Wilfried Weber

An engineered epigenetic transgene switch in mammalian cells

In multicellular systems cell identity is imprinted by epigenetic regulation circuits, which determine the global transcriptome of adult cells in a cell phenotype–specific manner. By combining two repressors, which control each others expression, we have developed a mammalian epigenetic circuitry able to switch between two stable transgene expression states after transient administration of two alternate drugs. Engineered Chinese hamster ovary cells (CHO-K1) showed toggle switch–specific expression profiles of a human glycoprotein in culture, as well as after microencapsulation and implantation into mice. Switch dynamics and expression stability could be predicted with mathematical models. Epigenetic transgene control through toggle switches is an important tool for engineering artificial gene networks in mammalian cells.

Emerging biomedical applications of synthetic biology

Synthetic biology aims to create functional devices, systems and organisms with novel and useful functions on the basis of catalogued and standardized biological building blocks. Although they were initially constructed to elucidate the dynamics of simple processes, designed devices now contribute to the understanding of disease mechanisms, provide novel diagnostic tools, enable economic production of therapeutics and allow the design of novel strategies for the treatment of cancer, immune diseases and metabolic disorders, such as diabetes and gout, as well as a range of infectious diseases. In this Review, we cover the impact and potential of synthetic biology for biomedical applications.

Macrolide-based transgene control in mammalian cells and mice

Heterologous mammalian gene regulation systems for adjustable expression of multiple transgenes are necessary for advanced human gene therapy and tissue engineering, and for sophisticated in vivo gene-function analyses, drug discovery, and biopharmaceutical manufacturing. The antibiotic-dependent interaction between the repressor (E) and operator (ETR) derived from an Escherichia coli erythromycin-resistance regulon was used to design repressible (EOFF) and inducible (EON) mammalian gene regulation systems (E.REX) responsive to clinically licensed macrolide antibiotics (erythromycin, clarithromycin, and roxithromycin). The EOFF system consists of a chimeric erythromycin-dependent transactivator (ET), constructed by fusing the prokaryotic repressor E to a eukaryotic transactivation domain that binds and activates transcription from ETR-containing synthetic eukaryotic promoters (PETR). Addition of macrolide antibiotic results in repression of transgene expression. The EON system is based on E binding to artificial ETR-derived operators cloned adjacent to constitutive promoters, resulting in repression of transgene expression. In the presence of macrolides, gene expression is induced. Control of transgene expression in primary cells, cell lines, and microencapsulated human cells transplanted into mice was demonstrated using the E.REX (EOFF and EON) systems. The macrolide-responsive E.REX technology was functionally compatible with the streptogramin (PIP)–regulated and tetracycline (TET)–regulated expression systems, and therefore may be combined for multiregulated multigene therapeutic interventions in mammalian cells and tissues.

Drug-sensing hydrogels for the inducible release of biopharmaceuticals

Drug-dependent dissociation or association of cellular receptors represents a potent pharmacologic mode of action for regulating cell fate and function. Transferring the knowledge of pharmacologically triggered protein-protein interactions to materials science will enable novel design concepts for stimuli-sensing smart hydrogels. Here, we show the design and validation of an antibiotic-sensing hydrogel for the trigger-inducible release of human vascular endothelial growth factor. Genetically engineered bacterial gyrase subunit B (GyrB) (ref. 4) coupled to polyacrylamide was dimerized by the addition of the aminocoumarin antibiotic coumermycin, resulting in hydrogel formation. Addition of increasing concentrations of clinically validated novobiocin (Albamycin) dissociated the GyrB subunits, thereby resulting in dissociation of the hydrogel and dose- and time-dependent liberation of the entrapped protein pharmaceutical VEGF(121) for triggering proliferation of human umbilical vein endothelial cells. Pharmacologically controlled hydrogels have the potential to fulfil the promises of stimuli-sensing materials as smart devices for spatiotemporally controlled delivery of drugs within the patient.

Synthetic ecosystems based on airborne inter- and intrakingdom communication

Intercellular communication within an organism, between populations, or across species and kingdoms forms the basis of many ecosystems in which organisms coexist through symbiotic, parasitic, or predator–prey relationships. Using multistep airborne communication and signal transduction, we present synthetic ecosystems within a mammalian cell population, in mice, or across species and kingdoms. Inter- and intrakingdom communication was enabled by using sender cells that produce volatile aldehydes, small vitamin-derived molecules, or antibiotics that diffuse, by gas or liquid phase, to receiver cells and induce the expression of specific target genes. Intercellular and cross-kingdom communication was shown to enable quorum sensing between and among mammalian cells, bacteria, yeast, and plants, resulting in precise spatiotemporal control of IFN-β production. Interconnection of bacterial, yeast, and mammalian cell signaling enabled the construction of multistep signal transduction and processing networks as well as the design of synthetic ecosystems that mimic fundamental coexistence patterns in nature, including symbiosis, parasitism, and oscillating predator–prey interactions.

A synthetic mammalian gene circuit reveals antituberculosis compounds

Synthetic biology provides insight into natural gene-network dynamics and enables assembly of engineered transcription circuitries for production of difficult-to-access therapeutic molecules. In Mycobacterium tuberculosis EthR binds to a specific operator (OethR) thereby repressing ethA and preventing EthA-catalyzed conversion of the prodrug ethionamide, which increases the resistance of the pathogen to this last-line-of-defense treatment. We have designed a synthetic mammalian gene circuit that senses the EthR–OethR interaction in human cells and produces a quantitative reporter gene expression readout. Challenging of the synthetic network with compounds of a rationally designed chemical library revealed 2-phenylethyl-butyrate as a nontoxic substance that abolished EthRs repressor function inside human cells, in mice, and within M. tuberculosis where it triggered derepression of ethA and increased the sensitivity of this pathogen to ethionamide. The discovery of antituberculosis compounds by using synthetic mammalian gene circuits may establish a new line of defense against multidrug-resistant M. tuberculosis.

A synthetic time-delay circuit in mammalian cells and mice

Time-delay circuitries in which a transcription factor processes independent input parameters can modulate NF-κB activation, manage quorum-sensing cross-talk, and control the circadian clock. We have constructed a synthetic mammalian gene network that processes four different input signals to control either immediate or time-delayed transcription of specific target genes. BirA-mediated ligation of biotin to a biotinylation signal-containing VP16 transactivation domain triggers heterodimerization of chimeric VP16 to a streptavidin-linked tetracycline repressor (TetR). At increasing biotin concentrations up to 20 nM, TetR-specific promoters are gradually activated (off to on, input signal 1), are maximally induced at concentrations between 20 nM and 10 μM, and are adjustably shut off at biotin levels exceeding 10 μM (on to off, input signal 2). These specific expression characteristics with a discrete biotin concentration window emulate a biotin-triggered bandpass filter. Removal of biotin from the culture environment (input signal 3) results in time-delayed transgene expression until the intracellular biotinylated VP16 pool is degraded. Because the TetR component of the chimeric transactivator retains its tetracycline responsiveness, addition of this antibiotic (input signal 4) overrides biotin control and immediately shuts off target gene expression. Biotin-responsive immediate, bandpass filter, and time-delay transcription characteristics were predicted by a computational model and have been validated in standard cultivation settings or biopharmaceutical manufacturing scenarios using trangenic CHO-K1 cell derivatives and have been confirmed in mice. Synthetic gene circuitries provide insight into structure–function correlations of native signaling networks and foster advances in gene therapy and biopharmaceutical manufacturing.

Gas-inducible transgene expression in mammalian cells and mice

We describe the design and detailed characterization of a gas-inducible transgene control system functional in different mammalian cells, mice and prototype biopharmaceutical manufacturing. The acetaldehyde-inducible AlcR-PalcA transactivator-promoter interaction of the Aspergillus nidulans ethanol-catabolizing regulon was engineered for gas-adjustable transgene expression in mammalian cells. Fungal AlcR retained its transactivation characteristics in a variety of mammalian cell lines and reversibly adjusted transgene transcription from chimeric mammalian promoters (PAIR) containing PalcA-derived operators in a gaseous acetaldehyde-dependent manner. Mice implanted with microencapsulated cells engineered for acetaldehyde-inducible regulation (AIR) of the human glycoprotein secreted placental alkaline phosphatase showed adjustable serum phosphatase levels after exposure to different gaseous acetaldehyde concentrations. AIR-controlled interferon-β production in transgenic CHO-K1-derived serum-free suspension cultures could be modulated by fine-tuning inflow and outflow of acetaldehyde-containing gas during standard bioreactor operation. AIR technology could serve as a tool for therapeutic transgene dosing as well as biopharmaceutical manufacturing.

Pharmacologic transgene control systems for gene therapy

Pharmacologic transgene‐expression dosing is considered essential for future gene therapy scenarios. Genetic interventions require precise transcription or translation fine‐tuning of therapeutic transgenes to enable their titration into the therapeutic window, to adapt them to daily changing dosing regimes of the patient, to integrate them seamlessly into the patients transcriptome orchestra, and to terminate their expression after successful therapy. In recent years, decisive progress has been achieved in designing high‐precision trigger‐inducible mammalian transgene control modalities responsive to clinically licensed and inert heterologous molecules or to endogenous physiologic signals. Availability of a portfolio of compatible transcription control systems has enabled assembly of higher‐order control circuitries providing simultaneous or independent control of several transgenes and the design of (semi‐)synthetic gene networks, which emulate digital expression switches, regulatory transcription cascades, epigenetic expression imprinting, and cellular transcription memories. This review provides an overview of cutting‐edge developments in transgene control systems, of the design of synthetic gene networks, and of the delivery of such systems for the prototype treatment of prominent human disease phenotypes. Copyright

A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells

Growth and differentiation of multicellular systems is orchestrated by spatially restricted gene expression programs in specialized subpopulations. The targeted manipulation of such processes by synthetic tools with high-spatiotemporal resolution could, therefore, enable a deepened understanding of developmental processes and open new opportunities in tissue engineering. Here, we describe the first red/far-red light-triggered gene switch for mammalian cells for achieving gene expression control in time and space. We show that the system can reversibly be toggled between stable on- and off-states using short light pulses at 660 or 740 nm. Red light-induced gene expression was shown to correlate with the applied photon number and was compatible with different mammalian cell lines, including human primary cells. The light-induced expression kinetics were quantitatively analyzed by a mathematical model. We apply the system for the spatially controlled engineering of angiogenesis in chicken embryos. The system’s performance combined with cell- and tissue-compatible regulating red light will enable unprecedented spatiotemporally controlled molecular interventions in mammalian cells, tissues and organisms.