Publikationen

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide. © The Author 2006. Published by Oxford University Press. All rights reserved.

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 patient's 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 © 2006 John Wiley & Sons, Ltd.

Nutrient and oxygen availability are key metabolic parameters for biopharmaceutical manufacturing. In order to enable mammalian cells to manifest their intracellular nutrient and oxygen levels we engineered a genetic sensor circuitry which converts signals impinging on the cellular redox balance into a robust reporter gene expression readout. Capitalizing on the Streptomyces coelicolor redox control system, consisting of REX modulating ROP-containing promoters in an NADH-dependent manner, we designed a mammalian dual sensor transcription control system by fusing REX to the generic VP16 transactivation domain of Herpes simplex, which reconstitutes an artificial transactivator (REDOX) able to bind and activate chimeric promoters assembled by placing a ROP operator module 5′ of a minimal eukaryotic promoter (PROP). When nutrient levels were low and resulted in depleted NADH pools REDOX-dependent PROP-driven expression of secreted (human-secreted alkaline phosphatase; SEAP) or intracellular (Renilla reniformis luciferase; rLUC) reporter genes was high as a consequence of increased REDOX-PROP affinity. Conversely, at hypoxic conditions leading to high intracellular NADH levels, strongly reduced REDOX-PROP interaction mediated low-level transgene expression in Chinese hamster ovary (CHO-K1) cells. Other molecules (for example, 2,4-dinitrophenol, cyanide or hydrogen peroxide) which are known to imbalance the intracellular NADH/NAD+ poise could also be detected using the REDOX-PROP sensor circuitry. REDOX's sensor capacity (nutrient and oxygen levels) operated seamlessly in transgenic CHO-K1 cell derivatives adapted for growth in serum-free suspension cultures and enabled precise monitoring of the population's metabolic state. As the first genetic metabolic sensor designed for mammalian cells, REDOX may foster advances in process development and biopharmaceutical manufacturing. © 2006 Elsevier Inc. All rights reserved.

With a rate exceeding 90% in cattle, artificial insemination (AI) is the prime reproduction technology in stock farming. AI success is expected to increase with extended persistence of sperms in utero. In order to enable controlled sperm release during artificial insemination we have designed two strategies for the automated microencapsulation of bovine spermatozoa in either alginate-Ca2+ or cellulose sulfate (CS)-poly-diallyldimethyl ammonium chloride (pDADMAC) capsules using standard encapsulation hardware. Animal protein- and citric acid-free sperm extenders and encapsulation protocols have been developed to ensure encapsulation compatible with sperm physiology. Bovine spermatozoa have showed high motility rates inside CS-pDADMAC-based capsules, were preserved by standard cryoconservation and rescued with high viability/motility following disintegration of the thawed capsules. CS-pDADMAC-based capsules break up within 72 h after addition of either purified cellulase or cellulase-filled alignate-Ca2+ capsules. The controlled release, associated with the microencapsulation of bovine spermatozoa, may be a promising approach to increase the success rate of artificial insemination. © 2005 Elsevier B.V. All rights reserved.

Five new derivatives of the polycyclic alumosiloxalic [Ph2SiO]8[Al(O)OH]4 have been synthesized by replacement of the protic hydrogen atoms on the hydroxy-groups attached to the aluminium atoms by the divalent group 14 elements germanium, till and lead. The compounds can be divided in those with one metal atom per alumosiloxane moiety, [Ph2SiO]8[Al(O)OH]2[AlO2]M (M=Ge, Sn), and those with complete substitution of the protic hydrogen atoms by metal atoms like [Ph2SiO]8[AlO2]4M2 (M= Sn, Pb). Always one element of the series Ge, Sn, Ph is missing in the two types of compounds. Crystal structure analyses of [Ph2SiO]8[Al(O)OH]2[AlO2]2M∙2 C4H8O2 (M= Ge (1), Sn (2a)), [Ph2SiO]8[Al(O)OH]2[AlO2]2Sn∙2THF(2b) and [Ph2SiO]8[AlO2]4M2 (M= Sn (3) Pb (4)) have been performed elucidating either polycyclic basket-type (1, 2a, 2b) or closed polyhedral structures (3, 4).

The siloxyalanes [(Et3SiOAlH2)2AlH3]n (1), [tBuMe2SiOAlH2]2 (2), [(tBuMe2SiO)2AlH]2 (3), and [(tBuMe2SiO)5Al2H] (4) have been synthesized and characterized by IR and NMR spectroscopic means. By X-ray diffraction studies on single crystals it was established that molecule 1 is a coordination polymer in the solid state with tetrameric units [Et3SiOAlH2]4 (almost linear Al∙∙∙Al∙∙∙Al∙∙∙Al arrangement) interlinked by Al2H6 groups to form a one-dimensional chain (formal description with neutral building blocks). Compound 3 is dimeric through Al-O contacts with a cis orientation of the hydride ligands with respect to the central Al2O2 cycle. Compound 4 may be described as a double adduct between the two Lewis acid-base compounds (tBuMe2SiO)2AlH and Al(OSiMe2tBu)3. The bulkiness of the substituents on the silicon atoms plays an important role in the aggregation of the compounds. Hydride bridging between molecules or within the molecular entities is only observed in compound 1.

Thermally and chemically durable hydrophobic oleophobic coatings, containing different ceramic particles such as SiO2, SiC, Al2O3, which can be alternative instead of Teflon, have been developed and applied on the aluminum substrates by spin-coating method. Polyimides, which are high-thermal resistant heteroaromatic polymers, were synthesized, and fluor oligomers were added to these polymers to obtain hydrophobic-oleophobic properties. After coating, Al surface was subjected to Taber-abrasion, adhesion, corrosion, and thermal tests. The effects of the particle size of ceramic powders, organic matrix, and heat on the coating material were investigated. Coating material was characterized by FTIR spectrophotometer. Surface properties and thermal resistance of the coating materials were investigated by SEM and TGA analyses. After thermal curing, contact angles of these coatings with H2O and n-hexadecane were measured. It was observed that coatings like ceramic particles are more resistant against scratch and abrasion than the other coatings. Also, they are harder than coatings, which do not include ceramic particles. It was seen that coatings, containing Fluorolink D10H, have high-contact angles with water and n-hexadecane.

The optical and electrical properties of two types of wet chemical processed tin doped indium oxide (ITO) films deposited by spin coating technique as well as of a commercial sputtered ITO film have been measured. The transmission and reflection spectra in the wavelength range 0.25 to 20 µm have been simulated using the Scout 2 software with different dielectric function models. The electrical parameters obtained from the modeling are compared with those obtained experimentally. The optical data of a low porosity (28%), low specific resistivity (ρ = 6.3 x 10-4 Ω cm) sol-gel film deposited from an ethylene glycol solution of In and Sri salts and sintered at 550 °C, as well as those of all Asahi Glass sputtered film (ρ = 1.9 x 10-4 Ω cm) have been successfully fitted with the Drude model and the electrical parameters deduced from the fitting are in good agreement with the measured ones. On the other hand, the best fit of the optical data of a thick single coating prepared using dispersed crystalline nanoparticles was obtained using the Bruggemann effective medium theory coupled with the extended Drude model. The electrical and structural parameters deduced from the fitting are also in good agreement with those measured. The results are discussed in terms of the morphology of the coatings.