Immunomimetic Designer Cells Protect Mice from MRSA Infection.

Many community- and hospital-acquired bacterial infections are caused by antibiotic-resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) predisposes humans to invasive infections that are difficult to eradicate. We designed a closed-loop gene network programming mammalian cells to autonomously detect and eliminate bacterial infections. The genetic circuit contains human Toll-like receptors as the bacterial sensor and a synthetic promoter driving reversible and adjustable expression of lysostaphin, a bacteriolytic enzyme highly lethal to S. aureus. Immunomimetic designer cells harboring this genetic circuit exhibited fast and robust sense-and-destroy kinetics against live staphylococci. When tested in a foreign-body infection model in mice, microencapsulated cell implants prevented planktonic MRSA infection and reduced MRSA biofilm formation by 91%. Notably, this system achieved a 100% cure rate of acute MRSA infections, whereas conventional vancomycin treatment failed. These results suggest that immunomimetic designer cells could offer a therapeutic approach for early detection, prevention, and cure of pathogenic infections in the post-antibiotic era.

Designing cell function: assembly of synthetic gene circuits for cell biology applications.

Synthetic biology is the discipline of engineering application-driven biological functionalities that were not evolved by nature. Early breakthroughs of cell engineering, which were based on ectopic (over)expression of single sets of transgenes, have already had a revolutionary impact on the biotechnology industry, regenerative medicine and blood transfusion therapies. Now, we require larger-scale, rationally assembled genetic circuits engineered to programme and control various human cell functions with high spatiotemporal precision in order to solve more complex problems in applied life sciences, biomedicine and environmental sciences. This will open new possibilities for employing synthetic biology to advance personalized medicine by converting cells into living therapeutics to combat hitherto intractable diseases.

Combinatorial protein dimerization enables precise multi-input synthetic computations.

Bacterial transcription factors (TFs) with helix-turn-helix (HTH) DNA-binding domains have been widely explored to build orthogonal transcriptional regulation systems in mammalian cells. Here we capitalize on the modular structure of these proteins to build a framework for multi-input logic gates relying on serial combinations of inducible protein-protein interactions. We found that for some TFs, their HTH domain alone is sufficient for DNA binding. By fusing the HTH domain to TFs, we established dimerization dependent rather than DNA-binding-dependent activation. This enabled us to convert gene switches from OFF-type into more widely applicable ON-type systems and to create mammalian gene switches responsive to new inducers. By combining both OFF and ON modes of action, we built a compact, high-performance bandpass filter. Furthermore, we were able to show cytosolic and extracellular dimerization. Cascading up to five pairwise fusion proteins yielded robust multi-input AND logic gates. Combinations of different pairwise fusion proteins afforded a variety of 4-input 1-output AND and OR logic gate configurations.

Evolution of molecular switches for regulation of transgene expression by clinically licensed gluconate.

Synthetic biology holds great promise to improve the safety and efficacy of future gene and engineered cell therapies by providing new means of endogenous or exogenous control of the embedded therapeutic programs. Here, we focused on gluconate as a clinically licensed small-molecule inducer and engineered gluconate-sensitive molecular switches to regulate transgene expression in human cell cultures and in mice. Several switch designs were assembled based on the gluconate-responsive transcriptional repressor GntR from Escherichia coli. Initially we assembled OFF- and ON-type switches by rewiring the native gluconate-dependent binding of GntR to target DNA sequences in mammalian cells. Then, we utilized the ability of GntR to dimerize in the presence of gluconate to activate gene expression from a split transcriptional activator. By means of random mutagenesis of GntR combined with phenotypic screening, we identified variants that significantly enhanced the functionality of the genetic devices, enabling the construction of robust two-input logic gates. We also demonstrated the potential utility of the synthetic switch in two in vivo settings, one employing implantation of alginate-encapsulated engineered cells and the other involving modification of host cells by DNA delivery. Then, as proof-of-concept, the gluconate-actuated genetic switch was connected to insulin secretion, and the components encoding gluconate-induced insulin production were introduced into type-1 diabetic mice as naked DNA via hydrodynamic tail vein injection. Normoglycemia was restored, thereby showcasing the suitability of oral gluconate to regulate in situ production of a therapeutic protein.

Design of programmable post-translational switch control platform for on-demand protein secretion in mammalian cells.

The development of novel strategies to program cellular behaviors is a central goal in synthetic biology, and post-translational control mediated by engineered protein circuits is a particularly attractive approach to achieve rapid protein secretion on demand. We have developed a programmable protease-mediated post-translational switch (POSH) control platform composed of a chimeric protein unit that consists of a protein of interest fused via a transmembrane domain to a cleavable ER-retention signal, together with two cytosolic inducer-sensitive split protease components. The protease components combine in the presence of the specific inducer to generate active protease, which cleaves the ER-retention signal, releasing the transmembrane-domain-linked protein for trafficking to the trans-Golgi region. A furin site placed downstream of the protein ensures cleavage and subsequent secretion of the desired protein. We show that stimuli ranging from plant-derived, clinically compatible chemicals to remotely controllable inducers such as light and electrostimulation can program protein secretion in various POSH-engineered designer mammalian cells. As proof-of-concept, an all-in-one POSH control plasmid encoding insulin and abscisic acid-activatable split protease units was hydrodynamically transfected into the liver of type-1 diabetic mice. Induction with abscisic acid attenuated glycemic excursions in glucose-tolerance tests. Increased blood levels of insulin were maintained for 12 days.

Controlling therapeutic protein expression via inhalation of a butter flavor molecule.

Precise control of the delivery of therapeutic proteins is critical for gene- and cell-based therapies, and expression should only be switched on in the presence of a specific trigger signal of appropriate magnitude. Focusing on the advantages of delivering the trigger by inhalation, we have developed a mammalian synthetic gene switch that enables regulation of transgene expression by exposure to the semi-volatile small molecule acetoin, a widely used, FDA-approved food flavor additive. The gene switch capitalizes on the bacterial regulatory protein AcoR fused to a mammalian transactivation domain, which binds to promoter regions with specific DNA sequences in the presence of acetoin and dose-dependently activates expression of downstream transgenes. Wild-type mice implanted with alginate-encapsulated cells transgenic for the acetoin gene switch showed a dose-dependent increase in blood levels of reporter protein in response to either administration of acetoin solution via oral gavage or longer exposure to acetoin aerosol generated by a commercial portable inhaler. Intake of typical acetoin-containing foods, such as butter, lychees and cheese, did not activate transgene expression. As a proof of concept, we show that blood glucose levels were normalized by acetoin aerosol inhalation in type-I diabetic mice implanted with acetoin-responsive insulin-producing cells. Delivery of trigger molecules using portable inhalers may facilitate regular administration of therapeutic proteins via next-generation cell-based therapies to treat chronic diseases for which frequent dosing is required.

Toward Photosynthetic Mammalian Cells through Artificial Endosymbiosis.

Photosynthesis in plants occurs within specialized organelles known as chloroplasts, which are postulated to have originated through endosymbiosis with cyanobacteria. In nature, instances are also observed wherein specific invertebrates engage in symbiotic relationships with photosynthetic bacteria, allowing them to subsist as photoautotrophic organisms over extended durations. Consequently, the concept of engineering artificial endosymbiosis between mammalian cells and cyanobacteria represents a promising avenue for enabling photosynthesis in mammals. The study embarked with the identification of Synechocystis PCC 6803 as a suitable candidate for establishing a long-term endosymbiotic relationship with macrophages. The cyanobacteria internalized by macrophages exhibited the capacity to rescue ATP deficiencies within their host cells under conditions of illumination. Following this discovery, a membrane-coating strategy is developed for the intracellular delivery of cyanobacteria into non-macrophage mammalian cells. This pioneering technique led to the identification of human embryonic kidney cells HEK293 as optimal hosts for achieving sustained endosymbiosis with Synechocystis PCC 6803. The study offers valuable insights that may serve as a reference for the eventual achievement of artificial photosynthesis in mammals.

Small-Molecule Regulators for Gene Switches to Program Mammalian Cell Behaviour.

Synthetic or natural small molecules have been extensively employed as trigger signals or inducers to regulate engineered gene circuits introduced into living cells in order to obtain desired outputs in a controlled and predictable manner. Here, we provide an overview of small molecules used to drive synthetic-biology-based gene circuits in mammalian cells, together with examples of applications at different levels of control, including regulation of DNA manipulation, RNA synthesis and editing, and protein synthesis, maturation, and trafficking. We also discuss the therapeutic potential of these small-molecule-responsive gene circuits, focusing on the advantages and disadvantages of using small molecules as triggers, the mechanisms involved, and the requirements for selecting suitable molecules, including efficiency, specificity, orthogonality, and safety. Finally, we explore potential future directions for translation of these devices to clinical medicine.

Programmable DARPin-based receptors for the detection of thrombotic markers.

Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing 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 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein.

Enhancing cell-based therapies with synthetic gene circuits responsive to molecular stimuli.

Synthetic biology aims to contribute to the development of next-generation patient-specific cell-based therapies for chronic diseases especially through the construction of sophisticated synthetic gene switches to enhance the safety and spatiotemporal controllability of engineered cells. Indeed, switches that sense and process specific cues, which may be either externally administered triggers or endogenous disease-associated molecules, have emerged as powerful tools for programming and fine-tuning therapeutic outputs. Living engineered cells, often referred to as designer cells, incorporating such switches are delivered to patients either as encapsulated cell implants or by infusion, as in the case of the clinically approved CAR-T cell therapies. Here, we review recent developments in synthetic gene switches responsive to molecular stimuli, spanning regulatory mechanisms acting at the transcriptional, translational, and posttranslational levels. We also discuss current challenges facing clinical translation of cell-based therapies employing these devices.

Hydratable Core-Shell Polymer Networks for Atmospheric Water Harvesting Powered by Sunlight.

The development of technologies to enable fresh water harvesting from atmospheric moisture could help overcome the problem of potable water scarcity. Here, an atmospheric water harvesting (AWH) device is assembled in a core-shell structure, with the core consisting of networks of alginate (Alg) and polyaniline (PANI) and the outer layer consisting of thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) modified with sulfonic acid groups (SPNIPAM) to increase the water adsorption at low relative humidity. The resulting hydrogel, modified with lithium chloride (LiCl) for increased water storage capacity (SPNIPAM-Li-PANIAlg), displays a similar lower critical solution temperature to pristine PNIPAM (32 °C) while affording a 15-fold higher water capture ratio, and releases water upon exposure to sunlight at intensities less than 1 kW m-2 . The developed AWH system is capable of harvesting 6.5 L of water per kilogram in a single daily absorption/desorption cycle under sunlight and can operate at relative humidity levels as low as 17% with no additional external energy input. The thermo-responsive hydrogel SPNIPAM-Li-PANIAlg exhibits excellent stability during natural sunlight-driven absorption/desorption cycles for at least 30 days, and allows sustainable harvesting of over 28.3 L kg-1 from a moisture-rich environment by means of multiple absorption/desorption cycles.

Bottom-up de novo design of functional proteins with complex structural features.

De novo protein design has enabled the creation of new protein structures. However, the design of functional proteins has proved challenging, in part due to the difficulty of transplanting structurally complex functional sites to available protein structures. Here, we used a bottom-up approach to build de novo proteins tailored to accommodate structurally complex functional motifs. We applied the bottom-up strategy to successfully design five folds for four distinct binding motifs, including a bifunctionalized protein with two motifs. Crystal structures confirmed the atomic-level accuracy of the computational designs. These de novo proteins were functional as components of biosensors to monitor antibody responses and as orthogonal ligands to modulate synthetic signaling receptors in engineered mammalian cells. Our work demonstrates the potential of bottom-up approaches to accommodate complex structural motifs, which will be essential to endow de novo proteins with elaborate biochemical functions, such as molecular recognition or catalysis.

Synthetic biology in multicellular organisms: Opportunities in nematodes.

Synthetic biology has mainly focused on introducing new or altered functionality in single cell systems: primarily bacteria, yeast, or mammalian cells. Here, we describe the extension of synthetic biology to nematodes, in particular the well-studied model organism Caenorhabditis elegans, as a convenient platform for developing applications in a multicellular setting. We review transgenesis techniques for nematodes, as well as the application of synthetic biology principles to construct nematode gene switches and genetic devices to control motility. Finally, we discuss potential applications of engineered nematodes.

5-Fluorouracil blocks quorum-sensing of biofilm-embedded methicillin-resistant Staphylococcus aureus in mice.

Antibiotic-resistant pathogens often escape antimicrobial treatment by forming protective biofilms in response to quorum-sensing communication via diffusible autoinducers. Biofilm formation by the nosocomial pathogen methicillin-resistant Staphylococcus aureus (MRSA) is triggered by the quorum-sensor autoinducer-2 (AI-2), whose biosynthesis is mediated by methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) and S-ribosylhomocysteine lyase (LuxS). Here, we present a high-throughput screening platform for small-molecular inhibitors of either enzyme. This platform employs a cell-based assay to report non-toxic, bioavailable and cell-penetrating inhibitors of AI-2 production, utilizing engineered human cells programmed to constitutively secrete AI-2 by tapping into the endogenous methylation cycle via ectopic expression of codon-optimized MTAN and LuxS. Screening of a library of over 5000 commercial compounds yielded 66 hits, including the FDA-licensed cytostatic anti-cancer drug 5-fluorouracil (5-FU). Secondary screening and validation studies showed that 5-FU is a potent quorum-quencher, inhibiting AI-2 production and release by MRSA, Staphylococcus epidermidis, Escherichia coli and Vibrio harveyi. 5-FU efficiently reduced adherence and blocked biofilm formation of MRSA in vitro at an order-of-magnitude-lower concentration than that clinically relevant for anti-cancer therapy. Furthermore, 5-FU reestablished antibiotic susceptibility and enabled daptomycin-mediated prevention and clearance of MRSA infection in a mouse model of human implant-associated infection.

Neurons differentiate magnitude and location of mechanical stimuli.

Neuronal activity can be modulated by mechanical stimuli. To study this phenomenon quantitatively, we mechanically stimulated rat cortical neurons by shear stress and local indentation. Neurons show 2 distinct responses, classified as transient and sustained. Transient responses display fast kinetics, similar to spontaneous neuronal activity, whereas sustained responses last several minutes before returning to baseline. Local soma stimulations with micrometer-sized beads evoke transient responses at low forces of ∼220 nN and pressures of ∼5.6 kPa and sustained responses at higher forces of ∼360 nN and pressures of ∼9.2 kPa. Among the neuronal compartments, axons are highly susceptible to mechanical stimulation and predominantly show sustained responses, whereas the less susceptible dendrites predominantly respond transiently. Chemical perturbation experiments suggest that mechanically evoked responses require the influx of extracellular calcium through ion channels. We propose that subtraumatic forces/pressures applied to neurons evoke neuronal responses via nonspecific gating of ion channels.

Sunlight-Controllable Biopharmaceutical Production for Remote Emergency Supply of Directly Injectable Therapeutic Proteins.

Biopharmaceutical manufacturing requires specialized facilities and a long-range cold supply chain for the delivery of the therapeutics to patients. In order to produce biopharmaceuticals in locations lacking such infrastructure, a production process is designed that utilizes the trigger-inducible release of large quantities of a stored therapeutic protein from engineered endocrine cells within minutes to generate a directly injectable saline solution of the protein. To illustrate the versatility of this approach, it is shown that not only insulin, but also glucagon-like peptide 1 (GLP-1), nanoluciferase (NLuc), and the model biopharmaceutical erythropoietin (EPO) can be trigger-inducibly released, even when using biologically inactive insulin as a carrier. The facilitating beta cells are engineered with a controllable TRPV1-mediated Ca2+ influx that induces the fusion of cytoplasmic storage vesicles with the membrane, leading to the release of the stored protein. When required, the growth medium is exchanged for saline solution, and the system is stimulated with the small molecule capsaicin, with a hand-warming pack, or simply by using sunlight. Injection of insulin saline solution obtained in this way into a type-1 diabetes mouse model results in the regulation of blood glucose levels. It is believed that this system will be readily adaptable to deliver various biopharmaceutical proteins at remote locations.

dCas9-mediated dysregulation of gene expression in human induced pluripotent stem cells during primitive streak differentiation.

CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9) from S. pyogenes as a platform for targeted epigenetic editing in stem cells to enhance the expression of the eomesodermin gene (EOMES) during differentiation. We observed, however, that the dCas9 protein itself exerts a potential non-specific effect in hiPSCs, affecting the cell's phenotype and gene expression patterns during subsequent directed differentiation. We show that this effect is specific to the condition when cells are cultured in medium that does not actively maintain the pluripotency network, and that the sgRNA-free apo-dCas9 protein itself influences endogenous gene expression. Transcriptomics analysis revealed that a significant number of genes involved in developmental processes and various other genes with non-overlapping biological functions are affected by dCas9 overexpression. This suggests a potential adverse phenotypic effect of dCas9 itself in hiPSCs, which could have implications for when and how CRISPR/Cas9-based tools can be used reliably and safely in pluripotent stem cells.

CelloSelect - A synthetic cellobiose metabolic pathway for selection of stable transgenic CHO cell lines.

Current protocols for generating stable transgenic cell lines mostly rely on antibiotic selection or the use of specialized cell lines lacking an essential part of their metabolic machinery, but these approaches require working with either toxic chemicals or knockout cell lines, which can reduce productivity. Since most mammalian cells cannot utilize cellobiose, a disaccharide consisting of two ß-1,4-linked glucose molecules, we designed an antibiotic-free selection system, CelloSelect, which consists of a selection cassette encoding Neurospora crassa cellodextrin transporter CDT1 and ß-glucosidase GH1-1. When cultivated in glucose-free culture medium containing cellobiose, CelloSelect-transfected cells proliferate by metabolizing cellobiose as a primary energy source, and are protected from glucose starvation. We show that the combination of CelloSelect with a PiggyBac transposase-based integration strategy provides a platform for the swift and efficient generation of stable transgenic cell lines. Growth rate analysis of metabolically engineered cells in cellobiose medium confirmed the expansion of cells stably expressing high levels of a cargo fluorescent marker protein. We further validated this strategy by applying the CelloSelect system for stable integration of sequences encoding two biopharmaceutical proteins, erythropoietin and the monoclonal antibody rituximab, and confirmed that the proteins are efficiently produced in either cellobiose- or glucose-containing medium in suspension-adapted CHO cells cultured in chemically defined media. We believe coupling heterologous metabolic pathways additively to the endogenous metabolism of mammalian cells has the potential to complement or to replace current cell-line selection systems.

A synthetic multifunctional mammalian pH sensor and CO2 transgene-control device.

All metabolic activities operate within a narrow pH range that is controlled by the CO2-bicarbonate buffering system. We hypothesized that pH could serve as surrogate signal to monitor and respond to the physiological state. By functionally rewiring the human proton-activated cell-surface receptor TDAG8 to chimeric promoters, we created a synthetic signaling cascade that precisely monitors extracellular pH within the physiological range. The synthetic pH sensor could be adjusted by organic acids as well as gaseous CO2 that shifts the CO2-bicarbonate balance toward hydrogen ions. This enabled the design of gas-programmable logic gates, provided remote control of cellular behavior inside microfluidic devices, and allowed for CO2-triggered production of biopharmaceuticals in standard bioreactors. When implanting cells containing the synthetic pH sensor linked to production of insulin into type 1 diabetic mice developing diabetic ketoacidosis, the prosthetic network automatically scored acidic pH and coordinated an insulin expression response that corrected ketoacidosis.

An overview of signaling pathways regulating YAP/TAZ activity.

YAP and TAZ are ubiquitously expressed homologous proteins originally identified as penultimate effectors of the Hippo signaling pathway, which plays a key role in maintaining mammalian tissue/organ size. Presently, it is known that YAP/TAZ also interact with various non-Hippo signaling pathways, and have diverse roles in multiple biological processes, including cell proliferation, tissue regeneration, cell lineage fate determination, tumorigenesis, and mechanosensing. In this review, we first examine the various microenvironmental cues and signaling pathways that regulate YAP/TAZ activation, through the Hippo and non-Hippo signaling pathways. This is followed by a brief summary of the interactions of YAP/TAZ with TEAD1-4 and a diverse array of other non-TEAD transcription factors. Finally, we offer a critical perspective on how increasing knowledge of the regulatory mechanisms of YAP/TAZ signaling might open the door to novel therapeutic applications in the interrelated fields of biomaterials, tissue engineering, regenerative medicine and synthetic biology.