Crucial aspects for maintaining rAAV stability.

The storage of rAAV vectors for gene therapy applications is critical for ensuring a constant product quality and defined amount of medication at the time of administration. Therefore, we determined the influence of different storage conditions on the physicochemical and biological properties of rAAV8 and rAAV9 preparations. Particular attention was paid to short-term storage, which plays a crucial role in both the manufacturing process and in clinical applications.

Factors affecting rAAV titers during triple-plasmid transient transfection in HEK-293 cells.

The efficiency of triple-plasmid transfection in recombinant Adeno-Associated Virus (rAAV) production was analyzed by examining two distinct HEK-293 cells lines. These were categorized as high producer (HP) and low producer (LP) based on their differing levels of productivity under identical conditions. Analysis of RNA expression levels of viral genes revealed disparities in plasmid derived gene expression between the cell lines.

Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer packaging HEK-293 cell lines used for rAAV production.

In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success.

Orthogonal characterization of rAAV9 reveals unexpected transgene heterogeneity.

Recombinant adeno-associated virus (rAAV) is the most widely used viral vector for in vivo human gene therapy. To ensure safety and efficacy of gene therapy products, a comprehensive analytical profile of the rAAVs is needed, which provides crucial information for therapeutic development and manufacturing. Besides information on rAAV quantities and possible contaminating DNA and protein species, assessing rAAV quality is of utmost importance.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.