Cornelia C. Weber

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.

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.

Multi-chromatic control of mammalian gene expression and signaling

The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical applications.

Engineered Streptomyces quorum-sensing components enable inducible siRNA-mediated translation control in mammalian cells and adjustable transcription control in mice.

Recent advances in functional genomics, gene therapy, tissue engineering, drug discovery and biopharmaceuticals production have been fostered by precise small‐molecule‐mediated fine‐tuning of desired transgenes.

A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1

We describe the design and detailed characterization of 6-hydroxy-nicotine (6HNic)-adjustable transgene expression (NICE) systems engineered for lentiviral transduction and in vivo modulation of angiogenic responses. Arthrobacter nicotinovorans pAO1 encodes a unique catabolic machinery on its plasmid pAO1, which enables this Gram-positive soil bacterium to use the tobacco alkaloid nicotine as the exclusive carbon source. The 6HNic-responsive repressor-operator (HdnoR-ONIC) interaction, controlling 6HNic oxidase production in A.nicotinovorans pAO1, was engineered for generic 6HNic-adjustable transgene expression in mammalian cells. HdnoR fused to different transactivation domains retained its ONIC-binding capacity in mammalian cells and reversibly adjusted transgene transcription from chimeric ONIC-containing promoters (PNIC; ONIC fused to a minimal eukaryotic promoter [Pmin]) in a 6HNic-responsive manner. The combination of transactivators containing various transactivation domains with promoters differing in the number of operator modules as well as in their relative inter-ONIC and/or ONIC-Pmin spacing revealed steric constraints influencing overall NICE regulation performance in mammalian cells. Mice implanted with microencapsulated cells engineered for NICE-controlled expression of the human glycoprotein secreted placental alkaline phosphatase (SEAP) showed high SEAP serum levels in the absence of regulating 6HNic. 6HNic was unable to modulate SEAP expression, suggesting that this nicotine derivative exhibits control-incompatible pharmacokinetics in mice. However, chicken embryos transduced with HIV-1-derived self-inactivating lentiviral particles transgenic for NICE-adjustable expression of the human vascular endothelial growth factor 121 (VEGF121) showed graded 6HNic response following administration of different 6HNic concentrations. Owing to the clinically inert and highly water-soluble compound 6HNic, NICE-adjustable transgene control systems may become a welcome alternative to available drug-responsive homologs in basic research, therapeutic cell engineering and biopharmaceutical manufacturing.

In vivo transduction of HIV-1-derived lentiviral particles engineered for macrolide-adjustable transgene expression

The molecular merger of latest‐generation transduction technologies with advanced transgene control modalities may foster decisive advances in therapeutic reprogramming of somatic cell phenotypes.

Quorum‐Sensing‐Based Toolbox for Regulatable Transgene and siRNA Expression in Mammalian Cells

Technologies for regulated expression of multiple transgenes in mammalian cells have gathered momentum for bioengineering, gene therapy, drug discovery, and gene‐function analyses. Capitalizing on recently developed mammalian transgene modalities (QuoRex) derived from Streptomyces coelicolor, we have designed a flexible and highly compatible expression vector set that enables desired transgene/siRNA control in response to the nontoxic butyrolactone SCB1. The construction‐kit‐like expression portfolio includes (i) multicistronic (pTRIDENT), (ii) autoregulated, (iii) bidirectional (pBiRex), (iv) oncoretro‐ and lentiviral transduction, and (v) RNA polymerase II‐based siRNA transcription‐fine‐tuning vectors for straightforward implementation of QuoRex‐controlled (trans)gene modulation in mammalian cells.

A general strategy for the production of difficult-to-express inducer-dependent bacterial repressor proteins in Escherichia coli.

Inducer-dependent prokaryotic transcriptional repressor proteins that originally evolved to orchestrate the transcriptome with intracellular and extracellular metabolite pools, have become universal tools in synthetic biology, drug discovery, diagnostics and functional genomics. Production of the repressor proteins is often limited due to inhibiting effects on the production host and requires iterative process optimization for each individual repressor. At the example of the Streptomyces pristinaespiralis-derived streptogramin-dependent repressor PIP, the expression of which was shown to inhibit growth of Escherichia coli BL21*, we demonstrate that the addition of the PIP-specific streptogramin antibiotic pristinamycin I neutralizes the growth-inhibiting effect and results in >100-fold increased PIP titers. The yield of PIP was further increased 2.5-fold by the engineering of a new E. coli host suitable for the production of growth-inhibiting proteins encoded by an unfavorable codon usage. PIP produced in the presence of pristinamycin I was purified and was shown to retain the antibiotic-dependent binding to its operator pir as demonstrated by a fluorescence resonance energy transfer (FRET)-based approach. At the example of the macrolide-, tetracycline- and arsenic-dependent repressors MphR(A), TetR and ArsR, we further demonstrate that the production yields can be increased 2- to 3-fold by the addition of the cognate inducer molecules erythromycin, tetracycline and As(3+), respectively. Therefore, the addition of inducer molecules specific to the target repressor protein seems to be a general strategy to increase the yield of this interesting protein class.

Inducible Gene Expression for Antibiotic Drug Discovery and Diagnostics

The rapid spreading of multi-drug-resistant human-pathogenic bacteria in industrialized countries and the thereto related increased morbidity and mortality urgently require decisive countermeasures to contain this imminent threat: (i) Discovery of novel anti-infectives has to outperform the emergence of novel resistance mechanisms, and the use of clinically licensed antibiotics in stock farming will have to be restricted or banned. Both of these key actions require highly sensitive and rapid technology to discover and validate novel antibiotic lead structures as well as detect trace amounts of illegal antibiotics in food samples including milk or meat. To increase the discovery rate for novel anti-infective lead structures, we have designed the Mammalian Antibiotic Sensor Technology (MAST), a mammalian cell-based screening platform with integrated cytotoxicity and bioavailability assessment of novel antibiotic structures. The basis of MAST are antibiotic biosensors derived from prokaryotic transcriptional regulators, which are highly responsive to potent antibiotic core structures of a desired class. In order to enforce the ban of certain antibiotics in food samples we developed an in vitro Biosensor ImmunoAssay (BIA) for which we have engineered prokaryote-derived biosensors into a cell-free test system for rapid and sensitive detection of antibiotics in biological samples like milk or serum.