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Genetic circuitry for personalized human cell therapy
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ScienceDirect Genetic circuitry for personalized human cell therapy Fabian Tolle1, Pascal Stu¨cheli1 and Martin Fussenegger1,2 Synthetic biology uses engineering principles to design and assemble biological components and systems for a variety of applications. On the basis of genetic engineering, synthetic gene switches can be interconnected to construct complex gene circuits, capable of sensing and integrating diverse input signals for precise spatiotemporal control of target gene expression in living cells. Designer cells can be equipped with advanced gene circuitry enabling them to react precisely to pre-programmed combinations of conditions, automatically triggering a specified response, such as therapeutic protein production. Such cells are promising therapeutic modalities for applications where traditional medical treatments have limitations. Herein, we highlight selected recent examples of designer cells with engineered gene circuits targeted toward applications in personalized human medicine. Addresses 1 Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland 2 Faculty of Science, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland Corresponding author: Fussenegger, Martin ([email protected])
Current Opinion in Biotechnology 2019, 59:31–38 This review comes from a themed issue on Tissue, cell and pathway engineering Edited by Marjan De Mey and Eveline Peeters
https://doi.org/10.1016/j.copbio.2019.02.003 0958-1669/ã 2018 Elsevier Ltd. All rights reserved.
Introduction The idea of cell therapy, in which living cellular material is injected or transplanted into a patient, dates back to at least the nineteenth century when Swiss doctor Paul Niehans experimented with injecting live cells obtained from sheep embryos, in an attempt to cure illnesses and rejuvenate his patients [1]. The field has come a long way since then. Indeed, the burgeoning development of methods for genetic manipulation and constitutive overexpression of transgenes has resulted in immense progress; for example, genetically engineered T-cells are emerging as powerful medicines in cancer therapy [2,3,4]. However, many potential applications of genetically engineered cells require the cells to sense an input www.sciencedirect.com
stimulus and modulate the strength, timing and cellular context of gene expression accordingly [5]. As depicted in Figure 1a, a current key challenge in cell engineering is to design and implement advanced input signal-responsive gene circuitry, in order to achieve precise spatiotemporal control of target gene activities in living cells [6]. This is not easy, because, in a therapeutic context, engineered cells have to perform under ever-changing and, therefore, uncertain conditions. In order to autonomously and reliably perform the target function, the effect of the cellular output has to be sensed, and the information entered into a feedback loop capable of re-adjusting the output strength [7]. In other words, the cells are required to monitor the status of their environment, and not only produce specific molecules on demand, but also adjust their performance to correct differences between the desired and actual states.
Building gene circuits to provide closed-loop-controlled designer cell activity Gene circuits consist of interconnected gene switches, which act as regulatory access points at which gene expression can be initiated, interrupted or terminated. Depending on the desired regulatory level and mechanism of action, different input signals can be applied (Figure 1a). Input methods may be influenced by properties such as cellmembrane permeability, specificity, spatiotemporal resolution, reversibility, and response time, which all have to be considered and weighed against each other, depending on the intended regulatory outcome. To date, the most commonly employed types of input stimuli are chemical triggers, such as ions, small organic molecules, or proteins. However, electromagnetic waves such as light have also found extensive use as inducers due to their superior spatiotemporal controllability [8,9]. Gene switches can be implemented at various regulatory levels (Figure 1b). (i) At the extracellular level, stimuli can be sensed by (artificial) receptors that turn on intracellular signaling cascades capable of amplifying and propagating signals into, for example, the nucleus [10,11]. (ii) At the DNA level, transcriptional control of gene expression can be modulated by, for example, controlling natural or artificial transcription factors [12,13]. (iii) Gene switches can also be implemented at the translational level through, for example, ligand-dependent ribozymes that regulate mRNA stability [14,15]. Multiplexed combinations of gene switches regulated by different input stimuli allow for the assembly of Boolean logic gates, which in turn can be elaborated into complex gene circuits with emergent properties [16]. Biologically Current Opinion in Biotechnology 2019, 59:31–38
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Figure 1
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Engineered gene circuits enable conditional closed-loop-controlled designer cell activity. (a) Gene circuits responsive to a variety of input signals, for example small molecules, proteins, or light, can be engineered, allowing for closedloop control of designer cell outputs. Therapeutic cells with embedded sensory, signal processing, and response capabilities can act as, for example smart drugs by adjusting cellular activities in response to environmental cues. (b) Gene switches act as access points where gene expression can be modulated to gain control over biological functions at different regulatory levels, such as signal transduction, transcription, or translation.
implemented logic gates are systems that can perform logical operations on one or more binary inputs, producing a single binary output, analogously to the way circuits and chips are designed for digital computers. By interconnecting networks of gene switches acting as logic gates, complex gene circuits can be designed that robustly compute one or more input stimuli, for example, the presence of specific biomarkers for a disease state, in order to generate a specific output, such as the production of a therapeutic protein [17]. Beyond simple detectors and inducible systems, signal integration circuits can be developed to react very precisely to pre-programmed sets of conditions, automatically triggering a defined reaction [18]. With the increasing complexity of interconnected sensory and regulatory gene circuits, it is becoming possible to tune cells for practical applications as therapeutic modalities in personalized human medicine [19]. Current Opinion in Biotechnology 2019, 59:31–38
Engineering mammalian cells towards applications in personalized human cell therapy Sensors for molecular diagnostics and therapy
The cell membrane constitutes an effective barrier against many external stimuli. To overcome this, transmembrane receptors transduce signaling information from outside the cell, while the signal itself does not have to cross the membrane physically. Natural receptors can be repurposed for some applications in cell engineering, but for many desirable input signals, no natural receptor is available [20]. To bridge this gap, an extracellular sensor platform based on a universal designer receptor scaffold has been engineered [21]. To demonstrate the scope of this ‘generalized extracellular molecule sensor’ (GEMS) platform, antibody fragments targeting a variety of different inputs, ranging from small molecules to www.sciencedirect.com
Genetic circuitry for personalized human cell therapy Tolle, Stu¨cheli and Fussenegger 33
proteins, have been fused to erythropoietin receptor (EpoR) extracellular and transmembrane domains. By exchanging the intracellular domains, artificial receptors activating a variety of different signaling pathways could be obtained. Based on the GEMS platform, a designer receptor for caffeine has been created (Figure 2a) [22]. Stimulation of designer cells with physiologically relevant concentrations of caffeine, generated by regular intake of beverages such as tea and coffee, resulted in caffeineinduced transgene expression. Type-2 diabetic mice implanted with these microencapsulated cells expressing glucagon-like peptide 1 (GLP1) showed substantially improved glucose homeostasis after coffee consumption. This approach of integrating therapy into the patient’s lifestyle, by coupling the expression of a therapeutic transgene to an inexpensive and non-toxic trigger such as caffeine, represents a promising step towards improving patient compliance. Another inexpensive and non-toxic class of exogenous trigger molecules is free fatty acids (FFAs); these are essential dietary components that also act as signal molecules. Designer cells harboring free fatty acidregulated transgene switches have been engineered and applied for the induction of a reporter gene after consumption of Swiss cheese [23]. The easy adaptability of the FFA-activated transgene switch to various mouse models supports the idea that it is a promising candidate for future diagnostic and therapeutic applications related to, for example, sensing and regulating pathological blood-fat levels. As diagnosis marks the beginning of any successful therapy, one crucial application area for designer cells is in sensing and diagnosing disease states. Toward this end, a synthetic biology-inspired biomedical tattoo using engineered cells has been developed [24]. The cells were programmed to produce the pigment melanin in response to persistently increased Ca2+ concentrations, as seen in hypercalcemia-associated cancer. The sensor system was validated in mice bearing subcutaneously implanted encapsulated engineered cells, resulting in cancer-celldependent generation of an optical signal detectable with the naked eye. Combining the intrinsic sensing properties of bacterial cells with the wireless connectivity of a microelectronic platform, an ingestible micro-bio-electronic device (IMBED) for in vivo sensing in the difficult-to-access gastrointestinal environment has been developed [25]. Genetically engineered heme-sensitive, light-generating biosensor bacteria have been integrated into a miniaturized luminescence readout device for the acute diagnosis of gastrointestinal bleeding. The blood-sensing capability of these engineered cells was demonstrated in a murine in vivo assay, while the IMBED was validated in a porcine model system for gastric bleeding. Considering the www.sciencedirect.com
modular nature of this platform, other whole-cell biosensors could be rapidly integrated to achieve minimally invasive detection of other biomarkers in the gastrointestinal tract. To enable safer cell-based cancer therapies, a new class of cell-contact-sensing device has been engineered to function effectively in human nonimmune cells [26]. Designer cells equipped with this system were capable of explicitly triggering the release of therapeutic output molecules upon sensing contact with a target cancer cell. Cell-based therapy with nonimmune cells represents a promising modality for cancer therapy, eliminating many of the risks associated with current approaches based on engineered immune cells. Combating bacterial infections
Designer cells have been applied very effectively for sensing and controlling bacterial infections. In a pioneering example, a closed-loop gene network capable of detecting and eliminating methicillin-resistant Staphylococcus aureus (MRSA) bacterial infections in mice was designed (Figure 2b) [27]. In this approach, a bacterium-sensing human Toll-like receptor-based genetic circuit was employed to regulate the expression of lysostaphin, a bactericidal enzyme that can kill most known strains of Staphylococci. Microencapsulated cell implants achieved full cure of acute MRSA infections in a mouse model in which conventional vancomycin treatment failed, providing a proof-of-concept that this approach is a viable option for the treatment of antibiotic-resistant pathogens. One reason for the gradual loss of efficacy observed with traditional antibiotics is the coordinated formation of biofilms. In an attempt to reduce biofilm formation, synthetic biology principles have been used to rationally engineer quorum-quenching cells capable of activating a twofold defense, consisting of the secretion of an inactivating enzyme to silence bacterial quorum sensing and an anti-biofilm effector to dissolve the biofilm matrix [28]. In an alternative approach, synthetic mammalian cells were programmed to sense signaling peptides secreted by various microbes and respond with the production of autoinducer-2 (AI2), a key signal molecule regulating bacterial group behavior [29]. Besides engineered mammalian cells, bacteria have also been genetically modified to kill-specific pathogens or to inhibit their virulence. An engineered Escherichia coli strain encoding an anti-biofilm enzyme showed prophylactic and therapeutic activity against Pseudomonas aeruginosa during gut infection in two animal models [30]. These studies open up new possibilities for future anti-infective strategies. Toward curing diabetes
Type 1 diabetes results from a loss of pancreatic insulinproducing beta cells, which are essential for the tight Current Opinion in Biotechnology 2019, 59:31–38
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Figure 2
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Selected examples of applications of engineered mammalian cells towards human personalized therapy. (a) The GEMS receptor platform for sensing of various inputs to activate multiple endogenous signaling pathways. GEMS have been engineered to sense proteins or small molecules such as caffeine [22] and activate different intracellular pathways such as JAK-STAT, thereby enabling, for example transgenic GLP1 expression for the treatment of diabetes. (b) Microencapsulated immunomimetic designer cells to protect mice from MRSA infections [27]. Upon sensing of extracellular MRSA cell-wall components (PGN and LPA) by Toll-like receptors (TLR) a signaling pathway is triggered, resulting in activation and translocation of AP-1 and NF-kB into the nucleus. These activated transcription factors then bind to a synthetic chimeric promoter containing AP-1 and NF-kB response elements, thereby inducing the expression of lysostaphin, a bacteriolytic therapeutic protein, fully curing the infected mice. (c) Smartphone-controlled insulin production in optogenetically engineered cells for semiautomatic glucose homeostasis in diabetic mice [35]. The wireless signal from a smartphone controls a light source, enabling the regulation of insulin production by optically stimulating engineered cells harboring a light-responsive optogenetic interface. Integration of an electronic glucometer enables sensing of the therapeutic output strength and transmission of digital feedback wirelessly to the smartphone. aCaffVHH, single-domain VHH camelid antibody against caffeine; IL6RB, interleukin-6 receptor subunit beta; STAT3, signal transducer and
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Genetic circuitry for personalized human cell therapy Tolle, Stu¨cheli and Fussenegger 35
control of blood glucose levels. Although transplantation of a human donor pancreas or islets into patients can be curative, there is a severe shortage of donors, and the procedure involves the risk of transplant rejection. Therefore, the use of patient-derived pancreatic beta cells constitutes an attractive approach for type 1 diabetes treatment. Two examples of differentiating pluripotent stem cells into somatic glucose-sensitive insulin-secreting beta-like cells have recently been reported. A programmable synthetic lineage-control network has been designed that is capable of differentiating human embryonic stem cells (hESCs) [31] and human induced pluripotent stem cells (hIPSCs) [32]. In an alternative approach, beta-cell-mimetic designer cells were created by engineering human HEK-293 cells and were able to correct insulin deficiency and thereby self-sufficiently abolish persistent hyperglycemia in type 1 diabetic mice [33]. Together, these examples demonstrate that genetic networks offer a robust methodology for the generation of insulin-secreting beta-like cells with numerous applications in basic research and regenerative medicine. To combat insulin-resistance, a self-adjusting synthetic gene circuit was designed to sense and reverse insulinresistance syndrome, and was shown to be effective in mouse models [34]. By functionally rewiring the mitogen-activated protein kinase (MAPK) signaling pathway, a synthetic insulin-sensitive transcription-control device was obtained. The synthetic insulin-sensing designer circuit can self-sufficiently distinguish between physiological and increased blood insulin levels and correspondingly express the insulin-sensitizing compound adiponectin. Because of the closed-loop nature of this insulin-sensor device, self-adjusting adiponectin production is achieved in response to the state of insulin resistance, thereby preventing adiponectin overdosing. With the constant progress in cell phone development, today’s smartphones represent very powerful mobile computers with great potential to be integrated into point-of-care mobile health solutions. As a further step in translating cell-based therapies into the clinic, a smartphone-controlled technological infrastructure for the semiautomatic treatment of diabetes in mice has been established [35]. As depicted in Figure 2c, in this setup, a remote-controllable home server was programmed to process wireless signals from a smartphone, enabling the regulation of insulin production by optically stimulating engineered cells harboring a light-responsive optogenetic interface. The system engineered in this work integrates an electronic glucometer transmitting data wirelessly to the smartphone, thereby combining the digitalized diagnostic accuracy of the sensor with the
efficiency of engineered cells to self-sufficiently produce and deliver insulin. In situ production of designer exosomes for the delivery of therapeutic cargo
In order to extend the types of therapeutic effector outputs generatable by implanted engineered cells, a system for the customized production of mRNA-containing designer exosomes has been created [36]. Exosomes are cell-derived vesicles that represent a promising transport vehicle for therapeutic cargo molecules, with the inherent ability to reach every site in the body and even cross the blood–brain barrier. Their potential for future applications has been demonstrated by the efficient production and delivery of a therapeutic mRNA, leading to attenuation of neurotoxicity and neuroinflammation, in an in vivo model of Parkinson’s disease. In situ production and distribution of therapeutic mRNAs by implanted designer cells holds great potential to fuel the currently rapidly evolving field of RNA therapeutics, which has so far been held back by the limitations of available delivery methods [37].
Challenges and outlook As biological systems are highly complex and often not fully understood, it can be challenging to rationally design gene circuits with predictable behavior. Typically, many empirically driven design-build-test iterations are needed until a functional setup can be established. To facilitate and speed up the development cycle, better in silico tools, such as design software and machine-learning algorithms would be extremely beneficial [38,39]. Especially, improved simulations of genetic circuits could aid in tuning effector strength, and represent a promising approach to better understand the effects of designed systems, for example, on host cell resources and metabolism [40]. Beside computer simulations, improved in vitro test systems that simulate the environment found in the desired application are of paramount importance as complementary assays to animal models [41]. Synthetic biology will significantly benefit from the tremendous progress that has been made in recent years around enabling technologies. (i) Improved highthroughput DNA synthesis methods allow for automated gene, and even genome, synthesis, substantially reducing the time scale in which new constructs can be generated and tested [42,43]. (ii) New programmable genome editing tools based on the CRISPR/Cas system are becoming available, facilitating the precise manipulation of genetic information and serving as programmable regulators [44,45,46]. (iii) Improved delivery systems such as viruses with larger DNA cargo capacity, for example, baculovirus-based vector systems, allow for the delivery of longer stretches of genetic material enabling, for example, multigene expression [47]. With the increasing
(Figure 2 Legend Continued) activator of transcription protein 3; PGN, peptidoglycan; LPA, lipoteichoic acid; AP-1, activator protein 1; NF-kB, nuclear factor kappa B; GTP, guanosine triphosphate; BphS, bacteriophytochrome; c-di-GMP, cyclic di-guanosine monophosphate; STING, stimulator of interferon genes. www.sciencedirect.com
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complexity of designer genetic circuitry, more refined computations and therefore a higher degree of functional control can be achieved, which, in turn, will translate into more effective and safer personalized treatment options in the future [48]. Despite the ever-increasing speed of development, there are still several research areas where further advances are needed. To obtain precise conditional control over cellular functions and to further decouple synthetic gene circuits from host metabolism and signaling machinery, a higher level of orthogonality to natural cellular processes is required. In particular, novel gene switches reactive to complementary types of input stimuli need to be developed. Keeping in mind the development and combination of artificial orthogonal components, one could envision the assembly of an entirely orthogonal central dogma, build on top (AEGIS) or genetic polymers of, for example, ‘artificial expanded genetic information systems’ with unnatural backbone chemistries [49,50]. By re-engineering DNA replication, transcription, and translation, artificial genetic programs could be isolated from host regulation, which would make it possible to expand the roles of these processes within the cell [51]. Last but not least, programmable cellular circuitry could be broadened to multicellular networks based on engineered intercellular communication, controlling the orchestrated behavior of entire cell ensembles [52,53,54–56].
Conclusion Beyond understanding fundamental biological phenomena, combining gene switches in new and complex ways can give rise to unprecedented cellular functions. The precise level of control over cellular outputs provided by genetic circuits, together with their robustness, makes engineered designer cells promising therapeutic entities with an enormous range of potential applications in personalized human medicine.
Conflict of interest statement Nothing declared.
Acknowledgements
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Work in the laboratory of M.F. is financially supported in part through a European Research Council (ERC) advanced grant and in part by the National Centre of Competence in Research (NCCR) for Molecular Systems Engineering. The authors would like to thank Leo Scheller for critical reading of the manuscript and helpful discussions.
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This paper describes a modular signaling platform used to engineer artificial genetic programs in which specific cell–cell contacts induce changes in cell adhesion. Despite their simplicity, these minimal intercellular programs were sufficient to yield assemblies with hallmarks of natural developmental systems. These results provide insights into the evolution of multicellularity and demonstrate the potential to engineer customized self-organizing tissues or materials. 54. Kim S, Kerns SJ, Ziesack M, Bry L, Gerber GK, Way JC, Silver PA: Quorum sensing can be repurposed to promote information
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ScienceDirect Genetic circuitry for personalized human cell therapy Fabian Tolle1, Pascal Stu¨cheli1 and Martin Fussenegger1,2 Synthetic biology uses engineering principles to design and assemble biological components and systems for a variety of applications. On the basis of genetic engineering, synthetic gene switches can be interconnected to construct complex gene circuits, capable of sensing and integrating diverse input signals for precise spatiotemporal control of target gene expression in living cells. Designer cells can be equipped with advanced gene circuitry enabling them to react precisely to pre-programmed combinations of conditions, automatically triggering a specified response, such as therapeutic protein production. Such cells are promising therapeutic modalities for applications where traditional medical treatments have limitations. Herein, we highlight selected recent examples of designer cells with engineered gene circuits targeted toward applications in personalized human medicine. Addresses 1 Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland 2 Faculty of Science, University of Basel, Mattenstrasse 26, CH-4058 Basel, Switzerland Corresponding author: Fussenegger, Martin ([email protected])
Current Opinion in Biotechnology 2019, 59:31–38 This review comes from a themed issue on Tissue, cell and pathway engineering Edited by Marjan De Mey and Eveline Peeters
https://doi.org/10.1016/j.copbio.2019.02.003 0958-1669/ã 2018 Elsevier Ltd. All rights reserved.
Introduction The idea of cell therapy, in which living cellular material is injected or transplanted into a patient, dates back to at least the nineteenth century when Swiss doctor Paul Niehans experimented with injecting live cells obtained from sheep embryos, in an attempt to cure illnesses and rejuvenate his patients [1]. The field has come a long way since then. Indeed, the burgeoning development of methods for genetic manipulation and constitutive overexpression of transgenes has resulted in immense progress; for example, genetically engineered T-cells are emerging as powerful medicines in cancer therapy [2,3,4]. However, many potential applications of genetically engineered cells require the cells to sense an input www.sciencedirect.com
stimulus and modulate the strength, timing and cellular context of gene expression accordingly [5]. As depicted in Figure 1a, a current key challenge in cell engineering is to design and implement advanced input signal-responsive gene circuitry, in order to achieve precise spatiotemporal control of target gene activities in living cells [6]. This is not easy, because, in a therapeutic context, engineered cells have to perform under ever-changing and, therefore, uncertain conditions. In order to autonomously and reliably perform the target function, the effect of the cellular output has to be sensed, and the information entered into a feedback loop capable of re-adjusting the output strength [7]. In other words, the cells are required to monitor the status of their environment, and not only produce specific molecules on demand, but also adjust their performance to correct differences between the desired and actual states.
Building gene circuits to provide closed-loop-controlled designer cell activity Gene circuits consist of interconnected gene switches, which act as regulatory access points at which gene expression can be initiated, interrupted or terminated. Depending on the desired regulatory level and mechanism of action, different input signals can be applied (Figure 1a). Input methods may be influenced by properties such as cellmembrane permeability, specificity, spatiotemporal resolution, reversibility, and response time, which all have to be considered and weighed against each other, depending on the intended regulatory outcome. To date, the most commonly employed types of input stimuli are chemical triggers, such as ions, small organic molecules, or proteins. However, electromagnetic waves such as light have also found extensive use as inducers due to their superior spatiotemporal controllability [8,9]. Gene switches can be implemented at various regulatory levels (Figure 1b). (i) At the extracellular level, stimuli can be sensed by (artificial) receptors that turn on intracellular signaling cascades capable of amplifying and propagating signals into, for example, the nucleus [10,11]. (ii) At the DNA level, transcriptional control of gene expression can be modulated by, for example, controlling natural or artificial transcription factors [12,13]. (iii) Gene switches can also be implemented at the translational level through, for example, ligand-dependent ribozymes that regulate mRNA stability [14,15]. Multiplexed combinations of gene switches regulated by different input stimuli allow for the assembly of Boolean logic gates, which in turn can be elaborated into complex gene circuits with emergent properties [16]. Biologically Current Opinion in Biotechnology 2019, 59:31–38
32 Tissue, cell and pathway engineering
Figure 1
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Engineered gene circuits enable conditional closed-loop-controlled designer cell activity. (a) Gene circuits responsive to a variety of input signals, for example small molecules, proteins, or light, can be engineered, allowing for closedloop control of designer cell outputs. Therapeutic cells with embedded sensory, signal processing, and response capabilities can act as, for example smart drugs by adjusting cellular activities in response to environmental cues. (b) Gene switches act as access points where gene expression can be modulated to gain control over biological functions at different regulatory levels, such as signal transduction, transcription, or translation.
implemented logic gates are systems that can perform logical operations on one or more binary inputs, producing a single binary output, analogously to the way circuits and chips are designed for digital computers. By interconnecting networks of gene switches acting as logic gates, complex gene circuits can be designed that robustly compute one or more input stimuli, for example, the presence of specific biomarkers for a disease state, in order to generate a specific output, such as the production of a therapeutic protein [17]. Beyond simple detectors and inducible systems, signal integration circuits can be developed to react very precisely to pre-programmed sets of conditions, automatically triggering a defined reaction [18]. With the increasing complexity of interconnected sensory and regulatory gene circuits, it is becoming possible to tune cells for practical applications as therapeutic modalities in personalized human medicine [19]. Current Opinion in Biotechnology 2019, 59:31–38
Engineering mammalian cells towards applications in personalized human cell therapy Sensors for molecular diagnostics and therapy
The cell membrane constitutes an effective barrier against many external stimuli. To overcome this, transmembrane receptors transduce signaling information from outside the cell, while the signal itself does not have to cross the membrane physically. Natural receptors can be repurposed for some applications in cell engineering, but for many desirable input signals, no natural receptor is available [20]. To bridge this gap, an extracellular sensor platform based on a universal designer receptor scaffold has been engineered [21]. To demonstrate the scope of this ‘generalized extracellular molecule sensor’ (GEMS) platform, antibody fragments targeting a variety of different inputs, ranging from small molecules to www.sciencedirect.com
Genetic circuitry for personalized human cell therapy Tolle, Stu¨cheli and Fussenegger 33
proteins, have been fused to erythropoietin receptor (EpoR) extracellular and transmembrane domains. By exchanging the intracellular domains, artificial receptors activating a variety of different signaling pathways could be obtained. Based on the GEMS platform, a designer receptor for caffeine has been created (Figure 2a) [22]. Stimulation of designer cells with physiologically relevant concentrations of caffeine, generated by regular intake of beverages such as tea and coffee, resulted in caffeineinduced transgene expression. Type-2 diabetic mice implanted with these microencapsulated cells expressing glucagon-like peptide 1 (GLP1) showed substantially improved glucose homeostasis after coffee consumption. This approach of integrating therapy into the patient’s lifestyle, by coupling the expression of a therapeutic transgene to an inexpensive and non-toxic trigger such as caffeine, represents a promising step towards improving patient compliance. Another inexpensive and non-toxic class of exogenous trigger molecules is free fatty acids (FFAs); these are essential dietary components that also act as signal molecules. Designer cells harboring free fatty acidregulated transgene switches have been engineered and applied for the induction of a reporter gene after consumption of Swiss cheese [23]. The easy adaptability of the FFA-activated transgene switch to various mouse models supports the idea that it is a promising candidate for future diagnostic and therapeutic applications related to, for example, sensing and regulating pathological blood-fat levels. As diagnosis marks the beginning of any successful therapy, one crucial application area for designer cells is in sensing and diagnosing disease states. Toward this end, a synthetic biology-inspired biomedical tattoo using engineered cells has been developed [24]. The cells were programmed to produce the pigment melanin in response to persistently increased Ca2+ concentrations, as seen in hypercalcemia-associated cancer. The sensor system was validated in mice bearing subcutaneously implanted encapsulated engineered cells, resulting in cancer-celldependent generation of an optical signal detectable with the naked eye. Combining the intrinsic sensing properties of bacterial cells with the wireless connectivity of a microelectronic platform, an ingestible micro-bio-electronic device (IMBED) for in vivo sensing in the difficult-to-access gastrointestinal environment has been developed [25]. Genetically engineered heme-sensitive, light-generating biosensor bacteria have been integrated into a miniaturized luminescence readout device for the acute diagnosis of gastrointestinal bleeding. The blood-sensing capability of these engineered cells was demonstrated in a murine in vivo assay, while the IMBED was validated in a porcine model system for gastric bleeding. Considering the www.sciencedirect.com
modular nature of this platform, other whole-cell biosensors could be rapidly integrated to achieve minimally invasive detection of other biomarkers in the gastrointestinal tract. To enable safer cell-based cancer therapies, a new class of cell-contact-sensing device has been engineered to function effectively in human nonimmune cells [26]. Designer cells equipped with this system were capable of explicitly triggering the release of therapeutic output molecules upon sensing contact with a target cancer cell. Cell-based therapy with nonimmune cells represents a promising modality for cancer therapy, eliminating many of the risks associated with current approaches based on engineered immune cells. Combating bacterial infections
Designer cells have been applied very effectively for sensing and controlling bacterial infections. In a pioneering example, a closed-loop gene network capable of detecting and eliminating methicillin-resistant Staphylococcus aureus (MRSA) bacterial infections in mice was designed (Figure 2b) [27]. In this approach, a bacterium-sensing human Toll-like receptor-based genetic circuit was employed to regulate the expression of lysostaphin, a bactericidal enzyme that can kill most known strains of Staphylococci. Microencapsulated cell implants achieved full cure of acute MRSA infections in a mouse model in which conventional vancomycin treatment failed, providing a proof-of-concept that this approach is a viable option for the treatment of antibiotic-resistant pathogens. One reason for the gradual loss of efficacy observed with traditional antibiotics is the coordinated formation of biofilms. In an attempt to reduce biofilm formation, synthetic biology principles have been used to rationally engineer quorum-quenching cells capable of activating a twofold defense, consisting of the secretion of an inactivating enzyme to silence bacterial quorum sensing and an anti-biofilm effector to dissolve the biofilm matrix [28]. In an alternative approach, synthetic mammalian cells were programmed to sense signaling peptides secreted by various microbes and respond with the production of autoinducer-2 (AI2), a key signal molecule regulating bacterial group behavior [29]. Besides engineered mammalian cells, bacteria have also been genetically modified to kill-specific pathogens or to inhibit their virulence. An engineered Escherichia coli strain encoding an anti-biofilm enzyme showed prophylactic and therapeutic activity against Pseudomonas aeruginosa during gut infection in two animal models [30]. These studies open up new possibilities for future anti-infective strategies. Toward curing diabetes
Type 1 diabetes results from a loss of pancreatic insulinproducing beta cells, which are essential for the tight Current Opinion in Biotechnology 2019, 59:31–38
34 Tissue, cell and pathway engineering
Figure 2
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Selected examples of applications of engineered mammalian cells towards human personalized therapy. (a) The GEMS receptor platform for sensing of various inputs to activate multiple endogenous signaling pathways. GEMS have been engineered to sense proteins or small molecules such as caffeine [22] and activate different intracellular pathways such as JAK-STAT, thereby enabling, for example transgenic GLP1 expression for the treatment of diabetes. (b) Microencapsulated immunomimetic designer cells to protect mice from MRSA infections [27]. Upon sensing of extracellular MRSA cell-wall components (PGN and LPA) by Toll-like receptors (TLR) a signaling pathway is triggered, resulting in activation and translocation of AP-1 and NF-kB into the nucleus. These activated transcription factors then bind to a synthetic chimeric promoter containing AP-1 and NF-kB response elements, thereby inducing the expression of lysostaphin, a bacteriolytic therapeutic protein, fully curing the infected mice. (c) Smartphone-controlled insulin production in optogenetically engineered cells for semiautomatic glucose homeostasis in diabetic mice [35]. The wireless signal from a smartphone controls a light source, enabling the regulation of insulin production by optically stimulating engineered cells harboring a light-responsive optogenetic interface. Integration of an electronic glucometer enables sensing of the therapeutic output strength and transmission of digital feedback wirelessly to the smartphone. aCaffVHH, single-domain VHH camelid antibody against caffeine; IL6RB, interleukin-6 receptor subunit beta; STAT3, signal transducer and
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Genetic circuitry for personalized human cell therapy Tolle, Stu¨cheli and Fussenegger 35
control of blood glucose levels. Although transplantation of a human donor pancreas or islets into patients can be curative, there is a severe shortage of donors, and the procedure involves the risk of transplant rejection. Therefore, the use of patient-derived pancreatic beta cells constitutes an attractive approach for type 1 diabetes treatment. Two examples of differentiating pluripotent stem cells into somatic glucose-sensitive insulin-secreting beta-like cells have recently been reported. A programmable synthetic lineage-control network has been designed that is capable of differentiating human embryonic stem cells (hESCs) [31] and human induced pluripotent stem cells (hIPSCs) [32]. In an alternative approach, beta-cell-mimetic designer cells were created by engineering human HEK-293 cells and were able to correct insulin deficiency and thereby self-sufficiently abolish persistent hyperglycemia in type 1 diabetic mice [33]. Together, these examples demonstrate that genetic networks offer a robust methodology for the generation of insulin-secreting beta-like cells with numerous applications in basic research and regenerative medicine. To combat insulin-resistance, a self-adjusting synthetic gene circuit was designed to sense and reverse insulinresistance syndrome, and was shown to be effective in mouse models [34]. By functionally rewiring the mitogen-activated protein kinase (MAPK) signaling pathway, a synthetic insulin-sensitive transcription-control device was obtained. The synthetic insulin-sensing designer circuit can self-sufficiently distinguish between physiological and increased blood insulin levels and correspondingly express the insulin-sensitizing compound adiponectin. Because of the closed-loop nature of this insulin-sensor device, self-adjusting adiponectin production is achieved in response to the state of insulin resistance, thereby preventing adiponectin overdosing. With the constant progress in cell phone development, today’s smartphones represent very powerful mobile computers with great potential to be integrated into point-of-care mobile health solutions. As a further step in translating cell-based therapies into the clinic, a smartphone-controlled technological infrastructure for the semiautomatic treatment of diabetes in mice has been established [35]. As depicted in Figure 2c, in this setup, a remote-controllable home server was programmed to process wireless signals from a smartphone, enabling the regulation of insulin production by optically stimulating engineered cells harboring a light-responsive optogenetic interface. The system engineered in this work integrates an electronic glucometer transmitting data wirelessly to the smartphone, thereby combining the digitalized diagnostic accuracy of the sensor with the
efficiency of engineered cells to self-sufficiently produce and deliver insulin. In situ production of designer exosomes for the delivery of therapeutic cargo
In order to extend the types of therapeutic effector outputs generatable by implanted engineered cells, a system for the customized production of mRNA-containing designer exosomes has been created [36]. Exosomes are cell-derived vesicles that represent a promising transport vehicle for therapeutic cargo molecules, with the inherent ability to reach every site in the body and even cross the blood–brain barrier. Their potential for future applications has been demonstrated by the efficient production and delivery of a therapeutic mRNA, leading to attenuation of neurotoxicity and neuroinflammation, in an in vivo model of Parkinson’s disease. In situ production and distribution of therapeutic mRNAs by implanted designer cells holds great potential to fuel the currently rapidly evolving field of RNA therapeutics, which has so far been held back by the limitations of available delivery methods [37].
Challenges and outlook As biological systems are highly complex and often not fully understood, it can be challenging to rationally design gene circuits with predictable behavior. Typically, many empirically driven design-build-test iterations are needed until a functional setup can be established. To facilitate and speed up the development cycle, better in silico tools, such as design software and machine-learning algorithms would be extremely beneficial [38,39]. Especially, improved simulations of genetic circuits could aid in tuning effector strength, and represent a promising approach to better understand the effects of designed systems, for example, on host cell resources and metabolism [40]. Beside computer simulations, improved in vitro test systems that simulate the environment found in the desired application are of paramount importance as complementary assays to animal models [41]. Synthetic biology will significantly benefit from the tremendous progress that has been made in recent years around enabling technologies. (i) Improved highthroughput DNA synthesis methods allow for automated gene, and even genome, synthesis, substantially reducing the time scale in which new constructs can be generated and tested [42,43]. (ii) New programmable genome editing tools based on the CRISPR/Cas system are becoming available, facilitating the precise manipulation of genetic information and serving as programmable regulators [44,45,46]. (iii) Improved delivery systems such as viruses with larger DNA cargo capacity, for example, baculovirus-based vector systems, allow for the delivery of longer stretches of genetic material enabling, for example, multigene expression [47]. With the increasing
(Figure 2 Legend Continued) activator of transcription protein 3; PGN, peptidoglycan; LPA, lipoteichoic acid; AP-1, activator protein 1; NF-kB, nuclear factor kappa B; GTP, guanosine triphosphate; BphS, bacteriophytochrome; c-di-GMP, cyclic di-guanosine monophosphate; STING, stimulator of interferon genes. www.sciencedirect.com
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36 Tissue, cell and pathway engineering
complexity of designer genetic circuitry, more refined computations and therefore a higher degree of functional control can be achieved, which, in turn, will translate into more effective and safer personalized treatment options in the future [48]. Despite the ever-increasing speed of development, there are still several research areas where further advances are needed. To obtain precise conditional control over cellular functions and to further decouple synthetic gene circuits from host metabolism and signaling machinery, a higher level of orthogonality to natural cellular processes is required. In particular, novel gene switches reactive to complementary types of input stimuli need to be developed. Keeping in mind the development and combination of artificial orthogonal components, one could envision the assembly of an entirely orthogonal central dogma, build on top (AEGIS) or genetic polymers of, for example, ‘artificial expanded genetic information systems’ with unnatural backbone chemistries [49,50]. By re-engineering DNA replication, transcription, and translation, artificial genetic programs could be isolated from host regulation, which would make it possible to expand the roles of these processes within the cell [51]. Last but not least, programmable cellular circuitry could be broadened to multicellular networks based on engineered intercellular communication, controlling the orchestrated behavior of entire cell ensembles [52,53,54–56].
Conclusion Beyond understanding fundamental biological phenomena, combining gene switches in new and complex ways can give rise to unprecedented cellular functions. The precise level of control over cellular outputs provided by genetic circuits, together with their robustness, makes engineered designer cells promising therapeutic entities with an enormous range of potential applications in personalized human medicine.
Conflict of interest statement Nothing declared.
Acknowledgements
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Work in the laboratory of M.F. is financially supported in part through a European Research Council (ERC) advanced grant and in part by the National Centre of Competence in Research (NCCR) for Molecular Systems Engineering. The authors would like to thank Leo Scheller for critical reading of the manuscript and helpful discussions.
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References and recommended reading
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Genetic circuitry for personalized human cell therapy Tolle, Stu¨cheli and Fussenegger 37
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38 Tissue, cell and pathway engineering
This paper describes a modular signaling platform used to engineer artificial genetic programs in which specific cell–cell contacts induce changes in cell adhesion. Despite their simplicity, these minimal intercellular programs were sufficient to yield assemblies with hallmarks of natural developmental systems. These results provide insights into the evolution of multicellularity and demonstrate the potential to engineer customized self-organizing tissues or materials. 54. Kim S, Kerns SJ, Ziesack M, Bry L, Gerber GK, Way JC, Silver PA: Quorum sensing can be repurposed to promote information
Current Opinion in Biotechnology 2019, 59:31–38
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