Dept. Gene Regulation and Differentiation (RDIF)
http://hdl.handle.net/10033/6823
Abt. Genregulation udn Differenzierung (RDIF)2024-03-28T15:23:46ZRecombinant protein expression by targeting pre-selected chromosomal loci
http://hdl.handle.net/10033/621043
Recombinant protein expression by targeting pre-selected chromosomal loci
Nehlsen, Kristina; Schucht, Roland; da Gama-Norton, Leonor; Krömer, Wolfgang; Baer, Alexandra; Cayli, Aziz; Hauser, Hansjörg; Wirth, Dagmar
Abstract Background Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. Results We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context. Conclusion RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.
2009-12-14T00:00:00ZTowards rational engineering of cells: Recombinant gene expression in defined chromosomal loci
http://hdl.handle.net/10033/621033
Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci
Nehlsen, Kristina; da Gama-Norton, Leonor; Schucht, Roland; Hauser, Hansjörg; Wirth, Dagmar
2011-11-22T00:00:00ZTowards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data
http://hdl.handle.net/10033/621011
Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data
Martins, José P; Santos, Jorge M; Almeida, Joana M d; Filipe, Mariana A; de Almeida, Mariana V T; Almeida, Sílvia C C; Água-Doce, Ana; Varela, Alexandre; Gilljam, Mari; Stellan, Birgitta; Pohl, Susanne; Dittmar, Kurt; Lindenmaier, Werner; Alici, Evren; Graça, Luís; Cruz, Pedro E; Cruz, Helder J; Bárcia, Rita N
Abstract Introduction Standardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP). Methods The adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed. Results The isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties. Conclusions We have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.
2014-01-17T00:00:00ZDNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4).
http://hdl.handle.net/10033/620842
DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4).
Quentmeier, Hilmar; Eberth, Sonja; Romani, Julia; Weich, Herbert A; Zaborski, Margarete; Drexler, Hans G
Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation.
2012-01-17T00:00:00Z