Biology subjects

Mueller, L.

Publications and source records attributed to Mueller, L..

At least 19 recordsLinked to original sources

bioRxiv2026

A Framework for NGN1-Induced Sensory Neuron Differentiation for Disease Modelling and Drug Screening

Background: Neuropathic pain is a burdensome, difficult-to-treat, and highly heterogeneous condition with limited therapeutic options, underscoring the need for robust and reproducible human disease models. Human induced pluripotent stem cell (iPSC)-derived sensory neurons provide a promising platform for patient specific disease modelling and drug screening; however, their translational use is hampered by variability in differentiation efficiency, cellular composition, and functional maturation across protocols and cell lines. Methods: Here, we present a standardized and potentially scalable framework for NGN1 driven differentiation of human iPSCs into sensory neurons. Building on a previously published two step protocol (1), we systematically deconstructed and optimized each stage of differentiation across a large panel of genetically diverse iPSC lines. Results: We identified robust parameters for neural crest like cell (NCLC) generation, established a flow cytometry-based quality control strategy for NCLCs, and defined optimal combinations of seeding density and lentiviral multiplicity of infection to maximize sensory neuron progenitor yield. To improve culture homogeneity, we compared antimitotic selection strategies and demonstrated that tightly timed Ara-C treatment combined with low progenitor seeding density yields consistently pure sensory neuron cultures. We further evaluated maturation under physiologically relevant glucose conditions and performed a systematic review of media compositions to derive two defined maturation media. Morphological, immunocytochemical, transcriptomic, and electrophysiological analyses revealed that time in culture is a major determinant of maturation, while specific supplements such as prostaglandin E2; (PGE2) selectively enhance transcriptional signatures associated with nociceptor identity without substantially altering global network activity. Bulk RNA sequencing demonstrated broad expression of sensory neuron and pain related markers and gene programs across conditions, with long term maturation and PGE2; treatment showing the highest similarity to human dorsal root ganglion reference data. Functional assessment using multi electrode arrays enabled the detection of donor specific electrophysiological phenotypes, including reproducible hyperexcitability in small fiber neuropathy patient derived lines. Conclusions: This study establishes a modular, reproducible NGN1 based differentiation workflow with integrated quality checkpoints that accommodates iPSC line to line variability. The framework provides a practical foundation for translational sensory neuron research, patient specific disease modelling, and scalable drug screening applications.

neuroscience↗

bioRxiv2026

A chemogenetic CRISPR knockout screen identifies a ubiquitin-mediated calcineurin-BMP pathway relay in T cell activation

T cell activation is dependent on calcineurin signalling, yet how this pathway integrates with other regulatory systems remains incompletely understood. Here, we exploit the dual pharmacology of FK506, which inhibits calcineurin while also releasing FKBP12-mediated repression of BMP receptors, to dissect signalling crosstalk during T cell responses. A comparative genome-wide CRISPR knockout screen using FK506 and Cyclosporin A revealed that FK506 uniquely engages BMP pathway components and ubiquitin regulatory networks. Functional analyses in Jurkat and primary human T cells showed that FK506 induces BMP receptor-dependent activation of SMAD1/5/8 and triggers a rapid remodelling of K48- and K29-linked ubiquitin chains. Proteomics further demonstrated selective ubiquitination of immune signalling proteins and ubiquitin regulators, linking BMP pathway activation to proteasome-dependent turnover and non-proteolytic ubiquitin signalling. In primary T cells, BMP receptor signalling enhance calcineurin-driven activation, while changes in ubiquitin conjugation modulate this response, thereby establishing an integrated calcineurin-BMP-ubiquitin relay. Together, our results uncover a context-dependent signalling axis in which BMP activation and ubiquitin dynamics fine-tune calcineurin-mediated T cell signalling.

cell biology↗

bioRxiv2026

Multi-level Analysis of Codon Usage Patterns Reveals Systematic Optimization of Oncogenic Gene Expression in Pancreatic Cancer

BackgroundCodon usage bias, the non-random usage of synonymous codons in coding sequences, represents a fundamental feature of genomic organization that has been largely understudied in cancer biology. Pancreatic ductal adenocarcinoma (PDAC), the predominant subtype of pancreatic cancer, is characterized by aggressive disease progression and limited therapeutic options, necessitating novel approaches to understand its molecular pathogenesis. Leveraging publicly available single-cell RNA sequencing data, we performed comprehensive codon usage analyses across different cellular populations in PDAC. ResultsEmploying a variety of computational codon usage indices uncovered the connections between cancer-specific cellular state features and codon usage signatures. Our findings reveal that malignant pancreatic cells express genes with significantly higher GC content, demonstrate preferential usage of optimal codons through increased frequency of preferred synonymous codons, and exhibit a marked preference for more cost-effective amino acids. Analysis of transcript-level bulk RNA-seq data from PDAC tumors revealed that these codon optimization patterns extend to alternative isoform usage, with highly expressed isoforms displaying increased codon optimality and enhanced mRNA stability. ConclusionThese codon usage-dependent adaptations operating at both gene expression and transcript isoform levels may enable malignant cells to enhance gene expression rates, potentially leading to increased translational efficiency and protein production. These insights into the codon usage landscape of PDAC may provide potential biomarkers for disease monitoring and treatment response prediction.

cancer biology↗

bioRxiv2026

The impact of crystallographic plane orientation as an unexplored terrain in hemocompatible material design

Thrombogenicity causes significant complications in the application of blood-contacting implants, requiring strategies to prevent adverse coagulation reactions. The thrombotic responses to the foreign surfaces are mainly driven by surficial factors such as surface energy, topography, and electrochemical interactions. Although anticoagulation therapies reduce the risks of clotting, patients might still encounter bleeding complications. Therefore, rather than high-risk anticoagulation therapies to counteract coagulation, it is essential to ensure hemocompatibility through the materials intrinsic properties. Endothelialization is crucial in preventing thrombotic complications, with various strategies explored for facilitating endothelial cell adhesion and proliferation. We investigated the impact of crystallographic anisotropy on endothelial and blood cell interactions on four main planes (A-, C-, M-, and R-planes) of single crystalline alumina (-Al2O3, sapphire). Employing advanced surface characterization techniques, including SIMS, KPFM and Zeta potential measurements, our study sheds light on the hemocompatibility of biomaterials considering anisotropic effects. We elucidated that the A-plane of alumina promotes endothelialization and suppresses platelet activation in contrast to other crystallographic planes. Our investigation into cell-surface interactions provides valuable insights and contributes to the advanced biomaterial design, ultimately leading to enhanced clinical outcomes.

bioengineering↗

bioRxiv2025

C-terminal lysine residues localise NLRP10 at lipid compartments and govern NLRP10 oligomer formation

NLRP10 is an atypical member of the NOD-like receptor (NLR) family because it lacks a leucine-rich repeat domain at its C-terminus. Recently, NLRP10 has been shown to form an inflammasome, however, the mechanism and the trigger of activation remain elusive. Here, we show that in human epithelial cells and keratinocytes NLRP10 oligomerises in response to the stressor m-3M3FBS. NLRP10 co-localises with ASC upon overexpression, but ASC nucleation and recruitment were different to other NLRPs. While neither ATP hydrolysis nor the pyrin domain were required, the C-terminal tail region (aa 584-655) was both necessary and sufficient for oligomerisation. Generation of chimeric proteins showed that the function of the tail is conserved between human and mouse NLRP10 in respect to aggregation but convers different protein stability. Changes in the subcellular localisation of NLRP10 and oligomerization were dependent on the presence of evolutionarily conserved lysine residues in the tail region, which localise the majority of NLRP10 at lipid droplets. Our study identifies the C-terminal basic tail of NLRP10 as a key regulatory element for oligomerisation and localisation at lipid interfaces. These findings underline differences of NLRP10 activation in respect to other inflammasome forming NLRPs and suggest a role of lipids in NLRP10 activation.

immunology↗

bioRxiv2025

Focal adhesion pathway inhibition is the central axis of macrophage phenotypic responses to monoclonal antibody therapy in aggressive lymphoma via high-throughput screening and high-content imaging

High-grade B-cell lymphoma (HGBCL) frequently arises as a refractory or relapsed state of diffuse large B-cell lymphoma (DLBCL) and is associated with poor outcomes due to multi-drug resistance and hallmark oncogenic translocations. To identify novel therapeutic strategies, we developed a dual high-throughput screening (HTS) and high-content imaging (HCI) macrophage-tumour co-culture platform that quantifies antibody-dependent and antibody-independent cellular phagocytosis (ADCP/AICP) across a 1,241-compound library. Using GFP+ HGBCL cells and mCherry+ macrophages, we validated our methodology through time-resolved phenotypic profiling, Euclidean distance-based analysis, and hit compound prioritisation. Pathway interrogation revealed focal adhesion as a central hub of macrophage phenotypic modulation, highlighting focal adhesion kinase (FAK/PTK2) as a candidate therapeutic target. Pharmacological inhibition with PF-562271 enhanced phagocytic activity, altered macrophage morphology, and synergised with anti-CD20 monoclonal antibodies in vitro and ex vivo. In vivo, Rituximab plus PF-562271 significantly reduced lymphoma burden and prolonged survival in xenograft models. Collectively, our work demonstrates that HTS/HCI-driven phenotypic profiling of tumour-associated macrophages can uncover actionable therapeutic combinations and nominates FAK inhibition as a promising strategy to potentiate antibody immunotherapy in HGBCL.

cell biology↗

bioRxiv2025

ZOnally magnified Oblique Multi-slice (ZOOM) for cardiac diffusion weighted imaging in vivo

PurposeCardiac diffusion weighted imaging (cDWI) commonly utilizes 2D-selective RF-pulses (such as ZOOMit) to avoid aliasing artifacts when reducing the field of view (FOV). These RF-pulses, which may require parallel transmit capability, typically take several 10s of milliseconds, prolonging the echo time (TE) of the imaging sequence. Conversely, slice selection using ZOnally-magnified Oblique Multi-slice (ZOOM) (i.e. tilting the excitation relative to the refocusing RF-pulse) is an alternative for reducing FOV while shortening TE. We hypothesized that ZOOM, with appropriate choices for the RF-pulse tilt angle and thickness, can be reliably used as an alternative to ZOOMit for cardiac Diffusion Tensor Imaging (cDTI). MethodsWe scanned phantoms using full FOV and ZOOM with various parameters chosen from a 2-dimensional grid of angles and thicknesses. We identified a subset of 5 ZOOM parameters for in vivo cDTI experiments. We performed cDTI on healthy volunteers on a Siemens Prisma (6 subjects) and a Connectom (6 subjects) MR system using the 5 identified ZOOM settings, as well as ZOOMit (Prisma only) and no ZOOM (Connectom only). We compared mean diffusivity (MD), fractional anisotropy (FA), and secondary eigenvector angle (|E2A|) between scans. ResultsAverage MD, and standard deviation of MD and FA, were significantly reduced by ZOOM compared to no ZOOM on Connectom. We found no significant differences in any averaged diffusion measures between ZOOMit and ZOOM on Prisma. ConclusionCombining ZOOM with cardiac diffusion weighted imaging allows for TE to be considerably shortened (from 79ms to 67ms) while reducing FOV to avoid aliasing artifacts.

biophysics↗

bioRxiv2025

Prior cocaine use disrupts identification of hidden states by single units and neural ensembles in orbitofrontal cortex

The orbitofrontal cortex (OFC) is critical to identifying task structure and to generalizing appropriately across task states with similar underlying or hidden causes. This capability is at the heart of OFCs proposed role in a network responsible for cognitive mapping, and its loss can explain many deficits associated with OFC damage or inactivation. Substance use disorder is defined by behaviors that share much in common with these deficits, such as an inability to modify learned behaviors in the face of new information about undesired consequences. One explanation for this similarity would be if addictive drugs impacted the ability of OFC to recognize underlying similarities, hidden states, that allow information learned in one setting to be used in another. To explore this possibility, we trained rats to self-administer cocaine and then recorded single unit activity in lateral OFC as these rats performed in an odor sequence task consisting of unique and shared positions. In well-trained controls, we observed chance decoding of sequence at shared positions and near chance decoding even at unique positions, reflecting the irrelevance of distinguishing these positions in the task. By contrast, in cocaine-experienced rats, decoding remained significantly elevated, particularly at the positions that had superficial sensory differences that were collapsed in controls across learning. These neural differences were accompanied by increases in behavioral variability at these positions. A tensor component analysis showed that this effect of reduced generalization after cocaine use also extended across positions in the sequences. These results show that prior cocaine use disrupts the normal identification of hidden states by OFC.

neuroscience↗

bioRxiv2025

Proteomic and structural comparison between cilia from primary ciliary dyskinesia patients with a DNAH5 defect

Primary ciliary dyskinesia (PCD) is a genetic disorder that affects the motile cilia in various organs, leading to recurrent respiratory infections, subfertility, and laterality abnormalities. Traditional diagnostic methods include high-speed video microscopy, immunofluorescence staining, electron microscopy, and genetic screening. However, how different disease-causing variants in the same PCD gene affect clinical presentation, as well as ciliary composition and structure, is not well understood. We investigated the effect of various mutations in DNAH5, an axonemal dynein heavy chain, using mass spectrometry and cryo-electron tomography. We demonstrated differences in the axonemal composition for patients with dnah5 mutations. Furthermore, we showed that reductions in some ciliary components are patient specific. Some of them, KIAA1430, CFAP97, and DTHD1, were not previously recognized as components of human respiratory motile cilia. Finally, we demonstrated that some differences in protein abundance between wild-type and PCD samples can be observed in the 96-nm repeated unit of the axoneme. Our results suggest that many axonemal components are affected in the DNAH5-defective samples. Additionally, our results highlight that disparate mutations have seemingly distinctive consequences for the axonemal composition.

cell biology↗

bioRxiv2025

Robust constrained weighted least squares for in vivo human cardiac diffusion kurtosis imaging

SummaryRobust estimation with convexity constraints significantly improves signal fitting for in vivo human cardiac diffusion kurtosis imaging. PurposeCardiac diffusion tensor imaging (cDTI) is an emerging technique to investigate the microstructure of heart tissue. At sufficiently high b-values, additional information on microstructure can be observed, but the data require a representation beyond DTI such as diffusion kurtosis imaging (DKI). cDTI is highly prone to image corruption, which researchers usually attempt to handle with shot-rejection. However, this can be handled more generally with robust estimation techniques. Recent work has also demonstrated the need to perform constrained fitting for DKI, as fitted parameters can otherwise violate necessary constraints on the signal behaviour, causing significant errors in estimated measures. MethodsWe developed robust constrained weighted least squares (RCWLS) by combining robust estimation with convexity constraints specifically for DKI. Using in vivo cardiac DKI data from 11 healthy volunteers collected with a Connectom scanner, we tested various combinations of fitting techniques, with/without robustness and with/without constraints. ResultsRCWLS was the only tested technique that convincingly showed radial kurtosis to be larger than axial kurtosis for all subjects, something that is expected in myocardium due to increased restrictions to diffusion in the plane perpendicular to the primary myocyte direction. RCWLS also showed the best correction of corrupted regions in diffusion parameter maps for individual subjects. ConclusionFitting techniques utilizing both robust estimation and constraints are essential to facilitate applicability of in vivo cardiac DKI.

physiology↗

bioRxiv2025

Cardiac diffusion kurtosis imaging in the human heart in vivo using 300mT/m gradients

PurposeDiffusion tensor imaging (DTI) is commonly used in cardiac diffusion magnetic resonance imaging (dMRI). However, the tissues microstructure (cells, membranes, etc.) restricts the movement of the water molecules, making the spin displacements deviate from Gaussian behaviour. This effect may be observed with diffusion kurtosis imaging (DKI) using sufficiently high b-values (b > 450 s/mm2), which are presently outside the realm of routine cardiac dMRI due to the limited gradient strength of clinical scanners. The Connectom scanner with Gmax = 300 mT/m enables high b-values at echo times (TE) similar to DTI on standard clinical scanners, therefore facilitating cardiac DKI in humans. MethodsCardiac-gated, second-order motion-compensated dMRI was performed with bmax = 1350 s/mm2 in 10 healthy volunteers on a 3T MRI scanner with Gmax = 300 mT/m. The signal was fitted to a cumulant expansion up to and including the kurtosis term and diffusion metrics such as fractional anisotropy (FA), mean diffusivity (MD), mean kurtosis (MK), axial kurtosis (AK), and radial kurtosis (RK) were calculated. ResultsWe demonstrate deviation of the signal from monoexponential decay for b-values > 450 s/mm2 (MK = 0.32 {+/-} 0.03). Radial kurtosis (RK = 0.35 {+/-} 0.04) was observed slightly larger than axial kurtosis (AK = 0.27 {+/-} 0.02), and the difference is statistically significant (RK - AK = 0.08 {+/-} 0.04, p = 2e - 4). ConclusionThis work demonstrates the feasibility of quantifying kurtosis effect in the human heart in vivo (at an echo time shorter than typical TEs reported for cardiac DTI), using high-performance gradient systems (which are 4-8 times stronger than on standard clinical scanners). Our work lays the foundation for exploring new biomarkers in cardiac dMRI beyond DTI.

biophysics↗

bioRxiv2024

Sex-specific role of epigenetic modification of a leptin upstream enhancer in the adipose tissue

ObjectiveMaternal hormonal status can have long-term effects on offspring metabolic health, likely regulated via epigenetic mechanisms. We elucidated the effect of maternal thyroid hormones on epigenetic regulation of Leptin (Lep) transcription in adipose tissue (AT) and investigated the role of DNA methylation at a Lep upstream enhancer (UE) in adipocyte biology. MethodsPregnant mice were treated with Triiodothyronine (T3). Offspring body composition and adipose tissue were analysed at 6 months (treatment: N = 8, control: N = 12). DNA methylation at the Lep UE and Lep mRNA levels in gonadal white adipose tissue (gWAT) and leptin serum levels were measured. The Lep UE was hypomethylated in murine preadipocytes using a dCas9-SunTag-TET1 system, followed by differentiation. In human visceral AT samples (N = 52) from the Leipzig Obesity BioBank (LOBB) LEP UE methylation and LEP mRNA levels were assessed alongside leptin serum levels and correlated with body fat percentage. Causal relationships were explored using mediation analysis. ResultsHigh maternal T3 levels reduced offspring body weight, total fat, and gWAT mass. Female offspring of T3-treated mothers showed lower Lep mRNA levels and higher Lep UE methylation compared to controls. CRISPR/dCas9 editing reduced Lep UE methylation by [~]20% in preadipocytes without altering Lep expression or lipid accumulation after differentiation. In humans, a mediation of sex-specific difference in body fat percentage via Lep UE methylation was found. ConclusionMaternal thyroid hormones might affect offspring gWAT Lep expression in a sex-specific manner, potentially driven by DNA methylation at the Lep UE, influencing body fat percentage.

genetics↗

bioRxiv2024

Orthosteric STING inhibition elucidates molecular correction of SAVI STING

STING is broadly implicated in diseases ranging from cancer, autoimmune disease, neurodegeneration to rare, monogenic diseases.1 Early drug discovery campaigns focused on STING activation as a promising platform for cancer immunotherapy yet failed in multiple clinical trials due to lack of efficacy thus far.2 Current research and development activities concentrate on STING inhibition for treating autoimmune disease and neuroinflammation. While the progression of STING activators into the clinic has been successful, the discovery and clinical progression of STING inhibitors remain elusive. Questions persist about the molecular properties needed to distinguish between a STING activator and inhibitor, particularly within SAVI disease, a monogenic autoinflammatory disease that renders STING constitutively active.3 Here we leverage an orthosteric STING activator and inhibitor from the same chemical series to discover that STING M271 is a critical residue for molecular activation. The M271CH3 NMR chemical shifts reveal a unique molecular signature for pharmacological or genetically driven activation and inhibition that is not captured by x-ray crystallography. Additionally, M271 directly interacts with the most common SAVI mutation, V155M, and using an orthosteric STING inhibitor, we show partial rescue and molecular correction of STING V155M. Finally, these data present insights into therapeutic STING molecular correction for treating SAVI patients. Our results elucidate an unappreciated structural interaction critical for STING modulation that could be utilized as a molecular diagnostic tool for drug discovery. Furthermore, we demonstrate for the first time how the therapeutic requirements of a molecular corrector differ from an orthosteric STING inhibitor, and why this is important for the SAVI disease population.

molecular biology↗

bioRxiv2024

Prior cocaine use diminishes encoding of latent information by orbitofrontal, but not medial, prefrontal ensembles

Maladaptive decision-making is a hallmark of substance use disorders, though how drugs of abuse alter neural representations supporting adaptive behavior remains poorly understood. Past studies show the orbitofrontal (OFC) and prelimbic (PL) cortices are important for decision making, tracking both task-relevant and latent information. However, previous studies have focused on how drugs of abuse impact the firing rates of individual units. More work at the ensemble level is necessary to accurately characterize potential drug-induced changes. Using single-unit recordings in rats during a multidimensional decision-making task and then applying population and ensemble level analyses, we show that prior use of cocaine altered the strength and structure of task-relevant and latent representations in the OFC, changes relatable to suboptimal decision making in this and perhaps other settings. These data expand our understanding of the neuropathological underpinnings of maladaptive decision-making in SUDs, potentially enabling enhanced future treatment strategies.

neuroscience↗

bioRxiv2022

MMR vaccination induces a trained immunity program characterized by functional and metabolic reprogramming of γδ T cells

The measles, mumps and rubella (MMR) vaccine protects against all-cause mortality in children, but the immunological mechanisms mediating these effects are poorly known. We systematically investigated whether MMR can induce long-term functional changes in innate immune cells, a process termed trained immunity, that could at least partially mediate this heterologous protection. In a randomized placebo-controlled trial, 39 healthy adults received either the MMR vaccine or a placebo. By using single-cell RNA-sequencing, we found that MMR caused transcriptomic changes in CD14-positive monocytes and NK cells, but most profoundly in {gamma}{delta} T cells. Surprisingly, monocyte function was not altered by MMR vaccination. In contrast, the function of {gamma}{delta} T cells was significantly enhanced by MMR vaccination, with higher production of TNF and IFN{gamma}, as well as upregulation of cellular metabolic pathways. In conclusion, we describe a new trained immunity program characterized by modulation of {gamma}{delta} T cell function induced by MMR vaccination. One-sentence summaryMMR vaccination induces cellular and metabolic reprogramming in {gamma}{delta} T cells towards a more active phenotype.

immunology↗

bioRxiv2022

A Vaccinia-based system for directed evolution of GPCRs in mammalian cells

Low stability and poor expression levels are inherent in many G protein-coupled receptors, hindering structural and biophysical analyses. Directed evolution in bacterial or yeast display systems has been successfully used to overcome both limitations in some cases. Yet, some receptors cannot be tackled in microbial systems, due to their complex molecular composition or due to unfavorable ligand properties. Here, we report an approach to evolve G protein-coupled receptors in mammalian cells. To achieve clonality of individual mutants, and a rather uniform gene copy number, both prerequisites for strict genotype-phenotype linkage in mammalian cells, we developed a highly efficient viral transduction system based on Vaccinia virus. Using rational design of synthetic DNA libraries, we first evolved neurotensin receptor 1 for high stability and expression, reaching levels similar or higher to what has been achieved in bacterial systems. Second, using parathyroid hormone 1 receptor we demonstrate that also receptors with complex molecular architectures and large ligands can be readily evolved in mammalian cells. Furthermore, in our system, the physiological signaling environment in mammalian cells can be employed to evolve functional receptor properties. We obtained fully functional receptor variants exhibiting increased allosteric coupling between the ligand binding site and the G protein interface, resulting in higher signaling efficacy. Thus, our approach provides new means to readily improve the biophysical properties of receptors in a mammalian cellular environment. Moreover, it opens the possibility to modulate receptor signaling and to gain further insights into the intricate molecular interplay required for GPCR activation.

biochemistry↗

bioRxiv2022

The Cilialyzer - a freely available open-source software for a standardised identification of impaired mucociliary activity facilitating the diagnostic testing for PCD

Background and ObjectivePrimary ciliary dyskinesia (PCD) is a rare genetic disorder causing a defective ciliary structure, which predominantly leads to an impaired mucociliary clearance and associated airway disease. As there is currently no single diagnostic gold standard test, PCD is diagnosed by a combination of several methods comprising genetic testing and the examination of the ciliary structure and function. Among the approved diagnostic methods, only high-speed video microscopy (HSVM) allows to directly observe the ciliary motion and therefore, to directly assess ciliary function. In the present work, we present our recently developed freely available open-source software - termed "Cilialyzer", which has been specifically designed to support and facilitate the analysis of the mucociliary activity in respiratory epithelial cells captured by high-speed video microscopy. MethodsIn its current state, the Cilialyzer software enables clinical PCD diagnosticians to load, preprocess and replay the image sequences with a feature-rich replaying module facilitating the commonly performed qualitative visual assessment of ciliary function. The image processing methods made accessible through an intuitive user interface allow clinical specialists to comfortably compute the ciliary beating frequency (CBF), the activity map and the "frequency correlation length" - an observable getting newly introduced. Furthermore, the Cilialyzer contains a simple-to-use particle tracking interface to determine the mucociliary transport speed. ResultsThe Cilialyzer is fully written in the Python programming language and freely available under the terms of the MIT license. The proper functioning of the computational analysis methods constituting the Cilialyzer software is demonstrated by using simulated and representative sample data from clinical practice. ConclusionsThe Cilialyzer serves as a useful clinical tool for standardised PCD diagnostics and provides new quantitative information awaiting to be clinically evaluated using cohorts of PCD. As the Cilialyzer is freely available under the terms of a permissive open-source license, it serves as a ground frame for further development of computational methods aiming at the quantification and automation of the analysis of mucociliary activity captured by HSVM.

biophysics↗

bioRxiv2022

Angelman syndrome patient neuron screen identifies a potent and selective clinical ASO targeting UBE3A-ATS with long lasting effect in cynomolgus monkey

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of neuronal E3 ligase UBE3A with no available treatment. Restoring UBE3A levels via downregulation of the paternally cis-acting long non-coding antisense transcript (UBE3A-ATS) is a potential disease modifying. Developing molecules targeting human UBE3A-ATS is challenging because it is expressed only in neurons and lacks animal species sequence conservation. To overcome this, we performed a library screen of locked-nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) on AS patient-derived neurons, identifying initial sequences. Further optimization led to the identification of the ASO, RO7248824, which selectively and potently reduces UBE3A-ATS, while concomitantly upregulating the UBE3A mRNA and protein. These properties held true in both human AS patient- and neurotypical-, as well as cynomolgus monkey-derived neurons. In vivo use of tool molecules in wild-type (WT) and AS Ube3am-/p+ mice, revealed a steep relationship between UBE3A-ATS knock-down and UBE3A mRNA/protein upregulation, whereby an almost 90% downregulation was needed to achieve a 50% upregulation, respectively. This relationship was confirmed in cynomolgus monkeys. Whereby, repeated lumbar intrathecal administrations of RO7248824 was well tolerated without adverse in-life effects or tissue pathology and produced a robust, long lasting (up to 3 months) paternal reactivation of UBE3A mRNA/protein across key monkey brain regions. Our results demonstrate that AS human pluripotent stem cell neurons serve as an excellent translational tool and furthermore LNA-modified ASOs exhibit excellent drug-like properties. Sustained efficacy translated to infrequent, intrathecal dosing and serves as the basis for the ongoing clinical development of RO7248824 for AS. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/495066v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1c2a97eorg.highwire.dtl.DTLVardef@1059954org.highwire.dtl.DTLVardef@95835aorg.highwire.dtl.DTLVardef@1e2adea_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO From AS patient blood to a neuronal screen, identifies clinical ASO with excellent in vivo properties. (1) Patients were recruited. (2) Whereby blood was reprogrammed into hIPSC and subsequently differentiated into neurons. (3) ASOs were designed and screened on human neurons to downregulate the UBE3A-ATS likely via directed RNase H Cleavage of Nascent Transcripts. (4a) RO7248824 was identified that potently and selective reduces UBE3A-ATS, concomitantly with upregulating the UBE3A sense transcript and protein which was used for in vitro pk/pd. (4b) In parallel tool murine ASO were used demonstrate in vivo POC.(5) Pivotal nonhuman primate studies to monitor safety and predict the human dose. (6) RO7248824 is in AS clinical trial. C_FIG One Sentence SummaryFrom angelman syndrome human neuron screen to cynomolgus monkey proof of concept identifies the clinical molecule RO7248824

neuroscience↗