Dr. Claudia Fink-Straube

Recombinant adeno-associated viral (rAAV) vectors are a leading platform for in vivo gene therapy, valued for their excellent safety, broad serotype diversity, and scalable production. Targeted delivery through capsid display of ligands holds great promise, yet current retargeting strategies often rely on extensive capsid re-engineering and restrict the use of ligands incompatible with intracellular expression systems. Here, we present a modular AAV retargeting platform that, for the first time, employs the SpyTag/SpyCatcher system via genetic integration into the AAV2 capsid. SpyTag is a small peptide that forms a covalent, irreversible bond with its protein partner, SpyCatcher, allowing site-specific ligand coupling under physiological conditions. Inserting SpyTag into surface-exposed capsid sites enabled postassembly functionalization of AAVs with SpyCatcher-fused targeting proteins. As proof of concept, we used SpyCatcher fusions with designed ankyrin repeat proteins (DARPins) specific for EGFR, EpCAM, and HER2. This conferred highly specific transduction of corresponding cancer cell lines with minimal off-target activity. Therapeutic potential was demonstrated by delivering a suicide gene, inducing selective cancer cell killing upon prodrug administration. This “one-fits-all” platform allows rapid and flexible retargeting without significantly altering the underlying vectors genome or production process. It supports the incorporation of large or complex ligands not amenable to genetic fusion and facilitates high-throughput preclinical evaluation strategies. By uniting capsid engineering with modular ligand display, our approach provides a scalable and versatile framework for precision gene delivery, broadening the applicability of rAAV in both therapeutic and discovery settings.

Hydrogels are natural/synthetic polymer-based materials with a large percentage of water content, usually above 80 %, and are suitable for many application fields such as wearable sensors, biomedicine, cosmetics, agriculture, etc. However, their performance is susceptible to environmental changes in temperature, relative humidity, and mechanical deformation due to their aqueous and soft nature. We investigate the mechanical response of both filled and unfilled alginate/gellan hydrogels using a combined axial-torsional rheometric approach with cylindrical samples of large length/diameter ratio under controlled temperature and relative humidity. Dynamic Mechanical Analysis (DMA) is performed on the same specimen in both torsion and extension under identical experimental conditions. This rheometric approach ensures consistent initial and boundary conditions, which are essential for a reliable estimation of viscoelastic moduli G* and E*, and their dependence on temperature, frequency, and relative humidity. Our findings indicate that humidity critically affects the mechanical response of the material due to sample volume shrinkage, necessitating corrections to the viscoelastic moduli. We also find temperature plays a role only at low/medium relative humidity values. The inclusion of fillers leads to a modest increase in the elasticity of the hydrogel, probably due to restricted water diffusion out of the sample. In connection with the latest, unfilled samples in breaking tests present only slippage due to twist-induced surface water excess, opposite to breakage events shown by filled samples, probably linked to restricted water diffusion.

Selenium sulfide, the active ingredient of traditional antidandruff shampoos, is industrially produced from selenium dioxide (SeO2) and hydrogen sulfide (H2S) under acidic conditions. This reaction can also be carried out with natural H2S and H2S generated by sulfate-reducing bacteria (SRB). These bacteria are robust and, by relying on their conventional growth medium, also thrive in “waste” materials, such as a mixture of cabbage juice and compost on the one side, and a mixture of spoiled milk and mineral water on the other. In these mixtures, SRB are able to utilize the DL-lactate and sulfate (SO42−) present naturally and produce up to 4.1 mM concentrations of H2S in the gas phase above a standard culture medium. This gas subsequently escapes the fermentation vessel and can be collected and reacted with SeO2 in a separate compartment, where it yields, for instance, pure selenium sulfide, therefore avoiding the need for any cumbersome workup or purification procedures. Thus “harvesting” H2S and similar (bio-)gases produced by the fermentation of organic waste materials by suitable microorganisms provides an elegant avenue to turn dirty waste into valuable clean chemical products of considerable industrial and pharmaceutical interest. © 2025 by the authors.

Herein, a study dealing with a progress on palladium (Pd) electrocatalysts for an efficient glycerol electrooxidation in model aqueous and real fermentation solutions with special focus on some physicochemical parameters (e.g., the impact of adsorption stage of multiple species, presence of oxygen, influence of anodic limits and Pd-size) was conducted. During the course of investigations by tandem of an optical oxygen minisensor and cyclic voltammetry a significant impact of oxygen on the efficiency of glycerol electrooxidation on Pd electrocatalysts at alkaline pH in model aqueous and yeast fermentation media was revealed. The obtained knowledge was used for the optimization of an assay utilizing Pd-sensing layers for glycerol determination and quantification in yeast fermentation medium. Received results showed a satisfactory agreement with a control measurement carried out by gas chromatography mass-spectrometry.

Glycerol is a widely used signaling bioanalyte in biotechnology. Glycerol can serve as a substrate or product of many metabolic processes in cells. Therefore, quantification of glycerol in fermentation samples with inexpensive, reliable, and rapid sensing systems is of great importance. In this work, an amperometric assay based on one-step designed electroplated functional Pd layers with controlled design was proposed for a rapid and selective measurement of glycerol in yeast fermentation medium. A novel assay utilizing electroplated Pd-sensing layers allows the quantification of glycerol in yeast fermentation medium in the presence of interfering species with RSD below 3% and recoveries ranged from 99 to 103%. The assay requires minimal sample preparation, viz. adjusting of sample pH to 12. The time taken to complete the electrochemical analysis was 3 min. Remarkably, during investigations, it was revealed that sensitivity and selectivity of glycerol determination on Pd sensors were significantly affected by its adsorption and did not depend on the surface structure of sensing layers. This study is expected to contribute to both fundamental and practical research fields related to a preliminary choice of functional sensing layers for specific biotechnology and life science applications in the future.

Peptide drugs have seen rapid advancement in biopharmaceutical development, with over 80 candidates approved
globally. Despite their therapeutic potential, the clinical translation of peptide drugs is hampered by challenges
in production yields and stability. Engineered bacterial therapeutics is a unique approach being explored to overcome
these issues by using bacteria to produce and deliver therapeutic compounds at the body site of use. A key advan‑
tage of this technology is the possibility to control drug delivery within the body in real time using genetic switches.
However, the performance of such genetic switches suffers when used to control drugs that require post‑translational
modifications or are toxic to the host. In this study, these challenges were experienced when attempting to establish
a thermal switch for the production of a ribosomally synthesized and post‑translationally modified peptide antibiotic,
darobactin, in probiotic E. coli. These challenges were overcome by developing a thermo‑amplifier circuit that com‑
bined the thermal switch with a T7 RNA Polymerase. Due to the orthogonality of the Polymerase, this strategy
overcame limitations imposed by the host transcriptional machinery. This circuit enabled production of pathogen‑
inhibitory levels of darobactin at 40 °C while maintaining leakiness below the detection limit at 37 °C. Furthermore,
the thermo‑amplifier circuit sustained gene expression beyond the thermal induction duration such that with only
2 h of induction, the bacteria were able to produce pathogen‑inhibitory levels of darobactin. This performance
was maintained even in physiologically relevant simulated conditions of the intestines that include bile salts and low
nutrient levels

Herein, a rapid electrochemical screening of yeasts (Saccharomyces cerevisiae) in vitro mode depending on their optical density, cultivation time and growth medium used was conducted in 3 min by palladium nanoparticles (Pd-NPs)-modified electrodes. Pd-NPs-modified electrodes operated in cyclic voltammetry mode at low scan rates, i.e. 5–20 mV/s supported a low oxidative process in the yeast extracellular matrix. The electrochemical screening relied on an efficient electrooxidation of secondary metabolites, i.e. organohydrazines formed in the extracellular medium as a result of microbial activity of yeast cells. More importantly, during the study the impact of fundamental parameters, viz. type of the matrix and pH on electroanalytical response of Pd-NPs-based electrodes in real fermentation medium was investigated in detail. The efficiency of the proposed in vitro electrochemical screening of yeast extracellular matrix was not affected by pH of the samples or composition of the multicomponent medium, but more likely exclusively depended on the presence of organohydrazines. The potential of this electroanalytical approach towards profiling of the extracellular matrix of Saccharomyces cerevisiae was compared with results obtained by gas chromatography mass-spectrometry (GC–MS) and genetically encoded biosensor (ro-GFP2) assays.

Herein an assay toward a rapid and reliable profiling of extracellular matrix of Escherichia coli (E. coli) utilizing a tandem of GC-MS as a tool for definition of the exact chemical nature of low molecular weight compounds and cyclic voltammetry for their high throughput detection is presented. Briefly, during a set of investigations the formation of glycerol in the extracellular matrix (ECM) of E. coli at physiological relevant conditions of cells was revealed. Based on the obtained knowledge, the electrochemical protocol allowing both qualitative and quantitative analyses of glycerol in E. coli ECMs at palladium ink-modified screen printed electrodes with precision values (RSD) <10 % and recovery rates ranged from 98 % to 102 % was proposed. The provided protocol for a rapid electrochemical profiling of the bacterial ECMs can readily be used as a guideline for the controlled electroanalysis of target electroactive signaling analytes in complex biological samples.

Hydrogel precursors that crosslink within minutes are essential for the development of cell encapsulation matrices and their implementation in automated systems. Such timescales allow sufficient mixing of cells and hydrogel precursors under low shear forces and the achievement of homogeneous networks and cell distributions in the 3D cell culture. The previous work showed that the thiol-tetrazole methylsulfone (TzMS) reaction crosslinks star-poly(ethylene glycol) (PEG) hydrogels within minutes at around physiological pH and can be accelerated or slowed down with small pH changes. The resulting hydrogels are cytocompatible and stable in cell culture conditions. Here, the gelation kinetics and mechanical properties of PEG-based hydrogels formed by thiol-TzMS crosslinking as a function of buffer, crosslinker structure and degree of TzMS functionality are reported. Crosslinkers of different architecture, length and chemical nature (PEG versus peptide) are tested, and degree of TzMS functionality is modified by inclusion of RGD cell-adhesive ligand, all at concentration ranges typically used in cell culture. These studies corroborate that thiol/PEG-4TzMS hydrogels show gelation times and stiffnesses that are suitable for 3D cell encapsulation and tunable through changes in hydrogel composition. The results of this study guide formulation of encapsulating hydrogels for manual and automated 3D cell culture.