Steffen Honrath


Last Name

Honrath

First Name

Steffen

ORCID

0009-0003-7513-8872

Organisational unit

03811 - Leroux, Jean-Christophe / Leroux, Jean-Christophe

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Publications 1 - 6 of 6
  • Rapid selection of transgenic mammalian cells via diphtheria toxin resistance
    Item type: Journal Article
    Scherer, David; Honrath, Steffen; Leroux, Jean-Christophe; et al. (2025)
    Trends in Biotechnology
    The ability to generate stable transgenic mammalian cell lines is crucial to the investigation of gene functions and the production of recombinant proteins. Mammalian cells can be readily transfected in cell culture settings via both viral and nonviral vectors to induce transgene expression. However, there is an unmet need for efficient selection of transfected cells, since current methods involve rather inefficient antibiotic selection protocols or require the coexpression of fluorescent marker proteins, followed by laborious cell-sorting procedures. Thus, our aim was to implement a rapid and efficient selection approach for transgene-expressing human cells, using an engineered diphtheria toxin (DT) resistance-based selection, referred to as selecDT. We demonstrated that selecDT is expressed on the cell surface, provides efficient protection from DT by inactivating its uptake receptor, and, therefore, enables selection. SelecDT allows for greater selection efficiency in a more rapid timeline compared with conventional antibiotic methods. Thus, the resistance described herein may positively impact biotechnological processes.
  • Hurdles to healing: Overcoming cellular barriers for viral and nonviral gene therapy
    Item type: Review Article
    Honrath, Steffen; Burger, Michael; Leroux, Jean-Christophe (2025)
    International Journal of Pharmaceutics
    Gene delivery offers great potential for treating various diseases, yet its success requires overcoming several biological barriers. These hurdles span from extracellular degradation, reaching the target cells, and inefficient cellular uptake to endosomal entrapment, cytoplasmic transport, nuclear entry, and transcription limitations. Viruses and non-viral vectors deal with these barriers via different mechanisms. Viral vectors, such as adenoviruses, adeno-associated viruses, and lentiviruses use natural mechanisms to efficiently deliver genetic material but face limitations including immunogenicity, cargo capacity, and production complexity. Nonviral vectors, including lipid nanoparticles, polymers, and protein-based systems, offer scalable and safer alternatives but often fall short in overcoming intracellular barriers and achieving high transfection efficiencies. Recent advancements in vector engineering have partially overcome several of these challenges. Ionizable lipids improve endosomal escape while minimizing toxicity. Biodegradable polymers balance efficacy with safety, and engineered protein systems, inspired by viral or bacterial entry mechanisms, integrate multifunctionality for enhanced delivery. Despite these advances, challenges, particularly in achieving robust in vivo translatability, scalability, and reduced immunogenicity, remain. This review synthesizes current knowledge of cellular barriers and the approaches to overcome them, providing a roadmap for designing more efficient gene delivery systems. By addressing these barriers, the field can advance toward safer, and more effective therapies.
  • Isolation of bacterial extracellular vesicles from raw samples using a portable microstructured electrochemical device
    Item type: Journal Article
    Mantella, Valeria; Bienz, Siiri; Brigger, Finn; et al. (2025)
    Drug Delivery and Translational Research
    Bacterial extracellular vesicles (EVs) are nanosized vesicles released by both Gram-negative and Gram-positive bacteria, playing critical roles in microbial communication, host-pathogen interactions, and immune modulation. Despite their significance in research and clinical applications, conventional isolation methods, such as ultracentrifugation (UC), are often slow, labor-intensive, and susceptible to contamination. In this study, we evaluated a novel portable microstructured electrochemical device (PMED) designed for rapid and selective bacterial EV isolation directly from biological samples. Using immunoaffinity-based capture and voltage-triggered release, the device-isolated EVs from Gram-negative Escherichia coli (E. coli), Gram-positive Lactobacillus fermentum (Lb. fermentum) culture supernatants and from urine samples spiked with E. coli , showing superior purity compared to UC. Characterization through nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and Western blot confirms enhanced selectivity and reduced contaminants. Functional assays demonstrated that device-isolated Lb. fermentum EVs selectively activated Toll-like receptor 4 (TLR4) without triggering TLR2, unlike UC-isolated EVs, suggesting a more refined immunomodulatory effect. These findings highlight the device’s translational potential for EV-based diagnostics, particularly for noninvasive urinary tract infection detection, and its broader applications in studying bacterial communication and immune regulation.
  • Closing the gap: Nonviral TFAMoplex transfection boosted by bZIP domains compared to AAV-mediated transduction
    Item type: Journal Article
    Honrath, Steffen; Heussi, Miguel; Beckert, Lukas; et al. (2025)
    Molecular Therapy - Nucleic Acids
    The TFAMoplex is a nanoparticulate gene delivery system based on the mitochondrial transcription factor A (TFAM) protein, which can be engineered with various functional domains to enhance plasmid DNA transfection. In this study, we aimed at improving the TFAMoplex system by incorporating basic leucine zipper (bZIP) domains, derived from the cyclic AMP (cAMP)-responsive element-binding protein (CREB), which are known to bind DNA upon dimerization. Additionally, we screened bZIP domains of other proteins (i.e., transcription regulator protein BACH1, cyclic AMP-dependent transcription factor ATF-3, and basic leucine zipper transcriptional factor ATF-like BATF) under challenging transfection conditions, identifying the bZIP domain of BACH1, bZIPBACH1, as particularly effective in enhancing the TFAMoplex performance, reducing the half-maximal effective concentration by more than 2-fold. We show that bZIP domains facilitate interactions with the cell membrane as single proteins and thus increase the cell association of TFAMoplexes. Finally, we compared the optimized bZIPBACH1-TFAMoplex to adeno-associated viruses (AAVs) regarding in vitro transfection efficiency and transgene expression levels. While AAVs achieved higher transfection efficiency based on the number of transfected cells, both the original and improved TFAMoplex constructs surpassed AAVs in transgene expression per cell.
  • The intracellular journey of DNA delivered with the protein-based transfection system TFAMoplex and Lipofectamine
    Item type: Journal Article
    Greitens, Christina; Honrath, Steffen; Maurer, Philip; et al. (2025)
    International Journal of Pharmaceutics
    In non-viral DNA delivery, assessing the cellular trafficking of DNA is crucial to understand how transfection systems interact with the cellular machinery. Using a combination of innovative and established imaging assays and flow cytometry, we characterize DNA delivery in HeLa cells with three different transfection systems: (i) the original protein-based TFAMoplex, using the mitochondrial transcription factor A (TFAM) for DNA compaction, (ii) a modified version of the TFAMoplex incorporating a basic leucine zipper (bZIP) domain, (TFAMoplex-bZIP), and (iii) Lipofectamine. Particularly, DNA cell association, uptake, endosomal rupture, cytoplasmic delivery and reporter gene expression are assessed. TFAMoplex-bZIP mediated delivery of Cy3-labeled DNA shows significantly higher cell association compared to the original TFAMoplex and Lipofectamine (15.6, 6.6. and 0.53 Cy3 objects per cell, respectively). However, the highest DNA internalization efficiency is achieved with Lipofectamine (21.8 % vs. ∼15 % for TFAMoplex-based systems). All transfection systems induce endosomal rupture and the formation of barrier-to-autointegration factor (BAF)-positive nucleoprotein structures, termed BAF clusters. With TFAMoplex-bZIP, BAF clusters are numerous, and significantly more cells show reporter gene expression (46.7 %) compared to Lipofectamine (8.7 %). However, reporter protein intensity is highest with Lipofectamine. The described high-precision characterization tools of DNA delivery might be applied to other non-viral transfection agents to identify their bottlenecks in detail.
Publications 1 - 6 of 6