Expression of glycoproteins with excellent pharmacokinetic properties on the novel CAP-Go expression platform

Due to their clinical importance, the development of therapeutic proteins has accelerated immensely over the past years. However, the expression of highly glycosylated recombinant therapeutic proteins like for example blood coagulation factors or serum proteins, has remained a challenging task. C1-Inhibitor (C1-Inh) holds 7 N- and 8 O-glycosylation sites. Plasma derived C1-Inh (Berinert) as well as recombinant C1-Inh from transgenic rabbits (Ruconest) are approved for the treatment of acute attacks in patients with hereditary angioedema. However, the recombinant product shows a dramatically reduced serum half-life in pharmacokinetic studies in comparison to the plasma derived counterparts. We have developed the CAP-Go protein expression platform to confer optimal glycosylation to complex glycoproteins like C1-Inh. The CAP-Go.1 cell line has been modified to facilitate expression of proteins with fully sialylated N-glycans. Recombinant proteins like human alpha-antitrypsin or human placental alkaline phosphatase produced with CAP-Go.1 show a significantly prolonged serum half-life in rats. However, expression of rhC1-Inh in CAP-Go.1 cells has no positive impact on the pharmacokinetic profile. Expression of rhC1-Inh in CAP-Go.2 cells, which in addition addresses the O-linked glycosylation patterns, results in a significantly increased serum half-life compared to its counterpart and is actually indistinguishable from the plasma-purified protein. C1-Inh expressed in CAP-Go.1 or CAP-Go.2 cells show a similar reduction of terminal galactose on N-glycans, but their O-glycans differ. O-glycan analysis shows that rhC1-Inh expressed by CAP-Go.2 cells contains only core1 O-glycan structures, highly comparable to plasma-derived Berinert. Our results indicate that in addition to N-glycosylation, also the structure and composition of O-linked glycans plays a crucial role for pharmacokinetic properties of glycoproteins. In conclusion, rhC1-Inh expressed from CAP-Go.2 cells, which have been optimized for the expression of N- and O-glycosylated proteins, display glycan patterns closely similar to plasma-derived C1-Inh. The resulting molecule has a significantly prolonged serum half-life as compared to C1-Inh generated on a conventional human cell line. Our new recombinant molecule matches serum-derived C1-Inh in all aspects analyzed: specific activity, serum half-life, and glycosylation pattern and offers the advantage of being producible at large scale on a safe platform

Strain localization as a key to reconciling experimentally derived flow-law data with dynamic models of continental collision

Abstract. : Published strength profiles predict strength discontinuities within and/or at the base of continental crust during compression. We use finite element models to investigate the effect of strength discontinuities on continental collision dynamics. The style of deformation in model crust during continued subduction of underlying mantle lithosphere is controlled by: (1) experimental flow-law data; (2) the crustal geotherm; (3) strain localization by erosion; (4) strain-softening and other localization effects. In the absence of erosion and other factors causing strain localization, numerical models with typical geothermal gradients and frictional/ductile rheologies predict diffuse crustal deformation with whole-scale detachment of crust from mantle lithosphere. This prediction is at odds with earlier model studies that only considered frictional crustal rheologies and showed asymmetric, focused crustal deformation. Without localization, model deformation is not consistent with that observed in small collisional orogens such as the Swiss Alps. This suggests that strain localization by a combination of erosion and rheological effects such as strain softening must play a major role in focusing deformation, and that strength profiles derived under constant strain rates and uniform material properties cannot be used to infer crustal strength during collision dynamic

Engineering of exosomes for targeted delivery of therapeutic microRNAs

Exosomes are small membrane vesicles secreted from most cell types. They contain a range of proteins, lipids, mRNAs and microRNAs (miRNA) and are naturally taken up by cells in order to deliver these contents to recipient cells. Increased understanding of this process as well as advances in bioengineering has led to investigations into the use of exosomes as targeted vehicles to transport therapeutic non-coding RNAs, proteins or drug molecules directly across cellular barriers into recipient cells. miRNAs are small non-coding RNA molecules, which play a key role in mediating biological function due to their prominent role in gene regulation. Deregulation of miRNAs is a common feature in cancer, suggesting that these molecules could serve as targets for therapeutic intervention by restoring or inhibiting their cellular function. However, various biological barriers including in vivo nuclease degradation, fibrous nature of tumors, and miRNA-induced immune response drastically hinder their bioavailability. In the context of these complex settings, exosomal delivery may display a novel strategy for targeted delivery of RNA therapeutics. We have recently identified and characterized novel pro-apoptotic miRNAs (Kleemann et al 2016, Flum et al 2017), which are down-regulated in cancer cells suggesting promising potential for therapeutic approaches. In the current study we conducted an initial evaluation of the novel concept to enable targeted delivery of small therapeutic RNAs using exosomes as biological transport systems. CEVECs amniocyte production cells (CAP) are utile human expression hosts highly suitable for the production of glycosylated biotherapeutic proteins. However, there are currently no reports as to whether this human cell line may as well produce extracellular vesicles suitable for targeted delivery of therapeutics. In order to evaluate these cells as production hosts for exosomes, we initially cultivated parental CAP cells and were able to isolate exosomes by centrifugation. Exosomal preparations were examined for vesicle identity, size, morphology and concentration using dynamic light scattering, flow cytometry, western blotting and electron microscopy. In order to be able to visualize exosomes and track targeted delivery in subsequent experiments, we engineered CAP cells to stably overexpress a CD63-GFP-fusion protein. This overexpression of a fluorescent transmembrane protein present on exosomes enabled the tracing of produced exosomes using flow cytometry. To functionally analyze isolated exosomes regarding their potential to delivery small therapeutic agents, these fluorescently labeled exosomes were further engineered to overexpress therapeutic, pro-apoptotic and control miRNAs by stable genome integration into CAP parental cells. Resulting preparations of engineered exosomes were analyzed for modified miRNA content in comparison to parental cells using qPCR. Currently the cells are further engineered to overexpress modified surface receptors to facilitate targeted uptake by tumor cells. Fluorescently labelled exosomes carrying therapeutic miRNAs and surface receptors will finally be co-cultured with target cells to analyze the potential of engineered exosomes to crossing membrane barriers and reliably deliver therapeutic miRNAs to recipient cells. The current study pursues the novel therapeutic concept of using exosomes as delivery vehicle for small non-coding RNAs molecules. We present data which assess for the first time the potential application of exosomes produced by a human production host for biotherapeutics. In addition to these current engineering and delivery approaches, future application of this cell therapy will heavily depend upon mass production of these delivery vectors and production issues will therefore gain increasing importance. Kleemann M, Bereuther J, Fischer S, Marquart K, Hänle S, Unger K, Jendrossek V, Riedel C, Handrick R, Otte K (2016) Investigation on tissue specific effects of pro-apoptotic miR-147b as a potential biomarker in ovarian cancer prognosis. Oncotarget 8 (12), 18773-18791 Flum M, Kleemann M, Schneider H, Weis B, Fischer S, Handrick R, Otte K (2017) miR-217-5p induces apoptosis by directly targeting PRKCI, BAG3, ITGAV and MAPK1 in colorectal cancer cells. J Cell Commun Signal, doi: 10.1007/s12079-017-0410-x

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

Differential virus restriction patterns of rhesus macaque and human APOBEC3A: Implications for lentivirus evolution

AbstractThe human apolipoprotein B mRNA editing enzyme catalytic peptide-like 3 (APOBEC3; A3) family of proteins (A3A-H) are known to restrict various retroviruses and retroelements, but the full complement of rhesus macaque A3 proteins remains unclear. We report the isolation and characterization of the hA3A homologue from rhesus macaques (rhA3A) and show that the rhesus macaque and human A3 genes are orthologous. RhA3A is expressed at high levels in activated CD4+ T cells, is widely expressed in macaque tissues, and is degraded in the presence of the human immunodeficiency virus (HIV-1) and simian–human immunodeficiency virus (SHIV) genomes. Our results indicate that rhA3A is a potent inhibitor of SHIVΔvif and to a lesser extent HIV-1Δvif. Unlike hA3A, rhA3A did not inhibit adeno-associated virus 2 (AAV-2) replication and L1 retrotransposition. These data suggest an evolutionary switch in primate A3A virus specificity and provide the first evidence that a primate A3A can inhibit lentivirus replication

Chronological aging leads to apoptosis in yeast

During the past years, yeast has been successfully established as a model to study mechanisms of apoptotic regulation. However, the beneficial effects of such a cell suicide program for a unicellular organism remained obscure. Here, we demonstrate that chronologically aged yeast cultures die exhibiting typical markers of apoptosis, accumulate oxygen radicals, and show caspase activation. Age-induced cell death is strongly delayed by overexpressing YAP1, a key transcriptional regulator in oxygen stress response. Disruption of apoptosis through deletion of yeast caspase YCA1 initially results in better survival of aged cultures. However, surviving cells lose the ability of regrowth, indicating that predamaged cells accumulate in the absence of apoptotic cell removal. Moreover, wild-type cells outlast yca1 disruptants in direct competition assays during long-term aging. We suggest that apoptosis in yeast confers a selective advantage for this unicellular organism, and demonstrate that old yeast cells release substances into the medium that stimulate survival of the clone

An AIF orthologue regulates apoptosis in yeast

Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1)

Guidelines and Recommendations on Yeast Cell Death Nomenclature

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cellular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the definition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death routines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the authors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the progress of this vibrant field of research

Guidelines and recommendations on yeast cell death nomenclature

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research

Endogene induced programmed cell death in Saccharomyces cerevisiae

Apoptose ist eine evolutionär konservierte Form des programmierten Zelltodes und äußerst wichtig für die Entwicklung von Vielzellern, eine Störung der Apoptose führt zu verschiedensten Krankheiten. In dieser Arbeit wurden neue Mediatoren der Apoptose in Hefe identifiziert und charakterisiert. Unter anderem das Homologe des Apoptosis Inducing Factor in der Bäckerhefe, Ynr074c oder Aif1. Ebenso wie das Säuger AIF ist Aif1p in den Mitochondrien lokalisiert und transloziert nach einem proapoptotischen Reiz in den Kern. Rekombinant aufgereinigtes Aif1p ist in der Lage die DNA in Hefezellkernen und Plasmid DNA zu degradieren. Eine Disruption des AIF1 Gens vermindert die Sensibilität der Hefezellen gegenüber Wasserstoffperoxid und altersinduzierter Apoptose. Umgekehrt führt die Überexpression von Aif1p zu einer erhöhten Apoptoserate nach einer Induktion des Zelltods durch Wasserstoffperoxid. Dieser Effekt wird vermindert durch eine Disruption der Hefecaspase (YCA1) oder Cyclophilin A. Außerdem konnte gezeigt werden, dass Aif1p nicht nur ein proapoptotisches Protein ist, sondern auch essentiell bei oxidativen Phosphorylierung ist, was Aif1p zu einem Janus ähnlichem Protein allegorisiert, mit wichtigen Funktionen im Leben und im Sterben.Apoptosis is an evolutionary conserved form of cell death crucial for metazoan development, and dysfunction of apoptosis leads to several diseases. In this thesis the identification and characterization of new mediators of yeast apoptosis is shown. These include amongst others the yeast apoptosis inducing factor homologue Ynr074cp, or Aif1p. Similar to mammalian AIF, Aif1p is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Aif1p degrades yeast nuclei and plasmid DNA. AIF1 disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, over-expression of Aif1p strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. It is also shown that yeast Aif1p is not only a proapoptotic protein, but is also essential for oxidative phosphorylation, which allegorizes Aif1 as a Janus-like molecule with important functions in life and death of cells