Prof. Dr. Wilfried Weber

The vessel-stabilizing effect of angiopoietin-1 (Ang1)/ Tie2 receptor signaling is a potential target for pro-angiogenic therapies as well as anti-angiogenic inhibition of tumor growth. We explored the endothelial and vascular specific activities of the Ang1 monomer, i.e. dissociated from its state as an oligomer. A truncated monomeric Ang1 variant (i.e. ΔAng1) containing the isolated fibrinogen-like receptor-binding domain of Ang1 was created and recombinantly produced in insect cells. ΔAng1 ligated the Tie2 receptor without triggering its phosphorylation. Moreover, monomeric ΔAng1 was observed to bind α5β1 integrin with similar affinity compared with Tie2. Unexpectedly, in vitro treatment of endothelial cells with ΔAng1 showed some of the known effects of full-length Ang1, including inhibition of basal endothelial cell permeability and stimulation of cell adhesion as well as activation of MAPKs. Local treatment of the microvasculature of the developing chicken chorioallantoic membrane with the ΔAng1 protein led to profound reduction of the mean vascular length density, thinning of vessels, and reduction of the number of vessel branching points. Similar effects were observed in side-by-side experiments with the recombinant full-length Ang1 protein. These effects of simplification of the vessel branching pattern were confirmed through local gene transfer with lentiviral particles encoding ΔAng1 or full-length Ang1. Together, our findings suggest a potential use for exogenous Ang1 in reducing rather than increasing vascular density. Furthermore, we show that the isolated receptor-binding domain of Ang1 is capable of mediating some effects of full-length Ang1 independently of Tie2 phosphorylation, possibly through integrin ligation. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

The dramatically increasing prevalence of multi-drug-resistant human pathogenic bacteria and related mortality requires two key actions: (i) decisive initiatives for the detection of novel antibiotics and (ii) a global ban for use of antibiotics as growth promotants in stock farming. Both key actions entail technology for precise, high-sensitive detection of antibiotic substances either to detect and validate novel anti-infective structures or to enforce the non-use of clinically relevant antibiotics. We have engineered prokaryotic antibiotic response regulators into a molecular biosensor configuration able to detect tetracycline, streptogramin, and macrolide antibiotics in spiked liquids including milk and serum at ng/mL concentrations and up to 2 orders of magnitude below current Swiss and EC threshold values. This broad-spectrum, class-specific, biosensor-based assay has been optimized for use in a storable ready-to-use and high-throughput-compatible ELISA-type format. At the center of the assay is an antibiotic sensor protein whose interaction with specific DNA fragments is responsive to a particular class of antibiotics. Binding of biosensor protein to the cognate DNA chemically linked to a solid surface is converted into an immuno-based colorimetric readout correlating with specific antibiotics concentrations. © 2004 Wiley Periodicals, Inc.

Background: 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. Methods: Capitalizing on well-evolved quorum-sensing regulatory networks in Streptomyces coelicolor we have designed a mammalian regulation system inducible by the non-toxic butyrolactone SCB1. Fusion of the S. coelicolor SCB1 quorum-sensing receptor ScbR to the human Kox-1-derived transsilencing domain reconstituted a mammalian transsilencer (SCS) able to repress transcription from SCS-specific operator-containing promoters in a reverse SCB1-adjustable manner. Results: This quorum-sensing-derived mammalian transgene control system (Q-ON) enabled precise SCB1-specific fine-tuning of (i) desired transgene transcription in a variety of mammalian/human cell lines and human primary cells, (ii) small interfering RNA-mediated posttranscriptional knockdown (siRNA) in mammalian cells, and (iii) dosing of a human glycoprotein in mice. Conclusions: As exemplified by Q-ON technology, bacterial quorum-sensing regulons may represent a near-infinite source for the design of mammalian gene control systems compatible with molecular interventions relevant to future gene therapy and tissue engineering scenarios. Copyright © 2004 John Wiley & Sons, Ltd.

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.

Inducible transgene expression technologies are of unmatched potential for biopharmaceutical manufacturing of unstable, growth-impairing and cytotoxic proteins as well as conditional metabolic engineering to improve desired cell phenotypes. Currently available transgene dosing modalities which rely on physical parameters or small-molecule drugs for transgene fine-tuning compromise downstream processing and/or are difficult to implement technologically. The recently designed gas-inducible acetaldehyde-inducible regulation (AIR) technology takes advantage of gaseous acetaldehyde to modulate product gene expression levels. At regulation effective concentrations gaseous acetaldehyde is physiologically inert and approved as food additive by the Federal Drug Administration (FDA). During standard bioreactor operation, gaseous acetaldehyde could simply be administered using standard/existing gas supply tubing and eventually eliminated by stripping with inducer-free air. We have determined key parameters controlling acetaldehyde transfer in three types of bioreactors and designed a mass balance-based model for optimal product gene expression fine-tuning using gaseous acetaldehyde. Operating a standard stirred-tank bioreactor set-up at 10 L scale we have validated AIR technology using CHO-K1-derived serum-free suspension cultures transgenic for gas-inducible production of human interferon-β (IFN-β). Gaseous acetaldehyde- inducible IFN-β production management was fully reversible while maintaining cell viability at over 95% during the entire process. Compatible with standard bioreactor design and downstream processing procedures AIR-based technology will foster novel opportunities for pilot and large-scale manufacturing of difficult-to-produce protein pharmaceuticals. © 2005 Elsevier Inc. All rights reserved.

Capitalizing on components evolved to metabolize ethanol in Aspergillus nidulans, we previously designed the first molecular gas-gene expression interface using gaseous acetaldehyde as the major inducer. This fungus-derived acetaldehyde-inducible gene regulation (AIR) system operated perfectly and enabled precise and reversible transgene expression dosing in a variety of mammalian cells. We now validate the use of mainstream cigarette smoke typically containing acetaldehyde at regulation-effective nontoxic concentrations as a non-invasive modality to adjust transgene transcription in mammalian cells and mice. Indeed, tobacco smoke-induced expression fine-tuning of AIR-driven transgenes was successful in mammalian cells. Even mice implanted with cells transgenic for AIR-controlled SEAP (human secreted alkaline phosphatase) production showed serum SEAP levels correlating with inhaled tobacco smoke doses. Tobacco smoke-controlled gene expression may foster clinical opportunities as well as advances in understanding smoke-related pathologies. © 2005 Wiley Periodicals, Inc.