Molekulare Grundlagen der episomalen Replikation: Charakterisierung zirkulärer, nichtviraler Vektoren

Der gerichtete und effiziente Transfer eines therapeutischen Gens in eine somatische Zelle ohne toxische oder immunogene Nebenwirkungen ist eines der wichtigsten Ziele in der Gentherapie. Der in dieser Arbeit verwendete nichtvirale, extrachromosomal replizierende Vektor basiert auf den Eigenschaften einer „Scaffold/Matrix Attachment Region“ (S/MAR) und nutzt durch deren Affinität zur Kernmatrix den endogenen Replikationsapparat der Wirtszelle. Die transkriptionsbedingte Konformationsänderung der S/MAR-Sequenz führt dabei zu einer stabilen episomalen Langzeitreplikation. Ein Coexpressionssystem zweier extrachromosomal replizierender Vektoren konnte etabliert werden, welches über den Selektionsdruck regulierbar ist und dadurch zur Expression eines funktionellen IgG-Moleküls führt. Zudem konnten durch die Verwendung eines sequenzspezifischen Rekombinasesystems die prokaryontischen Sequenzen aus dem Vektor deletiert werden. Dadurch wurde die stabile, selektions- druckunabhängige Expression des episomal replizierenden Vektors gewährleistet.A successful gene therapy depends on the efficient transfer of therapeutic relevant genes in a somatic cell without any toxic or immunogenic side-effects. Therefore, this work is focussed on a non-viral, extrachromosomal replicating vector. This vector is based on a so called "scaffold/matrix attachment region" (S/MAR) which guides the vector to the nuclear matrix where the endogenous replication machinery is localized. The transcriptional mediated conformational change of the S/MAR- sequence leads to a stable episomal long-term replication. Additionally, a two-vector co-replicating system has been established, that can be regulated through differential selection leading to a functional IgG-molecule. A sequence specific recombinase-mediated system has been used to delete the prokaryotic vector sequences. This guarantees the stable expression independent of any selection from the episomal replicating vector

Recombinant protein expression by targeting pre-selected chromosomal loci

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

The Path from Nasal Tissue to Nasal Mucosa on Chip: Part 1—Establishing a Nasal In Vitro Model for Drug Delivery Testing Based on a Novel Cell Line

In recent years, there has been a significant increase in the registration of drugs for nasal application with systemic effects. Previous preclinical in vitro test systems for transmucosal drug absorption studies have mostly been based on primary cells or on tumor cell lines such as RPMI 2650, but both approaches have disadvantages. Therefore, the aim of this study was to establish and characterize a novel immortalized nasal epithelial cell line as the basis for an improved 3D cell culture model of the nasal mucosa. First, porcine primary cells were isolated and transfected. The P1 cell line obtained from this process was characterized in terms of its expression of tissue-specific properties, namely, mucus expression, cilia formation, and epithelial barrier formation. Using air–liquid interface cultivation, it was possible to achieve both high mucus formation and the development of functional cilia. Epithelial integrity was expressed as both transepithelial electrical resistance and mucosal permeability, which was determined for sodium fluorescein, rhodamine B, and FITC-dextran 4000. We noted a high comparability of the novel cell culture model with native excised nasal mucosa in terms of these measures. Thus, this novel cell line seems to offer a promising approach for developing 3D nasal mucosa tissues that exhibit favorable characteristics to be used as an in vitro system for testing drug delivery systems

Performance of Genomic Bordering Elements at Predefined Genomic Loci

Eukaryotic DNA is organized into chromatin domains that regulate gene expression and chromosome behavior. Insulators and/or scaffold-matrix attachment regions (S/MARs) mark the boundaries of these chromatin domains where they delimit enhancing and silencing effects from the outside. By recombinase-mediated cassette exchange (RMCE), we were able to compare these two types of bordering elements at a number of predefined genomic loci. Flanking an expression vector with either S/MARs or two copies of the non-S/MAR chicken hypersensitive site 4 insulator demonstrates that while these borders confer related expression characteristics at most loci, their effect on chromatin organization is clearly distinct. Our results suggest that the activity of bordering elements is most pronounced for the abundant class of loci with a low but negligible expression potential in the case of highly expressed sites. By the RMCE procedure, we demonstrate that expression parameters are not due to a potential targeting action of bordering elements, in the sense that a linked transgene is directed into a special class of loci. Instead, we can relate the observed transcriptional augmentation phenomena to their function as genomic insulators

Enhancing pre-clinical research with simplified intestinal cell line models

Two-dimensional culture remains widely employed to determine the bioavailability of orally delivered drugs. To gain more knowledge about drug uptake mechanisms and risk assessment for the patient after oral drug admission, intestinal in vitro models demonstrating a closer similarity to the in vivo situation are needed. In particular, Caco-2 cell-based Transwell® models show advantages as they are reproducible, cost-efficient, and standardized. However, cellular complexity is impaired and cell function is strongly modified as important transporters in the apical membrane are missing. To overcome these limitations, primary organoid-based human small intestinal tissue models were developed recently but the application of these cultures in pre-clinical research still represents an enormous challenge, as culture setup is complex as well as time- and cost-intensive. To overcome these hurdles, we demonstrate the establishment of primary organoid-derived intestinal cell lines by immortalization. Besides exhibiting cellular diversity of the organoid, these immortalized cell lines enable a standardized and more cost-efficient culture. Further, our cell line-based Transwell®-like models display an organ-specific epithelial barrier integrity, ultrastructural features and representative transport functions. Altogether, our novel model systems are cost-efficient with close similarity to the in vivo situation, therefore favoring their use in bioavailability studies in the context of pre-clinical screenings

Cytological, molecular, cytogenetic, and physiological characterization of a novel immortalized human enteric glial cell line

Enteric glial cells (EGCs), the major components of the enteric nervous system (ENS), are implicated in the maintenance of gut homeostasis, thereby leading to severe pathological conditions when impaired. However, due to technical difficulties associated with EGCs isolation and cell culture maintenance that results in a lack of valuable in vitro models, their roles in physiological and pathological contexts have been poorly investigated so far. To this aim, we developed for the first time, a human immortalized EGC line (referred as ClK clone) through a validated lentiviral transgene protocol. As a result, ClK phenotypic glial features were confirmed by morphological and molecular evaluations, also providing the consensus karyotype and finely mapping the chromosomal rearrangements as well as HLA-related genotypes. Lastly, we investigated the ATP- and acetylcholine, serotonin and glutamate neurotransmitters mediated intracellular Ca2+ signaling activation and the response of EGCs markers (GFAP, SOX10, S100β, PLP1, and CCL2) upon inflammatory stimuli, further confirming the glial nature of the analyzed cells. Overall, this contribution provided a novel potential in vitro tool to finely characterize the EGCs behavior under physiological and pathological conditions in humans