TheraVision: Engineering platform technology for the development of oncolytic viruses based on herpes simplex virus type 1.

Viruses are able to efficiently penetrate cells, multiply, and eventually kill infected cells, release tumor antigens, and activate the immune system. Therefore, viruses are highly attractive novel agents for cancer therapy. Clinical trials with first generations of oncolytic viruses (OVs) are very promising but show significant need for optimization. The aim of TheraVision was to establish a broadly applicable engineering platform technology for combinatorial oncolytic virus and immunotherapy. Through genetic engineering, an attenuated herpes simplex virus type 1 (HSV1) was generated that showed increased safety compared to the wild-type strain. To demonstrate the modularity and the facilitated generation of new OVs, two transgenes encoding retargeting as well as immunomodulating single-chain variable fragments (scFvs) were integrated into the platform vector. The resulting virus selectively infected epidermal growth factor receptor (EGFR)-expressing cells and produced a functional immune checkpoint inhibitor against programmed cell death protein 1 (PD-1). Thus, both viral-mediated oncolysis and immune-cell-mediated therapy were combined into a single viral vector. Safety and functionality of the armed OVs have been shown in novel preclinical models ranging from patient-derived organoids and tissue-engineered human in vitro 3D tumor models to complex humanized mouse models. Consequently, a novel and proprietary engineering platform vector based on HSV1 is available for the facilitated preclinical development of oncolytic virotherapy.

Quantification of statin effects on hepatic cholesterol synthesis by transient (13)C-flux analysis.

The present work is the first to deal with the determination of cholesterol synthesis rates in primary rat hepatocytes using transient (13)C-flux analysis. The effects of statins on cholesterol biosynthesis and central carbon fluxes were quantified at a therapeutic concentration of 50 nM atorvastatin using carbon-labeled glutamine. The flux through the cholesterol pathway decreased from 0.27 to 0.08 mmol/l(cv)h in response to the administration of the hypolipidemic drug. Isotopic steady state was reached within 4h in the central carbon metabolism but not in the cholesterol pathway, regardless of whether atorvastatin was administered or not. Marked channeling was observed for the symmetrical tricarboxylic acid cycle intermediates, succinate and fumarate. Non-stationary (13)C-based flux identification delivers both intracellular fluxes and intermediate levels, which was for the first time utilized for investigating systems-level effects of the administered drug by quantifying the flux control of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part I. Experimental observations.

An experimental set-up for acquiring metabolite and transient (13)C-labeling data in mammalian cells is presented. An efficient sampling procedure was established for hepatic cells cultured in six-well plates as a monolayer attached to collagen, which allowed simultaneous quenching of metabolism and extraction of the intracellular intermediates of interest. Extracellular concentrations of glucose, amino acids, lactate, pyruvate, and urea were determined by GC-MS procedures and were used for estimation of metabolic uptake and excretion rates. Sensitive LC-MS and GC-MS methods were used to quantify the intracellular intermediates of tricarboxylic acid cycle, glycolysis, and pentose phosphate pathway and for the determination of isotopomer fractions of the respective metabolites. Mass isotopomer fractions were determined in a transient (13)C-labeling experiment using (13)C-labeled glucose as substrate. The absolute amounts of intracellular metabolites were obtained from a non-labeled experiment carried out in exactly the same way as the (13)C-labeling experiment, except that the media contained naturally labeled glucose only. Estimation of intracellular metabolic fluxes from the presented data is addressed in part II of this contribution.

Author Correction: 3D biodegradable scaffolds of polycaprolactone with silicate-containing hydroxyapatite microparticles for bone tissue engineering: high-resolution tomography and in vitro study.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

3D biodegradable scaffolds of polycaprolactone with silicate-containing hydroxyapatite microparticles for bone tissue engineering: high-resolution tomography and in vitro study.

To date, special interest has been paid to composite scaffolds based on polymers enriched with hydroxyapatite (HA). However, the role of HA containing different trace elements such as silicate in the structure of a polymer scaffold has not yet been fully explored. Here, we report the potential use of silicate-containing hydroxyapatite (SiHA) microparticles and microparticle aggregates in the predominant range from 2.23 to 12.40 µm in combination with polycaprolactone (PCL) as a hybrid scaffold with randomly oriented and well-aligned microfibers for regeneration of bone tissue. Chemical and mechanical properties of the developed 3D scaffolds were investigated with XRD, FTIR, EDX and tensile testing. Furthermore, the internal structure and surface morphology of the scaffolds were analyzed using synchrotron X-ray µCT and SEM. Upon culturing human mesenchymal stem cells (hMSC) on PCL-SiHA scaffolds, we found that both SiHA inclusion and microfiber orientation affected cell adhesion. The best hMSCs viability was revealed at 10 day for the PCL-SiHA scaffolds with well-aligned structure (~82%). It is expected that novel hybrid scaffolds of PCL will improve tissue ingrowth in vivo due to hydrophilic SiHA microparticles in combination with randomly oriented and well-aligned PCL microfibers, which mimic the structure of extracellular matrix of bone tissue.

A perfusion bioreactor system efficiently generates cell-loaded bone substitute materials for addressing critical size bone defects.

Critical size bone defects and non-union fractions are still challenging to treat. Cell-loaded bone substitutes have shown improved bone ingrowth and bone formation. However, a lack of methods for homogenously colonizing scaffolds limits the maximum volume of bone grafts. Additionally, therapy robustness is impaired by heterogeneous cell populations after graft generation. Our aim was to establish a technology for generating grafts with a size of 10.5 mm in diameter and 25 mm of height, and thus for grafts suited for treatment of critical size bone defects. Therefore, a novel tailor-made bioreactor system was developed, allowing standardized flow conditions in a porous poly(L-lactide-co-caprolactone) material. Scaffolds were seeded with primary human mesenchymal stem cells derived from four different donors. In contrast to static experimental conditions, homogenous cell distributions were accomplished under dynamic culture. Additionally, culture in the bioreactor system allowed the induction of osteogenic lineage commitment after one week of culture without addition of soluble factors. This was demonstrated by quantitative analysis of calcification and gene expression markers related to osteogenic lineage. In conclusion, the novel bioreactor technology allows efficient and standardized conditions for generating bone substitutes that are suitable for the treatment of critical size defects in humans.

Nano-hydroxyapatite-coated metal-ceramic composite of iron-tricalcium phosphate: Improving the surface wettability, adhesion and proliferation of mesenchymal stem cells in vitro.

Thin radio-frequency magnetron sputter deposited nano-hydroxyapatite (HA) films were prepared on the surface of a Fe-tricalcium phosphate (Fe-TCP) bioceramic composite, which was obtained using a conventional powder injection moulding technique. The obtained nano-hydroxyapatite coated Fe-TCP biocomposites (nano-HA-Fe-TCP) were studied with respect to their chemical and phase composition, surface morphology, water contact angle, surface free energy and hysteresis. The deposition process resulted in a homogeneous, single-phase HA coating. The ability of the surface to support adhesion and the proliferation of human mesenchymal stem cells (hMSCs) was studied using biological short-term tests in vitro. The surface of the uncoated Fe-TCP bioceramic composite showed an initial cell attachment after 24h of seeding, but adhesion, proliferation and growth did not persist during 14 days of culture. However, the HA-Fe-TCP surfaces allowed cell adhesion, and proliferation during 14 days. The deposition of the nano-HA films on the Fe-TCP surface resulted in higher surface energy, improved hydrophilicity and biocompatibility compared with the surface of the uncoated Fe-TCP. Furthermore, it is suggested that an increase in the polar component of the surface energy was responsible for the enhanced cell adhesion and proliferation in the case of the nano-HA-Fe-TCP biocomposites.

A mouse bone marrow stromal cell line with skeletal stem cell characteristics to study osteogenesis in vitro and in vivo.

Bone marrow stromal cells (BMSCs) are composed of progenitor and multipotent skeletal stem cells, which are able to differentiate in vitro into osteocytes, adipocytes, and chondrocytes. Mouse BMSCs (mBMSCs) are a versatile model system to investigate factors involved in BMSC differentiation in vitro and in vivo as a variety of transgenic mouse models are available. In this study, mBMSCs were isolated and osteogenic differentiation was investigated in tissue culture and in vivo. Three out of seven independent cell isolates showed the ability to differentiate into osteocytes, adipocytes, and chondrocytes in vitro. In vitro multipotency of an established mBMSC line was maintained over 45 passages. The osteogenic differentiation of this cell line was confirmed by quantitative polymerase chain reaction (qPCR) analysis of specific markers such as osteocalcin and shown to be Runx2 dependent. Notably, the cell line, when transplanted subcutaneously into mice, possesses full skeletal stem cell characteristics in vivo in early and late passages, evident from bone tissue formation, induction of vascularization, and hematopoiesis. This cell line provides, thus, a versatile tool to unravel the molecular mechanisms governing osteogenesis in vivo thereby aiding to improve current strategies in bone regenerative therapy.

A systems biology approach to dynamic modeling and inter-subject variability of statin pharmacokinetics in human hepatocytes.

BACKGROUND: The individual character of pharmacokinetics is of great importance in the risk assessment of new drug leads in pharmacological research. Amongst others, it is severely influenced by the properties and inter-individual variability of the enzymes and transporters of the drug detoxification system of the liver. Predicting individual drug biotransformation capacity requires quantitative and detailed models. RESULTS: In this contribution we present the de novo deterministic modeling of atorvastatin biotransformation based on comprehensive published knowledge on involved metabolic and transport pathways as well as physicochemical properties. The model was evaluated on primary human hepatocytes and parameter identifiability analysis was performed under multiple experimental constraints. Dynamic simulations of atorvastatin biotransformation considering the inter-individual variability of the two major involved enzymes CYP3A4 and UGT1A3 based on quantitative protein expression data in a large human liver bank (n = 150) highlighted the variability in the individual biotransformation profiles and therefore also points to the individuality of pharmacokinetics. CONCLUSIONS: A dynamic model for the biotransformation of atorvastatin has been developed using quantitative metabolite measurements in primary human hepatocytes. The model comprises kinetics for transport processes and metabolic enzymes as well as population liver expression data allowing us to assess the impact of inter-individual variability of concentrations of key proteins. Application of computational tools for parameter sensitivity analysis enabled us to considerably improve the validity of the model and to create a consistent framework for precise computer-aided simulations in toxicology.

Zebrafish embryos express an orthologue of HERG and are sensitive toward a range of QT-prolonging drugs inducing severe arrhythmia.

A wide range of drugs has been shown to prolong the QT interval of the electrocardiogram by blocking the pore-forming subunit of the rapidly activating delayed rectifier K+ channel, HERG (ether-à-go-go-related gene), sometimes leading to life-threatening arrhythmia. In this paper we describe cloning, sequence, and expression of the zebrafish orthologue of HERG, Zerg. Further, we studied effects of Zerg inhibition in zebrafish embryos caused by drugs or by an antisense approach. Zerg is expressed specifically in both heart chambers of zebrafish embryos, is composed of six transmembrane domains, and shows an especially high degree of amino acid conservation in the S6 and pore domain (99% identity). Several QT-prolonging drugs added to the bathing medium elicited bradycardia and arrhythmia in zebrafish embryos. The arrhythmia induced ranged from an atrioventricular 2:1 block, the ventricle beating half as often as the atrium, to more severe irregular arrhythmia with higher concentrations of the drugs. These effects were highly specific, reproducible, and rapid, e.g., 10 microM astemizole caused a 2:1 heartbeat within a minute after addition of the compound in all the embryos studied. Morpholino antisense oligonucleotides targeting Zerg were injected into zebrafish embryos and elicited similar dose-sensitive and specific arrhythmia as the QT-prolonging drugs, suggesting an evolutionarily conserved role for Erg in regulating heartbeat rate and rhythm. Further, we identified a mutation in the Per-Arnt-Sim domain of the Zerg channel in the breakdance mutant, also characterized by a 2:1 atrioventricular block. In conclusion, the zebrafish could be a tractable model organism for the study of Erg function and modulation but might also have a value in the field of cardiovascular pharmacology, e.g., as an early preclinical model for testing drugs under development for potential QT prolongation.