicated by orange crosses), as well as complexes thereof. Binding induces STAT3-mediated
gene expression of therapeutic proteins or the reporter protein human secreted placental
a) schematic of different classes of generated AMBERs
c) homodimeric receptors
b) screening of homodimeric and heterodimeric receptors against xFDPs and plasma
f)
d) heterodimeric receptors
g)
e) tandem receptors
h)
1 10-3 1 10-2 1 10-1 1 1000
20
40
60
80
100
xFDP concentration [µg/mL]
SEAP
[UL-1
]
B4A7A6G1scFvRR120-receptor
1 10-3 1 10-2 1 10-1 1 1000
20
40
60
80
100
SEAP
[UL-1
]
B4 + scFvA7 + scFvA6 + scFvG1 + scFvRR120-receptor
xFDP concentration [µg/mL]1 10-3 1 10-2 1 10-1 1 100
0
20
40
60
80
100
xFDP concentration [µg/mL]
SEAP
[UL-1
]
G1-A7-tandemG1-B4-tandemG1-G1-tandemscFv-A7-tandemscFv-B4-tandemscFv-G1-tandemRR120-receptor
1 10-3 1 10-2 1 10-1 1 1000
40
80
120
160
plasma concentration [%]
SEA
P [U
L-1]
B4A7A6G1scFvRR120-receptor
1 10-3 1 10-2 1 10-1 1 1000
40
80
120
160
plasma concentration [%]
SEA
P [U
L-1]
B4 + scFvA7 + scFvA6 + scFvG1 + scFvRR120-receptor
1 10-3 1 10-2 1 10-1 1 1000
40
80
120
160
plasma concentration [%]
SEA
P [U
L-1]
G1-A7-tandemG1-B4-tandemG1-G1-tandemscFv-A7-tandemscFv-B4-tandemscFv-G1-tandemRR120-receptor
SEAP
STAT3
heterodimeric receptor homodimeric tandem-receptorhomodimeric receptor
(i) (ii) (iii)
A12-A12
A10-A10
B12-B12
C4-C4
A12-scF
v
A10-F12
B12-F8
C4-A11
A12-G
1
A10-C11
B12-A6
C4-A7
A12-E11
A10-D11
B12-F10
C4-F9
A12-F6
A10-B4
B12-D12
C4-B10
A12-C4
A10-A12
B12-A10
C4-B12
A12-A11
A10-scF
v
B12-F12
C4-F8
A12-A7
A10-G
1
B12-C11
C4-A6
A12-F9
A10-E11
B12-D11
C4-F10
A12-B10
A10-F6
B12-B4
C4-D12
A12-B12
A10-C4
RR1200
10
20
30
40
fold
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ctio
n
xFDP plasma
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Tobias Strittmatter 30/240
alkaline phosphatase (SEAP). (i) Homodimeric receptors present one binding moiety only
and rely on symmetrically presented epitopes on the target. (ii) Heterodimeric receptors
display two different binding moieties, similarly to (iii) homodimeric tandem receptors,
which comprise two binders that are fused on top of the receptor chains in a head-to-tail
fashion. b) Combinations of different receptors comprising different binding domains were
tested for their response to purified xFDP protein (blue dots) as well as plasma (orange
dots). Values are normalized to uninduced samples of the same receptors and represent
means ± SD of triplicates. c)-e) Functional systems started to respond well below the
clinically relevant concentration of 0.3 µg/mL (dotted line). f)-h) When tested for reactivity
against human plasma the sensitivity of the system increased remarkably. c)-h) Solid lines
are non-linear fits. Values for the highest concentration were excluded for the fit in
subfigure e)-f) to improve the fits for lower to medium concentrations. The RR120 receptor
serves as a negative control. For details, see the main text. The x-axis is log-scale, and all
values are means ± SEM of N=3 independent experiments performed in triplicates.
Human plasma was tested to evaluate the performance of the system in a more native
setting and to investigate responses to other species of FDPs that were underrepresented in
the DARPin screen. In the case of plasma induction, we found weak responses for 37 (17.6 %)
and strong responses for 49 (23 %) of the 210 receptors. Among the plasma-responsive
receptor combinations, 77% included one of four DARPins (A6, A7, B4, F6). We could not find
any correlation of HTRF signal intensity from DARPin selection with either the number of
functional receptors or the resulting receptor signal strength in a homodimeric setup (fig. S6).
More stringent selection of DARPins on the basis of HTRF values therefore seems not to
influence the likelihood of finding a functional receptor. However, receptor activity in a
homodimeric setting seems to be predictive for the total number of functional receptor
combinations (fold induction >2). Functional receptors in some cases comprised DARPins that
showed aggregation behavior as recorded by SEC for the A7- and F9-DARPins (fig. S2),
indicating that auto-dimerization of the binder does not necessarily limit its usefulness in a
receptor.
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Tobias Strittmatter 31/240
Characterization of candidate AMBERs
We characterized a set of four DARPin binders comprising candidate DARPins A6, A7,
B4 and the less active DARPin G1 as well as the scFv in a homodimeric receptor configuration.
Addition of increasing concentrations of xFDPs revealed an OFF-type switching behavior for
receptors based on A6, A7 and B4 DARPins (fig. 2c). While this could be explained by inhibition
of auto-dimerization by ligand binding, only the A7 DARPin showed such characteristics
during SEC analysis. Therefore, we speculate that the observed OFF-type switching behavior
is caused by the binding of large antigens to both receptor chains, thereby reducing basal
(“leaky”) activity by stabilizing pre-formed receptor dimers in an inactive conformation.
In contrast, we recorded ON-type switching behavior for the same DARPins with up to
110-fold induction in response to human plasma (fig. 2f). These results suggest successful
expression and display of DARPin-AMBERs. The increase in activity towards plasma indicates
that these DARPins are activated by another protein that is not present, or is present only at
a trace level, in the xFDP preparation (compare fig. S1).
We next examined the four candidate DARPins in combination with the scFv as
heterodimeric receptor pairs. In contrast to the homodimeric configuration, these receptors
dose-dependently respond to xFDPs with inductions of up to 25-fold and EC50 values of 0.15,
0.19 and 0.12 µg/mL for DARPins A6, A7 and B4, respectively (fig. 2d). The measured EC50
values for xFDPs are well below the pathological threshold for D-dimer protein of 0.3 µg/mL
in blood (Froehling et al. 2007) (indicated by a dotted line in fig. 2 c-e). Induction with plasma
yielded similar dose responses to those in the homodimeric configuration (fig. 2g). However,
the heterodimeric setup also allows for homodimeric receptors at the cell surface, and the
latter most likely dominate or at least strongly contribute to the response in the case of
plasma induction.
Binding affinities of DARPins A6, A7, B4 and G1 as well as the scFv were quantified by
surface plasmon resonance (SPR) (fig. S7). Due to the symmetric structure of the target
molecules, we stipulated a heterogenous binding modality resulting in two distinct binding
constants. All binding constants, except for the G1-DARPin, were within the nanomolar range
measuring 169/244 nM for the scFv and 14/37 nM, 44/91 nM, 23/59 nM and 602/1030 nM
for DARPins A6, A7, B4 and G1, respectively. Affinities for the G1-DARPin were up to 10- to
Results
Tobias Strittmatter 32/240
50-fold lower compared to those of other DAPRPins which may explain the low performance
of the G1-DARin in a receptor context.
Generating monomeric tandem AMBERs
After identifying functional DARPin binders and scFv combinations in the homodimeric
and heterodimeric screen, we fused all 16 combinations of tested DARPins A7, B4, G1 and the
scFv to the receptor scaffold in a tandem orientation (see fig. 2a, iii). Screening of
homodimeric tandem receptors against xFDPs revealed three out of 16 (19 %) receptors to
be sensitive (fig. S8); all three contain a DARPin in combination with the scFv (fig. S8c,d). All
but one of the receptors based on DARPins (89 %) and two (29 %) of the scFv-DARPin
combinations were responsive to plasma. Notably, we found a working receptor for each of
the DARPins successfully tested before (fig. S8a,b). In contrast to the working DARPin tandem
receptors that contain a tandem repeat of a single DARPin, scFv alone did not respond to
neither plasma nor xFPDs in such a configuration, in accordance with the results for the
respective homodimeric receptors.
In a dose-response assay, DARPin-only tandem receptors detected xFDPs in a dose-
dependent manner, albeit with lower sensitivity compared to heterodimeric scFv
combinations, showing estimated EC50 values of 4.3 and 1.2 µg/mL for the G1-A7 and G1-B4
tandem receptors, respectively (fig. 2 e). Tandem receptors comprising DARPins and the scFv
were similarly sensitive as their heterodimeric counterparts, showing EC50 values of 0.31
µg/mL (vs 0.19 µg/mL) for the scFv-A7 receptor and 0.25 µg/mL (vs 0.12 µg/mL) for the scFv-
B4 receptor. Induction with plasma led to increased signal intensities for all tandem receptors,
even for the previously non-responding G1-G1 tandem receptor. These results indicate that
the tandem receptors exhibit full functionality and mostly reflect the features of the
corresponding heterodimeric receptors, while at the same time being more compact. The
question of whether tandem configurations also allow for different ligands to be detected will
be addressed later.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 33/240
Figure 3: Overview of processes involved in fibrinolysis. Fibrinogen forms a head-to-head
dimer via the E-domain (blue) that is covalently linked via di-sulfide bonds. Upon activation
with thrombin, fibrinogen oligomerizes via interactions of its D-domains (green) with the E-
d) state of cross linking affects induction
e) attribution of ligand specificity of each receptor
a) Overview of fibrinolytic processes
b) fibrinogen induces AMBERs c) inhibitors of coagulation block induction
Fibrinogen
D-domainE-domain
disulf ide bond
factor XIIIa
tridegin
non-cross-linked fibrin f ibre non-cross-linkedDD-E complex
cross-linked fibrin
plasmin
plasmin
TXA
TXA
thrombin
plasma
plasma +
hirudin
plasma +
hepari
n
fibrin
ogen
fibrin
ogen + hiru
din
fibrin
ogen + hepa
rin
uninduced
0
5
10
15
SEAP
[U*L
-1]
B4B4 + scFvG1-B4scFv-B4
10g/m
L
2g/m
L
uninduced
10g/m
L
2g/m
L
uninduced
10g/m
L
2g/m
L
uninduced
10g/m
L
2g/m
L
uninduced
0
5
10
15
SEAP
[U*L
-1]
B4B4 + scFvG1-B4scFv-B4
uninduced
no thrombin
thrombin and fib
rinogen
+ TXA
+ trideg
in
+ plasmin
+ plasmin + tri
degin
uninduced
no thrombin
thrombin and fib
rinogen
+ TXA
+ trideg
in
+ plasmin
+ plasmin + tri
degin
uninduced
no thrombin
thrombin and fib
rinogen
+ TXA
+ trideg
in
+ plasmin
+ plasmin + tri
degin
uninduced
no thrombin
thrombin and fib
rinogen
+ TXA
+ trideg
in
+ plasmin
+ plasmin + tri
degin0.0
0.5
1.0
1.5
2.0
2.5
norm
alize
d S
EAP
[RU]
B4 B4 + scFv G1-B4 scFv-B4(i)
uninduced
no thrombin
thrombin and fib
rinogen
+ TXA
+ trideg
in
+ plasmin
+ plasmin + tri
degin0.0
0.5
1.0
1.5no
rmal
ized
SEA
P [R
U](i i) AMBER
B4+ specific induction
(ii) AMBERB4/scFv
:
D-dimers
(i) AMBERB4
:
fragment E
(iii)AMBERG1-B4
:
FDPs
(iv) AMBERscFv-B4
:
xFDPs
45nm 6.5nm 10nm
Results
Tobias Strittmatter 34/240
domain to form an insoluble non-cross-linked fibrin mesh. The mesh is stabilized by factor
XIIIa, which introduces isopeptide bonds between adjacent D-domains. Fibrinolysis is
mediated by plasmin, which cleaves fibrin into smaller soluble fragments. Depending on
whether the fibrin was cross-linked prior to plasmin digestion, different types of fragments
arise. Degradation products include fragment E comprising the E-domain of fibrinogen
(blue), fragment D (green) and cross-linked D-dimers (green, indicated by orange crosses),
as well as complexes thereof. b) Reactivity of receptors towards fibrinogen was tested in a
minimal system using two different concentrations of fibrinogen and thrombin. c) Reactivity
towards fibrinogen and plasma can be blocked by addition of known anti-coagulants
heparin and hirudin. d) Cross-linking of fibrinogen is mediated by factor XIII (FXIII) and
blocked by its inhibitor tridegin. Reporter expression is modulated by addition of tridegin or
plasmin and TXA in a minimal system comprising purified fibrinogen and thrombin. Reporter
activity is normalized to a sample with constitutive expression of the reporter under the
same conditions to compensate for unspecific alterations. ii) Values of i) for B4+scFv are
normalized to values for B4 to emphasize differences in reporter expression between both
receptor configurations. All values are means ± SD of representative experiments in
triplicate. e) Summary of suggested receptor specificities. AMBERB4 may preferentially bind
to fragment E of fibrin, AMBERB4/scFv may bind to cross-linked D-dimers complexed with
fragment E (DD-E complex) or fragment E alone (because some parts are also presented in
the AMBERB4 configuration), AMBERG1-B4 may bind partially to non-cross-linked fibrin
degradation products (FDPs), and AMBERscFv-B4 favors cross-linked FDPs (xFDPs).
Linking fibrinogen activation to induction
To characterize the impact of potentially under-represented species of xFDPs in the
initial DARPin selection and to exclude off-target receptor activation, we next investigated
the receptor sensitivity to plasma. For all subsequent characterizations we focused on the
homodimeric B4-receptor (AMBERB4; PhCMV-B4-receptor-pA), the heterodimeric B4+scFv-
receptor (AMBERB4/scFv; PhCMV-B4-receptor-pA + PhCMV-scFv-receptor-pA), the tandem G1-B4-
receptor (AMBERG1-B4; PhCMV-G1-B4-receptor-pA), and the scFv-B4-receptor (AMBERscFv-B4;
PhCMV-scFv-B4-receptor-pA).
During coagulation and subsequent degradation of the fibrin mesh, various
fibrinogen-related complexes are formed (fig. 3a). In general, possible degradation products
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 35/240
that could serve as inducers of AMBER receptors include fragment E of fibrin, D-E and DD-E
complex, as well as D-dimers and larger, partially digested degradation products containing
the same epitopes. Fragment E of fibrin has a diameter of approximately 4 nm and a length
of 13 nm, while fragment D is at least 10 nm long and 5 nm wide, with D-dimer accordingly
being of 20 nm length and 5 nm diameter (Kollman et al. 2009).
We found that in addition to xFDPs and plasma, all four receptors could also be dose-
dependently induced by purified fibrinogen in the presence of FCS (fig. 3b). Plasmin
degradation products of fibrinogen, i.e., fragment D (fig. S9a) or fragment E (fig. S9b), or a
mixture of fragments D and E (Fig. S9c), did not trigger induction. We found that extensive
plasmin digestion of fibrinogen in the presence of thrombin decreased the signaling of
AMBERs, which indicates that high plasmin concentrations degrade fibrinogen faster than it
can be activated by thrombin, and these degradation products cannot be detected by the
receptors (fig. S10a).
Taken together, these results show that neither native fibrinogen nor its plasmin
cleavage products are inducers of AMBERB4, AMBERB4/scFv or AMBERG1-B4. Furthermore, the
activity of AMBERB4, AMBERB4/scFv and AMBERG1-B4 pre-incubated with fibrinogen for 24 h was
unchanged by additional incubation with plasmin for another 24 h (fig. S10b). This could
indicate that either the degradation products initially bound by the receptors cannot be
processed any further or that binding of (x)FDPs to the receptors blocks plasmin cleavage. In
accordance with these results, induction with xFDPs was also not modulated by plasmin (fig.
S10c).
To confirm that fibrinogen is the active molecule in plasma preparations, we
fractionated plasma by size-exclusion chromatography (SEC) to separate the proteins based
on their hydrodynamic radius. The run-times of the fractions that activated AMBERB4 and
AMBERB4/scFv corresponded well with reported run-times for fibrinogen (fig. S11a). The
presence of fibrinogen in the active fractions was confirmed by polyacrylamide gel-
electrophoresis followed by mass spectrometry (fig. S11b).
Furthermore, fibrinogen- and plasma-mediated induction was directly linked to
coagulation, because the induction was blocked by direct (hirudin) or indirect (heparin)
inhibition of thrombin. Both inhibitors specifically repressed induction of the system upon
addition of either 0.5 % (v/v) plasma or 10 µg/mL fibrinogen (fig. 3c, S12a).
Results
Tobias Strittmatter 36/240
Taken together, these results establish thrombin and thrombin-containing FCS as
potent drivers of coagulation directly linked to receptor activation without off-target activity.
At this point, we wondered whether the system could be used to evaluate anti-
coagulant agents. Indeed, AMBERB4, AMBERB4/scFv and AMBERG1-B4 showed the expected dose-
related response to the thrombin inhibitor argatroban, in good agreement with previous
results for argatroban used against fibrin-bound thrombin (Berry et al. 1994) (fig. S12b). The
prodrug ximelagatran remained ineffective in our test up to concentrations of 100 µM (fig.
S12c).
Finally, we tested whether differences in fibrin preparations affect receptor
sensitivity, using insoluble, dried fibrin resuspended in cell culture media containing FCS (fig.
S12d) and fibrin that was solubilized at acidic pH and precipitated by neutralization (fig. S12e).
In both cases, we recorded a bell-shaped induction of all receptors tested (AMBERB4,
AMBERB4/scFv, AMBERG1-B4 and AMBERscFv-B4) with peak inductions at around 0.3 mg/mL and 3-
10 µg/mL, respectively. Given the mechanism of receptor activation, we expect each receptor
chain to bind to a separate ligand at high concentrations, thus stabilizing the receptor in an
inactive conformation and giving rise to a bell-shaped induction curve. Interestingly, the
response to each compound varied among the receptors. While this may be partly due to
differences in compound composition and epitope accessibility, it also raises the possibility
that the receptors bind to different proteins or protein complexes that are present at
different concentrations in each preparation.
D-dimer- and fragment E-mediated activity
To investigate potential differences in receptor specificity and to further exclude off-
target activity we tried to identify the active molecule(s) for candidate receptors AMBERB4,
AMBERB4/scFv, AMBERG1-B4 and AMBERscFv-B4. We hypothesized that the receptors can
distinguish fibrin-derived fragment D, fragment E, D-dimer and DD-E complexes (compare fig.
3a). DD-E complex and D-dimers originate from the degradation of mature fibrin formed by
cross-linking of activated fibrinogen molecules via isopeptide bonds catalyzed by factor XIIIa
(Lorand et al. 1968; Gupta et al. 2016). The leech-derived peptide inhibitor tridegin reportedly
blocks factor XIIIa activity through direct inhibition (Bohm et al. 2014) and was therefore used
to reduce the concentrations of cross-linked species such as D-dimer and DD-E complexes.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 37/240
We investigated the effect of plasmin, the plasmin inhibitor tranexamic acid (TXA),
and tridegin on coagulation-mediated receptor activation. We used a setup without FCS and
initiated coagulation with fibrinogen and thrombin. Due to the unspecific effect of TXA on
SEAP expression (fig. S13), we normalized all samples to cells constitutively expressing the
reporter under the same conditions. Cells were washed with FCS-free medium before the
experiment to reduce residual serum components. However, as can be deduced from the
following results, trace amounts of plasmin as well as factor XIII remained, because inhibitors
reported to target these enzymes were still effective (fig. 3d, i). Possibly these enzymes are
even secreted by the cells themselves (Petryszak et al. 2016). While controls incubated with
only fibrinogen remained uninduced, addition of thrombin led to the expected increase of
SEAP reporter expression, indicating coagulation-induced receptor activation. We observed
stronger activation of tandem receptors AMBERG1-B4 and AMBERscFv-B4 than of homodimeric
or heterodimeric receptors AMBERB4 and AMBERB4/scFv (fig. 3d, i).
Interestingly, AMBERB4 activation was independent of TXA-mediated plasmin
inhibition. These results indicate that this receptor responds to early products of coagulation
(fig. 3d, i). In the presence of tridegin in the induction mix, AMBERB4 responded with an
increase of reporter expression, which also points towards more accessible, non-cross-linked
FDPs as inducers. This is consistent with the fact that AMBERB4 did not respond in previous
tests with D-dimer-enriched (fragment E depleted) cross-linked FDPs (compare fig. 2c).
Addition of plasmin again led to a decrease of signal intensities for all receptors; the decrease
was largest for AMBERB4 and AMBERB4/scFv (fig. 3d, i), likely because of excessive degradation
of fibrinogen by plasmin prior to coagulation (Hur et al. 2015) (compare fig. S9a). However,
further addition of tridegin to plasmin led to a marked increase of AMBERB4 activity
confirming the involvement of non-cross-linked degradation products (i.e. fragments D, E and
D-E complex) as ligands (fig. 3d, i). We also found earlier that the proteins detected by the
AMBERB4 and AMBERB4/scFv systems are most likely final products of plasmin digestion
(compare fig. S10b). Since fragment D is only symmetric if it is cross-linked, it only then
comprises two identical epitopes. Thus, fragment E is the sole candidate that fulfills all
requirements, and we conclude that DARPin-B4 (present in AMBERB4 and AMBERB4/scFv) binds
FDPs in a cross-linking-independent manner, and that possible binding partners are fragment
E as a single component or in D-E or DD-E complexes (fig. 3e, i).
Results
Tobias Strittmatter 38/240
Expression of heterodimeric AMBERB4/scFv also allows for the assembly of AMBERB4
homodimers on the cell surface. Hence, results for AMBERB4/scFv are overlaid with the
activation pattern of AMBERB4 (fig. 3d, i). We therefore normalized the SEAP levels of
AMBERB4/scFv to AMBERB4 to analyze the contribution of the scFv receptor (Fig. 3d, ii). AMBERB4
and AMBERB4/scFv responded similarly under all conditions examined, except for the
combination of plasmin and tridegin, for which we recorded a decrease in reporter expression
for the AMBERB4/scFv receptor configuration. This tridegin-mediated inhibition may imply that
the AMBERB4/scFv receptor relies on cross-linking. These results are consistent with earlier
findings that AMBERB4/scFv but not its AMBERB4 counterpart proved sensitive to purified cross-
linked FDPs, enriched in D-dimer protein (compare fig. 2c). We therefore conclude that the
scFv moiety within AMBERB4/scFv binds cross-linked D-dimer protein in the complex with
fragment E (DD-E complex), which in turns bind to the B4-DARPin (fig. 3e, ii). This specificity
of the scFv is in line with that reported in the original publication(Laroche et al. 1991).
Probably due to a low epitope count or steric hindrance, receptors based on the scFv alone
are not able to detect D-dimers or coagulation. Nevertheless, we can build functional
receptors that combine DARPin binders and the scFv.
The tandem receptors AMBERG1-B4 and AMBERscFv-B4 were both sensitive to TXA
inhibition, indicating a dependence on plasmin activity to produce their respective ligands
(fig. 3d, i). While AMBERG1-B4 showed an increase in activity upon addition of tridegin, the
same treatment led to a decreased reporter output for AMBERscFv-B4, indicating binding to
non-cross-linked and cross-linked FDPs, respectively. Supplementation with plasmin again
resulted in decreased reporter expression and additional tridegin lowered the reporter
expression further. However, the effect of tridegin was notably stronger for AMBERscFv-B4 (fig.
3d, i). These results indicate that both tandem receptor configurations detect partially
digested, larger FDPs, with AMBERscFv-B4 preferentially binding cross-linked fragments and
AMBERG1-B4 preferentially binding non-cross-linked fragments (fig. 3e, iii, iv).
In conclusion, these results reveal that the AMBERs in the analyzed set have different
sensitivities and specificities towards fibrin degradation products, thus providing an
illustration of the versatility of the AMBER platform.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 39/240
Generating mammalian designer cells for the detection of coagulation
To test the AMBER platform in a translationally relevant proof-of-concept format, we
generated mammalian designer cells to sense coagulation or xFDPs and respond by the
secretion of a therapeutic protein, TNK or hirudin. For this purpose, we employed the more
sensitive heterodimeric AMBERB4/scFv as well as the genetically more compact homodimeric
AMBERscFv-B4 tandem receptor (compare fig. 2d). Prior to generating stable cell lines, we
examined the sensitivity of different reporters containing two, four, 12, or 16 repeats of the
response element (fig. S14a), and selected a reporter element with four repeats.
We generated a plasmid for genomic integration of a reporter cassette based on the
four-repeat response element that drives induced expression of a secreted version of the
luminescent reporter nano-luciferase (Nluc) fused to the therapeutic protein TNK. We
genetically linked expression of TNK to the reporter Nluc via a furin cleavage site (RARYKR)
(figure 4a). The furin site is cleaved in the Golgi apparatus to deliver an equimolar ratio of
both proteins to the supernatant, hence providing a direct correlation of reporter activity with
potential therapeutic effect. We selected stable monoclonal HEK293T cells that express the
STAT3 transcription factor alongside AMBERscFv-B4 and the Nluc and TNK reporter cassette
(termed HEKscFv-B4/TNK). We also generated a polyclonal control cell line expressing the
receptor for RR120 instead (HEKRR120R/TNK); this does not respond to any of the coagulation-
dependent stimuli (fig. S14b).
Correlation of Nluc and TNK activity was confirmed by measuring the activity of TNK
and Nluc in the supernatant of the monoclonal cell line HEKscFv-B4/TNK in response to xFDPs (fig.
4 b). With an onset time of six hours, the system was responding within the limits of a
transcription-based gene switch (fig. S14c-d). Additionally, the system could detect xFDPs at
levels as low as 0.1 µg/mL, well below the clinical disease threshold of 0.3 µg/mL and was also
sensitive to plasma (fig. S14e-f).
Results
Tobias Strittmatter 40/240
Figure 4: a) Schematic illustration of vectors used for the generation of stable coagulation-
sensing cells. Top: sensing and expression cassette for a secreted nano-luciferase (Nluc)
reporter fused to a murine Fc tag for enhanced stability in vivo (Nluc-mFc) and a
tenecteplase-mFc fusion construct (TNK-mFc). Equimolar production and separation of the
products during secretion is ensured by placing a furin cleavage site (RARYKR) between the
two proteins. Middle: construct for stable integration of the reporter cassette using the
Sleeping Beauty transposon system expressing a fluorescent protein (FP) to facilitate
fluorescence-activated cell sorting (FACS) coupled to antibiotic resistance markers for zeocin
(ZeoR) or blasticidin S (BlastR) for stable cell line selection. Bottom: construct for stable
a) schematic of reporter/TNK construct used
c) schematic of hirudin reporter construct d) schematic of hirudin activity assay
e) inhibition of coagulation by hirudin f) effect on thrombin clotting time (TCT)
b) activity of TNK correlates with reporter output
AMBERB4/scFv/hirudin B4
day 1: induction of hirudin expression
with xFDPs
4x STAT3-Op Igk-SS nLuc-mFc RARYKR TNK-mFc
4x STAT3-Op Igk-SS nLuc-mFc p2a natSS-Hirudin-HM2
FP-ZeoR
nLuc-T
NK
5’ DR
3’ DR
FP-BlastR
receptor
5’ DR
3’ DR
STAT3
+ 1g/m
L xFDPs
uninduced
30
35
40
45
Thro
mbi
n C
lotti
ng T
ime
[sec
]
p=0.0016
00.0
10.0
20.0
60.2
00.6
42.0
0
heparin
0
2
4
6
8
10
-5.0 105
0.0
5.0 105
1.0 106
1.5 106
2.0 106
2.5 106
SEAP
[U*L
-1]
nLucSEAP
nLuc Luminescence [A
U]
xFDPs [µg/mL]
01.0
03.1
610
.0131
.64
100.0
431
6.31
1000
.18
3162
.56
1000
0.00
0
1 106
2 106
3 106
4 106
5 106
0
2
4
6
8
xFDP concentration [ng/mL]
Nlu
c Lu
min
esce
nce
[AU]
Nluc activity
TNK activtiy [U
/L]
TNK activity
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 41/240
integration of the receptors as well as an overexpression module for signal transducer and
activator of transcription 3 (STAT3) transcription factor. DR, direct repeat; Nluc, nano-
luciferase; TNK, tenecteplase (htPA variant); mFc, murine antibody fragment for increased
stability. b) Measurements of xFDP- induced expression of reporter Nluc and TNK from
HEKscFv-B4/TNK cells show the two are well correlated and legitimates the use of Nluc
reporter production as a proxy for TNK activity. 45 000 cells were seeded per well and
incubation was done for 24 h. We subsequently induced those cells for 24 h with the
indicated concentrations of xFDP protein or plasma. Nluc and TNK activity were measured
in the supernatant. TNK activity of untreated cells was subtracted from the observed values.
c) Schematic illustration of the vector for the generation of the monoclonal stable cell line
mHEKB4/scFv/hirudin for inducible hirudin-HM2 production. d) Workflow to assess hirudin-
HM2 efficacy in cell culture. As in b), mHEKB4/scFv/hirudin cells were induced with xFDP
protein on day 1. Supernatant of induced mHEKB4/scFv/hirudin cells was mixed with 0.5 %
(v/v, final) plasma on day 2 and dispensed on coagulation-sensing cells expressing
AMBERB4. SEAP reporter expression from sensor cells and Nluc activity of
mHEKB4/scFv/hirudin are measured in the same sample with a multiplate reader on day 3.
e) Hirudin-HM2-induced inhibition of coagulation is sensed by reporter cells equipped with
AMBERB4 and expressing SEAP in response to coagulation. mHEKB4/scFv/hirudin cells show
a clear correlation of induction by higher concentrations of xFDP protein with reduced
coagulation. f) Thrombin time of mHEKB4/scFv/hirudin cells induced with xFDPs was
assessed in a clinical laboratory and compared to uninduced controls. Values are b) mean ±
SD of quadruplicate determinations, or e-f) means ± SD of triplicate determinations.
Preventing coagulation with hirudin-producing designer cells
In most cases, the prevention of coagulation is therapeutically preferable to the
induction of clot lysis. Anti-coagulants such as hirudin and heparin or its derivatives are
generally used, especially for long-term administration. We therefore set out to determine
the effectiveness of a designer cell-based treatment strategy for the production of hirudin. As
a first step, we re-designed the reporter construct used for inducible tenecteplase expression.
We replaced the tenecteplase and furin cleavage sequence with a p2a peptide for the
separation of the adjacent proteins during translation and used a codon-optimized sequence
Results
Tobias Strittmatter 42/240
encoding secreted hirudin-HM2 from Poecilobdella manillensis (Mexican medical leech) (fig.
4c). We generated a stable monoclonal HEK293T cell line (mHEKB4/scFv/hirudin) harboring the
new reporter construct alongside AMBERB4/scFv. Next, we developed an assay to measure the
efficacy of the therapeutic output by harnessing the AMBERB4-based coagulation reporter
system. Figure 4d illustrates the workflow used in this experiment. mHEKB4/scFv/hirudin cells were
induced for 24 h with increasing doses of xFDPs. The supernatant was transferred to reporter
cells transfected with AMBERB4 as well as a SEAP reporter cassette and mixed with a final
concentration of 0.5 % (v/v) plasma to assess the anticoagulant effect of secreted hirudin.
Purified hirudin and heparin were added as a control. Hirudin expression was sufficient to
reduce the coagulation-induced signal of the reporter cells by 3.3-fold (fig. 4e), and
significantly (p=0.0016) prolonged the thrombin clotting time (TCT) by 10 % in an established
clinical laboratory test (fig. 4f), thus demonstrating functional secretion of the therapeutic
protein.
Taken together, the above results show that our modular receptor system works as
expected; we can detect coagulation at an early stage by employing the AMBERB4 system, or
we can detect early as well as late biomarkers of thrombotic events by using an integrated
sensor incorporating the scFv (AMBERB4/scFv or AMBERscFv-B4). Furthermore, our proof-of-
concept designer cell lines show that AMBER receptors can be coupled to therapeutic protein
output: either tenecteplase to boost clot lysis or hirudin to prevent further coagulation. These
results indicate that the AMBER pipeline has translational potential for cell-therapy
approaches to treat recurrent thrombosis or embolism in patients at risk.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 43/240
Discussion In this work we have combined DARPin selection with our GEMS technology, and we
show that the resulting AMBER platform enables the generation of programmable receptors
with customizable sensitivity and specificity by screening a vast space of possible epitopes.
Such versatility is unprecedented in a receptor system for soluble ligands. We used the
created receptors to reliably detect FDPs, which are generated at the onset of blood
coagulation, in cell culture experiments. Furthermore, the reporter cells were successfully
used to evaluate the efficacy of anti-coagulant drugs. In another proof-of-concept, we
showed that secretion of a therapeutic protein alongside a luminescent reporter provides a
direct correlation of reporter activity with the expression of a therapeutic protein.
Notably, not all hits from the initial DARPin selection turned out to be good candidates
for building a functional receptor. This also appears to be the case for other binders described
in the literature, and emphasizes the importance of a diverse library as input for the AMBER
pipeline. In fact, with respect to induction by plasma, about 16 % of combinations with a
known scFv and 40 % of DARPin-only receptors were active in a heterodimeric receptor
configuration. Since about 83 % of these DARPin-only receptors are based on one out of four
of the initially characterized 19 DARPins, the overall success rate in finding suitable binders
was 21 %. With a symmetric target the generation of homodimeric receptors is of course
much more likely, and we expect the chance of finding such receptors for regular monomeric
targets to be lower. Nevertheless, the GEMS platform has been successfully used in
combination with pre-existing binders to detect the monomeric 26 kDa cancer antigen PSA
with the use of two scFvs (Scheller et al. 2018). Here, out of three scFvs tested, two were
shown to form a working receptor. As discussed earlier, we expect the number of working
combinations of FDP binders that satisfy the design constraints of the receptor architecture
to be lower due to the much greater size. While a large surface is not necessarily indicative of
successful binder generation, the number of non-overlapping epitopes is nevertheless
increased, and therefore so is the chance of finding suitable pairs of binders for receptor
generation. While one can use the overall design to build receptors similar to already reported
binders, an integrated pipeline to check a receptor library for activity in cell culture is easy to
parallelize and provides a time- as well as cost-efficient solution. Therefore, we expect the
versatile and highly streamlined DARPin technology in combination with the AMBER
technology to yield more receptors for a range of different targets. Targets may include
Discussion
Tobias Strittmatter 44/240
soluble cancer antigens, cytokines or even cell-surface epitopes. AMBERs might be especially
useful for targets for which no natural receptor is known and available sequences for scFvs or
other binders are limited. Modular switching of the signaling domains of the underlying
receptor scaffold has already been reported (Scheller et al. 2018) and this could be used to
accelerate research in systems and stem-cell biology to rewire endogenous signaling
cascades.
In addition, the selected anti-FDP DARPins may be used to build novel diagnostic tools
as well as therapeutics by expanding the available epitope landscape for the development of
ELISA-like assays or DARPin-coupled fibrin-targeted drugs for clot lysis. Detection of fragment
E could offer more dynamic measurements compared to other degradation products,
especially D-dimers, in a potential therapeutic setting due to the shorter serum half-life
(Ardaillou et al. 1977; Tanswell et al. 1993).
Finally, ribosome display offers a powerful method to generate a highly diverse binder
library through enrichment of high-affinity binders in a cell-free setup. Irrespective of the
generation method, fewer rounds of binder selection might be beneficial for finding
functional receptors. Although the average binder affinity increases with each cycle, the
covered epitope space might be reduced at the same time. Expanding the receptor platform
to include other types of binders that can be generated via similarly tunable procedures such
as monobodies (Karatan et al. 2004), HEAT-like repeat proteins (Guellouz et al. 2013) and
nanobodies (Skerra et al. 1988),(Ward et al. 1989) would further broaden the range of
possible targets and applications, especially for the detection of small molecules (Lesne et al.
2019). Combinations of different binder selection platforms might also be a promising
strategy for difficult targets. The benefits of such a combination are already visible in this
study in terms of the change in binding site preference that was observed upon employing an
scFv in combination with a DARPin.
We hope that our system will inspire the clinical development of GEMS-based cellular
therapeutics to improve the treatment of patients at risk of thrombosis by reducing harmful
delays in intervention. We envision that FDP-targeted AMBERs will be useful in the
development of novel anti-coagulants as well as therapeutic cells. Our data already
demonstrates the feasibility of producing suitable therapeutics in a human cell line,
suggesting that the AMBER pipeline will be applicable to cell-based therapies in next-
generation medicine.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 45/240
Acknowledgements
The authors thank Steven Schmitt for help with size-exclusion chromatography and
Ulrike Lanner as well as Alexander Schmidt from the Proteomics Core Facility (PCF) at the
University Basel for their support with mass spectroscopy. We also thank Jens Sobek from the
Functional Genomics Center Zurich (FGCZ) for providing affinity data. We are grateful to
Mariangela Di Tacchio, Alexandra Gumienny and Thomas Horn from the single-cell facility at
the D-BSSE for help with cell sorting. We thank David Fuchs, Adrian Bertschi, Viktor Hällman,
and Pratik Saxena, all from ETH Zurich, for critical readings of the manuscript and valuable
discussions. We thank Craig Hamilton from the University Bern and Birgit Dreier from the
University of Zurich for critical comments on the manuscript. Finally, we want to thank Astrid
Beerlage from the University Hospital Basel and Rebekka Strittmatter from the St. Elisabethen
Hospital Lörrach for advice on medical aspects.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 47/240
Materials & Methods
Chemicals and proteins
Native human D-dimer protein (ab98311), recombinant hirudin protein (ab201396)
and native human plasmin (ab90928) were purchased from Abcam plc. trans-4-
(Aminomethyl)cyclohexanecarboxylic acid (tranexamic acid, TXA, 857653), heparin sodium
salt from porcine intestine (H3149), lyophilized human plasma (P9523), active thrombin from
human plasma (SRP6557), and fibrin from human plasma (F5386) was purchased from Sigma-
Aldrich. Human fibrinogen fragment E (HCI-0150E) and human fibrinogen fragment D (HCI-
0150D) were purchased from Haematologic Technologies, LLC. Soluble human fibrin (FIB-S-
1.0MG) was purchased from Molecular Innovations Inc. Tridegin (opr0062) was purchased
from Covalab S.A.S.
Selection and screening of DARPin binders specific for FDP
To generate DARPin binders for human FDP protein, the biotinylated target protein
was immobilized on either MyOne T1 streptavidin-coated beads (Pierce) or Sera-Mag
neutravidin-coated beads (GE), depending on the selection round. Ribosome display
selections were performed essentially as described (Dreier & Pluckthun, 2012), using a semi-
automatic KingFisher Flex MTP96 well platform.
The library includes a mix of N3C-DARPins with randomized and non-randomized N-
and C-caps respectively, and successively enriched pools were cloned as intermediates in a
ribosome display specific vector (Schilling et al. 2014). Selections were performed over four
rounds with decreasing target concentration and increasing washing steps to enrich high-
affinity binders.
The final enriched pool was cloned as fusions into a bacterial pQE40 derivative vector
with a N-terminal MRGSH8-and C-terminal FLAG tag via unique BamHI x HindIII sites
containing lacIq for expression control. After transformation into E. coli XL1-blue, 380 single
DARPin clones were expressed in 96-well format and lysed by addition of a concentrated Tris-
HCL-based HT-Lysis buffer containing OTG, lysozyme and nuclease. These bacterial crude
extracts of single DARPin clones were subjected to homogeneous time-resolved fluorescence
(HTRF)-based screening to identify potential binders. Binding of the FLAG-tagged DARPins to
streptavidin-immobilized biotinylated FDP protein was measured using FRET (donor:
Materials & Methods
Tobias Strittmatter 48/240
streptavidin-Tb, acceptor: anti-FLAG-d2, Cisbio). Further HTRF cross reactivity measurements
against ‘No Target’ and ‘HSA’ allowed for discrimination of FDP-specific hits.
Among the identified binders, 32 were sequenced and 19 single clones were selected.
The DARPins were expressed on a small scale and purified using a 96-well IMAC column
(HisPurTM Cobald plates, Thermo Scientific). DARPins after IMAC purification were analyzed
at a concentration of 10 µM on a Superdex 75 5/150 GL column (GE Healthcare) using an
Aekta Micro system (GE Healthcare) with PBS containing 400 nM NaCl as the running buffer.
Absorbance at 280 nm was recorded. β-Amylase (200 kDa), bovine serum albumin (66 kDa),
carbonic anhydrase (29 kDa) and cytochrome c (12.4 kDa) were used as molecular mass
standards. 15 DARPins were identified as monomers.
Sequence analysis
Phylogenetic analysis of amino acid sequences was done with a simple phylogeny tool
(www.ebi.ac.uk/Tools/phylogeny/simple_phylogeny/). Sequence alignments were
performed using Clustal O (www.ebi.ac.uk/Tools/msa/clustalo/). Pairwise identity calculation
was carried out using the EMBL-Needle algorithm employing the BLOSUM62 matrix
(www.ebi.ac.uk/Tools/psa/emboss_needle/). All algorithms are part of the EMBL-EBI search
and sequence analysis tools API (Madeira et al. 2019).
Plasmid preparation
A comprehensive list of all plasmids used in this study can be found in table S1.
Plasmids were generated using conventional molecular cloning techniques.
Polymerase chain reaction (PCR) was performed using Phusion® polymerase (F530,
ThermoFisher Scientific) or Q5® high-fidelity polymerase (M0491, New England Biolabs, NEB)
following the manufacturers’ recommendations. Cleavage of PCR products and plasmids was
done with restriction endonucleases (New England Biolabs NEB, HF enzymes were used if
applicable) and ligated with T4 DNA ligase (EL0011, ThermoFisher Scientific).
SDS-PAGE and Coomassie staining
Samples were heated for 15 minutes at 70°C prior to loading. Up to 20 µL of sample
was loaded per well of a 12-well or 15-well polyacrylamide gel (Bolt 4-12% Bis-Tris Plus gels,
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 49/240
Invitrogen). SDS-PAGE was performed by using 1x Bolt MOPS SDS Running Buffer (Invitrogen)
in a XCell SureLock® Mini-Cell (Invitrogen) following the instructions of the manufacturer.
PageRuler Plus Prestained Protein Ladder (Invitrogen) was used as a size reference.
Gels were washed three times with water after the run to remove SDS from the running
buffer, then stained with Coomassie (17% (v/v) methanol, 3.3% (v/v) acetic acid, and 0.08%
(w/v) Coomassie Brilliant Blue G250) for >1 h at RT. Gels were washed and destained in water
for several days before use in mass-spectrometric analysis.
Cell culture
HEK293T/17 (HEK293T) cells (ATCC® CRL-11268™) were purchased from the American
Type Culture Collection and grown in high-glucose Dulbecco's modified Eagle’s medium
containing GlutaMAX (DMEM, high glucose, GlutaMAX Supp, 61965026, ThermoFisher
Scientific) supplemented with 10 % fetal bovine serum (FBS, F7524, Sigma-Aldrich) at 37°C in
a humidified incubator with an 7.5% CO2 atmosphere. Cells were grown in 10 mL of DMEM at
a seeding density of 1.5 * 106 cells in a 10 cm culture dish and kept at < 80 % confluency.
For transfection, cells were seeded in 15 mL of DMEM supplemented with 1x
penicillin/streptomycin (L0022, Biowest) in 96-well culture plates (15000 cells per well).
Stable HEK293T cells were generated using the Sleeping Beauty transposon system (Kowarz
et al. 2015). Donor plasmids bearing a selection marker (resistance genes for puromycin (pac),
blasticidin (bsr) or zeocin (ble)) driven by a constitutive promoter and flanked by recognition
sites of the Sleeping Beauty transposase were co-transfected with an expression plasmid for
Sleeping Beauty transposase. For selection of stable clones, the cell culture medium was
supplemented with puromycin (2 µg/mL), blasticidin (4 µg/mL) or zeocin (20 µg/mL) or
combinations of them, depending on the types of plasmids to be integrated.
Transfections and stable cell line generation
Transfection was done in a 96-well plate by mixing 200 ng of plasmid DNA and 1.2 µL
of 1 mg/mL polyethyleneimine (PEI, 24765-1, Polysciences Inc.) per well in FCS-free DMEM.
The mixture was incubated for 5-10 minutes, added to each well and incubated with the cells
overnight. For all transient expression experiments, 2 ng of each receptor plasmid was mixed
with 30 ng of plasmid pLS13 and 30 ng of pLS15. Amounts of DNA were adjusted to 200 ng
Materials & Methods
Tobias Strittmatter 50/240
using the inert filler plasmid pDF101. A comprehensive list of plasmids used in each
experiment can be found in Table S2.
For stable cell line generation, 6-well plates were used. Here 250 000 cells per well
were seeded and transfected 24 h later. A total of 1 µg of plasmid DNA was mixed with 6 µL
of 1 mg/mL PEI in FCS-free DMEM. For two vectors to be integrated, 300 ng of each
integration vector were mixed with 200 ng of Sleeping Beauty expression vector pTS395 and
200 ng of pDF101.
For three integration vectors, 200 ng of each integration vector was mixed with 200
ng of Sleeping Beauty expression vector pTS395 and 200 ng of pDF101.
Cells were grown for one day after transfection prior to start selection, depending on the
selection marker used, with a final concentration of 2 µg/mL of puromycin, 4 µg/mL of
blasticidine S or 20 µg/mL of zeocine.
Stable monoclonal cell lines were produced from mixed populations by FACS-
mediated single-cell seeding using a BD Aria III FACS.
Reporter Assays
To measure the activity of secreted human placental alkaline phosphatase (SEAP), the
increase of absorbance at 405 nm due to hydrolysis of para-nitrophenyl phosphate (pNPP) in
the cell culture supernatant was followed (1 reading per minute for 25 minutes). To this end,
60-100 µL of supernatant was collected from each well and heat-inactivated for 30 minutes
at 65 °C. 180 µL of assay reagent containing 100 µL 2x SEAP buffer (20 mM homoarginine,
1 mM MgCl2, 21% (v/v) diethanolamine, pH 9.8), 20 µL of pNPP substrate solution (20 mM, in
2x SEAP buffer) and 60 µL of water were added to 20 µL of heat-inactivated supernatant. The
absorbance at 405 nm at 37 °C was monitored with a Tecan M1000 multiplate reader (Tecan
AG) and used to calculate SEAP activity.
Cell culture supernatant was also used to determine the activity of secreted nano-
luciferase with the Nano-Glo® Luciferase Assay System (N1110, Promega). To this end, 7.5 µL
of cell culture supernatant was mixed with 7.5 µL buffer/substrate mix per well in a 384-well
black-well plate. Luminescence of the reporter was measured using a Tecan M1000 multiplate
reader (Tecan AG).
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Tobias Strittmatter 51/240
Size-exclusion chromatography of reconstituted plasma
Reconstituted human plasma was filtered through a 0.2 µm syringe filter to clear
liposomes and loaded on a self-packed Sephadex-200 column equilibrated with D-PBS
containing 2 mM EDTA to prevent clotting of the column. The run was performed in the same
buffer and a total of 133 fractions of 1 mL were collected. Selected fractions were further
tested for their activity in cell culture as well as analyzed with SDS-PAGE and western blotting.
Mass spectrometry
In-gel digestion of relevant SDS-PAGE bands was performed by using trypsin in 100
mM ammonium bicarbonate buffer. Samples were purified by using C18 columns, dried, and
resuspended in 20 µL of a suitable buffer. Concentrations were adjusted to be approximately
0.1 µg/µL and 1 µg of the solution was subjected to mass spectrometric analysis on an
Orbitrap Elite system.
tPA activity measurements
A commercial kit was used to assess tPA activity following the manufacturer’s
guidelines (Biovision, #K178-100) with an adaptation of the protocol for 384-well plates. Here,
we adjusted the amount of sample to 6 µL and scaled down the amount of inhibitor cocktail
and substrate mix accordingly to 2 µL each. Plates were centrifuged in-between steps at 250
rcf to collect samples at the bottom of the wells. We followed the protocol to determine the
activity of tenecteplase (TNK) by recording the absorbance of the emerging pNA product at
405 nm over 1 h. A calibration curve was prepared using authentic pNA.
Statistics
All analyses were done with GraphPad Prism 8.4. Representative graphs showing n = 3
biologically independent samples are presented as bar diagrams ± SD. Unless indicated
otherwise, no statistical analysis was performed. Graphs in figure 2 show the mean ± SEM of
N=3 independent experiments performed in triplicates. No statistical analysis was performed.
To determine the level of significance in figure 4f a two-tailed unpaired t-test was performed
(t = 7.585, df = 4) using GraphPad Prism 8.4 and the p value was calculated to be 0.0016.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 53/240
Supplementary Information Table S1: Plasmids used in this work
Plasmid Description Reference
005-546-
1295-
M_A6
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-A6
This work
005-546-
1295-
M_A10
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-A10
This work
005-546-
1295-
M_A12
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-A12
This work
005-546-
1295-
M_C11
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-C11
This work
005-546-
1295-
M_F9
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-F9
This work
005-546-
1296-
M_A11
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-A11
This work
005-546-
1296-
M_B4
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-B4
This work
005-546-
1296-
M_F6
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-F6
This work
005-546-
1296-
M_G1
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-G1
This work
Supplementary Information
Tobias Strittmatter 54/240
005-546-
1297-
M_A7
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-A7
This work
005-546-
1297-
M_B10
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-B10
This work
005-546-
1297-
M_B12
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-B12
This work
005-546-
1297-
M_C4
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-C4
This work
005-546-
1297-
M_D12
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-D12
This work
005-546-
1297-
M_E11
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-E11
This work
005-546-
1298-
M_D11
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-D11
This work
005-546-
1298-
M_F8
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-F8
This work
005-546-
1298-
M_F10
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-F10
This work
005-546-
1298-
M_F12
Bacterial expression plasmid bearing an inducible promoter for
expression of DARPin-F12
This work
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Tobias Strittmatter 55/240
pDF101 Inert filler plasmid bearing a bacterial T7 promoter driving an
inactive ribozyme (PT7-SpAL-sTRSVac)
(Auslander
et al. 2016)
pLS13 STAT3-reporter plasmid with 2 STAT3 response elements (RE)
followed by a minimal promoter driving expression of human
secreted placental alkaline phosphatase (SEAP) (RE2-PhCMVmin-
SEAP-pA)
(Schukur et
al. 2015)
pLS15 Mammalian expression vector bearing the coding sequence of the
human STAT3 transcription factor under control of an hCMV
promoter (PhCMV-STAT3-pA)
(Schukur et
al. 2015)
pMM1 Mammalian expression vector with a modified MCS (PhCMV-MCS-
pA; MCS, EcoRI-ATG-SpeI-NheI-BamHI-STOP-XbaI-HindIII-FseI-
pA).
(Muller et
al. 2017)
pTS379 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with an anti-D-dimer scFv described by
Laroche et al. 1991. pLeo644 (Scheller et al. 2018) was used as a
backbone. (PhCMV-Igk-scFv-EpoR-IL6st-pA)
This work
pTS380 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a DARPin as a D-dimer binding moiety.
Additional labels indicate the DARPin used as the binding moiety.
pLeo644 (Scheller et al. 2018) was used as a backbone (PhCMV-Igk-
DARPin-EpoR-IL6st-pA).
This work
pTS395 PhCMV-driven Sleeping Beauty transposase mammalian expression
vector (PhCMV-SB100-pA).
This work
pTS441 STAT3-reporter plasmid with 2 STAT3 response elements (RE)
followed by a minimal promoter driving expression of human
secreted placental alkaline phosphatase (SEAP) (RE2-PhCMVmin-
SEAP-pA) in pMM1 backbone.
This work
pTS566 STAT3-reporter plasmid with 4 STAT3 response elements (RE)
followed by a minimal promoter driving expression of human
secreted placental alkaline phosphatase (SEAP) (RE2-PhCMVmin-
SEAP-pA) in pMM1 backbone.
This work
Supplementary Information
Tobias Strittmatter 56/240
pTS810 STAT3-reporter plasmid with 12 STAT3 response elements (RE)
followed by a minimal promoter driving expression of human
secreted placental alkaline phosphatase (SEAP) (RE2-PhCMVmin-
SEAP-pA) in pMM1 backbone.
This work
pTS824 STAT3-reporter plasmid with 16 STAT3 response elements (RE)
followed by a minimal promoter driving expression of human
secreted placental alkaline phosphatase (SEAP) (RE2-PhCMVmin-
SEAP-pA) in pMM1 backbone.
This work
pTS835 Stable Sleeping Beauty integration vector bearing three cassettes;
an PhCMV-driven EpoR receptor equipped with the anti-D-dimer
scFv, an PSV40-driven STAT3 and a PRPBSA-driven selection cassette
encoding the blue fluorescent protein mTagBFP2 fused via a p2a
peptide sequence to a puromycin resistance gene. (PhCMV-Igk-
scFv-EpoR-IL6st-pA-PSV40-STAT3-pA-PRPBSA-mTagBFP2-p2a-PuroR-
pA)
This work
pTS863 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with DARPin-A7. pMM1 was used as a
backbone. (PhCMV-Igk-DARPinA7-EpoR-IL6st-pA)
This work
pTS864 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with DARPin-B4. pMM1 was used as a
backbone. (PhCMV-Igk-DARPinB4-EpoR-IL6st-pA)
This work
pTS865 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with DARPin-G1. pMM1 was used as a
backbone. (PhCMV-Igk-DARPinG1-EpoR-IL6st-pA)
This work
pTS866 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-A7 on top of
DARPin B4. pMM1 was used as a backbone. (PhCMV-Igk-DARPinA7-
DARPinB4-EpoR-IL6st-pA)
This work
pTS867 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-A7 on top of
This work
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Tobias Strittmatter 57/240
DARPin G1. pMM1 was used as a backbone. (PhCMV-Igk-DARPinA7-
DARPinG1-EpoR-IL6st-pA)
pTS868 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-B4 on top of
DARPin A7. pMM1 was used as a backbone. (PhCMV-Igk-DARPinB4-
DARPinA7-EpoR-IL6st-pA)
This work
pTS869 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-B4 on top of
DARPin G1. pMM1 was used as a backbone. (PhCMV-Igk-DARPinB4-
DARPinG1-EpoR-IL6st-pA)
This work
pTS870 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-G1 on top of
DARPin A7. pMM1 was used as a backbone. (PhCMV-Igk-DARPinG1-
DARPinA7-EpoR-IL6st-pA)
This work
pTS871 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-G1 on top of
DARPin B4. pMM1 was used as a backbone. (PhCMV-Igk-DARPinG1-
DARPinB4-EpoR-IL6st-pA)
This work
pTS872 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-A7 on top of
DARPin A7. pMM1 was used as a backbone. (PhCMV-Igk-DARPinA7-
DARPinA7-EpoR-IL6st-pA)
This work
pTS873 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-B4 on top of
DARPin B4. pMM1 was used as a backbone. (PhCMV-Igk-DARPinB4-
DARPinB4-EpoR-IL6st-pA)
This work
pTS874 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-G1 on top of
DARPin G1. pMM1 was used as a backbone. (PhCMV-Igk-DARPinG1-
DARPinG1-EpoR-IL6st-pA)
This work
Supplementary Information
Tobias Strittmatter 58/240
pTS914 Stable Sleeping Beauty integration vector bearing two cassettes;
a PhCMV-driven EpoR receptor equipped with DARPin B4 and a
PRPBSA-driven selection cassette encoding a selection marker for
blasticidin resistance. (PhCMV-Igk-DARPinB4-EpoR-IL6st-pA-
PRPBSA-BlastR-pA)
This work
pTS922 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with the anti-D-dimer scFv. pMM1 was used as
a backbone. (PhCMV-Igk-scFv-EpoR-IL6st-pA)
This work
pTS930 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of the anti-D-dimer scFv
on top of DARPin-A7. pMM1 was used as a backbone. (PhCMV-Igk-
scFv-DARPinA7-EpoR-IL6st-pA)
This work
pTS931 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of the anti-D-dimer scFv
on top of DARPin-B4. pMM1 was used as a backbone. (PhCMV-Igk-
scFv-DARPinB4-EpoR-IL6st-pA)
This work
pTS932 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of the anti-D-dimer scFv
on top of DARPin-G1. pMM1 was used as a backbone. (PhCMV-Igk-
scFv-DARPinG1-EpoR-IL6st-pA)
This work
pTS941 Stable Sleeping Beauty integration vector bearing three cassettes;
an PhCMV-driven EpoR receptor equipped with a tandem fusion of
the anti-D-dimer scFv on top of DARPin-B4, an PSV40-driven STAT3
and a RPBSA-driven selection cassette encoding the blue
fluorescent protein mTagBFP2 fused via a p2a peptide sequence
to a selection marker for puromycin resistance. (PhCMV-Igk-scFv-
DARPinB4-EpoR-IL6st-pA-PSV40-STAT3-pA-PPRBSA-mTagBFP2-p2a-
PuroR-pA)
This work
pTS942 Stable Sleeping Beauty integration vector bearing three cassettes;
an PhCMV-driven EpoR receptor equipped with an scFv against the
industrial dye reactive red (RR120) as described in Scheller et al
This work
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Tobias Strittmatter 59/240
2018, an PSV40-driven STAT3 and a PRPBSA-driven selection cassette
encoding the blue fluorescent protein mTagBFP2 fused via a p2a
peptide sequence to a selection marker for puromycin resistance.
(PhCMV-Igk-scFv(RR120)-EpoR-IL6st-pA-PSV40-STAT3-pA-PRPBSA-
mTagBFP2-p2a-PuroR-pA)
pTS992 Stable Sleeping Beauty integration vector bearing two cassettes;
four repeats of the STAT3 response element sequence followed
by a minimal promoter driving expression of a secreted nano-
luciferase fused to a murine Fc (mFc) tag separated by a cleavage
site for furin from an also Fc-stabilized copy of tenecteplase (TNK).
A second cassette drives expression of a selection marker for
zeocin resistance fused to the yellow fluorescent protein YPet via
a p2a peptide sequence.
(RE4-PhCMVmin-Igk-Nluc-mFc-Furin-TNK-mFc-pA-PRBSA-ZeoR-p2a-
YPet-pA)
This work
pTS2011 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with an scFv against the industrial dye reactive
red (RR120) as described in Scheller et al 2018. This plasmid was
used as negative control. pMM1 was used as a backbone. (PhCMV-
Igk-scFv(RR120)-EpoR-IL6st-pA)
This work
pTS2151 Stable Sleeping Beauty integration vector bearing two cassettes;
four repeats of the STAT3 operator sequence followed by a
minimal promoter driving expression of a secreted nano-
luciferase fused to a murine Fc (mFc) tag separated by a p2a site
that separates translation of Nluc and hirudin-HM2. A second
cassette drives expression of a selection marker for zeocin
resistance fused to the yellow fluorescent protein YPet via a p2a
peptide sequence.
(RE4-PhCMVmin-Igk-Nluc-mFc-p2a-HIRM2-pA-PRBSA-ZeoR-p2a-
YPet-pA)
Supplementary Information
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pTS2165 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a double tandem fusion of the anti-D-
dimer scFv. pMM1 was used as a backbone.
(PhCMV-D-dimer-scFv-D-dimer-scFv-receptor-pA)
This work
pTS2166 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-A7 on top of
the anti-D-dimer scFv. pMM1 was used as a backbone.
(PhCMV-DARPinA7-D-dimer-scFv-receptor-pA)
This work
pTS2167 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-B4 on top of
the anti-D-dimer scFv. pMM1 was used as a backbone.
(PhCMV-DARPinB4-D-dimer-scFv(tandem)-receptor-pA)
This work
pTS2168 Mammalian expression vector bearing a PhCMV-driven EpoR
receptor equipped with a tandem fusion of DARPin-G1on top of
the anti-D-dimer scFv. pMM1 was used as a backbone.
(PhCMV-DARPinG1-D-dimer-scFv(tandem)-receptor-pA)
This work
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Table S2: Plasmids transfected in each experiment
Details of the transfection protocol can be found in the methods.
Figure Plasmids used
1 Plasmid library for loop DARPins
2
b)
pTS380-A12: PhCMV-DARPinA12-receptor-pA
pTS380-B4: PhCMV-DARPinB4-receptor-pA
pTS380-D11: PhCMV-DARPinD11-receptor-pA
pTS380-C11: PhCMV-DARPinC11-receptor-pA
pTS380-F12: PhCMV-DARPinF12-receptor-pA
pTS380-A10: PhCMV-DARPinA10-receptor-pA
pTS380-D12: PhCMV-DARPinD12-receptor-pA
pTS380-F10: PhCMV-DARPinF10-receptor-pA
pTS380-A6: PhCMV-DARPinA6-receptor-pA
pTS380-B12: PhCMV-DARPinB12-receptor-pA
pTS380-F8: PhCMV-DARPinF8-receptor-pA
pTS380-B10: PhCMV-DARPinB10-receptor-pA
pTS380-F9: PhCMV-DARPinF9-receptor-pA
pTS380-A7: PhCMV-DARPinA7-receptor-pA
pTS380-A11: PhCMV-DARPinA11-receptor-pA
pTS380-C4: PhCMV-DARPinC4-receptor-pA
pTS380-F6: PhCMV-DARPinF6-receptor-pA
pTS380-E11: PhCMV-DARPinE11-receptor-pA
pTS380-G1: PhCMV-DARPinG1-receptor-pA
pTS379: PhCMV-D-dimer-scFv-receptor-pA
pTS2011: PhCMV-RR120 -scFv-receptor-pA
2 ng of the indicated receptor plasmids,
30 ng of each pLS13 and pLS15 used to build the reporter system,
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
2
c) – d)
f) – g)
pTS380-A6: PhCMV-DARPinA6-receptor-pA
pTS380-A7: PhCMV-DARPinA7-receptor-pA
pTS380-B4: PhCMV-DARPinB4-receptor-pA
Supplementary Information
Tobias Strittmatter 62/240
pTS380-G1: PhCMV-DARPinG1-receptor-pA
pTS379: PhCMV-D-dimer-scFv-receptor-pA
pTS2011: PhCMV-RR120 -scFv-receptor-pA
2 ng of the indicated receptor plasmids,
30 ng of each pLS13 and pLS15 used to build the reporter system,
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
2
e)
h)
pTS870: PhCMV-DARPinG1-A7(tandem)-receptor-pA
pTS871: PhCMV-DARPinG1-B4(tandem)-receptor-pA
pTS872: PhCMV-DARPinG1-G1(tandem)-receptor-pA
pTS930: PhCMV-D-dimer-scFv-DARPinA7(tandem)-receptor-pA
pTS931: PhCMV-D-dimer-scFv-DARPinB4(tandem)-receptor-pA
pTS932: PhCMV-D-dimer-scFv-DARPinG1(tandem)-receptor-pA
pTS2011: PhCMV-RR120-scFv-receptor-pA
2 ng of the indicated receptor plasmids,
30 ng of each pLS13 and pLS15 used to build the reporter system,
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
3
b)-d)
pTS864: PhCMV-DARPinB4-receptor-pA
pTS871: PhCMV-DARPinG1-B4(tandem)-receptor-pA
pTS922: PhCMV-D-dimer-scFv-receptor-pA
pTS931: PhCMV-D-dimer-scFv-DARPinB4(tandem)-receptor-pA
2 ng of the indicated receptor plasmids,
30 ng of each pLS13 and pLS15 used to build the reporter system,
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
4
a) – b)
Stable cell line HEKscFv-B4/TNK was generated using the following plasmids
pTS941: PhCMV-D-dimer-scFv-DARPinB4(tandem)-receptor-pA-PSV40-STAT3-pA-
PhCMV-TagBFP2-p2a-PuroR-pA
pcTS992: RE4-PhCMV-IgkSS-Nluc- mFc-RARYKR-TNK- mFc-PhCMV-ZeoR-p2a-YPet-pA
pcTS395: PhCMV-SleepingBeauty-transposase-pA
pDF101 to adjust plasmid DNA amount to 1 µg per transfection
4
c) – e)
Stable cell line mHEKB4/scFv/hirudin was generated using the following plasmids
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pTS835: PhCMV-D-dimer-scFv-receptor-pA-PSV40-STAT3-pA-PhCMV-TagBFP2-p2a-
PuroR-pA
pTS914: PhCMV-DARPinB4-receptor-pA-PhCMV-BlastR-pA
pTS2151: RE4-PhCMV-IgkSS-Nluc-mFc-p2a-HIRM2-pA-PhCMV-ZeoR-p2a-YPet-pA
pcTS395: PhCMV-SleepingBeauty-transposase-pA
pDF101 to adjust plasmid DNA amount to 1 µg per transfection
S3 Plasmid library for loop DARPins
S4
a)
pTS380-B4: PhCMV-DARPinB4-receptor-pA
pTS864: PhCMV-DARPinB4-receptor-pA
pTS380-B4: PhCMV-DARPinB4-receptor-pA
pTS379: PhCMV-D-dimer-scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S4
b)
pLeo615: PhCMV-RR120-scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S4
c)
e)
100 ng of each of the following plasmids was used per transfection
pSEAP2ctr: PSV40-SEAP-pA
pFS29: PSV40-mCherry-pA
S5 pTS380-A12: PhCMV-DARPinA12-receptor-pA
pTS380-B4: PhCMV-DARPinB4-receptor-pA
pTS380-D11: PhCMV-DARPinD11-receptor-pA
pTS380-C11: PhCMV-DARPinC11-receptor-pA
pTS380-F12: PhCMV-DARPinF12-receptor-pA
pTS380-A10: PhCMV-DARPinA10-receptor-pA
pTS380-D12: PhCMV-DARPinD12-receptor-pA
pTS380-F10: PhCMV-DARPinF10-receptor-pA
pTS380-A6: PhCMV-DARPinA6-receptor-pA
pTS380-B12: PhCMV-DARPinB12-receptor-pA
pTS380-F8: PhCMV-DARPinF8-receptor-pA
pTS380-B10: PhCMV-DARPinB10-receptor-pA
Supplementary Information
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pTS380-F9: PhCMV-DARPinF9-receptor-pA
pTS380-A7: PhCMV-DARPinA7-receptor-pA
pTS380-A11: PhCMV-DARPinA11-receptor-pA
pTS380-C4: PhCMV-DARPinC4-receptor-pA
pTS380-F6: PhCMV-DARPinF6-receptor-pA
pTS380-E11: PhCMV-DARPinE11-receptor-pA
pTS380-G1: PhCMV-DARPinG1-receptor-pA
pTS379: PhCMV-D-dimer-scFv-receptor-pA
pTS2011: PhCMV-RR120 -scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S5
a) – d)
pTS380-A12: PhCMV-DARPinA12-receptor-pA
pTS380-B4: PhCMV-DARPinB4-receptor-pA
pTS380-D11: PhCMV-DARPinD11-receptor-pA
pTS380-C11: PhCMV-DARPinC11-receptor-pA
pTS380-F12: PhCMV-DARPinF12-receptor-pA
pTS380-A10: PhCMV-DARPinA10-receptor-pA
pTS380-D12: PhCMV-DARPinD12-receptor-pA
pTS380-F10: PhCMV-DARPinF10-receptor-pA
pTS380-A6: PhCMV-DARPinA6-receptor-pA
pTS380-B12: PhCMV-DARPinB12-receptor-pA
pTS380-F8: PhCMV-DARPinF8-receptor-pA
pTS380-B10: PhCMV-DARPinB10-receptor-pA
pTS380-F9: PhCMV-DARPinF9-receptor-pA
pTS380-A7: PhCMV-DARPinA7-receptor-pA
pTS380-A11: PhCMV-DARPinA11-receptor-pA
pTS380-C4: PhCMV-DARPinC4-receptor-pA
pTS380-F6: PhCMV-DARPinF6-receptor-pA
pTS380-E11: PhCMV-DARPinE11-receptor-pA
pTS380-G1: PhCMV-DARPinG1-receptor-pA
pTS379: PhCMV-D-dimer-scFv-receptor-pA
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pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S6 pTS380-A12: PhCMV-DARPinA12-receptor-pA
pTS380-B4: PhCMV-DARPinB4-receptor-pA
pTS380-D11: PhCMV-DARPinD11-receptor-pA
pTS380-C11: PhCMV-DARPinC11-receptor-pA
pTS380-F12: PhCMV-DARPinF12-receptor-pA
pTS380-A10: PhCMV-DARPinA10-receptor-pA
pTS380-D12: PhCMV-DARPinD12-receptor-pA
pTS380-F10: PhCMV-DARPinF10-receptor-pA
pTS380-A6: PhCMV-DARPinA6-receptor-pA
pTS380-B12: PhCMV-DARPinB12-receptor-pA
pTS380-F8: PhCMV-DARPinF8-receptor-pA
pTS380-B10: PhCMV-DARPinB10-receptor-pA
pTS380-F9: PhCMV-DARPinF9-receptor-pA
pTS380-A7: PhCMV-DARPinA7-receptor-pA
pTS380-A11: PhCMV-DARPinA11-receptor-pA
pTS380-C4: PhCMV-DARPinC4-receptor-pA
pTS380-F6: PhCMV-DARPinF6-receptor-pA
pTS380-E11: PhCMV-DARPinE11-receptor-pA
pTS380-G1: PhCMV-DARPinG1-receptor-pA
pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S8
a)
b)
pTS863: PhCMV-DARPinA7-receptor-pA
pTS864: PhCMV-DARPinB4-receptor-pA
pTS865: PhCMV-DARPinG1-receptor-pA
pTS866: PhCMV-DARPinA7-B4(tandem)-receptor-pA
pTS867: PhCMV-DARPinA7-G1 (tandem)-receptor-pA
pTS868: PhCMV-DARPinB4-A7(tandem)-receptor-pA
pTS869: PhCMV-DARPinB4-G1(tandem)-receptor-pA
pTS870: PhCMV-DARPinG1-A7(tandem)-receptor-pA
Supplementary Information
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pTS871: PhCMV-DARPinG1-B4(tandem)-receptor-pA
pTS872: PhCMV-DARPinA7-A7(tandem)-receptor-pA
pTS873: PhCMV-DARPinB4-B4(tandem)-receptor-pA
pTS874: PhCMV-DARPinG1-G1(tandem)-receptor-pA
pTS2011: PhCMV-RR120-scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S8
c)
d)
pTS922: PhCMV-D-dimer-scFv-receptor-pA
pTS2166: PhCMV-DARPinA7-D-dimer-scFv(tandem)-receptor-pA
pTS2167: PhCMV-DARPinB4-D-dimer-scFv(tandem)-receptor-pA
pTS2168: PhCMV-DARPinG1-D-dimer-scFv(tandem)-receptor-pA
pTS930: PhCMV-D-dimer-scFv-DARPinA7(tandem)-receptor-pA
pTS931: PhCMV-D-dimer-scFv-DARPinB4(tandem)-receptor-pA
pTS932: PhCMV-D-dimer-scFv-DARPinG1(tandem)-receptor-pA
pTS2165: PhCMV-D-dimer-scFv-D-dimer-scFv(tandem)-receptor-pA
pTS2011: PhCMV-RR120-scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S9
a)-c)
pTS864: PhCMV-DARPinB4-receptor-pA
pTS871: PhCMV-DARPinG1-B4(tandem)-receptor-pA
pTS922: PhCMV-D-dimer-scFv-receptor-pA
pTS931: PhCMV-D-dimer-scFv-DARPinB4(tandem)-receptor-pA
pTS2011: PhCMV-RR120-scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S10
a)
pTS864: PhCMV-DARPinB4-receptor-pA
pTS922: PhCMV-D-dimer-scFv-receptor-pA
pTS871: PhCMV-DARPinG1-B4(tandem)-receptor-pA
pTS2011: PhCMV-RR120-scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
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S10
b)
c)
pTS864: PhCMV-DARPinB4-receptor-pA
pTS922: PhCMV-D-dimer-scFv-receptor-pA
pTS2011: PhCMV-RR120-scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S11
a)
pTS380-B4: PhCMV-DARPinB4-receptor-pA
pTS379: PhCMV-D-dimer-scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S12
a)
pTS2011: PhCMV-RR120-scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pTS931: PhCMV-D-dimer-scFv-DARPinB4(tandem)-receptor-pA
pSEAP2control: PSV40-SEAP-pA
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S12
b-c)
pTS864: PhCMV-DARPinB4-receptor-pA
pTS922: PhCMV-D-dimer-scFv-receptor-pA
pTS871: PhCMV-DARPinG1-B4(tandem)-receptor-pA
pTS2011: PhCMV-RR120-scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S12
d-e)
pTS864: PhCMV-DARPinB4-receptor-pA
pTS922: PhCMV-D-dimer-scFv-receptor-pA
pTS871: PhCMV-DARPinG1-B4(tandem)-receptor-pA
pTS931: PhCMV-D-dimer-scFv-DARPinB4(tandem)-receptor-pA
pTS2011: PhCMV-RR120-scFv-receptor-pA
pLS13 and pLS15 used to build the reporter system
pSEAP2control: PSV40-SEAP-pA
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S13 pTS2011: PhCMV-RR120-scFv-receptor-pA
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pLS13 and pLS15 used to build the reporter system
pSEAP2control: PSV40-SEAP-pA
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S14
a)
pTS380-B4: PhCMV-DARPinB4-receptor-pA
pTS379: PhCMV-D-dimer-scFv-receptor-pA
pTS539: PSV40-STAT3-pA
pLS13: RE2-phCMVmin-SEAP-pA
pTS441: RE2-phCMVmin-SEAP-pA
pTS566: RE4-phCMVmin-SEAP-pA
pTS810: RE12-phCMVmin-SEAP-pA
pTS824: RE16-phCMVmin-SEAP-pA
pDF101 to adjust plasmid DNA amount to 200 ng per transfection
S14
b)
Stable cell line HEKscFv-B4/TNK and HEKRR120/TNK were generated using the following
plasmids
HEKscFv-B4/TNK:
pTS941: PhCMV-D-dimer-scFv-DARPinB4(tandem)-receptor-pA-PSV40-STAT3-pA-
PhCMV-TagBFP2-p2a-PuroR-pA
pcTS992: RE4-PhCMV-IgkSS-Nluc-mFc-RARYKR-TNK-mFc-PhCMV-ZeoR-p2a-YPet-pA
HEKRR120/TNK:
pTS942: PhCMV-RR120-scFv-receptor-pA-PSV40-STAT3-pA-PhCMV-TagBFP2-p2a-
PuroR-pA
pcTS992: RE4-PhCMV-IgkSS-Nluc-mFc-RARYKR-TNK-mFc-PhCMV-ZeoR-p2a-YPet-pA
pcTS395: PhCMV-SleepingBeauty-transposase-pA
pDF101 to adjust plasmid DNA amount to 1 µg per transfection
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Supplementary Figures
Supplementary Figure 1: Result of non-reducing polyacrylamide gel-electrophoresis of the
target protein (“native human D-dimer protein”, Abcam, ab98311) used in all experiments,
done with a 6 % gel. Analysis suggests that the mixture consists primarily of D-dimer protein
with significant amounts of other proteins. The putative identity of the protein in each band
is shown in the right-hand column. M: PageRuler Prestained Protein Ladder, 10 to 180 kDa.
180 kDa D-dimer
Fragment D
D-E fragments (fragment Y)
high MW FD P, fibrin
Fragment E
hSA
M 10 µ
g2
µg
130 kDa
100 kDa
70 kDa
55 kDa
Supplementary Figures
Tobias Strittmatter 70/240
005-546-1296-M_F6 MRGSHHHHHHHHGSDLGKKLLEAATSGQDDEVRILMANGADVNAMDHWGWTPLHLAAIEG 60005-546-1295-M_F9 MRGSHHHHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNAQDIWGWTPLHLAAYAG 60005-546-1296-M_G1 MRGSHHHHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNAT--------------- 45005-546-1297-M_A7 MRGSHHHHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNAF--------------- 45005-546-1295-M_A6 MRGSHHHHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNAE--------------- 45005-546-1296-M_B4 MRGSHHHHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNAE--------------- 45 ************************ :******************
005-546-1296-M_F6 HQEIVEVLLKTGADVNAKDQWGATPLHLAAVVGHLEIV---------------------- 98005-546-1295-M_F9 HLEIVKVLLKTGADVNAYDDWGSTPLHLAAWIGHLEIVEVLLKAGADVNAWDVHGFTPLH 120005-546-1296-M_G1 ------------------DNWGDTPLHLAAWHGHLEIVEVLLKTGADVNAQDIIGATPLH 87005-546-1297-M_A7 ------------------DWYGTTPLHLAAFNGHLEIVEVLLKTGADVNAQDLFGNTPLH 87005-546-1295-M_A6 ------------------DWYGTTPLHLAAHNGHLEIVEVLLKTGADVNAQDLFGNTPLH 87005-546-1296-M_B4 ------------------DWYGTTPLHLAAHNGHLEIVEVLLKTGADVNAQDLFGNTPLH 87 * :* ******* ******
005-546-1296-M_F6 -----------EVLLKHGADVNAQDISGQTPFDLAAWHGNEDIAEVLQKAAKLNDYKDDD 147005-546-1295-M_F9 LAAIRGHLEIVEVLLKHGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAAKLNDYKDDD 180005-546-1296-M_G1 LAAIMGHLEIVEVLLKAGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAAKLNDYKDDD 147005-546-1297-M_A7 LAAWNGHLEIVEVLLKHGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAAKLNDYKDDD 147005-546-1295-M_A6 LAAYEGHLEIVEVLLKHGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAAKLNDYKDDD 147005-546-1296-M_B4 LAAWNGHLEIVEVLLKHGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAAKLNDYKDDD 147 ***** ******** *:****** :**********************
005-546-1296-M_F6 DK* 149005-546-1295-M_F9 DK* 182005-546-1296-M_G1 DK* 149005-546-1297-M_A7 DK* 149005-546-1295-M_A6 DK* 149005-546-1296-M_B4 DK* 149 ***
b) CLUSTAL O (1.2.4) multiple sequence alignment
a) Simple phylogenetic analysis of DARPin amino acid sequence
identity similarityF6 86.0 % 90.0 %F9 74.9 % 78.7 %G1 92.0 % 94.0 %A7 97.3 % 98.0 %A6 100.0 % 100.0 %B4 98.7 % 99.3 %
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
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Supplementary Figure 2: Characterization of DARPin binders. a) Simple phylogenetic
analysis of all DARPins based on their amino acid sequences. DARPins G1, F6, F9, A7, A6 and
B4 are boxed in red. DARPins G1, F6, F9, A7, A6 and B4 were further analyzed by b)
alignment of amino acid sequences using the Clustal Omega 1.2.4 algorithm and c)
similarity analysis using pairwise alignments employing the EMBOSS-needle algorithm.
Supplementary Figures
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Supplementary Figure 3: Size exclusion chromatography was performed to assess the
dimerization behavior of selected DARPins. Chromatograms in red were found to represent
non-monomeric candidates.
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Supplementary Figure 4: Control experiments to assess the cross-reactivity of receptors and
inputs as well as the toxicity of inducers. HEK293T cells were transfected overnight with the
indicated receptors and the pLS13 STAT3 reporter plasmid alongside pLS15 for STAT3
transcription factor overexpression or a constitutive SEAP expressing plasmid (pSEAP2ctr),
as described in the methods. a) None of the receptor scaffolds used is responsive to
erythropoetin (EPO), which is the natural ligand for the native EPO receptor. Also, the STAT3
pathway is not induced by EPO administration. Cells were incubated with 10 ng/mL EPO
a) EPO insensitivity of AMBERs b) RR120-receptor as negative control
c) productivity is not influenced by xFDPs d) xFDPs are non-toxic at relevant concentrations
e ) plasma influences cell productivity f ) plasma shows dose-dependent toxicity
pTS380-B4
pTS380-B4 +
pTS379
pTS864
reporte
r ctr
0
5
10
15
SEAP
[UL-1
]+ EPO (10 ng/mL)
uninduced
1 % plasm
a
20g/m
L fibrin
ogen
5 µg/m
L xFDPs
100 n
g/mL RR12
0
10 ng/m
L RR120
2 ng/m
L RR120
uninduced
0
5
10
15
20
SEAP
[UL-1
]
10g/m
L
5g/m
L
2g/m
L
1g/m
L
0.5g/m
L
uninduced
0
50
100
150
SEAP
[UL-1
]
10g/m
L
5g/m
L
2g/m
L
1g/m
L
0.5g/m
L
uninduced
0
10000
20000
30000
40000
50000re
soru
fin [F
U]
50%
25%
20%
10% 5% 2.5
% 1% 0%0
10000
20000
30000
40000
reso
rufin
[FU]
50%
25%
20%
10% 5% 2.5
% 1% 0%0
10
20
30
40
SEAP
[UL-1
]
Supplementary Figures
Tobias Strittmatter 74/240
protein in complete DMEM containing 10 % FCS for 24 h and the supernatant was sampled
for analysis of SEAP reporter activity. b) The RR120 receptor used as the negative control
exclusively responds to its ligand RR120 and is not activated by plasma, fibrinogen or xFDPs.
c-f) Productivity and viability of HEK293T cells were assessed in c) and e) in terms of
constitutive expression of SEAP and in d) and f) by measuring resazurin reduction, which
leads to production of the fluorescent dye resorufin, in parallel. HEK293T cells were
transfected overnight with pSEAP2ctr prior to incubation with c)-d) xFDPs or e)-f) plasma at
the indicated concentrations. At 24 h after induction, the supernatant was sampled and
SEAP activity was measured. After sampling, the medium was exchanged for 100 µL of
complete DMEM containing 10 % FCS and resazurin. Cells were incubated with resazurin for
approximately 30 minutes and 60 µL of medium was transferred to a clear-bottomed assay
plate for fluorescence recording. All values are means ± SD of triplicates.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 75/240
Supplementary Figure 5: Screening of homodimeric receptors. HEK293T cells were
transfected overnight with the indicated receptors and the pLS13 STAT3 reporter plasmid
alongside pLS15 for STAT3 transcription factor overexpression, as described in the methods.
Cells were incubated with 0.5 µg/mL xFDPs or 0.5 % (v/v) reconstituted human plasma in
complete DMEM containing 10 % FCS for 24 h, and supernatant was sampled for analysis
a) Overview
b)
d)
c)
A12-A12
A10-A10
B12-B12
C4-C4
A12-scF
v
A10-F12
B12-F8
C4-A11
A12-G
1
A10-C
11
B12-A6
C4-A7
A12-E11
A10-D
11
B12-F10
C4-F9
A12-F6
A10-B4
B12-D12
C4-B10
A12-C4
A10-A12
B12-A10
C4-B12
A12-A
11
A10-scF
v
B12-F12
C4-F8
A12-A7
A10-G
1
B12-C
11C4-A
6
A12-F9
A10-E11
B12-D
11
C4-F10
A12-B10
A10-F6
B12-B4
C4-D12
A12-B12
A10-C4
RR1200
20
40
60
SEAP
[U*L
-1]
plasma xFDP uninduced
A12-A12B4-B4
D11-D
11
C11-C
11
F12-F12
A10-A10
D12-D12
F10-F10A6-A6
F8-F8
B12-B12
B10-B10F9-F
9A7-A7
A11-A
11C4-C4
F6-F6
E11-E11G1-G
1
scFv
-scFv
A12-scFv
B4-A12
D11-B4
C11-D
11
F12-C11
A10-F12
D12-A10
F10-D12
A6-F10
F8-A6
B12-F8
B10-B12
F9-B10A7-F9
A11-A7
C4-A11F6-C
4
E11-F6
G1-E11
scFv
-G1
A12-G1
B4-scFv
D11-A
12
C11-B4
F12-D11
A10-C11
D12-F12
F10-A10
A6-D12
F8-F10
B12-A6
B10-F8
F9-B12
A7-B10
A11-F9C4-A7
F6-A11
E11-C4G1-F
6
scFv
-E11
A12-E11B4-G1
D11-scF
v
C11-A
12
F12-B4
A10-D11
D12-C11
F10-F12
A6-A10
F8-D12
RR120pS2ct
r0
20
40
60
SEAP
[U*L
-1]
xFDP uninducedplasma
B12-F10
B10-A6F9-F
8
A7-B12
A11-B
10C4-F9
F6-A7
E11-A11G1-C
4
scFv
-F6
A12-F6
B4-E11
D11-G1
C11-scF
v
F12-A12
A10-B4
D12-D11
F10-C11
A6-F12
F8-A10
B12-D12
B10-F10F9-A
6A7-F8
A11-B
12
C4-B10F6-F
9
E11-A
7
G1-A11
scFv
-C4
A12-C4B4-F6
D11-E
11
C11-G1
F12-scF
v
A10-A12
D12-B4
F10-D11
A6-C11
F8-F12
B12-A10
B10-D12
F9-F10A7-A6
A11-F8
C4-B12
F6-B10
E11-F9G1-A
7
scFv
-A11
A12-A11B4-C4
D11-F6
C11-E
11
F12-G1
A10-scFv
D12-A12
F10-B4
A6-D11
F8-C11
B12-F12
B10-A10
F9-D12
A7-F10
A11-A
6C4-F8
F6-B12
E11-B
10G1-F
9
scFv
-A7
RR120pS2ct
r
0
20
40
60
SEA
P [U
*L-1
]
plasma xFDP uninduced
A12-A7
B4-A11
D11-C4
C11-F6
F12-E11
A10-G1
D12-scFv
F10-A12A6-B4
F8-D11
B12-C11
B10-F12
F9-A10
A7-D12
A11-F10C4-A6
F6-F8
E11-B
12
G1-B10
scFv
-F9
A12-F9
B4-A7
D11-A
11
C11-C4
F12-F6
A10-E11
D12-G1
F10-scF
v
A6-A12F8-B
4
B12-D11
B10-C11
F9-F12
A7-A10
A11-D
12
C4-F10
F6-A6
E11-F8
G1-B12
scFv
-B10
A12-B10B4-F9
D11-A7
C11-A
11
F12-C4
A10-F6
D12-E11
F10-G1
A6-scFv
F8-A12
B12-B4
B10-D11
F9-C11
A7-F12
A11-A
10
C4-D12
F6-F10
E11-A6G1-F
8
scFv
-B12
A12-B12
B4-B10
D11-F9
C11-A7
F12-A11
A10-C4
D12-F6
F10-E11A6-G1
F8-scFvRR120
pS2ctr
0
20
40
60
SEAP
[U*L
-1]
xFDP uninducedplasma
Supplementary Figures
Tobias Strittmatter 76/240
of SEAP reporter activity. All values are means ± SD of triplicates. a) Overview of all
measurements. b-d) Subsets of a) of 70 combinations each. b) 1-70, c) 71-140, d) 141-210.
All panels include RR120 as a negative control and constitutive expression of SEAP as a
reference.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 77/240
Supplementary Figure 6: Correlation of receptor activation by plasma of DARPin-AMBERs
in homodimeric configuration measured by SEAP reporter expression with the
corresponding signal intensities from HTRF (left y-axis) as well as the number of functional
receptor combinations (fold induction >5) for coagulation or xFDP detection (right y-axis).
HTRF signals are single values while receptor activation is represented by mean values of
triplicate determinations.
0 5 10 15 202.0
2.5
3.0
3.5
4.0
0
5
10
15
20
receptor signal (SEAP) [U*L-1]
HTR
F ra
tio [A
U]
# functional receptor comb.
HTRF signal
number of functional receptors - plasma
number of functional receptors - xFDPs
Supplementary Figures
Tobias Strittmatter 78/240
Supplementary Figure 7: Affinities of binders were assessed by surface plasmon resonance
(SPR) measurements on a Biacore T200 (GE healthcare) using a CMD200M chip. Duplicates
of full kinetic measurements were performed and fitted with a heterogenous ligand (hetlig)
model using BIAevaluation software (GE healthcare). Flow cell 1 (FC1) served as reference
for FC2 and was coated with 205 RU of target protein. Each concentration was measured
twice in ascending and descending order.
0 50 100 150 2000
20
40
60
t [sec]
dRU
[RU
]
0 200 4000
20
40
60
t [sec]
dRU
[RU
]
0 200 4000
20
40
60
t [sec]
dRU
[RU
]
0 100 200 3000
10
20
30
40
t [sec]
dRU
[RU
]
0 200 4000
10
20
30
40
Lo
remip
sum
dolor
sita
met ,cons ect e tue r adipi s cin g elit,
t [sec]
dRU
[RU
]
a) scFv b) DARPin A6
c) DARPin A7
e) DARPin G1
d) DARPin B4
kON
-1/2 = 633,084.2 / 16,502.9 M-1s-1
kOFF
-1/2 = 0.381 / 0.017 s-1
Kd-1/2 = 602 / 1031 nM
RUmax
= 14.6 / 15.2
kON
-1/2 = 146,625.2 / 1,410,638.3 M-1s-1
kOFF
-1/2 = 0.013 / 0.062 s-1
Kd-1/2 = 91 / 44 nM
RUmax
= 2.6 / 20.8
kON
-1/2 = 1,421,090.6 / 181,810.2 M-1s-1
kOFF
-1/2 = 0.033 / 0.011 s-1
Kd-1/2 = 23 / 59 nM
RUmax
= 20.2 / 5.1
kON
-1/2 = 2,515,538 / 375,788.4 M-1s-1
kOFF
-1/2 = 0.035 / 0.014 s-1
Kd-1/2 = 14 / 37 nM
RUmax
= 12.0 / 13.2
kON
-1/2 = 103,836.1 / 48,723 M-1s-1
kOFF
-1/2 = 0.025 / 0.008 s-1
Kd-1/2 = 244 / 169 nM
RUmax
= 12.6 / 12.9
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 79/240
Supplementary Figure 8: Screening of tandem receptors based on DARPins only. (a-b) or
scFv-DARPin combinations (c-d). HEK293T cells were transfected overnight with the
indicated receptors and the pLS13 STAT3 reporter plasmid alongside pLS15 for STAT3
transcription factor overexpression, as described in the methods. a) and c) Cells were
incubated for 24 h in complete DMEM containing 10 % FCS and xFDPs at 0.5 µg/mL or
a) DARPin-based tandem receptors induced with plasma and xFDPs
b) fold induction of DARPin-based tandem receptors induced with plasma and xFDPs
c) scFv-based tandem receptors induced with plasma and xFDPs
d) fold induction of scFv-based tandem receptors induced with plasma and xFDPs
A7B4
A7G1
B4A7
B4G1
G1A7G1B4
A7A7
B4B4
G1G1
RR120pS2ctr
0
20
40
60
80
SEA
P [U
*L-1
]
plasma
xFDP
uninduced
A7B4
A7G1
B4A7
B4G1
G1A7G1B4
A7A7
B4B4
G1G1
RR120pS2ctr
0
10
20
30
fold
indu
ctio
n
plasma
xFDP
scFv
A7-scFv
B4-scFv
G1-scFv
scFv-A
7
scFv-B
4
scFv-G
1
scFv-s
cFv
RR1200
50
100
150
SEAP
[UL-1
]
plasma
xFDP
uninduced
scFv
A7-scFv
B4-scFv
G1-scFv
scFv-A
7
scFv-B
4
scFv-G
1
scFv-s
cFv
RR1200
10
20
30
fold
indu
ctio
n
plasma
xFDP
Supplementary Figures
Tobias Strittmatter 80/240
plasma at a final concentration of 0.5 % (v/v) prior to assessment of SEAP reporter activity.
b) and d) Fold induction was calculated for each receptor by normalizing samples induced
with plasma or xFDP to uninduced control samples. All values are means ± SD of triplicate
determinations.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 81/240
Supplementary Figure 9: Activation of receptors by plasmin degradation products of native
fibrinogen was assessed. HEK293T cells were transfected overnight with the indicated
a) fragment D of fibrinogen
b) fragment E of fibrinogen
c) mixture of fragment D and E of fibrinogen
1.000.3
20.1
00.0
01.0
00.3
20.1
00.0
01.0
00.3
20.1
00.0
01.0
00.3
20.1
00.0
01.0
00.3
20.1
00.0
01.0
00.3
20.1
00.0
00
10
20
30
40
fragment D of fibrinogen [ g/mL]
SEAP
[UL-1
]B4
B4 + scFv
G1-B4
scFv-B4
RR120-receptor
SEAP ctr
2.000.6
30.2
00.0
02.0
00.6
30.2
00.0
02.0
00.6
30.2
00.0
02.0
00.6
30.2
00.0
02.0
00.6
30.2
00.0
02.0
00.6
30.2
00.0
00
10
20
30
40
fragment E of fibrinogen [ g/mL]
SEAP
[UL-1
]
B4
B4 + scFv
G1-B4
scFv-B4
RR120-receptor
SEAP ctr
1.00 +
2.00
0.32 +
0.63
0.10 +
0.20.00
1.00 +
2.00
0.32 +
0.63
0.10 +
0.20.00
1.00 +
2.00
0.32 +
0.63
0.10 +
0.20.00
1.00 +
2.00
0.32 +
0.63
0.10 +
0.20.00
1.00 +
2.00
0.32 +
0.63
0.10 +
0.20.00
1.00 +
2.00
0.32 +
0.63
0.10 +
0.20.00
0
10
20
30
40
fragment D + E of fibrinogen [ g/mL]
SEAP
[UL-1
]
B4
B4 + scFv
G1-B4
scFv-B4
RR120-receptor
SEAP ctr
Supplementary Figures
Tobias Strittmatter 82/240
receptors and the pLS13 STAT3 reporter plasmid alongside pLS15 for STAT3 transcription
factor overexpression, as described in the methods. Cells were incubated for 24 h in
complete DMEM containing 10 % FCS and a) purified fragment D protein of fibrinogen at 1,
0.32, 0.1 or 0 µg/mL or b) purified fragment E protein of fibrinogen at 2, 0.63, 0.2 or 0 µg/mL
or c) a combination of both fragment D and E of fibrinogen at the indicated concentrations.
All values are means ± SD of triplicate determinations.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 83/240
a) plasmin degrades fibrinogen at high concentrations
1.00000.1
0000.0
1000.0
0100.0
0001.0
0000.1
0000.0
1000.0
0100.0
0001.0
0000.1
0000.0
1000.0
0100.0
0001.0
0000.1
0000.0
1000.0
0100.0
0000
10
20
30
40
50
plasmin acitivity [mU/mL]
SEAP
[U*L
-1]
B4B4 + scFv
G1-B4RR120-receptor
c) plasmin on xFDPs
b) plasmin on fibrinogen
1.000.3
20.1
00.0
0
uninduced
1.000.3
20.1
00.0
0
uninduced
1.000.3
20.1
00.0
0
uninduced
1.000.3
20.1
00.0
0
uninduced
1.000.3
20.1
00.0
0
uninduced
1.000.3
20.1
00.0
0
uninduced
1.000.3
20.1
00.0
0
uninduced
1.000.3
20.1
00.0
0
uninduced
0
10
20
plasmin activitiy [mU/mL]
SEAP
[U*L
-1]
B4 - day1B4 - day2 + plasminB4+scFv - day1B4+scFv - day2 + plasminRR120-receptor - day1RR120-receptor - day2 + plasmin
10.0
3.16 1.00.3
20.1
00.0
320.0
10.0
0320.01
uninduced
10.0
3.16 1.00.3
20.1
00.0
320.0
10.0
0320.01
uninduced
10.0
3.16 1.00.3
20.1
00.0
320.0
10.0
0320.01
uninduced
0
5
10
15
plasmin activitiy [mU/mL]
SEAP
[U*L
-1]
B4B4 + scFv
RR120-receptor
Supplementary Figures
Tobias Strittmatter 84/240
Supplementary Figure 10: Additional characterization of inducer molecules. HEK293T cells
were transfected overnight with the indicated receptors and the pLS13 STAT3 reporter
plasmid alongside pLS15 for STAT3 transcription factor overexpression, as described in the
methods. Cells were incubated for 24 h in complete DMEM containing 10 % FCS and the test
substance. a) Fibrinogen-mediated activity of AMBERB4, AMBERB4/scFv or AMBERG1-B4 is
reduced by high activity of plasmin at 1 mU/mL. Fibrinogen was incubated at 5 µg/mL
together with the indicated concentrations of plasmin. Sensitivity to plasmin depends on
the receptor configuration. b) Cells expressing AMBERB4 or AMBERB4/scFv or RR120 receptor
were activated with fibrinogen at 5 µg/mL for 24 h prior to replacing the medium with
complete DMEM containing no fibrinogen but the indicated activity of plasmin instead. No
difference in activation can be observed between different concentrations of plasmin. c)
xFDP-activated expression of SEAP reporter is independent of plasmin activity in the
supernatant. Cells were incubated with 1 µg/mL xFDP protein and supplemented with the
indicated activities of plasmin. All values are means ± SD of triplicate determinations.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 85/240
Supplementary Figure 11: Analysis of plasma fractionated by size-exclusion
chromatography. Size-exclusion chromatography (SEC) was used to assess the active
fraction of whole human plasma using a self-packed Sephadex 200 column. Plasma was
filtered through a 0.2 µm filter to remove most of the lipoprotein vesicles prior to loading.
a) UV detection of SEC experiment (blue) correlated with activity of each fraction in the cell
culture (red and orange). b) SDS-PAGE under denaturing conditions of relevant fractions
from the SEC experiment. Boxed bands were excised and subjected to mass spectrometry
for identification. The results of mass spectrometry are presented in boxes on the left-hand
side, including the molecular mass of the respective full-length protein along with the
a) activity analysis of size exclusion chromatography derived fractions
b) SDS-PAGE and mass-spectrometric analysis of relevant fractions
Mfract ion number - 6% denaturing SDS-PAGE
MDD 22 24 26 28 29 30 31 32 34 36 38 40
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 1300
5
10
15
20
25
30
35
40
45
0
10
20
30
40
50
60
fraction No.
SEAP
[U*L
-1]
UV-Absorbance
Activity - B4
Activity - B4 + scFv
UV-Absorbance [mA
U]
Supplementary Figures
Tobias Strittmatter 86/240
relative abundance of the indicated protein in the sample. Values of receptor activity in a)
are means ± SD of duplicate determinations.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 87/240
Supplementary Figure 12: a) Unspecific activation of receptors under the conditions used
in figure 3b is excluded by measuring the responses with RR120-receptor-transfected
HEK293T cells harboring the pLS13 STAT3 reporter plasmid and pLS15 for STAT3
a) fibrinogen and plasma controls
plasma -
0.5 %
plasma +
hirudin
plasma +
hepari
n
fibrin
ogen -
10 g/m
L
fibrin
ogen -
2 g/mL
fibrin
ogen + hiru
din
fibrin
ogen + hepa
rin
uninduced
plasma -
0.5 %
plasma +
hirudin
plasma +
hepari
n
fibrin
ogen -
10 g/m
L
fibrin
ogen -
2 g/mL
fibrin
ogen + hiru
din
fibrin
ogen + hepa
rin
uninduced
0
10
20
30SE
AP [U
*L-1
]RR120
pS2ctr
b) Argatroban as an inhibitor
heparin 0.0
01.0
05.0
010
.00
100.0
00
10
20
30
Argatroban [ M]
SEA
P [U
L-1]
B4
B4 + scFv
G1-B4
RR120-receptor
c) Ximelegatran as an inhibitor
0.00
1.00
5.00
10.00
100.0
00
10
20
30
Ximelagatran [ M]
SEAP
[UL-1
]B4
B4 + scFv
G1-B4
RR120-receptor
heparin
d) induction with solid, insoluble fibrin e) induction with solublized, insoluble fibrin
1.0 10-2 0.1 1.0 10.0 100.00
10
20
30
40
5050
100
150
fibrin concentration [ g/mL] (soluble)
SEAP
[UL-1
]
B4
B4 + scFv
G1-B4
scFv-B4
RR120-receptor
pS2ctr
1.0 10-2 0.1 1.0 10.00
5
10
15
50100150
fibrin concentration [mg/mL] (solid)
SEAP
[UL-1
]
B4
B4 + scFv
G1-B4
scFv-B4
RR120-receptor
pS2ctr
Supplementary Figures
Tobias Strittmatter 88/240
transcription factor overexpression. Cells were incubated for 24 h in complete DMEM
containing plasma or fibrinogen and either 1 µg/mL hirudin or 15 USP/mL heparin.
Inhibition of coagulation by the thrombin inhibitors b) argatroban and c) ximelegatran was
assessed by measuring the SEAP reporter production of cells expressing AMBERB4,
AMBERB4/scFv or AMBERG1-B4. A 10-dose response plot of fibrin on AMBERB4, AMBERB4/scFv,
AMBERG1-B4 or AMBERscFv-B4 revealed bell-shape-like activation patterns for d) an insoluble
fibrin preparation and e) a solubilized fibrin preparation that becomes insoluble at neutral
pH. All values are means ± SD of triplicates.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 89/240
Supplementary Figure 13: Reference samples for assessment of cross-linking and
fibrinolysis-modulating factors depicted in fig. 3d). HEK293T cells were transfected with
either the RR120 receptor in combination with pLS13 and pLS15 or pSEAP2ctr constitutively
expressing SEAP reporter. The latter showed an unspecific boost of cell productivity in
DMEM supplemented with 10 mg/mL TXA. All values are means ± SD of triplicate
determinations.
cell productivity is boosted by tranexamic acid
0
10
20
30
40
SEAP
[UL-1
]
RR120-receptor
SEAP ctr
FGNTHRB
TrideginTXAFXIII
plasmin
+ ++
+++
++
+
++
+
++
++
++
++
++
+
++
+
+
+ ++
+++
++
+
++
+
++
++
++
++
++
+
++
+
+
Supplementary Figures
Tobias Strittmatter 90/240
Supplementary Figure 14: Characterization of stable cell lines. a) Different reporter
constructs were evaluated for their inducibility with xFDPs in combination with
AMBERB4/scFv. HEK293T cells were transfected overnight with the indicated receptors and
the pLS13 STAT3 reporter plasmid alongside pLS15 for STAT3 transcription factor
overexpression, as described in the methods. Cells were incubated for 24 h in complete
DMEM containing 10 % FCS and the test substance. xFDPs were supplemented at the
a) reporter design for stable constructs b) stable RR120-receptor nLuc/TNK expression
c) time course with xFDP induction
e) dose-response against xFDP f) dose-response against plasma
d) time course with plasma induction
1 0.10.050.0
1 0 1 0.10.050.0
1 0 1 0.10.050.0
1 0 1 0.10.050.0
1 0 1 0.10.050.0
1 00
20
40
60
80
100
xFDP concentration [µg/mL]
SEAP
[U*L
-1]
pLS13
pTS441
pTS566
pTS810
pTS824
uninduced
0.125 µ
g/mL xF
DPs
0.5 µg/m
L xFDPs
2.0 µg/m
L xFDPs
0.05 %
plasma
0
1 105
2 105
3 105
4 105
5 105
0
1
2
3
4
nLuc
[AU]
TNK activtiy [m
U/m
L]
TNK activity
N.D. N.D. N.D. N.D. N.D.
nLuc luminescence
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 360.0
5.0 105
1.0 106
1.5 106
2.0 106
time after induction [h]
nLuc
Lum
ines
cenc
e [A
U]
pcTS59
pcTS60
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 360.0
5.0 105
1.0 106
1.5 106
2.0 106
time after induction [h]
nLuc
Lum
ines
cenc
e [A
U]
pcTS59
pcTS60
10-2 10-1 100 1010
2 105
4 105
6 105
8 105
xFDP concentration [ g/mL]
nLuc
Lum
ines
cenc
e [A
U]
pcTS59
pcTS60
10-2 10-1 1000
2 105
4 105
6 105
8 105
plasma concentration [%]
nLuc
Lum
ines
cenc
e [A
U]
pcTS59
pcTS60
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 91/240
indicated concentrations, ranging from 1 µg/mL to 0.01 µg/mL. b) The stable polyclonal cell
line HEKRR120/TNK bears the inactive RR120 receptor controlling a STAT3-driven reporter
construct expressing reporter nano-luciferase (Nluc) coupled via a furin cleavage site to the
therapeutic protein tenecteplase (TNK). 45 000 cells were seeded per well and incubation
was done for 24 h. We subsequently induced these cells for 24 h with the indicated
concentrations of xFDPs or plasma. Nluc and TNK activity were measured in the
supernatant. The TNK activity of untreated cells was subtracted from the observed values.
c)-f) Stable monoclonal HEKscFv-B4/TNK cells and polyclonal HEKRR120/TNK were seeded at 15 000
cells per well and induced 24 h after seeding. c)-d) Time courses of reporter gene expression
after induction with either c) 1 µg/mL xFDPs or d) 1 % (v/v) reconstituted human plasma.
Reporter gene activity was measured at the indicated time points post induction. e)-f) Dose
responses of HEKscFv-B4/TNK and HEKRR120/TNK upon incubation with the indicated amounts of
inducer. A √10-dilution series was prepared with e) 5 µg/mL to 0.011 µg/mL xFDPs. In f)
stable cells were induced with plasma ranging from 2 % to 0.003 % (v/v). Values in a) are
means ± SD of triplicate determinations, values in b) are means ± SD of quadruplicate
determinations, values in c-f) are cumulative values of three independent measurements
performed in triplicate, N=9, shown as mean ± SD.
Chapter 1: Programmable DARPin-based Receptors for the Detection and Treatment of Thrombotic Events
Tobias Strittmatter 93/240
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Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 99/240
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical
production in mammalian cells
Chapter 2 is based on a manuscript that has been submitted for publication. This work builds
upon work described in the master theses of Sabina Egli (ETH Stud. ID: 11-923-422) and
Richard Plieninger (ETH Stud. ID: 12-063-137), both of whom had been co-supervised by
Mingqi Xie and Martin Fussenegger.
Author Contributions
Tobias Strittmatter1, Mingqi Xie4 and Martin Fussenegger1,5 designed the project. Tobias
Strittmatter1, Adrian Bertschi1, Sabina Egli2, Richard Plieninger1, Daniel Bojar3 and Mingqi Xie
performed experiments and analyzed the results. Tobias Strittmatter1, Adrian Bertschi1,
Mingqi Xie and Martin Fussenegger1,5 wrote the manuscript.
Contributions to each figure
Tobias Strittmatter1: figures 1a-c, 2a,c,d, 3, 4
Sabina Egli2 and Mingqi Xie4: figure 1d
Adrian Bertschi1: figures 1e-f, 2b
Chia-wei Lin6: performed glycosylation analysis for figure 4c
Author Affiliations 1Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058
Basel, Switzerland 2Present address: Roche Pharma (Schweiz) AG, Basel, Switzerland 3Present address: University of Gothenburg, Gothenburg, Sweden 4School of Life Sciences, Westlake University, 310024 Hangzhou, Zhejiang, China 5Faculty of Science, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland 6University of Zurich, Zürich, Switzerland
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 101/240
Abstract
In this work, we designed and built a gene switch that employs metabolically inert L-
glucose to regulate transgene expression in mammalian cells via D-idonate-mediated control
of the bacterial regulator LgnR. To this end, we engineered a metabolic cascade in mammalian
cells to produce the inducer molecule D-idonate from its precursor L-glucose by ectopically
expressing the Paracoccus species 43P- derived catabolic enzymes LgdA, LgnH and LgnI. To
obtain ON- and OFF-switches, we fused LgnR to the human transcriptional silencer domain
KRAB and the viral trans-activator domain VP16, respectively. Thus, these artificial
transcription factors KRAB-LgnR or VP16-LgnR modulated cognate promoters containing
LgnR-specific binding sites in a D-idonate-dependent manner as a direct result of L-glucose
metabolism. In a proof-of-concept experiment, we show that the switches can control
production of the model biopharmaceutical rituximab in both transiently and stably
transfected HEK-293T cells, as well as CHO-K1 cells. Rituximab production reached 5.9 µg/mL
in stably transfected HEK-293T cells and 3.3 µg/mL in stably transfected CHO-K1 cells.
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 103/240
Introduction
The production of therapeutic biologics by engineered mammalian cells in bioreactors
is a key element of modern-day medicine to treat various diseases, which include but are not
limited to cancer (Reff et al. 1994; Pierpont et al. 2018; Muhsin et al. 2004), immune system
disorders (Belliveau 2005; Monaco et al. 2015) and inherited enzyme deficiencies (Brady et
al. 1973; Brady et al. 1974; Ioannou et al. 1992; Grabowski et al. 1998). Typically, the
production cassette is induced by small molecules after an initial growth phase in order to
reduce stress on the cells and to improve the purity of the product (Kopp et al. 2019; Huang
et al. 2014). For this purpose, various gene switches have been developed for the exogenous
control of gene expression in bacteria, yeast and mammalian cells in response to the addition
of small inducer molecules or even upon exposure to light (Kallunki et al. 2019; Mansouri et
al. 2019). However, some of these inducers can have undesired side effects (Ahler et al. 2013),
while in the case of light as an inducer, insufficient penetration especially into high-density
cultures can lead to inhomogeneous induction (Suh et al. 2003). Attempts to increase yields
have long been focused on process engineering improvements, but implementation of
induced gene expression in mammalian cells has recently attracted increasing interest (Lonza
2019). Reasons for this development include the greater ability of mammalian cells to
produce difficult-to-express proteins, such as large and complex recombinant proteins that
require post-translational modifications. Although bacterial and yeast production systems
have their advantages in productivity and efficiency, only mammalian cells are able to
efficiently introduce post-translational modifications that are essential for functionality of
some biopharmaceuticals (Amann et al. 2019). Post translational modifications such as
specific glycosylation patterns (Zhang et al. 2016; Stavenhagen et al. 2018), carboxylation
(Hansson et al. 2005), phosphorylation (Yalak et al. 2012) and sulfation (Mufarrege et al. 2020)
are unique to the production cell line and often require mammalian cell systems for
implementation. Especially for the production of therapeutics like clotting factors (Mannully
et al. 2018), functionalized AB as well as novel multispecific drugs (Deshaies 2020),
mammalian cell lines have become more important to ensure quality and safety of the
product. To date, the most commonly used mammalian cell lines employed for industrial
production of biopharmaceuticals are HEK-293T cells for human derived and CHO-K1 cells for
a multitude of recombinant proteins (Amann et al. 2019).
Introduction
Tobias Strittmatter 104/240
All living organisms show homochirality toward organic compounds, such as amino
acids and sugars, where L-amino acids and D-sugars are typically abundant in nature (Shimizu
et al. 2012). L-Glucose is the enantiomer of D-glucose, and provides the same sensation of
sweetness as the common D-enantiomer, but is inert to degradation in mammals (Rudney
1940; Levin et al. 1995). Therefore, humans can neither store nor metabolize L-glucose to
produce energy (Rudney 1940; Sasajima et al. 1979). Thus, L-glucose seems to be an ideal
candidate for orthogonal control of gene expression in mammalian cells. In this work, we
established a gene switch for L-glucose-controlled expression of a model biopharmaceutical
in standard mammalian cell lines based on the D-idonate-responsive bacterial transcriptional
regulator LgnR (Shimizu et al. 2012). LgnR is a member of the IclR family of bacterial repressors
that control antibiotic resistance, metabolism, quorum sensing, and pathogenicity (Molina-
Henares et al. 2006). In its native context, LgnR drives the expression of the lgn operon, which
contains five enzymes involved in L-glucose catabolism. Those enzymes catalyze a chain of
reactions that further degrade the L-glucose oxidation product L-gluconate to the common
metabolites glycerinaldehyde-3-phosphate and pyruvate. In the initial step of L-glucose
degradation, L-glucose is oxidized to yield L-gluconate. This reaction is mediated by the
dehydrogenase LgdA, which is mainly involved in the degradation of scyllo-inositol (KM = 3.70
± 0.4 mM / Kcat = 705 ± 12 min-1), but can utilize L-glucose as an alternative substrate with
lower efficiency (KM = 59.7 ± 5.7 mM / Kcat = 1040 ± 28 min-1) (Shimizu et al. 2012). Following
its production from L-glucose, L-gluconate is isomerized to D-idonate by L-gluconate
dehydrogenase (L-GnDH; LgnH) and 5-keto-L-gluconate reductase (LgnI). Notably, only three
enzymes are needed to form D-idonate (figure 1a), which controls LgnR activity by inducing
dissociation of LgnR from its promoter (Shimizu et al. 2014). By fusing LgnR to the trans-
activator virus protein 16 (VP16) from herpes simplex virus (Jonker et al. 2005) or the silencing
domain Krüppel associated box (KRAB), (Margolin et al. 1994) we were able to build an L-
glucose-responsive synthetic trans-activator or trans-silencer, respectively. The resulting
artificial transcription factors were combined with synthetic promoters that include binding
sites for LgnR.
Thus, by employing components from the L-glucose degradation system, artificial
LgnR-based transcription factors and synthetic promoters, we were able to build L-glucose-
sensitive genetic ON- and OFF-switches to control transgene expression, as measured in
terms of production of reporter proteins. We show that these switches can control
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 105/240
production of the model biopharmaceutical rituximab in both transiently transfected and
stably transfected HEK-293T cells, as well as CHO-K1 cells.
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 107/240
Results
Design of an L-glucose-responsive gene circuit
To implement L-glucose as an inducer of an orthogonal gene switch, we engineered
expression vectors for the three enzymes necessary for conversion of L-glucose into D-
idonate in mammalian cells. We also fused the D-idonate-responsive bacterial repressor
protein LgnR with either a KRAB silencer domain or a VP16 activator domain to obtain
synthetic D-idonate-controlled mammalian transcription factors. Since it is reported that the
canine sodium/myo-inositol transporter 1 (SMIT1; SLC5A3) is non-specifically permeable for
L-glucose (Hager et al. 1995), we also included SMIT1 in our setup. Thus, we tested whether
ectopic expression of SMIT1 would increase the induction fold of L-glucose-dependent
transgene expression. Co-expression of the KRAB-LgnR and VP16-LgnR fusions together with
the metabolic cascade would yield genetic L-glucose-dependent ON- (figure 1b, left) and OFF-
switches (figure 1b, right), respectively. For the ON-switch, binding of D-idonate by KRAB-
LgnR triggers the release of the trans-silencer from the operator site and hence enables
transcription. In contrast, the VP16-LgnR trans-activator triggers expression when bound to
the promoter in the uninduced state. Binding of the D-idonate ligand then causes the trans-
activator to dissociate from the DNA, shutting down expression of the transgene. Both
switches are triggered once D-idonate reaches sufficient levels in the cytosol of transfected
cells through the activity of the enzyme cascade.
Based on the DNA sequence of the intergenic region between LgnR and the adjacent
lgn gene cluster, we examined four potential binding motifs for LgnR by cloning them either
downstream of a constitutive promoter derived from simian virus 40 (PSV40) or upstream of a
minimal human cytomegalovirus promoter (PhCMVmin) (figure 1c). Binding motif 0 spans the
whole intergenic region, containing operator elements for both the lgn gene cluster (LgnA)
and the LgnR gene. Three shortened motifs contain either a binding sequence spanning the
described -35 and -10 elements controlling expression of LgnA of the lgn gene cluster (motif
A), the corresponding version on the LgnR operon (motif C) or a shortened sequence based
on motif C (motif B1). Two and three repeats of binding motifs A (A2, A3) and C (C2, C3) were
compared to their respective single binding sites (A1, C1).
Results
Tobias Strittmatter 108/240
Figure 1: a) Overview of the L-glucose degradation pathway producing D-idonate. In the
first step, LgdA uses NAD+ to oxidize L-glucose to L-1,5-gluconolactone, which
spontaneously hydrolyzes to form L-gluconate. In two subsequent oxidation and reduction
reactions at the C5 atom, L-gluconate is epimerized to D-idonate using NAD/NADH as a
b) schematic of ON/OFF switcha) L-glucose catabolism
d) LgnR based expression
LgdA
L-glucose L-glucono-1,5-lactone
L-gluconateL-5-ketogluconateD-idonate
NAD+ NADH
LgnHLgnI
NAD+
H2O
NADHNAD+
NADH
c) LgnR binding sites
KRAB-LgnR
D-idonate
ON-system OFF-system
VP16-LgnR
L-glucoseSMIT1
PCMVmin SEAPOLgnRPCMV SEAPOLgnR
f) toxicity of L-glucose inducere) toxicity of intermediates
agtgcttagttcatattatgggaaccaatcccatgtcaatcagtgaacacaaatttctttcatatagtaatactgtgtgccataatatgggatattgaagatcacgaatcaagtataatacccttggttagggtacagttagtcacttgtgtttaaagaaagtatatcattatgacacacggtattataccctataacttct
-35-35-10 -10
pMX157 (0)pMX212/220 (A) pMX215/223 (B)
pMX216/224 (C)
LgnRLgnA LgnA - LgnR intergenic region
PCMVmin SEAPOLgnR
PSV40 SEAPOLgnR
L-glu 10
0mM
L-gluco
nate 1m
M
D-idon
ate 1mMblank
0
1 106
2 106
3 106
4 106
Lum
ines
cenc
e [A
U]
HEK-293T CHO-K1
0.00
0.010.0
30.1
00.3
21.0
03.1
610
.0031
.6310
0.00
0
2000
4000
6000
8000
0
5000
10000
15000
L-Glucose [mM|
Res
oruf
in (H
EK
-293
T) [F
U]
HEK-293T Resorufin (C
HO
-K1) [FU
]
CHO-K1
pcDNA3.1 pMX181 (VP16-LgnR)
pMX157
pMX220
pMX221
pMX222
pMX223
pMX224
pMX225
pMX226
0
40
80
120
SE
AP
(UL-1 ) 0 A1 A2 A3 B1 C1 C2 C3
pcDNA3.1 pMX183 (KRAB-LgnR)
pMX212
pMX213
pMX214
pMX215
pMX216
pMX217
pMX218
010203040
SEA
P (U
L-1) A1 A2 A3 B1 C1 C2 C3
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 109/240
cofactor by the dehydrogenase LgnH and reductase LgnI, respectively. b) Schematic of the
L-glucose-responsive genetic circuit. L-Glucose (dark hexagon) enters the cell with the help
of transporters (e.g., SMIT1) and is metabolized by the ectopically expressed enzymatic
cascade depicted in a) to produce D-idonate (orange triangle). D-idonate binds the bacterial
repressor protein LgnR, which then dissociates from the operator sequence of a synthetic
promoter. left: LgnR can be fused to the mammalian trans-silencing domain KRAB to
generate a signal in response to D-idonate binding, yielding an ON-switch by induced de-
repression of the strong PhCMV promoter. right: Alternatively, a fusion construct of LgnR and
the trans-activator VP16 induces expression from a weaker minimal promoter derived from
PhCMV (PhCMVmin) in the absence of D-idonate. Accordingly, the signal is quenched if D-
idonates forces LgnR to dissociate from the promoter, yielding an OFF-switch for induced
inactivation of the promoter. c) Overview of the intergenic region of LgnR and the lgn gene
cluster. The regulatory protein LgnR controls expression of both operons. The -10 (green
arrow) and -35 (red and orange arrow) elements are highlighted in bold. Parts of the
intergenic region that are used in response elements to report LgnR binding are indicated
by blue bars labeled with the corresponding genetic constructs. d) HEK-293T cells were co-
transfected with 125 ng of reporter plasmids (pMX212-218 and pMX157 and pMX220-226)
with 375 ng of effector plasmids harboring VP16-LgnR (pMX181-PSV40-VP16-LgnR-pA) or
KRAB-LgnR (pMX183-PhEF1a-KRAB-LgnR-pA) fusions in 48-well plates. e) The impact on
productivity of HEK-293T and CHO-K1 cells was monitored by measuring the luminescence
of secreted NLuc reporter produced from a constitutive PPGK promoter after 48 h. 20 ng of
pTS1034 (PPGK-Igk-NLuc-pA) and 180 ng of filler plasmid pDF101 were co-transfected in 96-
well plates. The impact on viability was assessed by measuring the ability of cells to reduce
resazurin to resorufin. d) The viability of wild-type HEK-293T and CHO-K1 cells was assessed
by resazurin assay after incubation with different amounts of L-glucose for 48 h. All values
are mean ± SD of n = 3 biologically independent samples.
HEK-293T cells co-transfected with 125 ng of PhCMVmin-based reporter constructs and
375 ng of the VP16-LgnR fusion (pMX181) exhibited inducible reporter gene expression for all
four binding motifs. Sequences based on binding motif 0 and A showed strongest induction
(figure 1d, top) and a two-fold repeat of motif A (pMX221) was chosen for further
characterization of the OFF-switch. Reporter constructs containing binding motifs A, B and C
Results
Tobias Strittmatter 110/240
in combination with the constitutive PSV40 promoter (125 ng) were co-transfected with a
KRAB-LgnR fusion (pMX183, 375 ng) to evaluate their use in ON-switches. Here, the plasmid
containing binding motif B in pMX215 showed best inducibility and was used as LgnR-specific
operator site in all follow-up studies.
Testing of metabolic enzymes
Prior to testing the metabolic enzymes, the impact of intermediates of the metabolic
cascade on the cells was assessed. Toxicity was evaluated with the resazurin reduction assay
and metabolic integrity was assessed in terms of constitutive expression of NanoLuc
luciferase (NLuc) in transiently transfected HEK-293T and CHO-K1 (figure 1e) cells. None of
the compounds affected cell viability or productivity. Furthermore, the inducer L-glucose was
found to be non-toxic at concentrations up to 100 mM in the resazurin reduction assay (figure
1f).
To establish whether all components of the enzymatic L-glucose degradation pathway
are required to produce D-idonate in HEK293T cells, we used the LgnR ON-system and
introduced the respective enzymes in different combinations. We tested expression from two
constitutive promoters based on the strong promoter for human elongation factor 1 α (PhEF1α)
and the weaker promoter for human phosphoglycerate kinase (PPGK). We found increased
reporter protein expression from the D-idonate-responsive promoter only in the presence of
all three enzymes of the metabolic cascade (figure 2a). Intriguingly, only the use of the weaker
PPGK promoter, but not the stronger PhEF1α promoter, afforded a functional gene switch.
Additional expression of the transporter SMIT1 increased the fold switching in CHO-K1 cells,
in accordance with the anticipated facilitation of L-glucose transport across the plasma
membrane. To exclude unspecific effects, we tested the intermediates of the metabolic
cascade for induction of the system in HEK-293T cells (figure 2b, i) and CHO-K1 cells (figure
2b, ii). Here we found specific activation of reporter expression from the synthetic promoter
construct in combination with the KRAB-LgnR trans-silencer only when the final product of
the metabolic cascade, D-idonate, was added. In contrast, the complete ON-system was
triggered by L-glucose, L-gluconate and D-idonate, indicating the contribution of each
enzyme.
Taken together, these findings confirm that L-glucose and the ectopic L-glucose
degradation pathway are safe and effective for use in HEK-293T and CHO-K1 cells.
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 111/240
Figure 2: a) Combinations of metabolic enzymes LgdA, LgnH and LgnI, as well as SMIT1,
were tested for ON-switch functionality in HEK-293T and CHO-K1 cells. Enzymes are
expressed from PPGK promoters or PhEF1α promoters as described in the methods section.
Plasmids comprising the full ON-switch that were excluded from the transfection mix in this
experiment were replaced by filler plasmid pDF101. Induction is shown as the ratio of
induced to uninduced samples under each condition. b) The mechanism of action was
validated by transfecting i) HEK-293T and ii) CHO-K1 cells with either the promoter and
VP16-LgnR trans-activator alone (LgnR + rep) or the full ON-system. 65 ng of trans-activator
b) validation of induction mechanisma) essential parts for ON switch
i) HEK293-T cells ii) CHO-K1 cellsHEK-293T - PGK
HEK-293T - EF1aCHO-K1 - EF1a
CHO-K1 - PGK
LgnH/ILgdA
SMIT1
LgnH/I + LgdA
LgnH/I + SMIT1
LgdA + SMIT1
LgnH/I + SMIT1 +
LgdA
reporte
r ctr
0
1
2
3
4
fold
indu
ctio
n [R
U]
L-gluco
se 10
0mM
L-gluco
nate 1m
M
D-idon
ate 1mMblank
L-gluco
se 10
0mM
L-gluco
nate 1m
M
D-idon
ate 1mMblank
0
100
200
300
400
500
SEA
P [U
L-1 ]
ΔLgdA, ΔLgnH, ΔLgnIfull system
0
10
20
30
40
SEA
P [U
L-1 ]
ΔLgdA, ΔLgnH, ΔLgnIfull system
L-gluco
se 10
0mM
L-gluco
nate 1m
M
D-idon
ate 1mMblank
L-gluco
se 10
0mM
L-gluco
nate 1m
M
D-idon
ate 1mMblank
c) ON / OFF switch
i) HEK-293T cells ii) CHO-K1 cells
0.00
0.10
1.00
10.00
100.0
00
20
40
60
80
0
10
20
30
40
50
L-Glucose [mM]
SEA
P [U
L-1]
ON
OF
FS
EAP
[UL
-1]
0.00
0.10
1.00
10.00
100.0
00
100
200
300
400
500
0
10
20
30
40
50
L-Glucose [mM]
SEA
P [U
L-1]
ON
OF
FS
EAP
[UL
-1]
d) time course of inductioni) HEK-293T cells ii) CHO-K1 cells
0 12 24 36 48 60 72 84 96
10
20
30
time [h]
SE
AP
[UL-1
]
-
+
1.45
1.80
1.992.19 2.16 2.06
0 12 24 36 48 60 72 84 96
50
100
150
200
250
time [h]
SEA
P [U
L-1]
-
+1.42
1.551.59
1.63
1.18
Results
Tobias Strittmatter 112/240
pMX183 in and 15 ng of pMX335 reporter were co-transfected with 20 ng of NLuc
expressing plasmid pTS1052 and 120 ng of filler plasmid pDF101. Both setups were tested
with key compounds of the L-glucose degradation pathway. Activation was monitored by
measuring SEAP levels in the supernatant after induction for 48 h. c) The responses of the
ON-switch (left y-axis) and OFF-switch (right y-axis) to various concentrations of L-glucose
were tested in i) HEK-293T and ii) CHO-K1 cells. The medium was exchanged again after 24
h and SEAP reporter activity was measured after 48 h. d) Expression of SEAP reporter from
the ON-switch induced with 10 mM L-glucose was recorded over 4 days in i) HEK-293T and
ii) CHO-K1 cells. Twice the standard volume of medium was used to allow for prolonged
incubation. Induction ratios of induced versus uninduced samples are indicated above the
relevant time points. All values are mean ± SD of n = 3 biologically independent samples.
Functionality of ON- vs OFF-switch
We next evaluated the performance of the ON-system comprising the trans-silencer KRAB-
LgnR and compared it with the OFF switch based on the trans-activator VP16-LgnR. Both
systems responded dose-dependently to increased inducer concentrations (figures 2c).
Induction of the ON-switch generated a clear reporter signal above the background, starting
at 24 h after induction (figure 2d). This signal was more pronounced in CHO-K1 cells than in
HEK-293T, owing to the higher fold induction in the former cell line. Although the fold-change
in reporter expression was higher in the case of the OFF-switch, we considered that higher
absolute expression levels would be preferable for biopharmaceutical production, so we
focused on the ON-switch in subsequent experiments.
L-Glucose-induced rituximab production
For proof-of-concept, we applied the L-glucose system to the production of rituximab
as a model biopharmaceutical. For this purpose, we created a reporter cassette for L-glucose-
inducible production of rituximab by placing a genetic fusion construct of the light and heavy
chain of the antibody under control of the ON-switch. To ensure an equimolar ratio of the
secreted chains, as well as minimal disturbance of the original amino acid sequence, we linked
both chains via a modified furin cleavage site (KRRKR) followed by a T2A sequence, similarly
to a previous study (Fang et al. 2007) (figure 3a).
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 113/240
a) rituximab production system
c) transient rituximab production
b) YPet expression in transient transfections
i) dose-response curve ii) peak rituximab production
i) dose-response curve ii) peak fluorescence
HEK-293T CHO-K10
100
200
300
400
500av
erag
e Y
Pet f
luor
esce
nce
[FU
]4.92 x
1.23 x
uninduced
induced
HEK-293T CHO-K1
0.0
0.1
0.2
0.3
IgG
[g/
mL]
8.1 x
28.5 x
uninduced
induced
0.0000
0.01000.0
3200.1
0000.3
1601.0
0003.1
620
10.0000
31.6230
100.0
000
0
1020
30
40
100200300400500
L-Glucose [mM|
mea
n Y
Pet f
luor
esce
nce
[FU
]
HEK-293T
CHO-K1
0.000
0.0100.0
320.1
000.3
161.0
003.1
63
10.000
31.626
100.0
000.0000.005
0.0100.015
0.020
0.1
0.2
0.3
L-Glucose [mM|
IgG
[g/
mL]
HEK-293T
CHO-K1
D-Idonate-dependent ON-switch
KRAB-LgnRPhEF1a
Rituximab
HC LCT2A IRESFurin YPetPCMV OLgnR
L-Glucose to D-idonate conversion
LgnH P2A LgnIPPGKLgdAPPGK SMIT1PPGK
Results
Tobias Strittmatter 114/240
Figure 3: a) Schematics of constructs used for rituximab production. The same trans-silencer
as before was used in combination with a rituximab expression cassette coupled to the
expression of yellow fluorescent reporter YPet via an internal ribosome entry site (IRES).
Both light and heavy chains of rituximab are expressed in equimolar ratios via a furin
cleavage site fused to a T2A sequence to ensure a high-quality final antibody product. L-
Glucose-dependent expression of b) intracellular YPet reporter (as assessed by flow-
cytometry) and c) secreted rituximab (measured by ELISA for human IgG) from transiently
transfected HEK-293T and CHO-K1 cells was measured after 72 h of continuous induction
with 3.16 mM L-glucose. Plots in i) represent dose-response curves, while bar graphs in ii)
represent peak values at 3.16 mM L-glucose. Values above the bars indicate fold inductions
of induced versus uninduced samples. Values are mean ± SD of b) n = 3 and c) n = 2
biologically independent samples.
The use of this combination enables both chains to be secreted individually, since they are
separated upon translation by the T2A sequence. In addition, the furin cleavage site removes
all the additional amino acids but two from the C-terminal of the heavy chain upon transport
through the Golgi apparatus. The remaining basic amino acids are removed by
carboxypeptidases residing in the trans-Golgi during secretion. We added a yellow fluorescent
reporter protein (YPet) via an internal ribosome entry site (IRES) to the antibody sequence
(figure 3a) as a reporter of induction of the system. We next evaluated the dose-response
curves of YPet and rituximab production in HEK-293T and CHO-K1 cells. YPet fluorescence
(figure 3b) and rituximab concentration (figure 3c) increased concomitantly with increasing
concentration of L-glucose in transiently transfected HEK-293T and CHO-K1 cells. We
measured maximum YPet fluorescence (figure 3 b, ii) and rituximab production (figure 3 c, ii)
at 10 mM L-glucose and obtained peak rituximab production of around 0.25 µg/mL in HEK-
293T and approximately 0.015 µg/mL in CHO-K1 cells. Encouraged by these results, we next
sought to improve rituximab production by generating stable HEK-293T and CHO-K1 cell lines.
To this end, we engineered Sleeping Beauty transposon vectors (Kowarz et al. 2015) to
facilitate genomic integration of all four cassettes making up the ON-switch. Since the
expression levels of stably integrated transgenes can differ from those of transiently
expressed constructs, and at the same time the level of the trans-silencer construct KRAB-
LgnR seemed to be critical for the functionality of the ON-switch, we decided to screen both
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 115/240
the PhEF1α (HEK-293TEF1α and CHO-K1EF1α) and PPGK promoters (HEK-293TPGK and CHO-K1PGK) for
optimal expression of KRAB-LgnR. All the polyclonal cell lines examined showed superior
sensitivity and inducibility compared to transient transfection in both HEK-293T and CHO-K1
cells (figure 4a). Maximum induction of YPet fluorescence was reached at 1 mM L-glucose in
CHO-K1 cell lines (CHO-K1EF1α and CHO-K1PGK) and at 10 mM for HEK-293T cell lines (HEK-
293TEF1α and HEK-293TPGK) (figure 4a, ii). Rituximab production reached 2.4 µg/mL and 5.9
µg/mL in HEK-293TEF1α and HEK-293TPGK and 0.7 µg/mL and 3.3 µg/mL in CHO-K1EF1α and CHO-
K1PGK, respectively (figure 4b). IgG expression was increased up to 24-fold in HEK-293T and up
to 220-fold in CHO-K1 compared to transient expression, while at the same time reducing the
amount of inducer necessary for full induction of the system.
Finally, we assessed whether expression of the metabolic enzymes comprising the L-
glucose switch would alter the glycosylation pattern of the produced rituximab antibody upon
prolonged incubation with L-glucose. To this end, we selected polyclonal cell lines of HEK-
293T cells and CHO-K1 cells that stably express LgdA, LgnH, LgnI and SMIT1 from a PPGK
promoter while also expressing rituximab from a constitutive PhCMV promoter. Cells were
seeded and grown for three days as in previous experiments in standard culture medium or
in medium supplemented with 10 mM L-glucose. Glycosylation patterns were compared
between both culture conditions for both cell lines and found to be broadly similar (figure 4c).
Results
Tobias Strittmatter 116/240
Figure 4: a) intracellular YPet reporter and b) secreted rituximab from stable HEK-293T and
CHO-K1 cells was measured after 72 h of continuous induction with various concentrations
of L-glucose. i) Dose-response curves were measured and ii) peak values of YPet and IgG
production are depicted in separate bar graphs. Expression of the metabolic cascade is
driven by a PPGK promoter. Expression of the synthetic transcription factor KRAB-LgnR is
a) YPet expression in stable HEK-293T and CHO-K1 cells
b) rituximab production in stable HEK-293T and CHO-K1 cells
i) dose-response curves ii) peak rituximab production
c) Assessment of glycosylation of rituximab
i) dose-response curves ii) peak YPet fluorescence
HEK-293
T EF1a
HEK-293
T PGK
CHO-K
1 EF1a
CHO-K
1 PGK
0
1000
2000
3000
4000
5000
aver
age
YPe
t flu
ores
cen
ce [F
U]
1.48 x
1.35 x
uninduced
induced
9.90 x
4.27 x
0
2
4
6
IgG
[g/
mL]
20.65 x
9.82 x2.37 x
2.57 xuninduced
induced
HEK-293
T EF1a
HEK-293
T PGK
CHO-K
1 EF1a
CHO-K
1 PGK
0.0000
0.0100
0.0320
0.1000
0.3160
1.0000
3.1630
10.0000
31.6260
100.0
000
0
1000
2000
3000
4000
L-Glucose [mM|
mea
n Y
Pet f
luor
esce
nce
[FU
]
HEK-293TEF1a
HEK-293TPGK
CHO-K1EF1a
CHO-K1PGK
0.000
0.010
0.032
0.100
0.316
1.000
3.163
10.000
31.626
100.0
000
2
4
6
L-Glucose [mM|
IgG
[g/
mL]
HEK-293TEF1a
HEK-293TPGK
CHO-K1EF1a
CHO-K1PGK
i) HEK-293T
10-3 10-2 10-1 100 101 10210-3
10-2
10-1
100
101
102
rel. abundance (no L-glucose) [%]rel.
abu
ndan
ce (1
0 m
M L
-glu
cose
) [%
]
R2=0.9978
ii) CHO-K1
10-3 10-2 10-1 100 101 10210-3
10-2
10-1
100
101
102
rel. abundance. (no L-glucose) [%]rel.
abu
ndan
ce. (
10 m
M L
-glu
cose
) [%
]
R2=0.9947
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 117/240
controlled by a strong PhEF1α promoter (HEK-293TEF1α and CHO-K1EF1α) or a weaker PPGK
promoter (HEK-293TPGK and CHO-K1PGK). Values above bars indicate induction ratios
between induced and uninduced states. Values are mean ± SD of b) n = 3 and c) n = 2
biologically independent samples. c) Correlation plot of results from MS-analysis to assess
glycosylation patterns of rituximab produced in i) HEK-293T and ii) CHO-K1 cells in presence
or absence of 10 mM L-glucose. Relative abundance of glycan modifications of rituximab
produced without L-glucose were plotted on the x-axis and correlated to abundancies found
in L-glucose treated sample. Straight lines indicate identity (f(x)=x). Detailed analysis is
provided in tables S3 and S4.
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 119/240
Discussion
Here, we have designed, built and characterized a novel gene switch based on a
metabolic cascade starting from the small molecule L-glucose, which is functionally inert in
humans. We transferred all three enzymes, LgdA, LgnH and LgnI, needed for conversion of L-
glucose to D-idonate from Paracoccus species 43P into HEK-293T and CHO-K1 cells, and we
confirmed that D-idonate produced in these cells can control the bacterial regulator protein
LgnR. Thus, by fusing LgnR to the silencer domain KRAB or the trans-activator domain VP16,
we could build ON- and OFF-systems, respectively. In a proof-of-concept, the ON-system
could trigger expression of the model biopharmaceutical rituximab dose-dependently in
response to L-glucose in the cell culture medium without affecting glycosylation. Stable
integration of the engineered pathway afforded polyclonal HEK-293T and CHO-K1 cell lines in
which IgG production reached 5.9 µg/mL and 3.3 µg/mL, respectively.
A key feature of our system is the use of L-glucose as an extremely safe and orthogonal
inducer to control gene expression in mammalian cells. In addition, the use of a metabolic
cascade instead of a single-component switch could offer much greater opportunities for
expression control by adjusting the enzyme activity at each step of the process, enabling fine-
tuning of the system, and perhaps the incorporation of logic-based control.
Interestingly, performance of the ON-switch was much better in cells with stable expression
than in cells with transient expression. This is most likely due to epigenetic modifications of
the promoter region that reduce leaky expression and thereby increase the fold-switching
upon de-repression caused by dissociation of KRAB-LgnR from the promoter. Metabolic
adaptations of the host cell line, as well as suboptimal expression ratios of the components
in transient transfections, might also contribute to the increased functionality in the stable
setup. The cytosolic fluorescent reporter protein YPet provided an easy and convenient tool
for monitoring promoter activity by flow-cytometry. However, the fold-switching of the YPet
reporter signal was reduced in the stable system, possibly due to the long half-life of the
protein, which would lead to a build-up of background signal. If flow-cytometry is not
required, this issue could be avoided in future implementations of the system by using a
different reporter that is not a cytosolic fluorescent protein.
There are undoubtedly many ways in which the IgG production yield could be
increased from the levels obtained in this initial trial. Although both HEK-293T and CHO-K1
cell lines are routinely used for biopharmaceutical production (Dumont et al. 2016), the use
Discussion
Tobias Strittmatter 120/240
of a suspension cell line would drastically increase the cell density in the production process
and hence improve production capacity. Furthermore, the output could potentially be
boosted by additional integrations of the rituximab expression cassette via the orthogonal
PiggyBac transposase system (Yusa et al. 2011) or by the use of the GS-system (Bebbington
et al. 1992; Brown et al. 1992; Fan et al. 2012). The stable cell lines could be further optimized
by selection of monoclonal cell lines.
We believe the successful implementation of this L-glucose-triggered gene switch
further expands the available array of inducers by adding a non-toxic, functionally inert small
molecule, providing a new option for biopharmaceuticals production.
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 121/240
Acknowledgements
The authors thank Pascal Stücheli and Viktor Hällman for fruitful discussions and
critical readings of the manuscript and Chia-wei Lin from the Functional Genomic Center
Zurich for analysis of glycosylation patterns of rituximab samples. This work was financed by
the Swiss National Centre of Competence in Research (NCCR) for Molecular Systems
Engineering.
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 123/240
Materials & Methods
Cell culture
HEK-293T cells and CHO-K1 cells were cultured in 10 cm cell culture dishes in
Dulbecco’s modified Eagle’s medium (DMEM, Gibco, #61965-026) supplemented with 10 %
FCS, 50 Units/mL penicillin and 50 µg/mL streptomycin (Gibco, #15070-063) in a humidified
atmosphere containing 7.5 % CO2. The medium was supplemented with 150 µM L-proline for
culturing CHO-K1 cells. Cells were routinely split using trypsin-EDTA (Gibco, #25300-054)
every two days or upon reaching 70 % confluency, and reseeded at 1.5 million cells per plate.
For cell culture experiments, cells were seeded at 15 000 cells per well in 96-well plates
(Corning, #3599) or 250 000 cells per well in a 6-well plate (Corning, #3516).
Plasmid generation and transfection
Plasmids used for ectopic expression of transgenes were obtained by standard
molecular cloning techniques. All plasmids used in this study are listed in table S1.
For transfection of HEK-293T and CHO-K1 cells with plasmid DNA, we used
polyethyleneimine (PEI, 24765-1, Polysciences Inc.) at a PEI:DNA ratio of 6:1. 24 h prior to
transfection, cells were seeded as described in the previous section. A solution of 200 ng
plasmid DNA in 150 µL of serum-free DMEM medium was mixed with 1.2 µg of PEI (1 mg/mL
stock) for each well of a 96-well plate. Transfection mixes were incubated at room
temperature for 5-10 minutes. The solution was mixed again and added to the cells in a drop-
wise manner. Transfected cells were were incubated overnight (>12 h) using standard culture
conditions. The next morning, medium was replaced with standard culture medium (as
described above) supplemented with inducer molecules as necessary.
Data in figure 1d was obtained from cells transfected in 48-well plate format using a
PEI:DNA ratio of 3:1 followed by incubation for only 6 h with the transfection mix prior to
medium exchange.
Experiments with the ON-switch were done using pMX183 (65 ng), pMX335 (15 ng),
pTS2181 (20 ng), pTS2183 (50 ng) and pTS2185 (50 ng) while for the OFF-switch plasmids
pMX181 (40 ng), pMX221 (40 ng), pTS2181 (20 ng), pTS2183 (50 ng) and pTS2185 (50 ng) were
used. In cases were the ON-switch was used to produce rituximab (figure 2) the reporter
Materials & Methods
Tobias Strittmatter 124/240
plasmid pMX335 was replaced with the same amount of pTS2247 and trans-silencer construct
pMX183 was exchanged for pTS2174.
Generation of stably expressing cell lines
HEK-293T and CHO-K1 cells were seeded in 6-well plates and transfected as described
in the previous section by using 250 ng each of pTS2178, pTS2179 and pTS395 (PhCMV-SB100X-
pA) and the filler plasmid pDF101. Cells were selected with 2 µg/mL of puromycin (InvivoGen,
#ant-pr), 4 µg/mL blasticidin (InvivoGen, #ant-bl) in standard cell culture medium for 10 days.
The resulting stable cell lines were further modified by integration of the rituximab
production cassette including PhEF1α - or PPGK-driven trans-silencer KRAB-LgnR. 150 ng of
pTS2228 (PhEF1α) or pTS2230 (PPGK) was co-transfected with 200 ng of Sleeping Beauty
expression vector pTS395 and 650 ng of filler plasmid pDF101. Selection was performed in
standard cell culture medium supplemented with 2 µg/mL of puromycin (InvivoGen, #ant-pr),
4 µg/mL blasticidin (InvivoGen, #ant-bl) and 50 µg/mL Zeocin (InvivoGen, #ant-zn) for 10 days.
To analyze the impact of L-glucose on rituximab glycosylation, stably expressing HEK-293T
cells and CHO-K1 cells were generated by co-transfecting 200 ng of each sleeping beauty
vectors harboring PPGK-driven SMIT1 (pTS2178), LgdA, LgnH and LgnI (pTS2179) and a PhCMV-
driven rituximab-YPet expression cassette (pTS2248) together with 400 ng of a sleeping
beauty expression construct (pTS395). Cells were selected with 2 µg/mL of puromycin
(InvivoGen, #ant-pr), 4 µg/mL blasticidin (InvivoGen, #ant-bl) and 20 µg/mL Zeocin
(InvivoGen, #ant-zn) for 10 days.
Induction with L-glucose
Dose-response curves of reporter gene (SEAP or YPet) expression and rituximab
production were generated by incubating cells in standard cell culture medium (see above)
with the indicated concentrations of L-glucose in a 3.162-fold (√10) dilution series ranging
from 0.01 mM to 100 mM. A 1 M Stock solution of D-gluconate was prepared in D-PBS. D-
Idonate stock solution was prepared as 1 M D-idonic acid-1,4-lactone in D-PBS containing 1.1
M NaOH.
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 125/240
Assessment of reporter gene expression
Activity of the reporter protein, secreted alkaline placental phosphatase (SEAP), was
measured as previously described (Berger et al. 1988). Briefly, supernatant from cell culture
experiments was heat-treated for 30 min at 65 °C to inactivate endogenous phosphatases. 20
µL of heat-treated supernatant was mixed with 180 µL of assay reagent containing 60 µL of
water, 80 µL of 2x assay buffer (1 M diethanolamine pH 9.8, 0.5 mM MgCl2, 10 mM L-
homoarginine) and 20 µL of substrate solution (120 mM p-nitrophenyl phosphate in 2x assay
buffer). Reporter activity was assessed at 37 °C by following substrate turnover at 405 nm on
an Infinite M1000 microplate reader (Tecan Trading AG).
NanoLuc® luciferase activity was measured using a Nano-Glo® kit (Promega AG),
following the recommendations of the manufacturer. Volumes were adjusted to a total of 15
µL per reaction for use in a 384-well plate format.
For viability assays, the culture medium was replaced with medium containing 30
µg/mL (final) of resazurin. Cells were incubated for 15 minutes and fluorescence in the
supernatant was analyzed at 560 nm excitation and 590 nm emission on an Infinite M1000
microplate reader (Tecan Trading AG).
A yellow fluorescent protein (YPet) was used as a non-secreted, cytosolic reporter for
indirect read-out of rituximab expression. The coding sequence of YPet was coupled to the
coding sequence of rituximab with the help of an internal ribosome entry site (IRES).
Expression of YPet was recorded by flow-cytometry using 488 nm excitation and 510 nm
emission on a BD LSR Fortessa flow-cytometer equipped with a 96-well automatic sampler.
Cells were detached using 60 µL per well of trypsin-EDTA, incubated for 5-10 minutes at 37
°C, and then resuspended in 60 µL of D-PBS without Ca2+ (Gibco, #14190-094). 60 µL of the
cell suspension were measured in flow-cytometry experiments. Gates were adjusted based
on wild-type HEK-293T and CHO-K1 cells.
Analysis of flow-cytometry data was done using FlowJo 10 software.
Measurement of rituximab production
Levels of rituximab in cell culture media were measured using an enzyme-linked
immunosorbent assay (ELISA) kit for human IgG (Immunology Consultants Laboratory Inc., #E-
80G) following the manufacturer’s protocol. Samples from transient expression experiments
Materials & Methods
Tobias Strittmatter 126/240
were diluted 1:5, while samples from stable cell lines were diluted 1:20 in 1x assay buffer
before measurement.
Analysis of glycosylation patterns
Rituximab was produced for three days from polyclonal HEK-293T or CHO-K1 cell lines
in standard cell culture medium or in the presence of 10 mM L-glucose. Cell culture
supernatant was collected on day 3, centrifuged for 5 min at 500 rcf, filtered through a 0.2
µm syringe filter, supplemented with cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail
(Roche, #04693159001) and stored refrigerated until further use.
The culture media was concentrated and buffer exchanged with an Amicon
ultrafiltration column (100 000KDa MWCO, Millipore). Purification of IgGs from the
concentrated solution was done with using a Protein A HP SpinTrap column (cytiva,
#28903132) following the manufacturer’s instructions. All purified IgGs were used for
analysis. Proteins were reduced and alkylated following a filter-assisted sample preparation
(FASP) protocol as described previously (Hang et al. 2015). In brief samples were treated with,
dithiothreitol (DTT) in 50 mM Tris buffer (pH 8.5) at 37⁰C for 1 h, followed by alkylation with
65 mM iodoacetamide (IAA) in 50mM Tris-buffer at 37⁰C in the dark for 1 h. DTT and IAA were
removed by centrifugation and proteins were subsequently digested with 1ug of trypsin. After
trypsin-digestion, samples were desalted using a C18 Zip-Tip. Peptides were resuspended in
10 µL 2.5% acetonitrile with 0.1% formic acid before analysis on a Q-Exactive mass
spectrometer (Thermo Fischer Scientific) coupled to a nano-Acquity UPLC system (Waters).
2µL of the solution was loaded onto a Acclaim PepMap 100 trap column (75 μm Å~ 20 mm,
100 Å, 3 μm particle size) and separated on a nanoACQUITY UPLC BEH130 C18 column (75 μm
Å~ 150 mm, 130 Å, 1.7 μm particle size), at a constant flow rate of 300 nL min-1, with a column
temperature of 50 °C and a linear gradient of 2−32% acetonitrile/0.1% formic acid in 59 min,
and then 32-45% acetonitrile/0.1% formic acid in 10 min, followed by a sharp increase to 98%
acetonitrile in 2 min and then held isocratically for another 10 min. The mass spectrometer
was operated under data dependent acquisition (DDA), one scan cycle was comprised of a full
scan MS survey spectrum, followed by up to 12 sequential HCD MS/MS on the most intense
signals above a threshold of 1e4. Full-scan MS spectra (600–2000 m/z) were acquired in the
FT-Orbitrap at a resolution of 70,000 at 400 m/z, while HCD MS/MS spectra were recorded in
a FT-Orbitrap at a resolution of 35,000 at 400 m/z. HCD was performed with a target value of
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 127/240
1e5 and normalization collision energy 25 was applied. AGC target values were 5e5 for full
FTMS. For all experiments, dynamic exclusion was used with a single repeat count, 15 s repeat
duration, and 30 s exclusion duration.
Glycopeptide were analysed by Byonic 3.6 (Protein Metrics, USA) and inspected
manually. The quantification for each glycan form on IgG was performed as described
previously (Hang et al. 2015).
Statistical Analysis
No statistical analysis was performed. All data are presented as means ± standard
deviation (SD) of triplicates or duplicates as indicated.
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 129/240
Supplementary Information
Table S1: Plasmid Information
Plasmid Description Reference
pCDNA3.1 Filler plasmid used to replace PhCMV-driven active
components.
Invitrogen™
V79020
pMX157 Reporter construct used with VP16-LgnR. LgnR-operator
sequence (motif 0) is cloned 5’ of the minimal promoter
PhCMVmin (0-PhCMVmin-SEAP-pA).
This work
pMX181 Constitutive VP16-LgnR expression vector with a
constitutive PSV40 promoter (PSV40-VP16-LgnR-pA).
This work
pMX183 Constitutive KRAB-LgnR expression vector with a strong
PhEF1α promoter (PhEF1α-KRAB-LgnR-pA).
This work
pMX212 Reporter construct used with KRAB-LgnR. LgnR-operator
sequence (motif A1) is cloned 3’ of the constitutive
promoter PSV40 (PSV40-A1-SEAP-pA).
This work
pMX213 Reporter construct used with KRAB-LgnR. A tandem repeat
of LgnR binding motif A used in pMX212 is cloned 3’ of the
constitutive promoter PSV40 (PSV40-A2-SEAP-pA).
This work
pMX214 Reporter construct used with KRAB-LgnR. A three-fold
repeat repeat of LgnR binding motif A used in pMX212 is
cloned 3’ of the constitutive promoter PSV40 (PSV40-A3-SEAP-
pA).
This work
pMX215 Reporter construct used with KRAB-LgnR. LgnR-operator
sequence (motif B1) is cloned 3’ of the constitutive
promoter PSV40 (PSV40-B1-SEAP-pA).
This work
pMX216 Reporter construct used with KRAB-LgnR. LgnR-operator
sequence (motif C1) is cloned 3’ of the constitutive
promoter PSV40 (PSV40-C1-SEAP-pA).
This work
pMX217 Reporter construct used with KRAB-LgnR. A tandem repeat
of LgnR binding motif C used in pMX216 is cloned 3’ of the
constitutive promoter PSV40 (PSV40-C2-SEAP-pA).
This work
Supplementary Information
Tobias Strittmatter 130/240
pMX218 Reporter construct used with KRAB-LgnR. A three-fold
repeat repeat of LgnR binding motif C used in pMX216 is
cloned 3’ of the constitutive promoter PSV40 (PSV40-C3-SEAP-
pA).
This work
pMX220 Reporter construct used with VP16-LgnR. LgnR-operator
sequence (motif A1) is cloned 5’ of the minimal promoter
PhCMVmin (A1-PhCMVmin-SEAP-pA).
This work
pMX221 Reporter construct used with VP16-LgnR. A tandem repeat
of LgnR binding motif A used in pMX220 is cloned 5’ of the
minimal promoter PhCMVmin (A2-PhCMVmin-SEAP-pA).
This work
pMX222 Reporter construct used with VP16-LgnR. A three-fold
repeat of LgnR bindng motif A used in pMX220 is cloned 5’
of the minimal promoter PhCMVmin (A3-PhCMVmin-SEAP-pA).
This work
pMX223 Reporter construct used with VP16-LgnR. LgnR-operator
sequence (motif B1) is cloned 5’ of the minimal promoter
PhCMVmin (B1-PhCMVmin-SEAP-pA).
This work
pMX224 Reporter construct used with VP16-LgnR. LgnR-operator
sequence (motif C1) used in pMX224 is cloned 5’ of the
minimal promoter PhCMVmin (C1-PhCMVmin-SEAP-pA).
This work
pMX225 Reporter construct used with VP16-LgnR. A tandem repeat
of LgnR binding motif C used in pMX224 is cloned 5’ of the
minimal promoter PhCMVmin (C2-PhCMVmin-SEAP-pA).
This work
pMX225 Reporter construct used with VP16-LgnR. A three-fold
repeat of LgnR binding motif C used in pMX224 is cloned 5’
of the minimal promoter PhCMVmin (C3-PhCMVmin-SEAP-pA).
This work
pMX335 Reporter construct for the ON-switch, to be used with
KRAB-LgnR. LgnR-operator sequence used in pMX223 is
cloned 3’ of a strong constitutive PhCMV promoter (PhCMV- B1-
SEAP-pA)
This work
Chapter 2: Gene switch for L-glucose-induced biopharmaceutical production in mammalian cells
Tobias Strittmatter 131/240
pDF101 Inert filler plasmid bearing a bacterial T7 promoter driving
expression of an inactive ribozyme (PT7-SpAL-sTRSVac) used
to adjust plasmid amounts for transfections.
(Auslander et
al. 2016)
pTS1052 Vector for constitutive expression of secreted NanoLuc
luciferase (NLuc) and cytosolic blue fluorescent reporter
(mTagBFP) (PPGK-Igk-NLuc-P2A-mTagBFP-pA).
This work
pTS2174 Constitutive KRAB-LgnR expression vector (PhEF1α-KRAB-
LgnR-pA).
This work
pTS2178 Vector for Sleeping Beauty-mediated stable integration of
PPGK driven SMIT1 transporter and PRPBSA-driven Puromycine
and mTagBFP2 selection markers (PPGK-SMIT1-pA-PRPBSA-
mTagBFP2-P2A-PuroR-pA).
This work
pTS2179 Vector for Sleeping Beauty-mediated stable integration of
PPGK driven metabolic enzymes LgnH-P2A-LgnI and LgdA
alongside PRPBSA-driven Blasticidine and mRuby2 selection
markers (PPGK-LgnH-P2A-LgnI-pA-PPGK-LgdA-pA-PRPBSA-
mRuby2-P2A-BlastR-pA).
This work
pTS2180 Constitutive expression vector for PhEF1α driven SMIT1
expression (PhEF1α -SMIT1-pA).
This work
pTS2181 Constitutive expression vector for PPGK driven SMIT1
expression (PPGK-SMIT1-pA).
This work
pTS2182 Constitutive expression vector for PhEF1α driven LgdA
expression (PhEF1α -LgdA-pA).
This work
pTS2183 Constitutive expression vector for PPGK driven LgdA
expression (PPGK-LgdA-pA).
This work
pTS2184 Constitutive expression vector for PhEF1α driven LgnH and
LgnI expression (PhEF1α -LgnH-P2A-LgnI-pA).
This work
pTS2185 Constitutive expression vector for PPGK driven LgnH and LgnI
expression (PPGK-LgnH-P2A-LgnI-pA).
This work
pTS2228 Vector for Sleeping Beauty-mediated stable integration of
ON-system-controlled (PhCMV-B1) expression of rituximab
This work
Supplementary Information
Tobias Strittmatter 132/240
and yellow fluorescent reporter YPet. A second cassette
constitutes PhEF1α -driven expression of KRAB-LgnR. The
third expression cassette harbors a PhCMV-driven Zeocin
selection marker (PhCMV-B1-rituximab-IRES-YPet-pA-PPGK-
KRAB-LgnR-pA-PhCMV-ZeoR-pA).
pTS2230 Vector for Sleeping Beauty-mediated stable integration of
ON-system-controlled (PhCMV- B1) expression of rituximab
and yellow fluorescent reporter YPet. A second cassette
constitutes PPGK-driven expression of KRAB-LgnR. The third
expression cassette harbors a PhCMV-driven Zeocin selection
marker (PhCMV- B1-rituximab-IRES-YPet-pA-PPGK-KRAB-LgnR-
pA-PhCMV-ZeoR-pA).
This work
pTS2247 Production construct for L-glucose inducible production of
rituximab, to be used with KRAB-LgnR. LgnR-operator
sequence is cloned 3’ of a strong constitutive PhCMV
promoter. Both chains of rituximab are fused via a furin-T2A
sequence to ensure equimolar secretion of both chains and
a minimal addition of amino acids to the antibody
sequence. Yellow fluorescent reporter YPet is added via an
internal ribosome entry site (IRES) to allow for quick
assessment of induction (PhCMV-B1-rituximab_LC-Furin-T2A-
rituximab_HC-IRES-YPet-pA).
This work
pTS2248 Vector for Sleeping Beauty-mediated stable integration of
PhCMV-driven rituximab. Both chains of rituximab are fused
via a furin-T2A sequence to ensure equimolar secretion of
both chains and a minimal addition of amino acids to the
antibody sequence. Yellow fluorescent reporter YPet is
added via an internal ribosome entry site (IRES) to allow for
quick assessment of induction (PhCMV-B1-rituximab_LC-
Furin-T2A-rituximab_HC-IRES-YPet-pA-PhCMV-ZeoR-pA).
This work
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Tobias Strittmatter 133/240
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Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 137/240
Chapter 3: Controlling gene expression through mechanical cues
Chapter 3 describes a work in progress that has not yet been submitted for publication.
Author Contributions
Tobias Strittmatter1, Krzysztof Krawczyk2 and Martin Fussenegger1,3 designed the project.
Tobias Strittmatter1 performed the experiments, analyzed the results and wrote the
manuscript. Krzysztof Krawczyk2 performed initial experiments that are not covered in this
manuscript, provided plasmids and wrote the manuscript. Paul Argast1 and Peter Buchman1
adapted and constructed the shear stress induction device based on a previous publication.
Contributions to each figure
Tobias Strittmatter1: figures 1-4
Author Affiliations 1Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 2Present address: Novartis Pharma AG, CH-4002 Basel, Switzerland
3Faculty of Science, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 139/240
Abstract
In humans, mechanoperception contributes to the proper programming of cell fate during
embryonic development and also serves as the basis of touch and proprioception. Cellular
mechanoreceptors respond to their environment by mediating transient adjustments of ion
homeostasis or by triggering cellular responses through calcium-dependent alteration of gene
expression via specific signaling pathways such as the NFAT (nuclear factor of activated T-
cells) pathway. Application of mechanoreceptors to control gene expression in vivo have been
reported but their application requires supplements that need to be exogenously supplied.
Additionally, mechanoreceptors are drug targets for various diseases but current techniques
to study mechanically gated processes are often based on custom tailored microfluidic
systems which require special setups or have limited throughput. Here, we employ a
programmable, 96-well-format, shear-stress induction device to examine the effects of
imposing various mechanical loads on mammalian adherent cell lines, with the aim of
developing a platform to characterize shear-stress-triggered, calcium-mediated gene
expression. We show that a reporter construct utilizing the endogenous NFAT pathway
enables the detection of shear-stress-induced calcium responses. The presented method is
suitable for high-throughput experiments and provides a large tunable parameter space to
optimize conditions for different cell types. Our findings indicate that the device is an effective
tool to explore conditions in terms of frequency, intensity, intervals as well as extracellular
matrix composition alongside the evaluation of different combinations of mechanosensitive
proteins for mechanically activated gene expression. We also examined the influence of
culture-plate surface coatings and expression of accessory proteins on mechanically induced
gene expression. We believe our results can serve as a platform for further investigations into
shear stress-controlled gene expression in basic research and drug screening as well as the
development of truly autonomous mechanoperception for use in next-generation medicine.
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 141/240
Introduction
Mechanical cues are omnipresent in the surroundings of cells. Being it single cells of
procaryotic or eucaryotic origin, adherent cells or free-floating cells, mechanical input is
always present to guide decisions of propagation and survival. In humans these cues provide
crucial information from early stages of embryonic cell development to guide patterning of
the emerging embryo and aid in cell differentiation (Nonomura et al. 2018; Vining and
Mooney 2017) but also in the adult to sense the environment (e.g. through senses of touch,
hearing or proprioception) as well as to control various physiological processes e.g. blood
pressure (Xu et al. 2018).
Key mediators of mechanosensation are mechanosensitive proteins in the plasma
membrane of cells. Bacterial channels like the mechanosensitive channel of small or large
conductance (MscS or MscL, respectively) provide a simple blueprint for such proteins. MscS
and MscL both consist of only a single transmembrane protein that oligomerizes in the plasma
membrane of bacteria to form mechanically gated pores that are activated by local distortions
of the membrane and serve as safety valves to protect the cell from osmotic stress (Booth et
al. 2012). This simple setup triggered research MscS and MscL to study the structures and
underlying principles (Chang et al. 1998; Liu et al. 2009). Due to the much larger and more
complex structure along with the fact that the corresponding gene remained elusive for a
long time, the crystal structures of two major mechanosensors have been solved more
recently: Piezo1, the major candidate for proprioception and potentially blood pressure
regulation (Saotome et al. 2018; Wang et al. 2018) and TMC1, a likely mediator for hearing
(Pan et al. 2018).
The purpose of mechanosensitive channels is the detection of mechanical cues for
transient adjustments of ion homeostasis or triggering of cellular responses through calcium-
dependent processes. Mechanoreceptor signaling is involved in tissue reorganization and
cell-cell alignment as seen for Piezo1 (McHugh et al. 2010; Nonomura et al. 2018). Some
alternative pathways of mechanosensation were found to modulate gene expression through
transmitting mechanical cues from the outside of the cell directly to the nucleus (Kirby et al.
2018). Here, the components of the cytoskeleton, actin filaments, microtubules or
intermediate filaments would relay the mechanical stress from their anchoring points at the
plasma membrane onto the lamin scaffold inside the nucleus. The mechanical force is
ultimately relayed via Nesprin and Sun proteins that span the nuclear envelope onto the
Introduction
Tobias Strittmatter 142/240
chromatin where changes in chromatin accessibility are induced that lead to alterations in
gene expression.
On the other hand, the actin skeleton also contributes to the sensitivity of
mechanosensitive calcium channels by focusing mechanical stress sensed by the whole cell
directly onto the receptor (Zhang et al. 2015) or on focal adhesion points in the plasma
membrane (Hayakawa et al. 2008). This mechanism enables cells to adjust to changes in local
pressure and shear stress mediated by altered flow of extracellular fluids or changes in extra
cellular matrix (ECM) composition. In turns, changes in cell-cell contacts or adherence to the
ECM directly influence the mechanical properties of the tissue, which affects the ability of
each cell to sense mechanical cues.
This leaves us with several different pathways as well as mechanosensitive proteins
that could be harnessed for mechanically induced gene expression. To enable efficient
screening of multiple combinations of mechanosensitive components, we developed a
higher-throughput method to assess shear stress-induced gene expression. We employed this
method to explore ways to boost intrinsic as well as engineered mechanosensitivity of
mammalian cells to trigger gene expression from synthetic promoters. Parameters like
stimulus intensity, frequency, extra cellular calcium concentration and extra cellular matrix
composition as well as the presence of accessory proteins were assessed for their impact on
gene expression.
The presented findings serve as a platform for future explorations of mechano-
activated gene expression in mammalian cells.
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 143/240
Results
Endogenous and engineered depolarization activated gene switches
Calcium is one of the most important mediators for mechanical stress. Hence, most
mechanosensitive receptors and channels trigger changes in intracellular calcium either
directly or further downstream in the signal transduction cascade. We therefore tested a well-
established reporter construct for elevated cytosolic calcium based on NFAT-responsive
elements from the interleukin 4 (IL4) promoter cloned 5’ of a mammalian minimal promoter
(Xie et al., 2016) alongside a semi-orthogonal pathway based on an engineered calcium
sensitive tobacco etch virus (TEV) protease for use in our setup (figure 1a). In HEK-293T cells
addition of 40 mM KCl to the cell culture medium can be used to trigger a robust NFAT
response (figure 1b) most likely caused by depolarization-induced release of store-operated
calcium (Colella et al. 2008).
To enable orthogonal calcium responsive signaling that is independent of the NFAT
pathway we explored the capability of a synthetic mediator to trigger gene expression upon
KCl-mediated depolarization. This mediator consists of a split TEV protease and a calcium
responsive protein comprising a calmodulin (hCaM) moiety including the M13 helix. By fusing
the split TEV moieties on each end of the hCaM protein we expected the resulting protein to
fold up in response to calcium binding thereby reconstituting an active TEV protease similarly
to other designs (O'Neill et al. 2018b; Lee et al. 2017). To enable calcium responsive gene
expression, the reconstituted TEV protease cleaves off a tTA transcriptional activator that is
fused via a TEV cleavage site (TEVsite) to a transmembrane domain (figure 1a). Once the
TEVsite is cleaved, the tTA translocates to the nucleus activating gene expression from a
synthetic promoter equipped with 5’ response elements for tTA followed by a minimal
promoter derived from the human cytomegalo virus (PhCMV) that drives expression of a
secreted human plcental alkaline phosphatase (SEAP) reporter.
To test the TEV protease-based gene switch we first established activation of the
transcription factor in combination with a constitutively active TEV. To improve access of the
TEV protease to the membrane bound tTA, we tethered the protease to the plasma
membrane via a transmembrane domain (TM). Indeed, both, cytosolic as well as membrane
bound TEV yielded increased reporter gene expression (figure 1c). We wondered whether this
system could be also inverted by using the TEV as the mobile component while the tTA would
Results
Tobias Strittmatter 144/240
reside in the nucleus. To this end we fused the transsilencer domain KRAB to the N- or C-
terminus of the tTA transactivator separated by a TEV cleavage site. This setup gave rise to
increased reporter gene expression upon co-expression of a nuclear-targeted TEV yielding a
functional ON-type gene switch (figure 1d). However, such an inverted system would further
complicate the experimental setup making it inferior to the membrane bound tTA system. In
the next step we replaced the constitutive TEV protease by a split TEV fused to the calcium
binding protein calmodulin (hCaM). This yielded a fusion protein comprising the C-terminal
fragment of the TEV protease (CTEV), the hCaM protein, the M13 helix peptide necessary for
hCaM function and the N-terminal TEV (NTEV) fragment (CTEV-hCaM-M13-NTEV). Upon calcium
binding, the hCaM at the core of the protein would bind to the M13 peptide and undergo a
confirmational change that would bring the ends of the protein into close proximity,
reconstituting an active TEV protease. Indeed, we were able to record increased reporter
levels in response to KCl stimulation when co transfecting this hCaM-TEV construct with the
membrane-bound tTA construct (figure 1e).
We next tested a set of known modulators of the calcium response for their ability to
boost KCl-induced calcium signaling. To this end we co-transfected HEK-293T cells with the
established NFAT reporter construct together with either single channels or combinations
thereof to probe for possible synergistic effects. Although it provides lower absolute
expression levels then the TEV-tTA gene switch, the NFAT reporter requires only one but two
components making it the reporter of choice for this screen.
Amplifiers of calcium response
Among the tested channels we also selected the voltage sensitive motor protein
prestin that is involved in the sensation of hearing (Santos-Sacchi et al. 2017). Upon
depolarization, prestin contracts alongside the membrane axis causing membrane tension to
increase. We reasoned that the heightened membrane tension would increase open
probabilities of other calcium channels like transient receptor potential (TRP) channels (Shen
et al. 2015) leading to an overall sensitization of the cell. Voltage gated calcium channel (CaV)-
mediated calcium flux across the membrane amplifies the calcium response triggered by
depolarization events. Inward rectifying potassium channels (Kir) are responsible for
decreasing the membrane potential as well as improving restoration of the potential after
depolarization (Kim et al. 2004). A stronger membrane potential has been reported to
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 145/240
increase calcium responses from CaV channels and this setup was essential for the
functionality of electrically induced gene switches based on HEK-293T cells in our group
before (Krawczyk et al. 2020).
Figure 1: a) schematic representation of calcium signaling by endogenous (left box) or
engineered (right box) signaling pathways. Either pathway is triggered by elevated cytosolic
a) graphical abstract
TM_TEVsit
e_tTA
+ CTEV_h
CaM_M
13_N
TEV
TM_TEVsit
e_tTA
TM_TEVsit
e_tTA
+ TEV
reporte
r only
0
5
10
15
SEAP
[UL-1
] + 40 mM KCl
uninduced
e) Calmodulin-controlled TEV
b) NFAT reporter activation by KCl
d) invertedTEV
KRAB_TEVsite
_tTA
tTA_T
EVsite_K
RABrep o
nly0
20
40
SEAP
[UL-1
]
uninduced
+ TEV-NLS
NFAT reporter0
5
10
15
SEAP
[UL-1
]
uninduced
+ 40mM KCl
c) TEV-inducible expression
TM-TEVsite-tT
A
reporte
r only
0
25
50
75
100
125
SEAP
[UL-1
]
+ cyto. TEV
+ TM TEV
uninduced
mechanical stress
Ca2+
promoter GOI promoter GOI
orthogonal signaling
cleavage
membrane bound tTA
tTA
protease
cut site
TM
NFAT
CaM
Results
Tobias Strittmatter 146/240
calcium that is initiated by ion flux across a mechanosensitive channel like Piezo1 in the
plasma membrane. In the endogenous pathway the trimeric calcineurin A (CnA)-calcineurin
B (CnB)-calmodulin (CaM) complex is activated by calcium. The activated complex binds to
phosphorylated NFAT transcription factor and dephosphorylates it through the
phosphatase activity of CnA. Dephosphorylated NFAT can enter the nucleus and initiate
transcription from NFAT responsive promoters. In an alternative pathway we engineered a
membrane bound transcriptional activator based on the tetracycline transactivator (tTA)
protein. Here a calcium sensitive protease cleaves the tTA moiety from the membrane which
then translocates to the nucleus to initiate transcription from a corresponding synthetic
promoter. Additional channels like calcium release activated channels (CRACs) or store-
operated channels (SOC) that would amplify the calcium signal are not depicted for sake of
clarity. b) To test activity of the endogenous NFAT pathway we transfected HEK-293T cells
with a plasmid containing a NFAT responsive promoter (pMX59) that drives expression of
the reporter protein secreted human placental alkaline phosphatase (SEAP). We induced
theNFAT pathway by depolarization of the membrane by adding 40 mM of KCl to the cell
culture medium. c) Expression of a tTA responsive promoter in HEK-293T cells was recorded
in response to constitutively active tobacco etch virus (TEV) protease mediated cleavage of
a corresponding TEV cleavage site (TEVsite). A tTA transcriptional activator was fused to a
transmembrane domain (TM) via a TEVsite. Upon cleavage the tTA translocates to the
nucleus to initiate transcription. Cytosolic TEV (cyt. TEV) or membrane bound TEV (TM-TEV)
were tested. d) In an inverted setup the transsilencer domain of the Krüppel associated box
(KRAB) protein was fused to the N- or C-terminal end of a tTA via a TEVsite. Upon
localization of the TEV to the nucleus through means of an attached nuclear localization
signal (NLS) the transsilencer domain is cleaved off and tTA is able to initiate transcription.
e) By engineering a split TEV construct with its N-terminal (NTEV) or C-terminal (CTEV)
halves fused the human CaM (hCaM) protein and the M13 peptide that is essential for
stabilization of hCaM in its induced conformation, we could render TEV activity dependent
on calcium. All values are mean ± SD, N=3 biologically independent samples. Samples from
b) were taken after 24h of induction while samples from c)-e) were taken after 48h of
induction.
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 147/240
In contrast to calcium flowing into the cell from the extracellular side, store operated
calcium is a reservoir of calcium stored inside the ER which can be released into the cytosol,
triggering a calcium response. Therefore, store operated calcium works in synergy with
calcium flux across the plasma membrane. Members of the caffeine sensitive ryanoidin
receptor (RyR) family mediate calcium release from the ER (Lanner et al. 2010) while ATP
consuming pumps like sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (ATP2A1) are
counteracting the release by shuttling cytosolic calcium back into the ER especially in muscle
cells (Salazar-Cantu et al. 2016).
We assessed prestin, hCaV 1.2, Kir2.1, a mutant version of RyR2 (RyR2-D189E) and
ATP2A1 as single components as well as in combination for their ability to boost NFAT
controlled reporter gene expression in HEK-293T (figure 2a). The single biggest contributor
was found to be the expression of hCaV1.2 in all combinations. Interestingly, RyR2-D189E also
seemed to cause superior inducibility compared to the reporter control.
a) modulators of calcium response
b) mutants of RyR2 c) overexpression of NFATc1 d) overexpression of NFATc2
reporte
r ctr
Prestin
hCaV1.2
Kir2.1
RyR2
ATP2A1
Prestin + hCaV
Prestin + Kir
Prestin + RyR
2
Prestin + ATP2A1
hCaV +
Kir
hCaV +
RyR2
hCaV +
ATP2A1
Kir + RyR
2
Kir + ATP2A1
RyR2 +
ATP2A1
Prestin + hCaV
+ Kir
Prestin + hCaV
+ RyR2
Prestin + hCaV
+ ATP2A1
Prestin + Kir +
RyR2
Prestin + Kir +
ATP2A1
Prestin + RyR
2 + ATP2A1
hCaV +
Kir + RyR
hCaV +
Kir + ATP2A1
hCaV +
RyR2 + ATP2A1
Kir + RyR
2 + ATP2A1
Prestin + hCaV
+ Kir + RyR
2
Prestin + Kir +
RyR2 + ATP2A1
Prestin + hCaV
+ RyR2 +
ATP2A1
Prestin + hCaV
+ Kir + ATP2A1
Prestin + hCaV
+ Kir + RyR
2 + ATP2A1
0
20
40
60
80
SEAP
[UL-1
]
uninduced
+ 40mM KCl
RyR2-w
T + hCaV + Kir
RyR2-E
189D +
hCaV + Kir
RyR2-R
4497
C + hC
aV + Kir
reporte
r contro
l0
2
4
6
SEAP
[UL-1
] + 40 mM KCl
uninduced
10 ng
5 ng
1 ng
0.1 ng
0 ng
0
50
100
SEAP
[UL-1
] + 40mM KCl
uninduced
20 ng
10 ng
5 ng
0 ng
0
10
20
30
40
50
SEAP
[UL-1
] + 40mM KCl
uninduced
Results
Tobias Strittmatter 148/240
Figure 2: Modulators of calcium homeostasis were tested for their effect on NFAT-induced
gene expression. a) Prestin, human L-type voltage gated channel (hCaV1.2), inwardly
rectifying potassium channel 2.1 (Kir2.1), ryanodine receptor 2 (RyR2) and ATPase
sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1 (ATP2A1) were tested as single
components or in combination for their ability to enhance KCl-induced NFAT signaling. b)
Mutants of RyR2 were tested in combination with hCaV1.2 and Kir2.1 for increased
inducibility of NFAT-mediated gene expression. Key transcription factors of the NFAT
pathway (c) NFATc1 and (d) NFATc2 were tested for beneficial effects on KCl-induced gene
expression from an NFAT-responsive synthetic promoter. All values are mean ± SD, N=3
biologically independent samples.
The mutant version RyR2-D189E used in this setup was reported to result in higher
open probability (Jiang et al. 2010), however, mutant RyR2-R4496C was also found to yield
higher calcium sensitivity in previous studies (Fernandez-Velasco et al. 2009). We therefore
assessed next how the mutant versions compared to wildtype RyR2 in boosting the calcium
response (figure 2b). Indeed, both mutants showed increased calcium responses compared
to the wildtype with the strongest response being recorded using RyR2-R4496C.
We furthermore tested overexpression of key players of the NFAT pathway, NFATc1
and NFATc2 as a way to boost the sensitivity and robustness of the NFAT pathway in HEK-
293T cells. NFATc1 (figure 2c) and NFATc2 (figure 2d) both increased absolute expression
levels while slightly reducing fold switches when co-transfected with Kir2.1 and CaV1.2. We
argued that increased expression is favorable to enable detection of smaller changes in
calcium homeostasis in order to make the sometimes variable NFAT reporter performance
more robust.
Having characterized a working calcium responsive pathway in HEK-293T cells, we
now moved on to establish NFAT-mediated mechanically triggered gene expression through
application of shear stress in cell culture.
Population-based induction of shear stress through turbulent flow
Induction of shear stress on whole cell populations was achieved using a device
published for an shRNA screen targeting mechanosensitive proteins involved in calcium
signaling (Xu et al. 2018). The device consists of a 3D-printed array of 96 pistons adapted for
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 149/240
a regular 96-well format which is glued to the membrane of a loud speaker. The speaker in
turns is controlled by a signal generator based on an Arduino controller coupled to an
operational amplifier and a hi-fi stereo amplifier. When running, the pistons would move up
and down inside the cell culture wells to generate turbulent flow and hence shear stress on
the bottom of the well. First tests with the genetically encoded calcium indicator (GECI)
GCaMP6s (Chen et al. 2013) in cultured mouse neurons to record calcium signals in response
to mechanical stimulation proved the device was functional (Gaub et al. 2020).
a) shear-stress response dependent on frequency and reporter
pYL1pAT50
pHY30
pMX57
pMX58
tTA-base
d0
5
10
15
SEAP
[U/L
] 10 Hz
30 Hz
60 Hz
uninduced
b) shear-stress response of wT HEK
0
20
40
60
nLuc
/ SE
AP
[AU]
10 Hz
30 Hz
60 Hz
uninduced
4xNFAT-NLuc
c) mPiezo1 modulates response
0
20
40
60
nLuc
/ SE
AP
[AU]
10 Hz
30 Hz
60 Hz
uninduced
4xNFAT-NLuc
d) stable mPiezo1 modulates timing and duty-cycle dependent response
pcTS1 wT
0
20
40
60
80
100
nLuc
/ SE
AP
[AU]
pcTS1 wT
0
50
100
nLuc
/ SE
AP
[AU]
1.7 %
3.3 %
7.7 %
uninduced
(i) day 1 (ii) day 2
Results
Tobias Strittmatter 150/240
Figure 3: A 96-well piston device was tested for the ability to modulate calcium induced
gene expression upon application of shear stress generated through turbulent flow
produced by oscillating pistons. a) To find the best calcium responsive promoter, a set of
published synthetic reporter constructs was tested with different frequencies. Induction was
done for 5 sec every 5 min at indicated frequencies for 24h and compared to corresponding
uninduced samples. b) To account for unspecific effects a dual-reporter setup was
introduced using NFAT-responsive expression of secreted NanoLuc luciferase (nLuc) and
constitutive expression of SEAP from a weak constitutive promoter derived from the simian
virus 40 (PSV40). By normalization of nLuc activity to SEAP expression a more robust signal
was generated that showed a clear dose dependent increase of NFAT activity in HEK-293T
cells upon stimulation with the piston device. c) Mechanosensitive channel Piezo1 was
overexpressed in HEK-293T cells grown for additional 24h after transfection under standard
culturing conditions to allow for proper expression of the channel prior to stimulation as
done in b). d) To ensure more homogenous expression of Piezo1, we generated a stable HEK-
293T cell line expressing Piezo1 (pcTS1). Induction of the cell line either (i) directly after
transfection or (ii) after 24h of regeneration was assessed. All values are mean ± SD, N=3
biologically independent samples.
The used NFAT reporter construct is well characterized and proven to be sensitive to
depolarization induced perturbations in calcium homeostasis. However, stimuli from
mechanoreceptors might differ in terms of downstream signaling from KCl induced calcium
signaling. We therefore screened various published reporter constructs that employ different
combinations of NFAT binding sites derived from calcium responsive IL2 and IL4 promoters as
well as the serum response element (SRE) alongside our TEV-tTA circuit for their response to
different frequencies of shear stress inducing oscillations. We again found an IL4-derived
NFAT promoter (pMX57) to yield the biggest fold changes with a peak at 30 Hz induction
frequency (figure 3a). Unfortunately, the semi-orthogonal TEV-tTA-based gene switch that
proved to be sensitive to KCl-mediated depolarization failed to record induction from shear
stress. Hence, we focused on the NFAT pathway for further studies. Based on the promoter
used in the pMX57 construct we designed a new reporter cassette and switched the reporter
gene from SEAP to NanoLuc luciferase (nLuc). This improved sensitivity and enabled the use
of SEAP as a constitutive reference for internal normalization to compensate for possible
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 151/240
unspecific effects caused by the piston device. Functionality of the reporter system was
confirmed by shear stress induction (figure 3b).
We finally tested transient expression of Piezo1 in HEK-293T cells to boost
mechanosensitivity and hence NFAT-mediated mechanically triggered gene expression.
Piezo1 expression yielded higher overall expression but lower fold switches compared to cells
without overexpression of mechanoreceptors (figure 3c). Additionally, for Piezo1 transfected
cells we did not see an improvement when using 30 Hz or 60 Hz for induction.
a) NFAT overexpression improves signal
+ NFAT1/2
+ NFAT1
+ NFAT2
reporte
r only
0
50000
100000
150000
200000
nLuc
/ SE
AP [A
U]
uninduced
3.3 %
14.3 %
b) extracellular calcium levels
c) shear stress response in HEK-293T cells can be modulated by ECM and mechanoreceptors
d) shear stress response in CHO-K1 cells can be modulated by ECM and mechanoreceptors
1.8 m
M CaCl2
5 mM CaCl2
10 m
M CaCl2
0
200
400
600
nLuc
/ fL
uc
uninduced
+ KCl 40 mM
(i) HEK-293T (ii) HEK-293T + MscL (iii) HEK-293T + Piezo1
uncoated
+ 1:25
0 Matrig
el
+ 1:50
Matr
igel
+ 37.5
g/mL getre
x
+ 75g/m
L getrex
+ 150
g/mL getre
x0
5
10
15
nLuc
/ fL
uc
uninducedinduced
uncoated
+ 1:25
0 Matrig
el
+ 1:50
Matr
igel
+ 37.5
g/mL getre
x
+ 75g/m
L getrex
+ 150
g/mL getre
x0
200
400
600
800
nLuc
/ fL
uc
uncoated
+ 1:25
0 Matrig
el
+ 1:50
Matr
igel
+ 37.5
g/mL getre
x
+ 75g/m
L getrex
+ 150
g/mL getre
x0
200
400
600
800
(i) CHO-K1 (ii) CHO-K1 + MscL (iii) CHO-K1 + Piezo1
uninducedinduced
uncoated
+ 1:25
0 Matrig
el
+ 1:50
Matr
igel
+ 37.5
g/mL getre
x
+ 75g/m
L getrex
+ 150
g/mL getre
x0
5
10
15
uncoated
+ 1:25
0 Matrig
el
+ 1:50
Matr
igel
+ 37.5
g/mL getre
x
+ 75g/m
L getrex
+ 150
g/mL getre
x0
5
10
15
uncoated
+ 1:25
0 Matrig
el
+ 1:50
Matr
igel
+ 37.5
g/mL getre
x
+ 75g/m
L getrex
+ 150
g/mL getre
x0
200
400
600
800
1000
Results
Tobias Strittmatter 152/240
Figure 4: a) NFATc1 (NFAT1) and NFATc2 (NFAT2) were tested as single components or in
combination (NFAT1/2) in response to 5 sec each 2.5 min or 5 sec each 0.5 min of
stimulation corresponding to 3.3 % or 14.3 % duty cycle for improved shear stress induced
gene expression. To improve robustness of the system and streamline future experiments,
stable cell lines were generated by designing a vector construct comprising NFAT-responsive
nLuc expression, constitutive expression of firefly luciferase (fLuc) and a selection cassette
conferring puromycine resistance as well as promoting expression of additional NFATc1
transcription factor from an attached IRES element. Resulting stable cell lines were used to
b) test different concentrations of extracellular calcium for improved responses to KCl
induction or shear stress (10 sec each 20 sec or 33.3 % duty cycle) mediated gene expression
in c) HEK-293T or d) CHO-K1 cell lines grown on different extra cellular matrix (ECM)-
simulating compounds upon additional expression of mechanoreceptors (ii) MscL or (iii)
Piezo1. All values are mean ± SD, N=3 biologically independent samples.
Since we experienced expression of Piezo1 to be slow and heterogenous within a
transiently transfected cell population, we next selected a HEK-293T cell line stably expressing
Piezo1 (pcTS1). We speculated that cell-cell adherence might be a major contributor to
improved mechanosensitivity and we therefore used this cell line to both confirm prolonged
pre-induction cultivation to boost induction folds and to test different induction protocols
using induction times of 5 seconds every 5, 2.5 or 1 minutes (translating to duty cycles of 1.7,
3.3 and 7.7 %, respectively). We found inducibility on day 2 to be superior with higher duty
cycles promoting higher fold switches in engineered as well as wild-type cells (figure 3d).
Drawing from previous experiments (figure 2c-d), we tested expression of additional
NFATc1 and NFATc2 to improve mechanosensitivity of the NFAT pathway. Similar to what we
saw from KCl induction, expression of NFATc1 was able to improve fold switches
tremendously in response to shear stress induction in a dose dependent manner, finally
offering a working setup for population-based mechanically induced gene expression in HEK-
293T cells (figure 4a).
To simplify the experimental design, we created a reporter construct that contains the
already established NFAT-reporter cassette as well as a constitutively expressed firefly
luciferase driven by a phosphoglycerate kinase promoter (PPGK) replacing the SEAP reporter
used in previous assays. In addition to the two reporter cassettes, we added a PRPBSA-driven
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 153/240
selection cassette to the construct that expresses a polycistronic RNA comprising an infra-red
fluorescent protein (iRFP) coupled to a puromycin resistance gene (puroR) via a p2a sequence
but also contains the CDS for NFATc1 which is driven by an internal ribosome entry site (IRES)
(PRPBSA-iRFP-p2a-puroR-IRES-NFATc1). By using cytosolic versions of the luciferase reporters,
we also aimed to reduce dependency on the secretory pathway to avoid artifacts caused by
expression of multiple membrane proteins like the channels used in this study. We tested a
polyclonal stable HEK-293T cell line by assessing the effect of three different concentrations
of calcium in the cell culture medium. Standard DMEM culture medium contains 1.8 mM of
calcium. We tested whether higher concentrations of calcium in the medium provides a
stronger trigger for KCl-induced NFAT signaling. In fact, we found medium supplemented with
5 or 10 mM calcium to promote higher fold switches compared to the standard formulation
(figure 4b). From there on we changed the standard medium composition to also contain a
final concentration of 10 mM calcium during shear stress induction.
Further experiments were conducted to characterize shear stress-responses of the
stable HEK-293T reporter cell line when grown on different surface coatings (Matrigel or
geltrex) or when transfected with mechanoreceptors MscL or Piezo1 (figure 4c). Induction
was performed using 30 Hz and a 10-sec-on-20-sec-off protocol (translating to 33 % duty
cycle). While surface coatings were ineffective in changing inducibility in untransfected stable
cells (figure 4c, i), low-density Matrigel coatings showed improved fold-switches in cells
transfected with MscL (figure 4c, ii). However, this could not be reproduced in cells
transfected with Piezo1 (figure 4c, iii), possibly due to a shortened incubation time of 24h.
In contrast, when conducting the same set of experiments in a stable CHO-K1 cell line,
we found opposite effects. For one, cell surface coatings showed increased performance even
in untransfected cells, with 37.5 µg/mL geltrex showing highest but 150 µg/mL geltrex lowest
inducibility (figure 4d, i). On the other hand, this cell line showed a strong increase in fold-
switches when transfected with mechanoreceptors MscL (figure 4d, ii) or Piezo1 (figure 4d,
iii) with low-density Matrigel or 37.5 µg/mL geltrex providing the biggest increase while
responses from cells grown in uncoated wells stayed the same.
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 155/240
Discussion
This study provides a platform for further characterization of shear stress-triggered,
calcium-mediated gene expression in mammalian adherent cell lines. We present two
different gene circuits that can be used for this purpose based on the endogenous NFAT
pathway and an engineered split-TEV-calmodulin fusion protein which enables the detection
of KCl-induced calcium signals in a semi-orthogonal manner. Intriguingly, induction of calcium
signaling via shear stress seems to trigger different downstream pathways than KCl-mediated
depolarization as both methods induce a different set of known genetic reporters for calcium
signaling (figure 2a). The inability to record calcium responses via the TEV-tTA switch might
lie in a higher concentration of intracellular calcium needed for activation as well as prolonged
duration of calcium transients. The lack of an amplification step as it is typically seen in native
signaling pathways, including the NFAT pathway, furthermore makes the TEV-tTA switch
more dynamic but also less sensitive. Since a similar strategy was reported to work with low
fold switches in HEK-293T cells (Wang et al. 2017) but exceled in neurons (O'Neill et al. 2018a;
Wang et al. 2017), which exhibit much larger changes in calcium levels upon stimulation,
future iterations of the TEV-tTA design should aim to render the construct more sensitive to
work at fold changes that are within the range of non-neuronal cell types.
While the L-type calcium channel CaV1.2 was already successfully employed in
therapeutic settings using electrically induced or depolarized cells to treat diabetes and
chronic pain (Xie et al. 2016; Wang et al. 2018; Krawczyk et al. 2020), the potential of the
other big contributor to calcium signaling, RyR2 channels, has yet to be explored in this
regard. Since RyR2 channels synergize with the established CaV-Kir-combination one could
expect superior sensitivity of the resulting gene network. Each of the channel families again
contains additional members that could be characterized. Hence, the selection studied here
is by far not comprehensive. Because of the sophisticated architecture of some of the
channels that involves multiple large subunits (Zamponi et al. 2015), studying responses by
means of transient expression might be challenging.
The differences in reporter signals seen in response to shear stress versus KCl-
mediated depolarization already indicate different pathways being triggered by each
stimulation method. Therefore, reporters that tap into alternative signaling pathways e.g.
cytoskeleton-mediated pathways (Kirby et al. 2018; Zhang et al. 2015) might provide an
additional means to realize mechanically induced gene expression.
Discussion
Tobias Strittmatter 156/240
Intriguingly extracellular matrix-simulating coatings like matrigel and geltrex provided
a strong boost in inducibility when used in combination with mechanoreceptors MscL in HEK-
293T as well as MscL and Piezo1 in CHO-K1 cells. This might indicate a potential mechanism
of activation of these receptors and should be carefully considered in subsequent
experiments. Dependency of mechanosensitivity on ECM was reported before for Piezo1
(Gaub et al. 2017).
Future research should focus on expanding the repertoire and knowledge about
mechanosensitive proteins that could be used to boost shear stress induced gene expression
as well as filling in the gaps of the parameter space of the induction protocol. The device itself
would greatly benefit from a way to calibrate the intensities of the provided oscillations to
reduce variability between devices. A further splitting of the array into subgroups that could
be individually controlled would be useful, too. Finally, other ways of inducing shear stress
that are not based on turbulent flow but would include laminar flow as well, should be
explored. Here, a small propeller or turbine could be considered that could be individually
controlled and would allow for rotation in both directions to induce laminar or turbulent flow
from the same setup.
We are convinced, that the presented method for shear stress mediated gene
expression can be useful in applications beyond basic research for example in screenings for
compounds targeting mechanosensitive signaling pathways and receptors in a more holistic
model that does not look at calcium transients alone but includes the underlying signaling
cascades as well. On the other hand, mechanosensitive circuits could not only be used in
combination with exogenous stimulation but could also sense physiological stimuli like flow
rate inside the vasculature. In potential future-medicine applications, cells that contain
mechanosensitive gene circuits, could potentially be employed to detect aberrant levels of
blood pressure in the context of cardio-vascular diseases or as part of stent implants to report
on or even counteract harmful occlusion events.
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 157/240
Materials & Methods
Cell culture
HEK293-T cells and CHO-K1 cells were cultured in 10 cm cell culture dishes in
Dulbecco’s Modified Eagle Medium (DMEM, gibco, #61965-026) supplemented with 10 %
FCS, 50 Units/mL Penicillin and 50 µg/mL of Streptomycin (gibco, #15070-063) in a humidified
atmosphere containing 7.5 % CO2. Medium was supplemented with 150 µM of L-proline for
culturing of CHO-K1 cells. All cell lines were split using Trypsin-EDTA (gibco, #25300-054)
every two days or when growing to more than 70 % confluency and reseeded at 1.5 million
cells. For cell culture experiments, cells were seeded at indicated concentrations (15 000, 30
000, 45 000 or 60 000 cells per well) in 96-well plates (Corning, #3599) or 250 000 cells per
well in a 6-well plate (Corning, #3516) for generation of stable cell lines.
Plasmid generation and transfection
All plasmids were cloned by standard molecular cloning techniques. See table 1 for a
comprehensive list of plasmids and the genetic elements they carry. Details on amounts of
transfected plasmids for each experiment can be found in table 2.
Polyethyleneimine (PEI, 24765-1, Polysciences Inc.) was used for transfection of
plasmid DNA in a ratio of PEI:DNA of 6:1. 24 h prior to transfection cells were seeded as
described above. 200 ng or 1 000 ng was mixed with 150 µL or 500 µL of serum-free DMEM
and 1.2 µg or 6 µg PEI (1.2 µL or 6 µL of a 1 mg/mL stock solution) was added for transfecting
one well of a 96-well plate or 6-well plate, respectively. The mixture was incubated at room
temperature for 5-10 minutes before adding it to the cells. Cells were incubated overnight
(>12 h) under standard culturing conditions. Medium was replaced by standard culture
medium (as described above) the next morning and inducer molecules were added as
necessary.
Stimulation of shear stress using the piston device
A description of the device and along with a schematic drawing can be found in the
main text. Each device is controlled by an Arduino controller coupled to an operational
amplifier with manual potentiometer. The resulting signal (intervals of signals of 3-60 Hz) is
fed into the speaker via a commercial hi-fi stereo amplifier. A gyrostat sensor attached to the
Materials & Methods
Tobias Strittmatter 158/240
membrane of the speaker measures the force on the array during operation reporting
changes in acceleration in volt and thereby provides a means to calibrate and adjust induction
intensities. This setup allows for the generation of fully customizable stimulation patterns by
allowing for selection of specific frequencies, intensities and intervals.
Experiments with this shear stress induction device were carried out in standard cell
culture incubators. Pistons were cleaned before each use by incubation in 1 M NaOH for at
least 10 min. Pistons were rinsed with ddH2O and dried in a cell culture flow-hood with 30
min of UV-sterilization. Devices were pre-warmed for a couple of hours in the cell culture
incubator to allow condensation to evaporate.
Generation of stable cell lines
For generation of stable cell lines cells were seeded in 6-well plates and transfected
with donor vectors for the sleeping beauty transposase system as described above using 600
ng of donor vector and 400 ng of pTS395 (PhCMV-SB100X-pA). Selection was done using 1
µg/mL of puromycine (invivogen, #ant-pr) for at least 10 days.
HEK-293T cells stably expressing mammalian Piezo1 were generated by transfecting
pTS391 and pTS395 while HEK-293T and CHO-K1 cells carrying the NFAT-dependent NanoLuc
luciferase (NLuc) reporter, NFATc1 expression cassette and constitutive expression of firefly
luciferase (fLuc) were generated using pTS2258 and pTS395.
Assessment of reporter gene expression
Activity of the reporter protein secreted alkaline placental phosphatase (SEAP) was
performed as previously described (Berger et al. 1988). In short, supernatant from cell culture
experiments was heat treated for 30 min at 65 °C to inactivate endogenous phosphatases. 20
µL of heat-treated supernatant was mixed with 60 µL of water and 80 µL of 2x assay buffer (1
M diethanolamine pH 9.8, 0.5 mM MgCl2, 10 mM L-homoarginine). Reaction was started by
the addition of 20 µL substrate solution (120 mM p-nitrophenylphosphate in 2x assay buffer).
Reporter activity was assessed by measuring the reaction kinetics at 37 °C by following
substrate turn-over at 405 nm in an Infinite M1000 microplate reader (Tecan Trading AG).
NanoLuc Luficerase (NLuc) activity from cell culture supernatant was recorded as
recommended by the manufacturer. 7.5 µL of Nano-Glo® Assay Reagent was mixed with
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 159/240
7.5 µL of supernatant per well of a 384-well plate and luminescence was measured in an
Infinite M1000 microplate reader (Tecan Trading AG) with 100 ms integration time.
NanoLuc Luficerase (NLuc) and Firefly Luciferase (FLuc) activity was measured from
cell lysates using Nano-Glo® Luciferase Assay (Promega) and ONE-Glo™ Luciferase Assay
(Promega) kits. To this end supernatant of cells was aspirated and replaced by 100 µL of 1x
Passive Lysis Buffer (Promega). Cells were incubated for up to 30 min at room temperature
while shaking at 400 rpm. 7.5 µL of Nano-Glo® or One-Glo™ Assay Reagent was mixed with
7.5 µL of lysate per well of a 384-well plate. Luminescence was recorded with 100 ms
integration time on an Infinite M1000 microplate reader (Tecan Trading AG).
Statistical Analysis
No statistical analysis was performed. Generally, data is presented as bar graphs
representing means ± SD of N=3 or N=6 biologically independent samples.
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 161/240
Supplementary Information
Table S1: Plasmid Information
Plasmid Description Reference
mPiezo1-
IRES-
eGFP
Mammalian expression vector of mammalian Piezo1 coupled
to internal ribosome entry site (IRES)-mediated expression of
enhanced green fluorescent protein (eGFP) from a constitutive
promoter derived from the human cytomegalo virus (PCMV)
(PCMV-CaV1.2-pA)
(Coste et al.
2010)
hCav1.2 Mammalian expression vector for channel forming subunit α1
of mammalian voltage gated channel 1.2 (CaV1.2) of red
fluorescent protein from a constitutive promoter derived from
the human cytomegalo virus (PCMV) (PCMV-CaV1.2-pA)
(Helton, Xu,
& Lipscombe,
2005)
pKK5 Mammalian expression vector for inward rectifying potassium
channel 2.1 (Kir2.1) from a constitutive promoter derived from
the human cytomegalo virus (PCMV) (PCMV-Kir2.1-pA)
(Krawczyk et
al. 2020)
RyR2 Mammalian expression vector for expression of wild type
ryanodine receptor 2 (RyR2) from a constitutive promoter
derived from the human cytomegalo virus (PCMV) (PCMV-RyR2-
pA)
(Jiang et al.
2010)
RyR2-
E189D
Mammalian expression vector for expression of mutant
ryanodine receptor 2 E189D (RyR2-E189D) from a constitutive
promoter derived from the human cytomegalo virus (PCMV)
(PCMV-RyR2-E189D-pA)
(Jiang et al.
2010)
RyR2-
R4496C
Mammalian expression vector for expression of mutant
ryanodine receptor 2 R4496C (RyR2-R4496C) from a
constitutive promoter derived from the human cytomegalo
virus (PCMV) (PCMV-RyR2-R4496C-pA)
(Jiang et al.
2010)
pFS29 Mammalian expression vector for expression of red fluorescent
protein mCherry from a constitutive promoter derived from
simian virus 40 (PSV40) (PSV40-mCherry-pA)
(Sedlmayer
et al. 2018)
Supplementary Information
Tobias Strittmatter 162/240
pDF101 Inert filler plasmid bearing a bacterial T7 promoter driving an
inactive ribozyme (PT7-SpAL-sTRSVac)
(Auslander et
al. 2018)
pFOX6 Mammalian expression vector for expression of red fluorescent
protein tdTomato from a constitutive promoter derived from
the human cytomegalo virus (PCMV) (PCMV-tdTomato-pA)
unpublished
pFOX6 Mammalian expression vector for expression of yellow
fluorescent protein YPet from a constitutive promoter derived
from the human cytomegalo virus (PCMV) (PCMV-YPet-pA)
unpublished
pSEAP2-
Control
Constitutive SEAP expression vector (PSV40-SEAP-pA). Clonetech
pMX57 Reporter plasmid for NFAT-induced expression of SEAP from a
NFAT responsive synthetic promoter containing five response
elements derived from the IL4 promoter (PNFAT5). (PNFAT5-
Citrine-2A-SEAP-pA)
(Xie et al.
2016)
pMX58 Reporter plasmid for NFAT-induced expression of SEAP from a
NFAT responsive synthetic promoter containing seven
response elements derived from the IL4 promoter (PNFAT7).
(PNFAT7-Citrine-2A-SEAP-pA)
(Xie et al.
2016)
pDA326 Reporter plasmid for tTA induced expression of yellow
fluorescent protein citrine and SEAP from a TREB1 promoter. In
order to reduce leakiness of the induction a destabilizing
ribozyme (sTRSV) is added to the 3’ of the construct. (PTREBI-
Citrine-2A-SEAP-sTRSV-pA)
(Auslander et
al. 2018)
pPST218 Mammalian expression vector for expression of tobacco etch
virus protease (TEV) from a constitutive promoter derived from
the human cytomegalo virus (PCMV) (PCMV-TEV-pA)
unpublished
pLeo7 Mammalian expression vector for expression of tobacco etch
virus protease (TEV) bound to the membrane by secretion
signal (SS) mediated insertion of the transmembrane domain
(TM) of the platelet-derived growth factor receptor (PDGFR)
from a synthetic constitutive promoter made up of parts from
unpublished
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Tobias Strittmatter 163/240
a promoter derived from the human cytomegalo virus, the
chicken beta-actin promoter and the rabbit beta-globin splice
acceptor (CAG; PCAG) (PCAG-SS-TM-TEV-pA)
pMM545 Mammalian expression vector for expression of yellow
fluorescent protein citrine from a constitutive promoter
derived from the human cytomegalo virus (PCMV) (PCMV-citrine-
pA)
unpublished
pVH288 Mammalian expression vector for expression of yellow
fluorescent protein citrine and SEAP from the constitutive
phosphoglycerate kinase promoter (PPGK) (PPGK-Citrine-p2A-
SEAP-pA)
Haellmann et
al.
(unpublished)
pYL01 Calcium sensitive reporter plasmid expressing SEAP reporter
protein.
(Liu et al.
2018)
pHY30 Calcium sensitive reporter plasmid expressing SEAP reporter
protein.
(Ye et al.
2011)
pAT50 Calcium sensitive reporter plasmid expressing SEAP reporter
protein.
(Tastanova et
al. 2018)
pTS75 PCMV-driven mammalian expression vector for a calpain-
induced tTA transactivator. tTA is inserted and anchored in the
plasma membrane via a secretion signal (SS) fused to a
transmembrane domain (TM) and a linker sequence containing
a calpain cleavage site (CCS). (PCMV-SS-TM-CCS-tTA)
This work
pTS87 Mammalian expression vector for expression of rat calpain
from a weak constitutive promoter derived from simian virus
40 (PSV40). (PSV40-calpain-pA)
This work
pTS90 Mammalian expression vector for expression of the small
calpain subunit from a weak constitutive promoter derived
from simian virus 40 (PSV40). (PSV40-calpain-pA)
This work
pTS96 PCMV-driven mammalian expression vector for a potential TEV-
induced tTA transactivator. tTA is localized to the cytosol via a
nuclear export signal (NES) fused via linker sequence containing
This work
Supplementary Information
Tobias Strittmatter 164/240
a TEV cleavage site (TEVsite) to a tTA carrying a nuclear
localization signal (NLS). (PCMV-NES-TEVsite-tTA-NLS)
pTS99 PCMV-driven mammalian expression vector for a TEV-induced
tTA transactivator. tTA is inserted and anchored in the plasma
membrane via a secretion signal (SS) fused to a transmembrane
domain (TM) and a linker sequence containing a TEV cleavage
site (TEVsite). (PCMV-SS-TM-TEVsite-tTA)
This work
pTS229 PCMV-driven mammalian expression vector for a split TEV
construct based on a human calmodulin (hCaM) that is fused to
the M13-peptide. C-terminal and N-terminal split TEV
fragments (CTev and NTev, respectively) are fused to the N-
terminus and C-terminus of the hCaM core protein. (PCMV-CTev-
hCaM-M13-NTev)
This work
pTS304 Mammalian expression vector of tobacco etch virus protease
(TEV) localized to the nucleus via a nuclear localization signal
(NLS) driven by a constitutive promoter derived from the
human cytomegalo virus (PCMV) (PCMV-TEV-NLS-pA)
This work
pTS305 PCMV-driven mammalian expression vector of TEV-induced tTA
transactivator. tTA-mediated expression is blocked by a KRAB
transsilencer domain fused to the N-terminus of tTA via a linker
sequence containing a TEV cleavage site (TEVsite) (PCMV-KRAB-
TEVsite-tTA)
This work
pTS307 PCMV-driven mammalian expression vector of TEV-induced tTA
transactivator. tTA-mediated expression is blocked by a KRAB
transsilencer domain fused to the C-terminus of tTA via a linker
sequence containing a TEV cleavage site (TEVsite) (PCMV-tTA-
TEVsite-KRAB)
This work
pTS391 Vector for stable integration of two expression cassettes
flanked by insertion and recognition sites of sleeping beauty
transposase (SB). Cassette one contains a PCMV-driven
mammalian Piezo1 (mPiezo1). Cassette two comprises a PRPBSA-
This work
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 165/240
driven blue fluorescent protein (BFP) coupled to a puromycin
resistance marker (puroR). (SB-(1)CMV-mPiezo1-pA-
(2)RPBSA_BFP_p2a_PuroR-pA-SB)
pTS395 Mammalian expression vector for SB100 sleeping beauty
transposase driven by a constitutive promoter derived from the
human cytomegalo virus (PCMV) (PhCMV-SB100-pA).
This work
pTS737 Mammalian expression vector for expression of prestin from a
constitutive promoter derived from the human cytomegalo
virus (PCMV) (PCMV-prestin-pA) prestin was isolated from cDNA
clone HsCD00436673 (Yang et al. 2011)
This work
pTS774 Mammalian expression vector for expression of
sarcoplasmic/endoplasmic reticulum calcium ATPase 1
(ATP2A1) from a constitutive promoter derived from the
human cytomegalo virus (PCMV) PCMV-ATP2A1-pA. ATP2A1 was
extracted from cDNA clone HsCD00731696.
This work
pTS775 Mammalian expression vector for expression of nuclear factor
and activator of transcription c1 (NFATc1) from a constitutive
promoter derived from the human cytomegalo virus (PCMV)
(PCMV-NFATc1-pA)
This work
pTS776 Mammalian expression vector for expression of nuclear factor
and activator of transcription c2 (NFATc2) from a constitutive
promoter derived from the human cytomegalo virus (PCMV)
(PCMV-NFATc2-pA)
This work
pTS1054 Mammalian expression vector for expression of bacterial
mechanosensitive channel of large conductance (MscL) from a
constitutive promoter derived from the human cytomegalo
virus (PCMV) (PCMV-MscL-pA)
This work
pTS2010 Reporter plasmid for NFAT-induced expression of secreted
nLuc reporter and blue fluorescent protein mTagBFP2 from a
NFAT responsive synthetic promoter containing four response
This work
Supplementary Information
Tobias Strittmatter 166/240
elements derived from the IL4 promoter (PNFAT4). (PNFAT4-SS-
nLuc-P2A-mTagBFP2-pA)
pTS2258 Vector for stable integration of three expression cassettes flanked by insertion and recognition sites of sleeping beauty transposase (SB). First cassette carries a reporter construct for NFAT-induced gene expression of cytosolic nLuc reporter coupled to blue fluorescent protein (mTagBFP2). Second cassette contains infra-red fluorescent reporter protein (iRFP) coupled to a resistance gene for puromycin (puroR) driven by a synthetic RPBSA promoter (PRBSA) and an ORF encoding nuclear factor and activator of transcription c1 (NFATc1) driven by an internal ribosome entry site (IRES). The third cassette enables expression of cytosolic firefly luciferase (fLuc) reporter for internal normalization of nLuc expression from a constitutive phosphoglycerate kinase promoter (PPGK). (SB-(1)PNFAT4-nLuc-P2A-mTagBFP2-pA-(2)RPBSA-iRFP-P2A-PuroR-IRES-NFATc1-pA-(3)PPGK-fLuc-pA-SB)
This work
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 167/240
Table S2: Plasmids transfected in each experiment
Details on the transfection procedure are given in the methods section.
Figure Plasmids used ng per well Goal of
experiment
1 b) pMX58
pDF101
50
ad. to 200
Verify KCl-induced
expression of
NFAT-responsive
promoter
1 c) pTS99
pPST218/pLeo7
pDA326
pFS29
100
0/10
40
ad. to 250
Test inducible TEV
reporter construct
with cytosolic TEV
and membrane
bound TEV
1 d) pTS305/307
pTS304
pDA326
pMM545
20
40
50
ad. to 250
Test inverted
KRAB-based TEV
reporter construct
1 e) pTS299
pTS99
pDA326
pFOX6
50
16.7
50
ad. to 250
Test calmodulin-
based split TEV
calcium reporter
construct
2 a) pTS737/hCaV/pKK5
RyR2-E189D
pTS774
pMX58
pDF101
0/5
0/7.5
0/10
50
ad. to 200
Test various
channel proteins
for improved
performance of
NFAT reporter
upon KCl
induction
2 b) RyR2/RyR2-E189D/RyR2-R4496C
hCaV
pKK5
5
50
12.5
Test mutant
versions of RyR2
channel protein
Supplementary Information
Tobias Strittmatter 168/240
pMX57
pFOX6
50
ad. to 200
for improved
performance of
NFAT reporter
upon KCl
induction
2 c) pTS775
hCaV
pKK5
pMX58
pDF101
20/10/5/0
5
5
50
ad. to 200
Test effects of
NFATc1
overexpression on
NFAT responses
with KCl induction
2 d) pTS775
hCaV
pKK5
pMX58
pDF101
10/5/1/0.1/0
5
5
50
ad. to 200
Test effects of
NFATc2
overexpression on
NFAT responses
with KCl induction
3 a) pMX57/58/pYL01/pHY30/pAT50
pDF101
50
ad. to 200
Screen for optimal
reporter construct
for shear stress
induced calcium-
dependent gene
expression
3 b) pTS2010
pSEAP2-Control
pDF101
50
50
ad. to 200
Dependency of
shear stress
induced NFAT-
mediated gene
expression on
frequency
3 c) mPiezo1-IRES-eGFP
pTS2010
pSEAP2-Control
pDF101
10
50
50
ad. to 200
Effect of mPiezo1
overexpression on
shear stress
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 169/240
induced gene
expression
3 d) pTS2010
pSEAP2-Control
pDF101
50
50
ad. to 200
Dependency of
shear stress
induced NFAT-
mediated gene
expression on
timing in stable
cell line and wT
cells
4 a) pTS2010
pVH288
pTS775
pTS776
pFS29
80
40
10/0
1/0
ad. to 200
Test effects of
NFATc1 or NFATc2
overexpression on
NFAT responses
under shear stress
4 c)-d) mPiezo1-IRES-eGFP/pTS1054/pFox8
pFS29
10
ad. to 200
Assess effects of
coatings and
overexpression of
mechanoreceptors
MscL and mPiezo1
on NFAT activity
Chapter 3: Controlling gene expression through mechanical cues
Tobias Strittmatter 171/240
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Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 177/240
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control
Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Chapter 4 describes a work in progress that has not yet been submitted for publication.
Author Contributions
Tobias Strittmatter1, Viktor Haellman1 and Martin Fussenegger1,3 designed the project and
wrote the manuscript. Tobias Strittmatter1 and Viktor Haellman1 contributed equally and
performed experiments, analyzed results and worked on the manuscript. Paul Argast1 and
Peter Buchman1 adapted and constructed the shear stress induction device based on a
previous publication.
Contributions to each figure
Tobias Strittmatter1 and Viktor Haellman1: figures 1-3, S1
Author Affiliations 1Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 2Present address: Novartis Pharma AG, CH-4002 Basel, Switzerland
3Faculty of Science, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 179/240
Abstract
Mechanical cues form the basis for many phenomena in biology. Mechanosensation enables
us to engage with our surroundings through senses of touch and hearing but also provides a
means for self-awareness (proprioception). Additionally, embryonic development and
patterning are aided by the mechanical properties of the surrounding tissue. In this chapter,
we explore what effects exogenous mechanical stimuli have on differentiation during germ
layer formation, with a specific focus on mesoderm differentiation. We specifically identified
mechanosensitive Wnt-β-catenin signaling to be modulated through shear stress leading to
alterations in lineage specification during ectoderm and mesoderm differentiation. We
further show that this effect is transient and reversible, and can be used to guide mesoderm
differentiation towards cardiac mesoderm in a stage-specific manner.
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 181/240
Introduction
Mechanical cues enable adults to probe their environment through the sense of touch
and hearing but also allow for proprioception and control of various other physiological
processes (e.g. blood pressure) (Xu et al. 2018). During embryogenesis, they guide the
fundamental processes of patterning of the embryo (Nonomura et al. 2018) and support
correct differentiation of cells to form functional tissues and organs (Vining et al. 2017).
Establishing the stage-specific role of mechanical cues during differentiation is not only
important for understanding embryonic development, but may also provide clues for more
efficient cell differentiation in vitro towards high-value cell types needed for regenerative
medicine, drug screening and disease modelling.
Wnt-β-catenin signaling is a highly conserved cell-cell communication system
regulating stem cell renewal, proliferation, and differentiation during embryogenesis and in
adult tissues to regulate tissue homeostasis (Logan and Nusse 2004). In embryonic stem cells
(ESCs), Wnt-β-catenin signaling levels are involved in regulating the transition between the
naïve and the primed ESC state, with high levels of Wnt-β-catenin signaling promoting
pluripotency and self-renewal in the naïve state (ten Berge et al. 2011; Xu et al. 2016).
Oppositely, in the primed state, Wnt-β-catenin signaling plays a crucial role in controlling
differentiation (Davidson et al. 2012; Kurek et al. 2015), where the temporal and stage-
specific activation Wnt-β-catenin signaling is involved in specifying the lineage trajectory
(figure 1a). For example, during the initial germ layer specification, activation of Wnt-β-
catenin directs the differentiating cell towards primitive streak specification (mesoderm and
endoderm progenitors state) (Loh et al. 2014; Loh et al. 2016), while no activation or
inactivation directs the cell towards ectoderm specification (Tchieu et al. 2017).
External mechanical forces (shear stress), lateral cell-cell interactions, and mechanical
properties (stiffness) of the extracellular matrix are putative factors that modulate Wnt-β-
catenin signaling in diverse cells and tissues. In endothelial cells and colon cancer cells, shear
stress inhibits Wnt-β-catenin signaling through the induction of Dickkopf Wnt signaling
pathway inhibitor 1 (DKK1) expression, an antagonist of the Wnt-β-catenin pathway (Li et al.
2016; Avvisato et al. 2007). Oppositely, studies in endothelial cells and osteoblasts have
shown that shear stress can also activate Wnt-β-catenin signaling potentially through
increased secretion of Wnt ligands (Cha et al. 2016; Cha et al. 2018) or reduced degradation
of β-catenin through increased phosphorylation of both AKT and glycogen synthase kinase 3β
Introduction
Tobias Strittmatter 182/240
(GSK-3β) (Norvell et al. 2004). In osteoblasts and mesenchymal stem cells, shear stress has
also been shown to increase Wnt-β-catenin signaling through disruption of cadherin/β-
catenin cell-cell interaction complexes, resulting in increased β-catenin nuclear translocation
(Norvell et al. 2004; Arnsdorf et al. 2009). During differentiation of ESCs, the relative
abundance of cadherin/β-catenin complexes, as determined by lateral cell-cell interactions
and the extracellular matrix's mechanical properties, contributes to reinforcing Wnt-β-
catenin signaling during subsequent differentiation (Azarin et al. 2012; Kinney et al. 2013;
Przybyla et al. 2016).
Various techniques exist to induce shear stress on cells utilizing microfluidic chips or
slides that allow for the application of tunable mechanical load on adherent or trapped
suspension cells in narrow channels. These techniques are often used in low-throughput
experiments to study single cells or small cell populations with great precision via atomic force
microscopy (Krieg et al. 2019), patch-clamp (Haswell et al. 2011) or microfluidic devices
(Huber et al. 2018). In this study, human induced pluripotent stem cells (hiPSCs) are exposed
to shear stress, i.e. turbulent flow, generated by a 96-well piston array adapted from a
previously developed device (Xu et al. 2018) (figure 1b). This device consists of a custom-
made 3D-printed array of 96 pistons driven by a membrane speaker controlled by an Arduino
controller coupled to an operational amplifier in series with a commercial hi-fi stereo
amplifier. Previous studies have demonstrated how this device can efficiently induce whole-
population shear stress stimulation on adherent cells grown in 384-well plates to screen for
mechanosensitive membrane receptors (Xu et al. 2018) or to study mechanically triggered
responses of isolated neurons in a 96-well format (Gaub et al. 2020)
When applied to hiPSCs during germ layer specification, we found differentiation of
hiPSC into ectoderm and mesoderm to be differentially affected by mechanical stimulation.
In contrast, stimulation during endoderm specification had no observable effect. To further
characterize the role that mechanical forces play in regulating Wnt-β-catenin signaling during
mesoderm (ME) specification, we generated a stable hiPSC reporter cell line equipped with
an established Tcf-3-responsive promoter (Molenaar et al 1996). By modulating shear stress
induction during ME specification, we show that shear stress can reversibly inhibit Wnt-β-
catenin signaling. Furthermore, we show that ME specification into paraxial or lateral
mesoderm lineages can be altered depending on the day of stimulation during differentiation.
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 183/240
Figure 1: a) iPSCs can differentiate into all three germ layers. While ectoderm derives from
early progenitor cells, endoderm and mesoderm lineages share cells from the primitive
streak as a common ancestor. Cells from the primitive streak can furthermore commit into
an anterior or middle primitive streak compartment with middle primitive streak giving rise
to lateral mesoderm and anterior primitive streak bifurcating into definite endoderm or
paraxial mesoderm. Wnt signaling inversely controls discrimination between paraxial and
lateral mesoderm. Paraxial mesoderm formation is dependent on Wnt signaling whereas
a) lineage committment
iPSC
primitivestreak
anter ior
middle
Wnt
Wnt
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10050
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Introduction
Tobias Strittmatter 184/240
formation of lateral mesoderm requires inhibition of Wnt. b) Schematic of a device for
induction of turbulent flow in cell culture. A 3D-printed array of 96 pistons is attached to the
membrane of a loud speaker that sits on top of a standard 96-well cell culture plate. A sine
signal (10 or 30 Hz) is generated by an Arduino controller in combination with a custom-
made operational amplifier that allows for adjustment of frequencies through a
potentiometer. The resulting signal is fed into a regular hi-fi stereo amplifier that drives the
speaker. The device is built into a chamber with connections for live support. During
operation the pistons move up and down inside each well generating turbulent flow. After
each run supernatant as well as RNA from cells can be harvested and analyzed. c)-e) hiPSCs
were differentiated using a commercial Tirlineage Differentiation Kit in the presence and
absence of turbulent flow and expression levels of selected representative genes were
measured using qPCR. Expression levels of key marker genes for c) ectoderm, d) mesoderm
and e) endoderm were normalized to an unstimulated control. f) A reporter hiPSC line for
activation of the beta-catenin/Wnt pathway was generated using an established Tcf-8-
responsive promoter driving expression of a cytosolic fast Fluorescent Timer (fast FT) and a
secreted NanoLuc luciferase (NLuc) reporter. fastFT matures from an early blue fluorescent
form into a red fluorescent reporter protein. Ratios of blue over red are plotted alongside
levels of secreted NLuc reporter. Activation of Wnt was simulated by titration of GSK-3
inhibitor CHIR99021. g) The reporter cell line characterized in f) was used to assess effects
of turbulent flow produced by the piston array on Wnt signaling over the course of three
days with daily media exchange. Values in b)-d) are means ± SD of N=4 biologically
independent samples. Values in e) are means ± SD of N=6 biologically independent samples
and values in f) are means ± SD of N=8 biologically independent samples. *: p < 0.05, **: p
< 0.01, ***: p < 0.001, ****: p < 0.0001.
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 185/240
Results
To assess the effect of shear stress during differentiation and how it affects lineage
commitment during generation of the three germ layers (ectoderm, endoderm and
mesoderm), hiPSCs were specified towards ectoderm for 5 days, endoderm for 3 days, or
mesoderm for 3 days (figure 1c-e). During differentiation, the cells were either left
undisturbed or cultured with turbulent flow applied by the piston array. Quantifying the
relative expression levels of key lineage markers in day 5 ectoderm cells revealed a distinctly
altered expression pattern suggesting increased differentiation into non-neural ectoderm
cells caused by the turbulent flow (figure 1c). Furthermore, day 3 mesoderm demonstrated
an apparent reduction of lateral mesoderm genes in favor of paraxial mesoderm (figure 1d).
Taken together, this suggests that piston array-induced shear stress might be directly
antagonizing Wnt-β-catenin signaling as both non-neural ectoderm and paraxial mesoderm
specification requires relatively lower levels of Wnt early during differentiation to deselect
alternative ectoderm or mesoderm lineages, respectively. This hypothesis is further
supported by the negligible influence that shear stress has on the expression levels in day 3
endoderm cells (figure 1e) since endoderm specification is comparably less dependent on the
levels of Wnt-β-catenin signaling.
To investigate the role of mechanical stress on Wnt-β-catenin signaling, we generated
a Wnt reporter hiPSC line based on an established Tcf-3-responsive promoter that controls
the expression of the secreted reporter protein NanoLuc luciferase (NLuc) coupled to a
fluorescent-timer (FT) fluorescent reporter protein. FT is expressed as a blue fluorescent
protein first (BFP) but changes fluorescence over time upon maturation into a red fluorescent
protein (RFP). The ratio of BFP over RFP can hence be used to study the dynamics of Wnt-β-
catenin signaling. The functionality of the reporter was confirmed by dose-dependent
inhibition of GSK-3 in stable polyclonal iPSCs by assessing both NLuc reporter activity as well
as BFP:RFP ratios (figure 1f). We further confirmed that mechano-regulation of Wnt-β-catenin
signaling is transient and reversible by applying piston stimulation on different days during
mesoderm differentiation (figure 1g). NLuc reporter activity was lower on days with
stimulation but recovered to levels similar to pre-induction when the device was turned off.
The established reversibility offers the option to apply mechano-regulation to modulate
lineage specification at specific stages during differentiation where Wnt-β-catenin signaling
is involved in regulating lineage bifurcations.
Results
Tobias Strittmatter 186/240
Figure 2: a) Wnt reporter levels were monitored for four days during undisturbed mesoderm
differentiation of hiPSCs. b) Modulation of wnt reporter activity upon stimulation with the
piston device (indicated by arrows on top of days of stimulation). NLuc reporter levels were
quenched reversibly at the day of induction. c) Expression levels of key mesoderm marker
genes were assessed by qPCR during undisturbed mesoderm differentiation using STEMdiff
Mesoderm Induction Media. Samples were taken as indicated every day over the course of
four days and compared to reference samples extracted on day 0. Groups contain genes
associated with (i) pan-mesoderm (brown), (ii) lateral/cardiac mesoderm (green) and (iii)
paraxial mesoderm and early somites (blue). Values in a) and b) represent means ± SD of
(i) primitive streak
rela
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expr
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b) reporter gene expression under shear stress induction
c) marker gene expression during undisturbed mesoderm differentiation
day 1day
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3day
4day
1day
2day
3day
4day
1day
2day
3day
4day
1day
2day
3day
40.0
0.51.01.52.0
rela
tive
lum
ines
cenc
e [R
LU] ind day 1
ind day 2
ind day 3 ind all days
ind day 4
a) NLuc during differentiation
day 1
day 2
day 3
day 4
0
200
400
600
800
Lum
ines
cenc
e [R
LU]
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 187/240
N=8 biologically independent samples whereas gene expression data in c) is represented as
means ± SD of N=4 biologically independent samples. *: p < 0.05, **: p < 0.01, ***: p <
0.001, ****: p < 0.0001.
To explore this hypothesis, we focused our efforts on mesoderm specification. We
first followed reporter expression of hiPSCs during mesoderm differentiation over four days
using a general mesoderm induction medium (figure 2a). NLuc reporter levels were increasing
through day 4 and additionally peaking on day 2. This pattern in Wnt-β-catenin signaling
presumably reflects changes in the cell population during the initial transition through the
primitive streak followed by the commitment to either paraxial or lateral mesoderm. In a
follow-up experiment, we assessed how stimulation with the piston device would alter
reporter expression (figure 2b). As seen before, cells undergoing mesoderm differentiation
showed reversible, mechano-induced quenching of NLuc reporter activity during stimulation.
Specifically, we observed quenching of the Wnt-β-catenin reporter activity upon shear stress
treatment on all but one day. The strongest reduction of Wnt-β-catenin signaling was
observed on day 2, which coincides with the highest signaling levels during undisturbed
differentiation (figure 2a). We speculated that the signaling levels are especially sensitive to
mechanical stimuli when at their peak. However, in contrast, the elevated reporter levels on
day 4 did not translate into higher sensitivity to shear stress. Instead, we found Wnt reporter
levels to be upregulated upon induction, suggesting that mechano-regulation of Wnt-β-
catenin signaling depends of the stage of differentiation.
We next sought to compare mesoderm differentiation with or without shear stress
induction. To this end, we first recorded expression patterns of key markers from a four-day
time course of undisturbed mesoderm differentiation of hiPSCs (figure 2c). We found that
iPSCs heterogeneously mature into a mixture of paraxial and lateral mesoderm-derived
lineages as indicated by elevated levels of lateral mesoderm markers FOXF1, HAND1, ISL1,
PRRX1, NKX2.5 and GATA4 and paraxial mesoderm markers TBX6, MSGN1, MEOX1, PAX3, and
FOXC2 on different days during the 4 days protocol. Accordingly, we observed the emergence
of potentially less mature mesoderm populations on day 2 and 3. For example, while markers
for paraxial mesoderm derived ventral (FOXC2) and dorsal (PAX3) somites peak on day 2 and
3, respectively, these populations seem to vanish by day 4. Furthermore, the cells also showed
high expression of a key marker of the lateral mesoderm-derived limb bud (PRRX1) on day 3,
Results
Tobias Strittmatter 188/240
which subsequently declined on day 4. In combination, this pattern is suggesting cardiac
mesoderm represented by marker genes GATA4 and NKX2.5 to be the predominant cell line
by day 4 of undisturbed differentiation with contaminations of early somites from the paraxial
mesoderm lineage and lateral mesoderm limb bud cells.
a) piston-assisted differentiation protocol - 3-day protocol
(i) primitive streak (iii) paraxial mesoderm(ii) lateral mesoderm
rela
tive
expr
essi
on le
vels
ctrd0-1d1-2d2-3d0-30.00.51.01.52.02.5 T
***
ctrd0-1d1-2d2-3d0-30.00.51.01.52.0 MIXL1
**
ctrd0-1d1-2d2-3d0-301234 TBX6
**** ***
**
ctrd0-1d1-2d2-3d0-30.00.51.01.52.0 MSGN1
*** *
ctrd0-1d1-2d2-3d0-30.00.51.01.52.0 HAND1
*
*
**
ctrd0-1d1-2d2-3d0-30.0
0.5
1.0
1.5 FOXF1
**
ctrd0-1d1-2d2-3d0-301234 TCF15
*****
ctrd0-1d1-2d2-3d0-30.00.51.01.52.02.5 MEOX1
ctrd0-1d1-2d2-3d0-30.00.51.01.52.02.5 ISL1
*** **
*
ctrd0-1d1-2d2-3d0-30.00.51.01.52.0 NKX2.5
***
ctrd0-1d1-2d2-3d0-30
1
2
3 GATA4*
*
ctrd0-1d1-2d2-3d0-30.00.51.01.52.0 FOXC2
ctrd0-1d1-2d2-3d0-30.0
0.5
1.0
1.5 PRRX1
*******
*
ctrd0-1d1-2d2-3d0-30.00.51.01.52.02.5 PAX3
*****
b) piston-assisted differentiation protocol - 4-day protocol
(i) primitive streak
rela
tive
expr
essi
on le
vels
(iii) paraxial mesoderm(ii) lateral mesoderm
ctrd0-1d1-2d2-3d3-4d0-402468 PAX3
*
ctrd0-1d1-2d2-3d3-4d0-4012345 PRRX1
******
****
****
ctrd0-1d1-2d2-3d3-4d0-4036
50100150 FOXC2
**
****
ctrd0-1d1-2d2-3d3-4d0-401234 GATA4
***
****
****
ctrd0-1d1-2d2-3d3-4d0-401234 NKX2.5
****
**
***
ctrd0-1d1-2d2-3d3-4d0-4036
2040 ISL1
****
******
ctrd0-1d1-2d2-3d3-4d0-402468
10 MEOX1
**
****
ctrd0-1d1-2d2-3d3-4d0-40.00.51.01.52.0 TCF15
** * *
ctrd0-1d1-2d2-3d3-4d0-40.00.51.01.52.0 HAND1
*** **
**
ctrd0-1d1-2d2-3d3-4d0-40.00.51.01.52.02.5 MSGN1
********
ctrd0-1d1-2d2-3d3-4d0-40
1
2
3 TBX6
** **
* *
****
ctrd0-1d1-2d2-3d3-4d0-40.00.51.01.52.0 MIXL1
****
ctrd0-1d1-2d2-3d3-4d0-40.00.51.01.52.02.5 T
****
***
ctrd0-1d1-2d2-3d3-4d0-40.00.51.01.52.02.5 FOXF1
*** * ******
****
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 189/240
Figure 3: Impact of piston stimulation (30 Hz for 10 sec, 25 % duty cycle) on mesoderm
differentiation using STEMdiff Mesoderm Induction Media was assessed by qPCR during
the a) 3-day or b) 4-day differentiation protocol. All samples were normalized to an
untreated control (ctr). (i) Pan-mesoderm (brown), (ii) lateral, cardiac, and limb bud
mesoderm (green), and (iii) paraxial mesoderm and early somite genes (blue) were
analyzed. Values in represent means ± SD of N = 4 biologically independent samples. *: p <
0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.
To test the impact on differentiation of piston-stimulation when run at different days,
we performed two independent experiments inducing cells on consecutive days over the
course of 3 (figure 3a) or 4 days (figure 3b). A switched-off array served as negative control
to quantify the relative changes in expression levels of key mesoderm markers. During the 3-
day run, pan-mesoderm markers T and MIXL1 stayed mostly constant with a slight increase
upon treatment on day 3. Lateral mesoderm differentiation seemed to be specifically
inhibited by shear stress induction on day two while being boosted by induction on day 3
(figure 3a, ii). In contrast, paraxial mesoderm commitment benefitted from induction during
day 1 or day 2 (figure 3a, iii). In the 4-day protocol, the specific drop in lateral mesoderm
markers upon induction on day 2 was less pronounced and only affected later genes, which
indicate that shear stress stimulation at this time hampered the progression of cells into the
lateral mesoderm lineage (figure 3b, ii). Oppositely, when shear stress was applied on day 3,
we observed increased expression of cardiac mesoderm markers ISL1 and NKX2.5 and limb
bud marker PRRX1. These results together with the higher levels of Wnt required to promote
mid versus anterior primitive streak and Wnt-β-catenin signaling inhibition required during
lateral mesoderm indicate that the cells are progressing through the primitive streak during
day 2 and subsequently specify towards lateral mesoderm on day 3. Hence, by transiently
applying shear stress on day 3 we can actively promote a more efficient transition towards
lateral mesoderm using the piston array. Shear stress induction on day 4 is similarly able to
promote upregulation of cardiac mesoderm markers ISL1 and NKX2.5 (figure 3b, ii).
Concurrent upregulation of GATA4 suggests that shear stress-mediated quenching of Wnt-β-
catenin signaling on day 4 might further aid in cardiac mesoderm differentiation (figure 3b,
ii). Activation or inhibition of Wnt-β-catenin signaling following lateral mesoderm
Results
Tobias Strittmatter 190/240
differentiation discriminates between subsequent specification into the limb bud or cardiac
mesoderm lineage, respectively. In agreement, we observed relatively lower upregulation of
limb bud marker PRRX1 in response to shear stress applied on day 4 compared to when shear
stress was applied on day 3 (figure 3b, ii).
To study effects of shear stress on genes directly associated with or know to influence
Wnt-β-catenin signaling, we assessed the relative expression levels of key genes involved in
cell adherence (figure S1a), the canonical Wnt pathway (figure S1b) as well as common
inhibitors (figure S1c) and activators (figure S1d) of Wnt-β-catenin. Intriguingly, we found that
continued shear stress induction over 2 days during mesoderm differentiation did not affect
the expression of any of the genes neither on the first nor the second day. This seems to
indicate that the observed effect on lineage commitment does not involve significant
alterations of the expression levels of the signaling components themselves but is purely
restricted to effects mediated by the existing signaling setup on a protein level.
Taken together, our data suggest that shear stress can be utilized to modulate lineage
commitment during ectoderm and mesoderm differentiation through alterations of Wnt
signaling. Furthermore, initial characterization of key genes directly associated with or known
to influence Wnt-β-catenin signaling suggest that the effect of shear stress is due to
phenotypic changes on a post-translational level rather than a transcriptional or post-
transcription level. However, full clarification of the exact mechanism will require additional
characterization.
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 191/240
Discussion
We here present a new way of modulating lineage commitment of iPSCs in cell culture
based on induction of turbulent flow generated by an oscillating piston array. The piston array
used in this study provides a traceless, robust, reversible and easy to implement induction
method. The method was validated using an established Wnt-responsive promoter construct
as well as gene expression analysis of key marker genes during the differentiation of all three
lineages with a specific focus on mesoderm differentiation. Quenching of reporter activity
exclusively on days of piston induction and selection against Wnt-dependent cell types
support the idea that the observed modulation is based on inhibition of Wnt signaling. This
effect was confirmed by alterations in lineage specification during mesoderm differentiation
exemplified by enhanced expression of key markers of cardiac mesoderm upon stimulation
on days which are reported to depend on Wnt inhibition.
Although the general time scale of lineage commitment seen in this setup diverges
slightly from established protocols, the sequence of the stages remains the same and likewise
the resulting lineages. However, based on our initial analysis of gene expression of markers
involved in Wnt signaling and cell adherence, we were unable to determine the underlying
mechanism causing the quenching of Wnt-β-catenin signaling nor can we conclusively exclude
supplementary effects on other pathways. Both issues should be characterized in-depth in
future studies. Furthermore, subsequent studies should also explore how shear stress
synergizes with modern differentiation protocols where highly optimized differentiation
media precisely direct each differentiation stages.
The arduino-based, flexible and easy to program controls in combination with tunable
oscillation frequencies and intensities allows the generation of basically any induction profile
of interest. The downside of this flexibility is the plethora of possible stimulation patterns
which prohibits efficient screening for optimal induction conditions. Hence, our data does
most likely not cover the optimal conditions and alternative modulation patterns might
further enhance the observed effect. Future work should therefore aim to establish a more
comprehensive parameter landscape and test induction on combinations of several days for
potential beneficial effects on lineage commitment. We already provide preliminary evidence
that differentiation into lateral mesoderm is enhanced by stimulation during mid primitive
streak exit on day 3 or during the limb bud versus cardiac mesoderm bifurcation stage on day
4. Therefore, a good starting point for further studies might be to test whether continued
Discussion
Tobias Strittmatter 192/240
stimulation from day 3 into day 4 can enrich for cardiac mesoderm or conversely, if
continuous stimulation on days 1 and 2 can enrich for paraxial mesoderm and somite
generation. Furthermore, since the level of Wnt signaling activation and inhibition is highly
stage dependent throughout human development future studies could also explore the
applicability of mechano-regulation to other differentiation stages and lineages.
The here described piston-based method is easy to implement for studying the effects
of shear stress on lineage commitment during differentiation of iPSCs into various lineages.
We hope that this technique will contribute to a broader understanding of the role of
mechanical cues in stem cell development as well as potentially facilitate the production of
high-quality cell lines for use in research as well as personalized medicine in the future.
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 193/240
Materials and Methods
Plasmid preparation
Plasmids were generated by digestion with standard restriction enzymes (New
England BioLabs), followed by dephosphorylation of the plasmid backbones with Antarctic
phosphatase (New England BioLabs, Cat. no. M0289), and then ligation of the backbones and
inserts with T4 DNA ligase (Thermo Scientific, Cat. no. EL0011). Phosphorylation of single
oligonucleotides was performed using T4 polynucleotide kinase (New England BioLabs, Cat.
no. M0201), according to the manufacturer’s instructions. Phosphorylation and annealing of
two oligonucleotides were performed using T4 polynucleotide kinase (New England BioLabs,
Cat. no. M0201) in T4 DNA ligase buffer (Thermo Scientific, Cat. no. B69). PCRs were
performed with Phusion high-fidelity DNA polymerase (Thermo Scientific, Cat. no. F530),
according to the manufacturer’s instructions. All plasmids used in this study are described in
Supplementary Table 1.
Cell culture
In house generated human induced pluripotent stem cells (hiPSCs) (Heng et al. 2013),
and derived stable lines, were propagated feeder-free in mTeSR1 medium (StemCell
Technologies, Cat. no. 85850) supplemented with NormocinTM (50 µg/mL, Invivogen, Cat.
no. ant-nr-1) on cell culture plates coated with Geltrex basement membrane matrix (Gibco,
Cat. no. A1413302). HiPSC lines were routinely passaged as aggregates using ReLeSR
(StemCell Technologies, Cat. no. 05872). All cells were maintained at 37°C in a humidified
atmosphere of 5% CO2 in air and routinely tested for mycoplasma contamination at an
accredited external sequencing facility (GATC Biotech AG).
To generate piggyBac transposon-mediated stable hiPSC lines, cells were seeded as
single cells and then transiently transfected by using Lipofectamine Stem (Invitrogen, Cat. no.
STEM00003) transfection reagent. In brief, 80% confluent hiPSCs were washed with PBS
(without calcium and magnesium, Gibco, Cat. no. 14190169) and dissociated to single cells
using StemPro Accutase (Gibco, Cat. no. A1110501) at 37°C for 5-10 min, or until the cells
were completely dissociated. Cells were harvested by using 4 volumes of DMEM/F12 (Gibco,
Cat. no. 31331028) and gently triturated to further dissociate them. The cell suspension was
centrifuged at 150 x g for 5 min, then the supernatant was aspirated, and the cells were gently
Materials and Methods
Tobias Strittmatter 194/240
resuspended in mTeSR1 supplemented with rho kinase inhibitor (Y-27632; 100 µM, StemCell
Technologies, Cat. no. 72304). Cells were counted and then seeded on Geltrex-coated multi-
well plates. After 20-24 h, the spent medium was aspirated. The cells were washed with PBS
and fresh mTeSR1 was added. Cells were then transiently transfected by mixing the required
amount of DNA with Opti-MEM reduced serum media (Gibco, Cat. no. 31985070).
Lipofectamine Stem was added to the solution and mixed thoroughly. The transfection
mixture was incubated for 10-12 min at room temperature and then added dropwise to the
cells. For optimal transfection results, cells were transfected within 30 min after removing the
rho kinase inhibitor. At 24 h after transfection, the medium was replaced with fresh mTeSR1.
At 48 h after transfection, selection medium was added, consisting of mTeSR1 supplemented
with puromycin (0.5 µg/mL, Invivogen, Cat. no. ant-pr-1) for 5 days before increasing selection
2x-times (1.0 µL puromycin) and continuing selection of an additional 7 days. Then, the cells
were assessed for transgene expression and differentiation potential.
Directed differentiation of hiPSCs
To assess mechano-regulation of Wnt/β-catenin signaling during differentiation into
different lineages, we used the STEMdiff Trilineage Differentiation Kit (StemCell Technologies,
Cat. no. 05230) to generate the three germ layers (mesoderm, endoderm and ectoderm). In
brief, hiPSCs grown to 80% confluency were dissociated and seeded as single cells in mTeSR1
supplemented with Y-27632 (100 µM) at a density of 5.0 x 104 cells/cm2 for mesoderm
differentiation or 1.0 x 105 cells/cm2 for endoderm and ectoderm differentiation on 50
µg/cm2 Geltrex-coated multi-well pates. At 18 h after seeding, spent medium was replaced
with fresh mTeSR1 for 6 h to recover cells. Then cells were washed with PBS and lineage
specification was induced as follows: mesoderm differentiation was specified by using
STEMDiff Trilineage Mesoderm Medium for 3 days with daily medium changes; endoderm
differentiation was specified by using STEMDiff Trilineage Endoderm Medium for 3 days with
daily medium changes; ectoderm differentiation was specified by using STEMDiff Trilineage
Ectoderm Medium for 5 days with daily medium changes.
For further characterization of mechano-regulation of Wnt/β-catenin signaling during
mesoderm differentiation, hiPSCs were seeded as described above and differentiated using
STEMdiff Mesoderm Induction Media (StemCell Technologies, Cat. no. 05221) for 3 to 4 days
with daily media medium changes.
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 195/240
Mechano-regulation of Wnt/β-catenin signaling
A schematic of the shear stress induction setup can be found in the main text (figure
1b) and is derived from a previously described device used to screen for mechanosensitive
genes in 384-well format (J. Xu et al. 2018). The setup consists of a device comprising a
custom-made 3D-printed array of 96 pistons that are attached to a membrane speaker
controlled by an Arduino controller that is linked to an operational amplifier. The Arduino can
be programmed to generate a pulses and intervals of a voltage signal that is converted into a
sine signal of 10-60 Hz by the operational amplifier circuit. The resulting interval-modulated
sine signal is fed into a commercial hi-fi stereo amplifier that drives the speaker. This setup
allows for the generation of fully customizable stimulation patterns by allowing for selection
of specific frequencies, intensities and intervals.
Experiments were done in standard cell culture incubators in humidified atmosphere
containing 5% CO2. Pistons were regenerated prior to use treatment with 1 M NaOH for at
least 10 min. Pistons were rinsed with ddH2O and dried in a cell culture flow-hood with 30
min of UV-sterilization. Devices were pre-warmed for a couple of hours in the cell culture
incubator to allow condensation to evaporate.
Initial experiments using the Trilineage Differentiation Kit (figure 1c-e) were done
using 10 Hz for 5 seconds every 5 minutes. After evaluation of cell viability settings were
adjusted to 30 Hz for 10 seconds every 30 seconds for all subsequent experiments.
Analytical assays
NLuc production was measured in cell culture supernatants with the Nano-Glo
Luciferase Assay System (Promega, Cat. no. N1110). In brief, 7.5 µL of each sample of secreted
Nluc was incubated with 7.5 µL Nano-Glo buffer/substrate mixture (50:1) for 5 min in black
384-well plates. Luminescence was measured with a Tecan Infinite M1000 PRO multiplate
reader (Tecan AG).
Gene expression profiling by RT-qPCR
A Quick-RNA mini prep kit (Zymo Research, Cat. no. R1054) was used for RNA isolation,
and 250 ng of purified RNA was used as a template for cDNA synthesis (High-Capacity cDNA
Materials and Methods
Tobias Strittmatter 196/240
Reverse Transcription Kit, Applied Biosystems, Cat. no. 4368814). Gene expression was
quantified using gene-specific primers and SsoAdvanced Universal SYBR Green Supermix (Bio-
Rad, Cat. no. 1725271) with an Eppendorf ep realplex Mastercycler (Eppendorf AG). Results
are presented as relative fold changes normalized to the expression of one or two
endogenous reference genes by using the DDCt method (Schmittgen & Livak, 2008). All qPCR
primers used in this study are listed in Supplementary Table 2.
Statistical analyses
All data represent the means ± SD of four or eight biologically independent samples
per condition. Statistical significance was assessed by unpaired t tests employing the two-
stage setup procedure of Benjamini, Krieger and Yekutieli using GraphPad Prism 9 software.
Significance is indicated as *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 197/240
Supplementary Information
Table S1: Plasmid Information
Plasmid Details Reference pCMV-hyPBase
Constitutive piggyBac expression vector (PhCMV-hyPBase-pA).
Sanger Institute, UK
pMM531
Vector encoding PhCMV-driven fast-type fluorescent timer (fastFT) (PhCMV-fastFT-pA)
(Chassin et al. 2019)
pTS1010 Vector encoding PhCMV-driven secreted Nluc expression vector (PhCMV-SP-Nluc-pA).
(Bojar, Fuhrer, & Fussenegger, 2019)
pTS1018 Cloning vector encoding a PhCMV-driven P2A sequence (PhCMV-P2A-pA). P2A was assembled by phosphorylating and annealing oligos OTS894 (5’ ctagTGGTGGTTC-TGGTGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGtGACGTGGAGGAGAACCCTGGACCTGCTAGCg 3’) and OTS895 (5’ gatcCGCTAGCAGGTCCAGGGTTCTCCTCCACGTCaCCAGCCTGCTTCAGCAGGCTGAAGTTAGTAGCTCCGCTTCCACCAGAACCACCa 3’) The annealed oligos were ligated into pTS1010 (SpeI/BamHI).
(Haellman et al. 2020) unpublished
pTS1019 piggyBac transposon vector with three insertion sites (A1, A2, A3) for stable transgene integration (5’ITR-A1-pA-A2-pA-A3-pA-3’ITR).
(Haellman et al. 2020) unpublished
pTS1024 Vector encoding PRPBSA-driven yellow fluorescent protein (YPet) coupled to a Puromycine resistance gene PuroR via a P2A sequence (PRPBSA-YPet-P2A-PuroR-pA)
(Haellman et al. 2020) unpublished
pTS1052 Vector encoding PPGK-driven secreted NLuc reporter coupled to a gene blue fluorescent protein (mTagBFP) via a P2A sequence (PPGK-SP-NLuc-P2A-mTagBFP-pA)
(Haellman et al. 2020) unpublished
pTS1053 Cloning vector encoding a fusion protein of P2A and a fast-type fluorescent timer (fastFT) (PhCMV-P2A-fastFT-pA). fastFT was excised from pMM531 (SpeI/BamHI) and ligated into pTS1018 (NheI/BamHI).
This work
pTS2106 Reporter vector encoding PTCF8-driven secreted Nluc expression coupled to a fast-type fluorescent timer (fastFT) fluorophore via P2A sequence (OTCF8-Pmin-SP-NLuc-P2A-fastFT-pA). P2A-fastFT was excised from pTS1053 (SpeI/BamHI) and ligated into pVH487 (NheI/BamHI).
This work
pTS2107 piggyBac transposon vector encoding a PTCF8-driven SP-Nluc-2A-fastFT and a constitutive YPet-2A-PuroR expression cassette (5’ITR-OTCF8-Pmin-SP-NLuc-P2A-fastFT-pA-PmPGK1-YPet-2A-PuroR-pA-3’ITR). OTCF8-Pmin-SP-NLuc-P2A-fastFT was excised from pTS2106 (MluI/HindIII) and ligated into the corresponding acceptor site A1 (MluI/HindIII) of pTS1019. PmPGK1 was excised from pTS1052 (MluI/EcoRI) and YPet-2A-PuroR was excised from pTS1024
This work
Supplementary Information
Tobias Strittmatter 198/240
(EcoRI/HindIII) and both fragments were jointly ligated into the corresponding acceptor site A3 (BsmBI) of pTS1019
pVH444 Reporter vector encoding Pmin-driven secreted Nluc expression
(Pmin-SP-NLuc-pA). Pmin was assembled by phosphorylating and annealing oligos OVH798 (5’ tcgaGTAGAGGGTATATAATGGAAGCTCGACTTCCAGGAGCTCTTCG- AAGCGG 3’) and OVH799 (5’ aattCCGCTTCGAAGAGCTCCTGGAAGTC- GAGCTTCCATTATATACCCTCTAC 3’). The annealed oligos were ligated into pTS1010 (XhoI/EcoRI).
This work
pVH487 Reporter vector encoding PTCF8-driven secreted Nluc expression (OTCF8-Pmin-SP-NLuc-pA). OTCF8 was assembled by phosphorylating and annealing oligos OVH862 (5’ cgcgTAGATCAAAGGGTAGCGTGGTAAGATCAAAGGGTAGCGTGGT- AAGATCAAAGGGTAGCGTGGTAAGATCAAAGGGTAGgtac 3’) and OVH863 (5’ CTACCCTTTGATCTTACCACGCTACCCTTTGATCTTACCAC- GCTACCCTTTGATCTTACCACGCTACCCTTTGATCTA 3’), and OVH864 (5’ CATAGATCAAAGGGTAGCGTGGTAAGATCAAAGGGTAGCGTGGTAAG- ATCAAAGGGTAGCGTGGTAAGATCAAAGGGGGC 3’) and OVH865 (5’ tcgaGCCCCCTTTGATCTTACCACGCTACCCTTTGATCTTACCACGCTAC- CCTTTGATCTTACCACGCTACCCTTTGATCTATGgtac 3’). The annealed oligos were ligated into pVH444 (MluI/XhoI).
This work
Oligonucleotides: Annealing base pairs contained in oligonucleotide sequences are shown in capital letters. Restriction endonuclease-specific sites and sticky-ends generated through oligonucleotide annealing are underlined. Abbreviations and additional information: P2A, self-cleaving peptide; 5’/3’ITR, 5’/3’inverted terminal repeat; hyPBase, hyperactive piggyBac transposase; Nluc, NanoLuc luciferase engineered from Oplophorus gracilirostris, OTCF8, T cell factor (TCF)-specific operator with 8 response elements; pA, polyadenylation signal; PhCMV, strong constitutive CMV promoter PCMVmin-1, strong hCMV minimal promoter; Pmin, pGL4.23-derived minimal promoter; PmPGK1, mouse phosphoglycerate kinase 1 promoter; PRPBSA, strong constitutive synthetic RPBSA promoter PuroR, puromycin resistance gene puromycin N-acetyl-transferase; SP, secretion signal peptide derived from IgG kappa light chain leader sequence; YPet, improved version of Venus derived from Aequorea victoria.
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 199/240
Table S2: List of primers for qRT-PCR
Gene Forward Primer Reverse Primer TBP TGTATCCACAGTGAATCTTGGTTG GGTTCGTGGCTCTCTTATCCTC GAPDH GTCTCCTCTGACTTCAACAGCG ACCACCCTGTTGCTGTAGCCAA GATA4 TCCCTCTTCCCTCCTCAAATT CAGCGTGTAAAGGCATCTG CER1 CAGGACAGTGCCCTTCAGCCA ACAGTGAGAGCAGGAGGTATGG CXCR4 CTCCTCTTTGTCATCACGCTTCC GGATGAGGACACTGCTGTAGAG EOMES AAATGGGTGACCTGTGGCAAAGC CTCCTGTCTCATCCAGTGGGAA FOXA2 GGAACACCACTACGCCTTCAAC AGTGCATCACCTGTTCGTAGGC FOXC2 CCTCCTGGTATCTCAACCACA GAGGGTCGAGTTCTCAATCCC FOXF1 AGCAGCCGTATCTGCACCAGAA CTCCTTTCGGTCACACATGCTG GSC GCACCATCTTCACTGACGAGCA TTTGGCGCGGCGGTTCTTAAAC HAND1 CAAGGATGCACAGTCTGGCGAT GCAGGAGGAAAACCTTCGTGCT ISL1 GCAGAGTGACATAGATCAGCCTG GCCTCAATAGGACTGGCTACCA MEOX1 GAGATTGCGGTAAACCTGGACC TCTGAACTTGGAGAGGCTGTGG MIXL1 GGAAGGATTTCCCACTCTGACG CCCGACATCCACTTGCGCGAG MSGN1 AGAGGGAGAAGCTCAGGATGAG GTGTCTGGATCTTGGTGAGAGG NKX2.5 AAGTGTGCGTCTGCCTTTCCCG TTGTCCGCCTCTGTCTTCTCCA PAX3 CTCCACGCTCCGGATAGTTC ATCTTGTGGCGGATGTGGTT PAX6 CTGAGGAATCAGAGAAGACAGGC ATGGAGCCAGATGTGAAGGAGG PRRX1 TGATGCTTTTGTGCGAGAAGA AGGGAAGCGTTTTTATTGGCT SIX1 AGGTCAGCAACTGGTTTAAGAACC GAGGAGAGAGTTGGTTCTGCTTG SOX1 GAGTGGAAGGTCATGTCCGAGG CCTTCTTGAGCAGCGTCTTGGT SOX10 ATGAACGCCTTCATGGTGTGGG CGCTTGTCACTTTCGTTCAGCAG SOX17 ACGCTTTCATGGTGTGGGCTAAG GTCAGCGCCTTCCACGACTTG T (Brachyury) CCTTCAGCAAAGTCAAGCTCACC TGAACTGGGTCTCAGGGAAGCA
TBX6 TCATCTCCGTGACAGCCTACCA CCGCAGTTTCCTCTTCACACGG TCF15 GCGGGCAGTGCCAAGGGCG CCCTCACCTTCAAGCAGCTGC TFAP2A GACCTCTCGATCCACTCCTTAC GAGACGGCATTGCTGTTGGACT
Supplementary Figures
Tobias Strittmatter 200/240
Supplementary Figures
Supplementary Figure 1: Perturbation of general factors of wnt signaling by piston
induction-mediated shear stress treatment over 2 days was assessed by qPCR. Genes
involved in a) cell adherence and b) beta-catenin signaling as well as c) genes encoding
a) cadherins and cadherin - interacting protein p120
day 1
day 2
0.00.51.01.52.0
E-cadherin
day 1
day 2
05
101520
N-cadherin
day 1
day 2
0.00.51.01.52.02.5
p120-catenin
rela
tive
expr
essi
on le
vels
b) beta-catenin - interacting proteins
day 1
day 2
0.00.51.01.52.0
beta-catenin
day 1
day 2
0500
100015002000
LEF1
day 1
day 2
0.00.51.01.5
CTNNBIP1
rela
tive
expr
essi
on le
vels
c) inhibitors of wnt signaling
day 1
day 2
0100020003000
DKK1
day 1
day 2
01000020000300004000050000
DKK4
rela
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expr
essi
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vels
d) wnt family
day 1
day 2
05
10152025
Wnt3
day 1
day 2
0100200300400
Wnt5b
day 1
day 2
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101520
Wnt9b
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day 2
050
100150200250
Wnt5a
day 1
day 2
0102030
Wnt3a
day 1
day 2
01000200030004000
Wnt6
day 1
day 2
0.00.51.01.52.02.5
Wnt4
day 1
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0500
10001500
Wnt8a
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uninducedshear stress
uninducedshear stress
uninducedshear stress
uninducedshear stress
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 201/240
known wnt inhibitors and c) genes from the wnt family itself were analyzed after 1 or 2 days
as indicated. Samples were normalized to an untreated reference before induction (d0).
Data is depicted as means ± SD of N=4 biologically independent samples.
Chapter 4: Mechano-Regulation of Wnt/β-catenin Signaling to Control Paraxial Versus Lateral Mesoderm Lineage Bifurcation
Tobias Strittmatter 203/240
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Discussion
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Discussion
This work covers different ways to control cellular behavior via classical receptor-
mediated signal transduction pathways, metabolic activation of a small molecule as well as
physical stimuli in the form of mechanical load and shear stress. Each approach contributes
to resolving prevalent issues of biological research on cell signaling and differentiation. The
following sections provide a short summary of each chapter and a discussion of the broader
implications of the results.
New receptors to expand the range of targetable disease
In the first chapter of this work, we presented a pipeline for the generation of novel
receptors based on the established EpoR scaffold. We used the pipeline to generate new
receptors to detect degradation products of fibrin from human blood following thrombotic
events. The receptors function as homodimers, heterodimers, or with tandem-binders fused
to the extracellular domains. Cells equipped with these receptors could also be modified to
secrete fibrinolytic as well as anti-coagulative therapeutics. This approach is not limited to a
single type of protein binder but can, in theory, be used in combination with any binder
selection platform of choice which expands the range of potential receptor ligands even
further. The presented work-flow does not only validate the initial GEMS platform design but
also increases reliability of receptor design in general. Although the presented data only
covers the generation of receptors against a single albeit highly relevant target, we are
confident that the results allow for an extrapolation on other targets as well. With the current
improvements, it is now possible to more reliably and predictably generate receptors against
a soluble target of choice.
A combination of binders derived from different selection strategies would increase
the amount and variation of available binders with different epitope preferences and
requirements for protein binding. In turn, an increasing number of binders also produces a
higher number of receptors and receptor combinations that need to be evaluated in a cell
culture screen. A more stringent selection for binders might therefore be considered to
reduce the size of the receptor library. However, such an approach would also dramatically
shrink the available epitope space. In addition, we did not find a correlation of binding affinity
and the success rate of a binder in a receptor context. Hence, affinities of selected binders
New receptors to expand the range of targetable disease
Tobias Strittmatter 208/240
seem to be less important than the epitope space that they cover. One way to “rescue” non-
dominant binders that attach to less-affine epitopes from a selection run is given by blocking
the dominant epitopes during selection (Tsui et al. 2002). This method could be used to
increase the amount of lower-affinity binders by adding the best binders of a first run to a
second round of selection against the same target. Skimming the binder pool with
combinatorial competition assays could be used to rid the library of “duplicate” binders i.e.
binders against the same epitope. However, neither of the approaches does account for the
steric context that the binders are introduced to once they are embedded in the receptor
scaffold. Yet, chances are that there are binders that bind the same epitope but do so in
different angles. Removing all duplicates might therefore also lead to elimination of binders
with just the necessary orientation. Here, the use of membrane-bound selection systems like
yeast-display (Uchanski et al. 2019) might help to increase the number of relevant binders by
screening them in a receptor-like context. In conclusion, priority should be given to
maintaining diversity of the binder pool instead of applying more stringent selection
conditions. Still, an increased variety also provokes a much larger library of receptors that has
to be assessed in cell-based assays.
Fortunately, the EpoR scaffold already provides a solution to this issue which is at least
applicable to larger targets. Binding of a larger protein to either or both of the extracellular
domains leads to a drop in residual activity that results in an OFF-type switch, as can be seen
in our evaluation of homodimeric receptors. Hence binding of the target in the native context
can be detected and functional receptors and their binders can be selected for further
characterization in a combinatorial screen. Modern automation solutions could aid in this task
to enable screening of larger libraries to reduce development time and increase robustness
of the presented pipeline. The unsophisticated cloning strategy would possibly even allow for
an integration into a lab automation environment for fully autonomous generation of
functional receptors.
While high-throughput binder selection now provides a vast space of possible
epitopes, the EpoR architecture itself remains limited by the underlying mechanism of
activation which requires the expression of two receptor chains that each need to bind to the
target individually. This involves the presence of two identical epitopes on the target molecule
with suitable spacing or the generation of two different receptor chains that are each
equipped with a different binding domain. While finding binders against a given target and
Discussion
Tobias Strittmatter 209/240
expression of two receptor chains in each cell is less of an issue for larger targets and easy-
to-transfect cell lines, detection of smaller molecules in primary cells might prove to be
difficult.
Unfortunately, the mechanism of activation of EpoR hampers its use for detection of
cell-surface markers and its signal transduction relies on endogenous signaling pathways.
However, complementary receptor architectures are available for use in cell-cell
communication that could potentially fill the gaps in EpoR-based signaling. The degree of
orthogonality depends on the signaling moieties of the respective receptor and can be
selected according to the desired application. For example, the modular extracellular sensor
architecture (MESA) (Schwarz et al. 2017) promises fully orthogonal signaling via the tTA
system but requires tedious fine tuning of expression levels of each component. However,
existing “off-the-shelf” MESA-receptors should be easy to integrate. Harnessing the signal
amplification capabilities of the cell’s native signal transduction cascades provides greater
sensitivity without the need for additional components. Chimeric G-proteins (Coward et al.
1999), GPCR-derived RASSLs (Redfern et al. 1999), chimeric antigen receptors (CARs) (Kuwana
et al. 1987) and synthetic Notch (synNotch) receptors (Morsut et al. 2016) all reroute a given
input signal to an existing endogenous pathway. In this context, the ability to reroute
additional inputs through the AMBER platform described in chapter 1 can be expected to
provide valuable synergistic effects. In combination with the existing receptor technologies,
it would be possible to specify disease conditions more precisely to reduce side-effects in
potential clinical applications of cell therapies. Also, it would be possible to mirror
developmental niches by mimicking the effects of more intricate growth factor combinations.
Obviously, the ultimate receptor for mammalian cell signaling applications is a small
monomeric receptor with a single transmembrane domain that would be activated by binding
of a single ligand molecule while downstream signaling would be modular to allow for entirely
orthogonal signaling or activation of native signal transduction pathways. Since there is no
such receptor to be found in nature, a possible solution might lie in a computationally
designed receptor scaffold. So far, fully de-novo designed new-to-nature receptors for
orthogonal cell signaling are still science-fiction. However, with recent progress in resolution
of protein structures in silico (Senior et al. 2020) and protein engineering (Yang et al. 2021) in
combination with ever improving computing power such an approach might lead to
functional designs in the not too far future.
Controlling cellular behavior with small molecules
Tobias Strittmatter 210/240
EpoR based receptors equipped with a single DARPin are as small as 2.5 kb while scFv
versions account for a 3 kb payload. Both receptor types could hence be used in gene therapy
vehicles like AAVs for example to enhance CAR-T cells by activation of the IL6/STAT3 pathway
(Li et al. 2018; Breuer et al. 2020) or in situ to provide protection of cardiomyocytes against
hypoxia-induced damage during ischemia through simulation of VEGF signaling (Parvin et al.
2014).
Controlling cellular behavior with small molecules
The second chapter of this thesis details the use of the inert-to-human small-molecule
inducer L-glucose in a novel gene switch. There are already a variety of small-molecule
induced gene switches that come in different flavors. ON- and OFF-type switches in
mammalian cells were reported using doxycycline (Gossen et al. 1992), vanillic acid (Gitzinger
et al. 2012), glucocorticoids (James et al. 2000) or cumate (Mullick et al. 2006) just to name a
few. Those systems are commonly used to control gene expression in vitro and there is a
growing number of in vivo applications (Gitzinger et al. 2012; Wang et al. 2020; Sakamoto et
al. 2020).
Compared to these examples, the use of L-glucose as inducer molecule is unique in
the sense that it does not employ the inducer molecule directly but relies on a metabolic
process to convert the pre-inducer into an active ligand. When designing the L-glucose gene
switch we found that there is no natural transcription factor or receptor available that would
be activated directly by L-glucose, hence L-glucose can be used as an orthogonal inducer.
Metabolic engineering typically aims to augment host cell function in order to increase
product yield and quality as detailed in the introduction. In this case, however, the metabolic
functions that were integrated did not target endogenous processes but were used to enable
conversion and detection of the small molecule inducer L-glucose, which makes this system
a metabolically activated gene switch. In a recent study from our group a new reporter system
for continuous monitoring of promoter activity was designed by introducing metabolic
functions into HEK-293T cells. Here, the production of the fluorescent dye betaxanthin was
possible by the ectopic expression of fungal enzymes leading to fluorescent cells upon
expression of the key enzyme of betaxanthin synthesis (Stucheli et al. 2020). Both studies
show-case a cross-kingdom implementation of synthetic biology and might prompt further
Discussion
Tobias Strittmatter 211/240
efforts in metabolic engineering in mammalian systems beyond improvements in
biopharmaceutical production to engineer artificial reporter systems and orthogonal gene
switches.
Implementing novel small-molecule induced gene switches is especially relevant since
inducers for some of the established gene switches are reported to affect cell viability and
productivity as seen for the doxycycline system (Moullan et al. 2015) or are not orthogonal
like the glucocorticoid-based gene switch. That said, there still remains a couple of gene
switches that show similar characteristics like the presented L-glucose gene circuit. Therefore,
we see the presented L-glucose gene switch as an extension of the existing toolbox that can
be freely combined with other gene switches that are controlled by e.g. vanillic acid or cumate
to control expression of multiple transgenes independently. A combination of small-molecule
inducers vanillic acid and benzoate was already used to control expression of a transgene
independently as well as in combination as part of a dual-input system (Xie et al. 2014).
Because the inducer L-glucose cannot be degraded by mammalian cells and gets
readily cleared from the blood stream through the kidneys, it is an ideal non-toxic compound
with respect to safety concerns (Raymer et al. 2003). On the downside of this quick clearance
rate and the resulting low concentrations of L-glucose in the blood stream, implementations
of L-glucose responsive gene switches for use in in vivo applications become challenging.
However, as we were able to show, these characteristics still allow for use of L-glucose in
closed systems like a bioreactor.
The orthogonal character of L-glucose metabolism also allows to speculate on the use
of L-glucose as an energy source to enable antibiotics free selection. By expression of the
three missing enzymes LgnE, LgnF and LgnG from Paraccocus sp.43P the L-glucose
degradation pathway could be completed. Thereby, L-glucose could be effectively converted
into pyruvate and glycerine-aldehyde-3-phosphate (GAP) which are common intermediates
of glycolysis and energy-rich substrates in eucaryotes. Transfecting cells with plasmids
encoding a transgene of choice that is genetically linked to the expression of all necessary
catabolic enzymes could enable the specific selection of transgenic cells under D-glucose
starvation and continuous supplementation with L-glucose. Such an orthogonal selection
process would omit the use of antibiotics and simplify downstream processing of
biopharmaceuticals. The use of other orthogonal energy-rich substrates that are cheaper and
easier to produce in higher quantities could further improve usability of the approach.
Mechanical cues to control gene expression and lineage commitment
Tobias Strittmatter 212/240
Mechanical cues to control gene expression and lineage commitment
In chapter 3 and 4 we studied effects of mechanical cues on calcium signaling and wnt
signaling, respectively. Calcium-activated nuclear factor and activator of transcription (NFAT)
was assessed besides an artificial orthogonal calcium-sensitive pathway for gene expression
based on the transcriptional activator tTA in combination with a protease from tobacco etch
virus (TEV). We also screened accessory receptors, channels and transcription factors
involved in calcium homeostasis for their potential to boost NFAT-activation. In order to
provide a higher-throughput protocol to study effects of shear stress, we implemented a
device that was based on a 3D-printed piston array attached to the membrane of a speaker
to allow for the generation of turbulent flow in a 96-well cell culture plate. We used this
device to explore the dependency of mechanically induced gene expression on extracellular
matrix density and composition as well as on the density of the cell population. Mechanical
cues generated from the same device were also used to modulate lineage commitment in
human induced pluripotent stem cells (hiPSCs).
In its native context, mechanosensation is involved in regulation of blood pressure
through sensation of shear stress in the vasculature (Wang et al. 2016). In addition,
mechanical properties of the nearby extracellular space are known cues in stem cell and tissue
development (Kerstein et al. 2013). More specifically, shear stress is a known modulator of
Wnt signaling (Arnsdorf et al. 2009; Jansen et al. 2010; Cha et al. 2016). Hence, we studied
the impact of mechanical stimulation on Wnt signaling by means of a stable iPSC reporter cell
line. In addition to a reversible quenching of Wnt signaling, we also found alterations in
differentiation into ectoderm and mesoderm lineages under standard differentiation
conditions. The presented protocol is non-invasive, does not require changes in growth factor
composition and can be easily combined with established methods.
Recent advances using focused ultrasound for remote gene expression based on
thermal effects in the cell might offer an alternative induction method for mechanical cues
that can be more readily applied to clinical settings (Liu et al. 2006). Control of gene
expression by temperature has been achieved through the use of heat-shock-protein (HSP)
70B-induced promoters (Miller et al. 2018). Alternatively, heat sensitive coiled-coil protein
domains derived from the bacterial regulator TlpA were successfully implemented to control
gene expression in a tunable manner (Piraner et al. 2017; Piraner et al. 2019). A rather
disputed (Meister 2016) study claimed induction of calcium responses in neurons by magnetic
Discussion
Tobias Strittmatter 213/240
ferritin tethered to the cytosolic side of the TRPV4 channel (Wheeler et al. 2016). Recent
experiments indicate that direct induction of heat-sensitive ion channels like transient
receptor potential cation channel subfamily V member 1 (TRPV1) might be feasible although
at temperatures not relevant for use in humans (Zhang et al. 2018).
Although thermal cues might seem more amenable to control cellular behavior in vivo
because the necessary technologies for thermal induction are omnipresent, thermally-
controlled gene switches suffer from unspecific or non-orthogonal induction. Hence the use
of such switches is limited to applications outside of the patient or to occasions when
systemically elevated temperatures are assessed i.e. in case of fever. On the other hand,
elevated temperatures were reported to promote differentiation of neural stem cells (Wang
et al. 2017) and hence can be considered save with respect to cell viability. Although, this
finding should not be generalized, temperature induced gene switches might indeed provide
a means for modulation of stem cell differentiation in vitro.
In contrast, purely mechanically induced gene expression offers the possibility to
detect endogenous signals while also allowing for specific induction of gene expression via
non-invasive artificial external stimuli like ultrasound or shear stress. Studies on
mechanogenetics for the remote control of cellular behavior have recently shown promising
results. In a first potential therapeutic application of ectopically expressed Piezo1, ultrasound
has been used to induce gene expression in T-cells to enhance their cytotoxic response to
cancer cells (Pan et al. 2018). However, this non-invasive stimulation method is still hampered
by requiring additional supplements (microbubbles) being present in the very vicinity of the
cells. Against this background, gene circuits and cells that are triggered by pure mechanical
stimulation in a therapy setting become indeed achievable. Such mechanogenetic systems
could be used in basic research as well as in next-generation medicine to monitor shear stress
in the patient’s body.
We hope that the results and methods described in chapter 3 and 4 facilitate research
on calcium signaling and cell differentiation including the channels that are involved as well
as on the role that cell-cell contact and cell adherence play. Since the shear stress used in this
experiment provides a general stimulus that works on the endogenous set of signaling
pathways, this method could be of special interest in stem cell research to omit the use of
costly growth factors used as supplements in established differentiation media. The so
reduced complexity would lead to a more robust differentiation protocol. Improving on the
Developments and future applications of mammalian synthetic biology
Tobias Strittmatter 214/240
generation of high-quality cells from patient-derived iPSCs which contribute to next-
generation personalized medicine in two ways. First, the generated cells could directly be
used in regenerative medicine. Second, the produced cell types could also be employed for
drug screening and testing in a patient-centered approach to account for patient-specific
anomalies in drug metabolism. Such processes are already examined for use in cardiovascular
disease (Paik et al. 2020) e.g. to assess liver toxicity (Gough et al. 2020) and rely on established
protocols for cell line differentiation.
Developments and future applications of mammalian synthetic biology
The projects presented in this thesis contribute to the overall goal of mammalian synthetic
biology of strengthening our understanding of cellular systems to ultimately being able to
improve (cell) therapies and biotechnological processes (figure III). Key to this goal is a way to
control cellular behavior in response to various inputs. In turns, controlling cellular behavior
through means of synthetic biology requires a solid understanding of the underlying
processes. To this end synthetic biology provides a rich toolbox of receptors, transcription
factors and response elements to alter and reroute cellular signaling to synthetic promoters.
In order to speed up the process of synthetic biological engineering, the BioBrick standard
(Constante et al. 2011) and the standardized European vector architecture (SEVA) (Silva-
Rocha et al. 2013) were established for genetic engineering of bacteria. SEVA is now
commonly used and continuously updated (Martinez-Garcia et al. 2020). Although it took a
while, similar initiatives were started that specifically cater to the needs of mammalian cells
with the goal of a unified genetic platform to stream-line synthetic biology-driven
engineering. To this end COmposable Mammalian Elements of Transcription (COMET) to
control gene expression via designed orthogonal transcription factors (Donahue et al. 2020)
were developed alongside a Versatile plasmid Architecture for Mammalian Synthetic Biology
(VAMSyB) (Haellman et al., unpublished) to provide a common vector and cloning strategy to
enhance reproducibility and exchange of modules between research groups. Lower costs and
increased turn-around times for custom gene synthesis are beginning to make in-house
cloning a thing of the past. Especially methods like Gibson cloning that require additional time
for synthesis of oligonucleotides in each step (Gibson et al. 2009) would be replaced by gene
synthesis approaches in the near future. Still, any modular cloning strategy that makes use of
Discussion
Tobias Strittmatter 215/240
an existing repertoire of parts and modules that can be recombined quickly with high
efficiency still has an edge against de novo synthesis. Therefore, we will keep seeing SEVA- or
VAMSyB-like platforms around for a while.
Recent improvements and adaptations of the CAR-architecture as well as the original
CARs themselves serve as prime examples of synthetic biology applications. Feasibility of
DARPins as binding moieties in CARs has been demonstrated (Hammill et al. 2015; Patasic et
al. 2020) and this now allows to target a growing range of diseases by CAR-T cell therapy. We
expect CAR-T cells to be used more commonly even outside cancer therapy to fight persistent
viral and fungal infections e.g. by HIV (Leibman et al. 2017; Patasic et al. 2020) or Aspergillus
fumigatus (Kumaresan et al. 2014; Seif et al. 2019). Additionally, immune-modulatory
interventions based on chimeric autoantibody receptor (CAAR)-T cells that target auto-
reactive B-cells (Ellebrecht et al. 2016) or CAR-Treg cells that bind an allergen and subsequently
quench over-shooting immune responses were described (Elinav et al. 2008; Brunstein et al.
2016). In this regard, we are confident that our AMBER system could aid in the development
of next-generation cell therapies to further improve specificity and safety of cell therapies.
Multiple studies have been performed already to reach this goal by implementing logic gates
on various levels of T-cell activation. For one, it was shown that the synNotch-platform could
functionally replace CARs in cytotoxic T-cell response, providing an alternative for targets that
are not accessible to the T-cell receptor (Roybal et al. 2016). In an extension to this work,
CARs could be conditionally expressed upon binding of synNotch receptors to cancer-defining
cell-surface markers (Roybal et al. 2016).
In split, universal, and programmable (SUPRA)-CARs, the T-cell receptor is truncated and
lacks an antigen binding domain. Instead, SUPRA-CARs are activated by exchangeable
adapters that bind to cell-surface antigens on one side and to the T-cell receptor on the other
side thereby enabling targeting of various different cell-surface markers by the same cell (Cho
et al. 2018). Recently, a system for protein logic gates, termed “colocalization-dependant
latching orthogonal cage/key proteins” (Co-LOCKR), was shown to recruit T-cells only if a
specified set of surface markers is present thereby performing logic operations on a protein
level directly on the cell (Lajoie et al. 2020). Both Co-LOCKR and the underlying concept used
in SUPRA-CARs could potentially be adapted for use in the AMBER platform. Here, the
extracellular domains of the EpoR could serve as binding sites for Co-LOCKR proteins to enable
protein logics to control receptor activity. Similar to SUPRA-CARs, the EpoR extracellular
Developments and future applications of mammalian synthetic biology
Tobias Strittmatter 216/240
domains could also recruit adaptors to mediate activation of the same receptor by multiple
different targets. Furthermore, SUPRA-CARs and protein logics could greatly benefit from the
addition of de novo designed protein logic gates to further widen the available combinatorial
space (Chen et al. 2020).
As an additional safety measure, timed activation of CARs was achieved by activation of
CAR-T cells upon binding of a small-molecule. In two different designs the small molecule
triggers either the complementation of the intracellular signaling domain (Wu et al. 2015) or
mediates the installation of an extracellular antigen binding moiety (Zajc et al. 2020). A similar
functionality could be generated with EpoR-based synthetic receptors to control expression
of the effector CARs by a small molecule or disease marker. Hence, the receptor platform
described in chapter 1 could be combined with any of the described CAR modifications by
providing an additional control layer for increased specificity and safety.
Induced gene expression provides advantages beyond the field of CAR-T cell therapy
and by now also sees some applications in biotechnological production. Simple logic gates are
used not only for the production of proteins that show extensive post-translational
modifications (Lonza 2019) but also in stem cell technologies like the opti-ox™ system to
control the differentiation of stem cells into a cell type of choice upon addition of doxycyline
(Pawlowski et al. 2017). A combination of multiple inducers at once was used in a synthetic
lineage control network described for the generation of beta-like cells from iPSCs (Saxena et
al. 2016). Such lineage control networks offer more robust differentiation and greater
homogeneity of the resulting cell population thereby improving the overall efficiency of the
process enabling commercial use of iPSC-derived cell lines for basic research and personalized
medicine in the future.
Most available orthogonal gene switches are based on bacterial regulators that lack
the signal amplification systems of most eucaryotic systems similar to the L-glucose gene
switch described in chapter 2. However, by now the use of dimerizing bacterial kinases was
demonstrated which offer orthogonal signal amplification through phosphorylation of
multiple effector proteins by a single activated kinase (Scheller et al. 2020; Maze et al. 2020).
Cell-based therapies are poised to transform the field of personalized medicine. A
number of proof-of-concept studies on cell-based therapies are lined up for clinical evaluation
already ranging from simple one-input-one-output systems for the detection and treatment
of gouty arthritis (Kemmer et al. 2010) to logic gates targeting psoriasis (Schukur et al. 2015),
Discussion
Tobias Strittmatter 217/240
allergic disease (Chassin et al. 2017) or liver damage (Bai et al. 2016). Progress in the field of
electrogenetics now enables a direct communication between electronic hardware and cells.
A pioneering study proved the feasibility of electrogenetic cell therapy for the treatment of
diabetes via induced gene expression as well as fast release of insulin from pre-formed
vesicles (Krawczyk et al. 2020).
Figure III: Synthetic biology can be used to engineer cells with new functionalities or to
augment existing cell lines. Engineered cells can be used in drug discovery to enable efficient
screening of compounds and assay toxicity (left), for direct use in patients to treat disease
(middle) or to produce and improve production of antibodies or enzyme-replacement
therapies (right).
Conclusion
Tobias Strittmatter 219/240
Conclusion The field of synthetic biology and related cellular therapies is advancing at a high pace.
With more genomes of species from all kingdoms of life being sequenced and more protein
structures being solved each year by the help of advanced automation techniques (Chavas et
al. 2015) we now know more parts and modules for synthetic biology-inspired applications
than ever before. Furthermore, this pace might even increase in the future since already
today protein structures can be predicted in silico with high accuracy (Senior et al. 2020) and
new gene switches can be designed computationally (Chen et al. 2020).
However, despite ongoing efforts to improve the underlying hardware used for
cellular implants (Bose et al. 2020) the number of real-world applications outside of stem cell
transplants is still limited. The approval of CAR-T cells and AAV-mediated gene therapies and
vaccines alongside major investments of big pharmaceutical companies might mark a turning
point in this regard with more money and human resources getting involved to accelerate the
transition of cell therapies from lab benches to patients.
In summary one can expect synthetic biology to have a growing impact on
biotechnological production of classic and modern biopharmaceuticals like antibodies and
gene therapy vehicles as well as on personalized medicine via the generation of patient-
derived disease models and autologous cell therapies in the future (figure III).
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Acknowledgements
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Acknowledgements I would like to thank …
… Professor Dr. Martin Fussenegger, for the opportunity to pursue my doctoral thesis
in his lab and all the helpful insights that I gathered during this time.
… my thesis committee, Professor Dr. Renato Paro, Professor Dr. Ivan Martin and
Professor Dr. Randall Platt for their great support and interesting discussions.
I also would like to thank Professor Dr. Niko Beerenwinkel for chairing the defense.
… all authors and co-workers for the fruitful collaborations, most important the
Plückthun group of University of Zurich and Benji and Krishna from the Müller lab of
D-BSSE.
... the single-cell facility of the BSSE, the functional genomics center Zürich (FGCZ) and
the proteomics facility of the University of Basel for their essential support and
expertise.
... the active members and alumni of the Fussenegger Group for a great time and many
(un-)interesting discussions.
... my family for their backing me in this adventure and for providing an anchor during
the PhD-life.
... Rebekka, for everything.
... Theo, for a little bit more.