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.

A Gene-Switch Platform Interfacing with Reactive Oxygen Species Enables Transcription Fine-Tuning by Soluble and Volatile Pharmacologics and Food Additives.

Synthetic biology aims to engineer transgene switches for precise therapeutic protein control in cell-based gene therapies. However, off-the-shelf trigger-inducible gene circuits are usually switched on by single or structurally similar molecules. This study presents a mammalian gene-switch platform that controls therapeutic gene expression by a wide range of molecules generating low, non-toxic levels of reactive oxygen species (ROS). In this system, KEAP1 (Kelch-like ECH-associated protein 1) serves as ROS sensor, regulating the translocation of NRF2 (nuclear factor erythroid 2-related factor 2) to the nucleus, where NRF2 binds to  antioxidant response elements (ARE) to activate the expression of a gene of interest. It is found that a promoter containing eight-tandem ARE repeats is highly sensitive to the low ROS levels generated by the soluble and volatile molecules, which include food preservatives, food additives, pharmaceuticals, and signal transduction inducers. In a proof-of-concept study, it is shown that many of these compounds can independently trigger microencapsulated engineered cells to produce sufficient insulin to restore normoglycemia in experimental type-1 diabetic mice. It is believed that this system greatly extends the variety of small-molecule inducers available to drive therapeutic gene switches.

Posttranslational Remote Control Mediated by Physical Inducers for Rapid Protein Release in Engineered Mammalian Cells.

Physical cues such as light, heat, or an electrical field can be utilized for traceless, on-demand activation of the expression of a desired therapeutic gene in appropriately engineered cells with excellent spatiotemporal resolution, good inducibility, and simple reversibility. A similar approach can be applied to build a depolarization-based protein secretion system that enables rapid release of a therapeutic protein pre-stored in intracellular vesicles in mammalian cells. Here, we present a protocol to create designer ß-cells that exhibit light-controllable rapid release (within 15 min) of a pre-synthesized proinsulin-nanoluciferase construct from vesicular stores. The construct is cleaved extracellularly to afford secreted insulin as a therapeutic protein and nanoluciferase as a reporter molecule. Such posttranslational remote control offers a much faster response than expression-based systems.

Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells.

Here, we present a gene regulation strategy enabling programmable control over eukaryotic translational initiation. By excising the natural poly-adenylation (poly-A) signal of target genes and replacing it with a synthetic control region harboring RNA-binding protein (RBP)-specific aptamers, cap-dependent translation is rendered exclusively dependent on synthetic translation initiation factors (STIFs) containing different RBPs engineered to conditionally associate with different eIF4F-binding proteins (eIFBPs). This modular design framework facilitates the engineering of various gene switches and intracellular sensors responding to many user-defined trigger signals of interest, demonstrating tightly controlled, rapid and reversible regulation of transgene expression in mammalian cells as well as compatibility with various clinically applicable delivery routes of in vivo gene therapy. Therapeutic efficacy was demonstrated in two animal models. To exemplify disease treatments that require on-demand drug secretion, we show that a custom-designed gene switch triggered by the FDA-approved drug grazoprevir can effectively control insulin expression and restore glucose homeostasis in diabetic mice. For diseases that require instantaneous sense-and-response treatment programs, we create highly specific sensors for various subcellularly (mis)localized protein markers (such as cancer-related fusion proteins) and show that translation-based protein sensors can be used either alone or in combination with other cell-state classification strategies to create therapeutic biocomputers driving self-sufficient elimination of tumor cells in mice. This design strategy demonstrates unprecedented flexibility for translational regulation and could form the basis for a novel class of programmable gene therapies in vivo.

Integrated compact regulators of protein activity enable control of signaling pathways and genome-editing in vivo.

Viral proteases and clinically safe inhibitors were employed to build integrated compact regulators of protein activity (iCROP) for post-translational regulation of functional proteins by tunable proteolytic activity. In the absence of inhibitor, the co-localized/fused protease cleaves a target peptide sequence introduced in an exposed loop of the protein of interest, irreversibly fragmenting the protein structure and destroying its functionality. We selected three proteases and demonstrated the versatility of the iCROP framework by validating it to regulate the functional activity of ten different proteins. iCROP switches can be delivered either as mRNA or DNA, and provide rapid actuation kinetics with large induction ratios, while remaining strongly suppressed in the off state without inhibitor. iCROPs for effectors of the NF-κB and NFAT signaling pathways were assembled and confirmed to enable precise activation/inhibition of downstream events in response to protease inhibitors. In lipopolysaccharide-treated mice, iCROP-sr-IκBα suppressed cytokine release ("cytokine storm") by rescuing the activity of IκBα, which suppresses NF-κB signaling. We also constructed compact inducible CRISPR-(d)Cas9 variants and showed that iCROP-Cas9-mediated knockout of the PCSK9 gene in the liver lowered blood LDL-cholesterol levels in mice. iCROP-based protein switches will facilitate protein-level regulation in basic research and translational applications.

Synthetic Gene Circuits for Regulation of Next-Generation Cell-Based Therapeutics.

Arming human cells with synthetic gene circuits enables to expand their capacity to execute superior sensing and response actions, offering tremendous potential for innovative cellular therapeutics. This can be achieved by assembling components from an ever-expanding molecular toolkit, incorporating switches based on transcriptional, translational, or post-translational control mechanisms. This review provides examples from the three classes of switches, and discusses their advantages and limitations to regulate the activity of therapeutic cells in vivo. Genetic switches designed to recognize internal disease-associated signals often encode intricate actuation programs that orchestrate a reduction in the sensed signal, establishing a closed-loop architecture. Conversely, switches engineered to detect external molecular or physical cues operate in an open-loop fashion, switching on or off upon signal exposure. The integration of such synthetic gene circuits into the next generation of chimeric antigen receptor T-cells is already enabling precise calibration of immune responses in terms of magnitude and timing, thereby improving the potency and safety of therapeutic cells. Furthermore, pre-clinical engineered cells targeting other chronic diseases are gathering increasing attention, and this review discusses the path forward for achieving clinical success. With synthetic biology at the forefront, cellular therapeutics holds great promise for groundbreaking treatments.

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.

Silicon-Based 3D Microfluidics for Parallelization of Droplet Generation.

Both the diversity and complexity of microfluidic systems have experienced a tremendous progress over the last decades, enabled by new materials, novel device concepts and innovative fabrication routes. In particular the subfield of high-throughput screening, used for biochemical, genetic and pharmacological samples, has extensively emerged from developments in droplet microfluidics. More recently, new 3D device architectures enabled either by stacking layers of PDMS or by direct 3D-printing have gained enormous attention for applications in chemical synthesis or biomedical assays. While the first microfluidic devices were based on silicon and glass structures, those materials have not yet been significantly expanded towards 3D despite their high chemical compatibility, mechanical strength or mass-production potential. In our work, we present a generic fabrication route based on the implementation of vertical vias and a redistribution layer to create glass-silicon-glass 3D microfluidic structures. It is used to build different droplet-generating devices with several flow-focusing junctions in parallel, all fed from a single source. We study the effect of having several of these junctions in parallel by varying the flow conditions of both the continuous and the dispersed phases. We demonstrate that the generic concept enables an upscaling in the production rate by increasing the number of droplet generators per device without sacrificing the monodispersity of the droplets.

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.

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.

An electrogenetic interface to program mammalian gene expression by direct current.

Wearable electronic devices are playing a rapidly expanding role in the acquisition of individuals' health data for personalized medical interventions; however, wearables cannot yet directly program gene-based therapies because of the lack of a direct electrogenetic interface. Here we provide the missing link by developing an electrogenetic interface that we call direct current (DC)-actuated regulation technology (DART), which enables electrode-mediated, time- and voltage-dependent transgene expression in human cells using DC from batteries. DART utilizes a DC supply to generate non-toxic levels of reactive oxygen species that act via a biosensor to reversibly fine-tune synthetic promoters. In a proof-of-concept study in a type 1 diabetic male mouse model, a once-daily transdermal stimulation of subcutaneously implanted microencapsulated engineered human cells by energized acupuncture needles (4.5 V DC for 10 s) stimulated insulin release and restored normoglycemia. We believe this technology will enable wearable electronic devices to directly program metabolic interventions.

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.

Shaping Synthetic Multicellular and Complex Multimaterial Tissues via Embedded Extrusion-Volumetric Printing of Microgels.

In living tissues, cells express their functions following complex signals from their surrounding microenvironment. Capturing both hierarchical architectures at the micro- and macroscale, and anisotropic cell patterning remains a major challenge in bioprinting, and a bottleneck toward creating physiologically-relevant models. Addressing this limitation, a novel technique is introduced, termed Embedded Extrusion-Volumetric Printing (EmVP), converging extrusion-bioprinting and layer-less, ultra-fast volumetric bioprinting, allowing spatially pattern multiple inks/cell types. Light-responsive microgels are developed for the first time as bioresins (µResins) for light-based volumetric bioprinting, providing a microporous environment permissive for cell homing and self-organization. Tuning the mechanical and optical properties of gelatin-based microparticles enables their use as support bath for suspended extrusion printing, in which features containing high cell densities can be easily introduced. µResins can be sculpted within seconds with tomographic light projections into centimeter-scale, granular hydrogel-based, convoluted constructs. Interstitial microvoids enhanced differentiation of multiple stem/progenitor cells (vascular, mesenchymal, neural), otherwise not possible with conventional bulk hydrogels. As proof-of-concept, EmVP is applied to create complex synthetic biology-inspired intercellular communication models, where adipocyte differentiation is regulated by optogenetic-engineered pancreatic cells. Overall, EmVP offers new avenues for producing regenerative grafts with biological functionality, and for developing engineered living systems and (metabolic) disease models.

Direct electrification of silicon microfluidics for electric field applications.

Microfluidic systems are widely used in fundamental research and industrial applications due to their unique behavior, enhanced control, and manipulation opportunities of liquids in constrained geometries. In micrometer-sized channels, electric fields are efficient mechanisms for manipulating liquids, leading to deflection, injection, poration or electrochemical modification of cells and droplets. While PDMS-based microfluidic devices are used due to their inexpensive fabrication, they are limited in terms of electrode integration. Using silicon as the channel material, microfabrication techniques can be used to create nearby electrodes. Despite the advantages that silicon provides, its opacity has prevented its usage in most important microfluidic applications that need optical access. To overcome this barrier, silicon-on-insulator technology in microfluidics is introduced to create optical viewports and channel-interfacing electrodes. More specifically, the microfluidic channel walls are directly electrified via selective, nanoscale etching to introduce insulation segments inside the silicon device layer, thereby achieving the most homogeneous electric field distributions and lowest operation voltages feasible across microfluidic channels. These ideal electrostatic conditions enable a drastic energy reduction, as effectively shown via picoinjection and fluorescence-activated droplet sorting applications at voltages below 6 and 15 V, respectively, facilitating low-voltage electric field applications in next-generation microfluidics.

A versatile bioelectronic interface programmed for hormone sensing.

Precision medicine requires smart, ultrasensitive, real-time profiling of bio-analytes using interconnected miniaturized devices to achieve individually optimized healthcare. Here, we report a versatile bioelectronic interface (VIBE) that senses signaling-cascade-guided receptor-ligand interactions via an electronic interface. We show that VIBE offers a low detection limit down to sub-nanomolar range characterised by an output current that decreases significantly, leading to precise profiling of these peptide hormones throughout the physiologically relevant concentration ranges. In a proof-of-concept application, we demonstrate that the VIBE platform differentiates insulin and GLP-1 levels in serum samples of wild-type mice from type-1 and type-2 diabetic mice. Evaluation of human serum samples shows that the bioelectronic device can differentiate between samples from different individuals and report differences in their metabolic states. As the target analyte can be changed simply by introducing engineered cells overexpressing the appropriate receptor, the VIBE interface has many potential applications for point-of-care diagnostics and personalized medicine via the internet of things.

An Efficient Ambient-Moisture-Driven Wearable Electrical Power Generator.

Existing devices for generating electrical power from water vapor in ambient air require high levels of relative humidity (RH), cannot operate for prolonged periods, and provide insufficient output for most practical applications. Here a heterogeneous moisture-driven electrical power generator (MODEG) is developed in the form of a free-standing bilayer of polyelectrolyte films, one consisting of a hygroscopic matrix of graphene oxide(GO)/polyaniline(PANI) [(GO)PANI] and the other consisting of poly(diallyldimethylammonium chloride)(PDDA)-modified fluorinated Nafion (F-Nafion (PDDA)). One MODEG unit (1 cm2 ) can deliver a stable open-circuit output of 0.9 V at 8 µA for more than 10 h with a matching external load. The device works over a wide range of temperature (-20 to +50 °C) and relative humidity (30% to 95% RH). It is shown that series and parallel combinations of MODEG units can directly supply sufficient power to drive commercial electronic devices such as light bulbs, supercapacitors, circuit boards, and screen displays. The (GO)PANI:F-Nafion (PDDA) hybrid film is embedded in a mask to harvest the energy from exhaled water vapor in human breath under real-life conditions. The device could consistently generate 450-600 mV during usual breathing, and provides sufficient power to drive medical devices, wearables, and emergency communication.

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.

Blood-Glucose-Powered Metabolic Fuel Cell for Self-Sufficient Bioelectronics.

Currently available bioelectronic devices consume too much power to be continuously operated on rechargeable batteries, and are often powered wirelessly, with attendant issues regarding reliability, convenience, and mobility. Thus, the availability of a robust, self-sufficient, implantable electrical power generator that works under physiological conditions would be transformative for many applications, from driving bioelectronic implants and prostheses to programing cellular behavior and patients' metabolism. Here, capitalizing on a new copper-containing, conductively tuned 3D carbon nanotube composite, an implantable blood-glucose-powered metabolic fuel cell is designed that continuously monitors blood-glucose levels, converts excess glucose into electrical power during hyperglycemia, and produces sufficient energy (0.7 mW cm-2 , 0.9 V, 50 mm glucose) to drive opto- and electro-genetic regulation of vesicular insulin release from engineered beta cells. It is shown that this integration of blood-glucose monitoring with elimination of excessive blood glucose by combined electro-metabolic conversion and insulin-release-mediated cellular consumption enables the metabolic fuel cell to restore blood-glucose homeostasis in an automatic, self-sufficient, and closed-loop manner in an experimental model of type-1 diabetes.

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.