Enhancement of rAAV titers via inhibition of the interferon signaling cascade in transfected HEK293 suspension cultures.

The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields. In our previous research, we analyzed global regulatory pathways and mRNA expression patterns associated with increased rAAV production in HEK293 cells. Our data revealed substantial variations in the expression patterns between cell lines with low (LP) and high-production (HP) rates. Moving to a deeper layer for a more detailed analysis of inflammation-related transcriptome data, we detected an increased expression of interferon-related genes in low-producing cell lines. Following upon these results, we investigated the use of Ruxolitinib, an interferon pathway inhibitor, during the transient production of rAAV in HEK293 cells as potential media additive to boost rAAV titers. Indeed, we find a two-fold increase in rAAV titers compared to the control when the interferon pathways were inhibited. In essence, this work offers a rational design approach for optimization of HEK293 cell line productivity and potential engineering targets, ultimately paving the way for more cost-efficient and readily available gene therapies for patients.

Indistinguishability of Identical Bosons from a Quantum Information Theory Perspective.

Using tools from quantum information theory, we present a general theory of indistinguishability of identical bosons in experiments consisting of passive linear optics followed by particle number detection. Our results do neither rely on additional assumptions on the input state of the interferometer, such as, for instance, a fixed mode occupation, nor on any assumption on the degrees of freedom that potentially make the particles distinguishable. We identify the expectation value of the projector onto the N-particle symmetric subspace as an operationally meaningful measure of indistinguishability, and derive tight lower bounds on it that can be efficiently measured in experiments. Moreover, we present a consistent definition of perfect distinguishability and characterize the corresponding set of states. In particular, we show that these states are diagonal in the computational basis up to a permutationally invariant unitary. Moreover, we find that convex combinations of states that describe partially distinguishable and perfectly indistinguishable particles can lead to perfect distinguishability, which itself is not preserved under convex combinations.

Scaling strategy for cell and gene therapy bioreactors based on turbulent parameters.

So far, power input has been used as the main parameter for bioreactor scale-up/-down in upstream process development and manufacturing. The rationale is that maintaining a consistent power input per unit volume should result in comparable mixing times at different scales. However, shear generated from turbulent flow may compromise the integrity of non-robust cells such as those used during the production of cell and gene therapies, which may lead to low product quality and yield. Of particular interest is the Kolmogorov length parameter that characterizes the smallest turbulent eddies in a mixture. To understand its impact on scale-up/-down decisions, the distribution of Kolmogorov length along the trajectory flow of individual particles in bioreactors was estimated in silico with the help of computational fluid dynamics simulations. Specifically, in this study the scalability of iPSC-derived lymphocyte production and the impact of shear stress across various differentiation stages were investigated. The study used bioreactors of volumes from 0.1 to 10 L, which correspond to the scales most used for parameter optimization. Our findings, which align with in vitro runs, help determine optimal agitation speed and shear stress adjustments for process transfer between scales and bioreactor types, using vertically-oriented wheel and pitched-blade impellers. In addition, empirical models specific to the bioreactors used in this study were developed. The provided computational analysis in combination with experimental data supports selection of appropriate bioreactors and operating conditions for various cell and gene therapy process steps.

Sulfate limitation increases specific plasmid DNA yield and productivity in E. coli fed-batch processes.

Plasmid DNA (pDNA) is a key biotechnological product whose importance became apparent in the last years due to its role as a raw material in the messenger ribonucleic acid (mRNA) vaccine manufacturing process. In pharmaceutical production processes, cells need to grow in the defined medium in order to guarantee the highest standards of quality and repeatability. However, often these requirements result in low product titer, productivity, and yield. In this study, we used constraint-based metabolic modeling to optimize the average volumetric productivity of pDNA production in a fed-batch process. We identified a set of 13 nutrients in the growth medium that are essential for cell growth but not for pDNA replication. When these nutrients are depleted in the medium, cell growth is stalled and pDNA production is increased, raising the specific and volumetric yield and productivity. To exploit this effect we designed a three-stage process (1. batch, 2. fed-batch with cell growth, 3. fed-batch without cell growth). The transition between stage 2 and 3 is induced by sulfate starvation. Its onset can be easily controlled via the initial concentration of sulfate in the medium. We validated the decoupling behavior of sulfate and assessed pDNA quality attributes (supercoiled pDNA content) in E. coli with lab-scale bioreactor cultivations. The results showed an increase in supercoiled pDNA to biomass yield by 33% and an increase of supercoiled pDNA volumetric productivity by 13 % upon limitation of sulfate. In conclusion, even for routinely manufactured biotechnological products such as pDNA, simple changes in the growth medium can significantly improve the yield and quality.

The use of simulation in medical ultrasound: Current perspectives on applications and practical implementation (WFUMB state-of-the-art paper).

Simulation has been shown to improve clinical learning outcomes, speed up the learning process, and improve trainee confidence, while taking the pressure off initial face-to-face patient clinical areas. The second part of The World Federation for Ultrasound in Medicine and Biology state-of-the-art paper on the use of simulators provides a general approach on the practical implementation. The importance of needs assessment before developing a simulation-based training program is outlined. We describe the current practical implementation and critically analyze how simulators can be integrated into complex task scenarios to train small or large groups. A wide range of simulation equipment is available especially for those seeking interventional ultrasound training, ranging from animal tissue models, simple synthetic phantoms, to sophisticated high-fidelity simulation platforms using virtual reality. Virtual reality simulators provide feedback and thereby allow trainees to not only to practice their motor skills and hand eye coordination but also to interact with the simulator. Future developments will integrate more elements of automated assessment and artificial intelligence, thereby enabling enhanced realistic training experience and improving skill transfer into clinical practice.

Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for gene therapy applications.

Human embryonal kidney cells (HEK-293) are the most common host cells used for transient recombinant adeno-associated virus (rAAV) production in pharmaceutical industry. To better cover the expected gene therapy product demands in the future, different traditional strategies such as cell line sub-cloning and/or addition of chemical substances to the fermentation media have been used to maximize titers and improve product quality. A more effective and advanced approach to boost yield can be envisaged by characterizing the transcriptome of different HEK-293 cell line pedigrees with distinct rAAV productivity patterns to subsequently identify potential gene targets for cell engineering. In this work, the mRNA expression profile of three HEK-293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. Mock runs using only transfection reagents were performed in parallel as a control. It finds significant differences in gene regulatory behaviors between the three cell lines at different growth and production stages. The evaluation of these transcriptomics profiles combined with collected in-process control parameters and titers shed some light on potential cell engineering targets to maximize transient production of rAAV in HEK-293 cells.

Optimizing VLP production in gene therapy: Opportunities and challenges for in silico modeling.

Over the past decades, virus-like particle (VLP)-based gene therapy (GT) evolved as a promising approach to cure inherited diseases or cancer. Tremendous costs due to inefficient production processes remain one of the key challenges despite considerable efforts to improve titers. This review aims to link genome-scale metabolic models (GSMMs) to cell lines used for VLP synthesis for the first time. We summarize recent advances and challenges of GSMMs for Chinese hamster ovary (CHO) cells and provide an overview of potential cell lines used in GT. Although GSMMs in CHO cells led to significant improvements in growth rates and recombinant protein (RP)-production, no GSMM has been established for VLP production so far. To facilitate the generation of GSMM for these cell lines we further provide an overview of existing omics data and the highest production titers so far reported.

The ultrasound use of simulators, current view, and perspectives: Requirements and technical aspects (WFUMB state of the art paper).

Simulation has been shown to improve clinical learning outcomes, speed up the learning process and improve learner confidence, whilst initially taking pressure off busy clinical lists. The World Federation for Ultrasound in Medicine and Biology (WFUMB) state of the art paper on the use of simulators in ultrasound education introduces ultrasound simulation, its advantages and challenges. It describes different simulator types, including low and high-fidelity simulators, the requirements and technical aspects of simulators, followed by the clinical applications of ultrasound simulation. The paper discusses the role of ultrasound simulation in ultrasound clinical training, referencing established literature. Requirements for successful ultrasound simulation acceptance into educational structures are explored. Despite being in its infancy, ultrasound simulation already offers a wide range of training opportunities and likely holds the key to a broader point of care ultrasound education for medical students, practicing doctors, and other health care professionals. Despite the drawbacks of simulation, there are also many advantages, which are expanding rapidly as the technology evolves.

Hardware Efficient Quantum Simulation of Non-Abelian Gauge Theories with Qudits on Rydberg Platforms.

Non-Abelian gauge theories underlie our understanding of fundamental forces in nature, and developing tailored quantum hardware and algorithms to simulate them is an outstanding challenge in the rapidly evolving field of quantum simulation. Here we take an approach where gauge fields, discretized in spacetime, are represented by qudits and are time evolved in Trotter steps with multiqudit quantum gates. This maps naturally and hardware efficiently to an architecture based on Rydberg tweezer arrays, where long-lived internal atomic states represent qudits, and the required quantum gates are performed as holonomic operations supported by a Rydberg blockade mechanism. We illustrate our proposal for a minimal digitization of SU(2) gauge fields, demonstrating a significant reduction in circuit depth and gate errors in comparison to a traditional qubit-based approach, which puts simulations of non-Abelian gauge theories within reach of NISQ devices.

Fluorinated Tetraarylethenes: Universal Tags for the Synthesis of Solid State Luminogens.

The aggregation-induced emission properties of tetraarylethenes (TAEs) have led to numerous applications in chemistry, biology, and materials science. Herein, we describe two fluorinated tetraarylethenes, which can be employed as universal tags for the synthesis of solid state luminogens. The tags are accessible in one or two steps from commercially available starting materials. Facile coupling reactions with ubiquitous substrates such as thiols, alcohols, amines, phosphines, aldehydes, and enamines allow preparing a wide range of TAE conjugates, including tagged amino acids, peptides, carbohydrates, steroids, and commercial polymers.

Assessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM.

In spite of continuous development of gene therapy vectors with thousands of drug candidates in clinical drug trials there are only a small number approved on the market today stressing the need to have characterization methods to assist in the validation of the drug development process. The level of packaging of the vector capsids appears to play a critical role in immunogenicity, hence an objective quantitative method assessing the content of particles containing a genome is an essential quality measurement. As transmission electron microscopy (TEM) allows direct visualization of the particles present in a specimen, it naturally seems as the most intuitive method of choice for characterizing recombinant adeno-associated virus (rAAV) particle packaging. Negative stain TEM (nsTEM) is an established characterization method for analysing the packaging of viral vectors. It has however shown limitations in terms of reliability. To overcome this drawback, we propose an analytical method based on CryoTEM that unambiguously and robustly determines the percentage of filled particles in an rAAV sample. In addition, we show that at a fixed number of vector particles the portion of filled particles correlates well with the potency of the drug. The method has been validated according to the ICH Q2 (R1) guidelines and the components investigated during the validation are presented in this study. The reliability of nsTEM as a method for the assessment of filled particles is also investigated along with a discussion about the origin of the observed variability of this method.

Proton-transfer-reaction mass spectrometry (PTR-MS) for online monitoring of glucose depletion and cell concentrations in HEK 293 gene therapy processes.

OBJECTIVES: The applicability of proton-transfer-reaction mass spectrometry (PTR-MS) as a versatile online monitoring tool to increase consistency and robustness for recombinant adeno-associated virus (rAAV) producing HEK 293 bioprocesses was evaluated. We present a structured workflow to extract process relevant information from PTR-MS data. RESULTS: Reproducibility of volatile organic compound (VOC) measurements was demonstrated with spiking experiments and the process data sets used for applicability evaluation consisted of HEK 293 cell culture triplicates with and without transfection. The developed data workflow enabled the identification of six VOCs, of which two were used to develop a soft sensor providing better real-time estimates than the conventional capacitance sensor. Acetaldehyde, another VOC, provides online process information about glucose depletion that can directly be used for process control purposes. CONCLUSIONS: The potential of PTR-MS for HEK 293 cell culture monitoring has been shown. VOC data derived information can be used to develop soft sensors and to directly set up new process control strategies.

Mixed-State Entanglement from Local Randomized Measurements.

We propose a method for detecting bipartite entanglement in a many-body mixed state based on estimating moments of the partially transposed density matrix. The estimates are obtained by performing local random measurements on the state, followed by postprocessing using the classical shadows framework. Our method can be applied to any quantum system with single-qubit control. We provide a detailed analysis of the required number of experimental runs, and demonstrate the protocol using existing experimental data [Brydges et al., Science 364, 260 (2019)SCIEAS0036-807510.1126/science.aau4963].

Matrix Product States: Entanglement, Symmetries, and State Transformations.

We analyze entanglement in the family of translationally invariant matrix product states (MPS). We give a criterion to determine when two states can be transformed into each other by local operations with a nonvanishing probability, a central question in entanglement theory. This induces a classification within this family of states, which we explicitly carry out for the simplest, nontrivial MPS. We also characterize all symmetries of translationally invariant MPS, both global and local (inhomogeneous). We illustrate our results with examples of states that are relevant in different physical contexts.

Subradiance via Entanglement in Atoms with Several Independent Decay Channels.

Spontaneous emission of atoms in free space is modified by the presence of other atoms in close vicinity inducing collective super- and subradiance. For two nearby atoms with a single decay channel the entangled antisymmetric superposition state of the two single excited states will not decay spontaneously. No such excited two-atom dark state exists, if the excited state has two independent optical decay channels of different frequencies or polarizations. However, we show that for an excited atomic state with N-1 independent spontaneous decay channels one can find a highly entangled N-particle dark state, which completely decouples from the vacuum radiation field. It does not decay spontaneously, nor will it absorb resonant laser light. Mathematically, we see that this state is the only such state orthogonal to the subspace spanned by the atomic ground states. Moreover, by means of generic numerical examples we demonstrate that the subradiant behavior largely survives at finite atomic distances including dipole-dipole interactions.

Training needs in adolescent medicine of practising doctors: a Swiss national survey of six disciplines.

OBJECTIVE: To assess and compare the training needs in adolescent medicine of doctors within 6 specialties as a basis for the development of pre/postgraduate and continuing medical education (CME) training curricula. DESIGN: Cross-sectional postal survey. SETTING: Switzerland. PARTICIPANTS: National, representative, random sample of 1857 practising doctors in 6 disciplines (general practitioners, paediatricians, gynaecologists, internists, psychiatrists, child psychiatrists) registered with the Swiss Medical Association. MAIN OUTCOME MEASURES: Perceived importance of and training interest in 35 topics related to adolescent medicine listed in a self-administered, anonymous questionnaire. RESULTS: A total of 1367 questionnaires were returned, representing a response rate of 73.9%. Clear interest in adolescent medicine was reported by 62.1% of respondents. Topics perceived to be the most important in everyday practice were functional symptoms (71.4%), acne (67.1%), obesity (64.6%), depression-anxiety (68.1%) and communication with adolescents (61.7%). Differences between disciplines were especially marked for gynaecologists, who expressed interest almost exclusively in medical topics specific to their field. In contrast, other disciplines commonly reported a keen interest in psychosocial problems. Accordingly, interest in further training was expressed mostly for functional symptoms (62.4%), eating disorders (56.3%), depression-anxiety (53.7%) and obesity (52.6%). Issues related to injury prevention, chronic disease and confidentiality were rated as low priorities. CONCLUSIONS: Regardless of discipline, Swiss primary care doctors expressed a strong interest in adolescent medicine. Continuing medical education courses should include both interdisciplinary courses and discipline-specific sessions. Further training should address epidemiological and legal/ethical issues (e.g. injury prevention, confidentiality, impact of chronic conditions).