Research

Efficient recombinant protein production depends on stable cell lines, especially for complex products like viruslike particles (VLPs) and adeno-associated viruses (AAVs) used in vaccines and gene therapy their use is aspirated. Conventional transfection methods often lead to inefficiencies and inconsistent product quality, making stable cell lines essential. However, developing these lines, particularly for large transgenes, is time-consuming and challenging. Current solutions, typically using Chinese hamster ovary (CHO) cells, may not meet the quality demands of VLPs and AAVs, which require human-like glycosylation. Therefore, a versatile, cell type-independent platform for stable cell line development is needed. Our system, based on baculoviral transduction of mammalian cells (BacMam), addresses this need. BacMam is cost-effective, scalable, and efficient, with key advantages: it doesn’t require high-biosafety labs, transduces various cell types, and integrates large DNA fragments into genomes. We developed the REMBAC platform (Rapid Efficient Manifold Baculovirus Transduction), enabling site-specific integration of large transgenes with customizable expression. This is particularly useful for cell-toxic proteins, and the system includes insulators to protect against host-cell silencing. REMBAC facilitates stable cell line development (SCLD) for a variety of biopharmaceutical applications, including VLP vaccines, AAV gene therapy vectors, and monoclonal antibodies. It combines BacMam’s versatility with homologous recombination for site-specific integration and uses the I-SceI homing endonuclease for precise transgene excision. A library of transfer vectors supports long-term, fine-tuned protein expression. This project aims to (i) optimize integration for model cell lines (HEK293 and HeLa) by adjusting homology region lengths, (ii) identify a genomic safe harbor (GSH) for HeLa cells, and (iii) characterize the transcriptional profiles of our plasmid toolbox. A major focus will be on genotypic and phenotypic characterization, verifying transgene integration, assessing copy number, checking for mutations or residual baculovirus sequences, and ensuring long-term cassette stability. We will also compare the growth and morphology of modified cells to wild-type cells to ensure the process does not negatively impact cell health. As proof of concept, we will compare REMBAC-based stable cell lines with conventional plasmid-based methods for producing influenza A VLPs and the therapeutic antibody Trastuzumab. We expect REMBAC to improve yield, consistency, and production time, demonstrating its broad potential for various biotechnological applications. Additionally, we plan to generate stable antigen-specific reporter cell lines using random genome integration. These reporter lines will simplify production by allowing easy identification and quantification of expression products and supporting potency testing during early production and clinical stages. In summary, this project aims to validate the REMBAC system’s efficiency and versatility for stable cell line development, optimize key components like homologous recombination sequences, and explore new genomic safe harbors. We aim to demonstrate REMBAC’s superiority over conventional methods in terms of efficiency and product quality while also providing valuable tools, such as stable reporter cell lines, for the biopharmaceutical industry.

PFAS are persistent organic compounds which consist of a hydrophilic head group and a hydrophobic alkyl chain of variable length (4-16) partially (poly-) or completely (per-) fluorinated. They are contaminants of soil and water, and can cause harm to the human health and the environment. PFAS have been widely used in industrial and commercial products such as fire-fighting foams, materials for cook-ware, high-temperature lubricants, ski wax, water repellant clothing and many more products since the 1940s. Various Pseudomonas spp. have been shown to degrade perfluoro-octane-sulfonate (PFOS) and it was observed that Pseudomonas strains have developed a strong tolerance to fluoride. In our project we will use a customized variant of the described method of Luan et al. (2013) to obtain Pseudomonas spp. mutants with improved PFAS degradation abilities. We will generate three plasmids encoding variants of DnaQ to provide strong, medium and weak mutator abilities. Pseudomonas. spp. obtained from strain collections or isolated from PFAS-contaminated environments (collaboration with AIT, Thomas Reichenauer) will be transformed with these plasmids and will be grown in inducing conditions, at the same time expressing inactive DnaQ protein which results in a mutator phenotype. Increasing concentrations of PFAS in the culture medium over time will lead to adaption by improved enzyme sets/degradation pathways in specific clones which can be isolated by high throughput screening methods and identified by whole genome sequencing. Once a feasible clone is identified, it can be grown in medium in absence of the inducer, so that expression of the inactive DnaQ protein is repressed or the obtained strain can be cured of the mutator plasmid, and genetic stability is restored. Cured strains will be tested for their PFAS reducing abilities in different environmental matrices like water or soil. Finally, degradation and transformation products emerging during PFAS-degradation will be analyzed by LC-HRMS/MS and LC-ion mobility-HRMS/MS.

Magnesium is an essential component of chlorophyll for plant physiology. Low Mg content in the leaves of grapevines reduces photosynthesis and, thus, glucose production and, consequently, lower wine quality. The right choice of rootstock is essential to alleviate this deficiency. However, the necessary Mg efficiency restricts the selection of the rootstocks, and in particular, the rootstocks that have been tried and tested in this country are less suitable. The deficiency can also be remedied by fertilizing the leaves, at least in the short term. But, the most sustainable solution would be to plant clones with an unproblematic Mg metabolism. An important grape variety for Austrian viticulture is mainly affected by Mag deficiency, namely Welschriesling (WR). The WR clones that are available for domestic viticulture all show more or less a weak Mg uptake. The variety has been used in viticulture for several centuries and was intensively cultivated and therefore exists in different genetic types. Since the old descriptions do not report this Mg deficiency, it is entirely conceivable that there is genomics in old genotypes that show average Mg utilization. Therefore, it would be necessary to look for genotypes that offer a better uptake and research it genetically. It is well known that crop phenotypic variation is shaped by their ancestors’ genetic variation and the selection and maintenance of collections of mutations. Moreo ver, most of this varia ti on is quan ti ta ti ve. Therefore, more than ever, an essential goal of genetics is to identify and use appropriate bio-markers for selection. In this way, appropriate biomarkers could be developed for the selection of WR, which enables a distinction between Mg-efficient and inefficient, which is very important for winegrowers. New clones with Mg efficiency would strengthen the local vine nurseries and viticulture and could also mean that vine material can be delivered to the neighbouring countries Hungary, Croa tia, Slovenia and Slovakia because the problem also exists there. Furthermore, this would result in a competitive advantage for domestic planting stock companies.