Generation of helper virus-free adeno-associated viral vector packaging/producer cell lines based on a human suspension cell line

The emerging number of clinical trials in the gene therapy field poses the challenge to the industry to produce viral vectors in a scalable, reproducible and cost-efficient manner. To address this issue, our CAP-GT platform comprises high density, serum free suspension cell lines that enable reproducible, scalable transfection and high titer production of viral vectors. An adeno-associated virus (AAV) based vector was the first approved gene therapy product in clinical applications. Attractive features of AAV as a gene therapy vector are e.g. its lack of pathogenicity and its ability to transduce dividing and non-dividing cells. Moving away from mainly targeting ultra-rare diseases and taking more common indications into focus will need to see significant improvements concerning productivity and consistent quality of AAV vector production in order to ensure supply. For this purpose, we are developing a helper virus-free packaging cell line that can easily be turned into a producer cell line by only one additional step of cell line development. Base of this packing cell line is the generation of a cell line with stable Tet-inducible expression of Rep proteins. Extensive screening of Rep expressing single cell clones resulted in clonal cell lines which produced high AAV titers upon induction and transfection of the missing components. In a next step, the adenoviral helper functions E2A and E4orf6 are introduced, due to their toxicity also under the control of a Tet-inducible promoter. In addition, the VA RNA is encoded by the same construct. Finally, a Tet-inducible capsid function and GFP as transgene flanked by the ITRs combined on one construct will be stably integrated resulting in a proof of principle producer cell line. This approach should enable a consistent quality production of AAV vectors that abolishes the drawbacks of transient transfection concerning reproducibility, consistency and high costs for GMP-grade DNA. Process optimization in regard to process duration, feeding strategy etc. is currently ongoing for further improving the vector yields

Knock‐out of multidrug efflux pump MexXY‐OprM results in increased susceptibility to antimicrobial peptides in Pseudomonas aeruginosa

Multidrug efflux systems of the resistance-nodulation-cell division family play a crucial role in resistance of Pseudomonas aeruginosa to a large variety of antibiotics. Here, we investigated the role of clinically relevant efflux pumps MexAB$^−$OprM, MexCD$^−$OprJ, and MexXY$^−$OprM in resistance against different cationic antimicrobial peptides (AMPs). Our results indicate that a knock-out in efflux pump MexXY-OprM increased susceptibility to some AMPs by two- to eightfold. Our data suggest a contribution of MexXY-OprM in resistance to certain AMPs in P. aeruginosa, which should be considered in the future development of new and highly active antimicrobial peptides to fight multidrug resistant infections

Oxidative stress response in pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative environmental and human opportunistic pathogen highly adapted to many different environmental conditions. It can cause a wide range of serious infections, including wounds, lungs, the urinary tract, and systemic infections. The high versatility and pathogenicity of this bacterium is attributed to its genomic complexity, the expression of several virulence factors, and its intrinsic resistance to various antimicrobials. However, to thrive and establish infection, P. aeruginosa must overcome several barriers. One of these barriers is the presence of oxidizing agents (e.g., hydrogen peroxide, superoxide, and hypochlorous acid) produced by the host immune system or that are commonly used as disinfectants in a variety of different environments including hospitals. These agents damage several cellular molecules and can cause cell death. Therefore, bacteria adapt to these harsh conditions by altering gene expression and eliciting several stress responses to survive under oxidative stress. Here, we used PubMed to evaluate the current knowledge on the oxidative stress responses adopted by P. aeruginosa. We will describe the genes that are often differently expressed under oxidative stress conditions, the pathways and proteins employed to sense and respond to oxidative stress, and how these changes in gene expression influence pathogenicity and the virulence of P. aeruginosa. Understanding these responses and changes in gene expression is critical to controlling bacterial pathogenicity and developing new therapeutic agents

The oxidative stress agent hypochlorite stimulates c-di-GMP synthesis and biofilm formation in Pseudomonas aeruginosa

The opportunistic human pathogen Pseudomonas aeruginosa is able to survive under a variety of often harmful environmental conditions due to a multitude of intrinsic and adaptive resistance mechanisms, including biofilm formation as one important survival strategy. Here, we investigated the adaptation of P. aeruginosa PAO1 to hypochlorite (HClO), a phagocyte-derived host defense compound and frequently used disinfectant. In static biofilm assays, we observed a significant enhancement in initial cell attachment in the presence of sublethal HClO concentrations. Subsequent LC-MS analyses revealed a strong increase in cyclic-di-GMP (c-di-GMP) levels suggesting a key role of this second messenger in HClO-induced biofilm development. Using DNA microarrays, we identified a 26-fold upregulation of ORF PA3177 coding for a putative diguanylate cyclase (DGC), which catalyzes the synthesis of the second messenger c-di-GMP – an important regulator of bacterial motility, sessility and persistence. This DGC PA3177 was further characterized in more detail demonstrating its impact on P. aeruginosa motility and biofilm formation. In addition, cell culture assays attested a role for PA3177 in the response of P. aeruginosa to human phagocytes. Using a subset of different mutants, we were able to show that both Pel and Psl exopolysaccharides are effectors in the PA3177-dependent c-di-GMP network

Oxidative Stress Response in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative environmental and human opportunistic pathogen highly adapted to many different environmental conditions. It can cause a wide range of serious infections, including wounds, lungs, the urinary tract, and systemic infections. The high versatility and pathogenicity of this bacterium is attributed to its genomic complexity, the expression of several virulence factors, and its intrinsic resistance to various antimicrobials. However, to thrive and establish infection, P. aeruginosa must overcome several barriers. One of these barriers is the presence of oxidizing agents (e.g., hydrogen peroxide, superoxide, and hypochlorous acid) produced by the host immune system or that are commonly used as disinfectants in a variety of different environments including hospitals. These agents damage several cellular molecules and can cause cell death. Therefore, bacteria adapt to these harsh conditions by altering gene expression and eliciting several stress responses to survive under oxidative stress. Here, we used PubMed to evaluate the current knowledge on the oxidative stress responses adopted by P. aeruginosa. We will describe the genes that are often differently expressed under oxidative stress conditions, the pathways and proteins employed to sense and respond to oxidative stress, and how these changes in gene expression influence pathogenicity and the virulence of P. aeruginosa. Understanding these responses and changes in gene expression is critical to controlling bacterial pathogenicity and developing new therapeutic agents

Human host defense peptide LL-37 stimulates virulence factor production and adaptive resistance in Pseudomonas aeruginosa.

A multitude of different virulence factors as well as the ability to rapidly adapt to adverse environmental conditions are important features for the high pathogenicity of Pseudomonas aeruginosa. Both virulence and adaptive resistance are tightly controlled by a complex regulatory network and respond to external stimuli, such as host signals or antibiotic stress, in a highly specific manner. Here, we demonstrate that physiological concentrations of the human host defense peptide LL-37 promote virulence factor production as well as an adaptive resistance against fluoroquinolone and aminoglycoside antibiotics in P. aeruginosa PAO1. Microarray analyses of P. aeruginosa cells exposed to LL-37 revealed an upregulation of gene clusters involved in the production of quorum sensing molecules and secreted virulence factors (PQS, phenazine, hydrogen cyanide (HCN), elastase and rhamnolipids) and in lipopolysaccharide (LPS) modification as well as an induction of genes encoding multidrug efflux pumps MexCD-OprJ and MexGHI-OpmD. Accordingly, we detected significantly elevated levels of toxic metabolites and proteases in bacterial supernatants after LL-37 treatment. Pre-incubation of bacteria with LL-37 for 2 h led to a decreased susceptibility towards gentamicin and ciprofloxacin. Quantitative Realtime PCR results using a PAO1-pqsE mutant strain present evidence that the quinolone response protein and virulence regulator PqsE may be implicated in the regulation of the observed phenotype in response to LL-37. Further experiments with synthetic cationic antimicrobial peptides IDR-1018, 1037 and HHC-36 showed no induction of pqsE expression, suggesting a new role of PqsE as highly specific host stress sensor

TypA is involved in virulence, antimicrobial resistance and biofilm formation in Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa is an important opportunistic human pathogen and is extremely difficult to treat due to its high intrinsic and adaptive antibiotic resistance, ability to form biofilms in chronic infections and broad arsenal of virulence factors, which are finely regulated. TypA is a GTPase that has recently been identified to modulate virulence in enteric Gram-negative pathogens. Results: Here, we demonstrate that mutation of typA in P. aeruginosa resulted in reduced virulence in phagocytic amoebae and human macrophage models of infection. In addition, the typA mutant was attenuated in rapid cell attachment to surfaces and biofilm formation, and exhibited reduced antibiotic resistance to ß-lactam, tetracycline and antimicrobial peptide antibiotics. Quantitative RT-PCR revealed the down-regulation, in a typA mutant, of important virulence-related genes such as those involved in regulation and assembly of the Type III secretion system, consistent with the observed phenotypes and role in virulence of P. aeruginosa. Conclusions: These data suggest that TypA is a newly identified modulator of pathogenesis in P. aeruginosa and is involved in multiple virulence-related characteristics.Other UBCNon UBCReviewedFacult

Human CAP cells represent a novel source for functional, miRNA-loaded exosome production.

Exosomes represent a promising delivery tool for nucleic acid-based pharmaceuticals. They are highly suitable for transporting therapeutic miRNAs to tumor cells, due to their natural membrane components. Further, exosomes are capable of effectively protecting nucleic acids against ribonucleases and enable the delivery of their content through cell membranes. However, no suitable production host for miRNA containing exosomes of non-tumorigenic origin has yet been identified. In this study we engineered an immortalised human amniocyte cell line (CAP® cells), whose exosomes were enriched and characterised. The cell line modifications not only enabled the production of GFP-labelled but also pro-apoptotic miRNA containing exosomes without negative influence on host cell growth. Furthermore, we demonstrated that pro-apoptotic miRNA containing CAP exosomes are taken up by ovarian cancer cells. Strikingly, delivery of functional exosomal miRNA led to downregulation of several reported target genes in the treated tumor cells. In summary, we revealed CAP cells of non-tumorigenic origin as a novel and efficient exosome production host with the potential to produce functional miRNA-loaded exosomes

Time-killing of <i>P. aeruginosa</i> PAO1 by antibiotics ciprofloxacin (A) or gentamicin (B) in the absence or presence of LL-37.

<p>Mid-log phase bacterial cultures were incubated with either 20 µg/ml LL-37 (filled circles) or without LL-37 (open squares) for 2 h. Following dilution of bacterial cultures to 10⁷ cells/ml and addition of 3-fold MIC concentrations of antibiotics ciprofloxacin (0.18 µg/ml) or gentamicin (1.5 µg/ml), colony forming units at indicated time points were determined using the optimized drop plate method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082240#pone.0082240-Herigstad1" target="_blank">[27]</a>. Experiments were performed in triplicate. The figure shows representative results of one experiment. Error bars indicate standard deviations of 10 spots per sample plated out on two different agar plates (n = 10).</p