Novo Nordisk (Germany)

An integrated upstream process was developed for the high-level production of a low-immunogenic L-asparaginase mutant (EcaII P40S/S206C) in Escherichia coli, combining high-cell-density fed-batch cultivation with controlled induction and post-induction nutrient feeding. Biomass accumulation was achieved by exponential substrate feeding, followed by pH-stat-based nutrient supply during the expression phase. Induction protocols were optimized using biomass-specific IPTG dosing (µmol IPTG/gcell), applied either as a pulse or via exponential feeding. This combined strategy resulted in a maximum volumetric enzyme activity of 134,516 U/L and a specific productivity of 3387 U/gcell, corresponding to 91-fold and 24-fold increases, respectively, compared with standard batch cultivation. Overall, these results indicate that the coordinated control of feeding strategy and induction conditions substantially improves both volumetric and cell-specific productivity of recombinant L-asparaginase and provides a robust upstream framework for the efficient manufacture of improved enzyme variants.

ABSTRACTWell-characterised mouse models of disease may provide valuable insights into pathophysiology. This study characterises the Townes mouse model of sickle cell disease (SCD) and establishes a time window in which the disease is present but does not progress significantly in terms of severity. We examined Townes mice with the HbAA, HbAS, and HbSS genotypes from young (4 weeks) to mature (5 months) stages of life to assess the disease state at different ages and any progression. We conducted blood tests, histological organ damage evaluations, and metabolic assessments to identify a suitable time frame for study based on welfare considerations. Townes HbSS mice displayed key SCD features such as anaemia, haemolysis, thromboinflammation and organ pathology. Notably, these manifestations remained relatively stable over the study period, indicating a stable phase suitable for conducting intervention studies. Mice with HbAS and HbAA genotypes served as comparative controls, showing minimal to no pathology throughout. These findings are valuable for future research on SCD and may ultimately lead to the development of more effective treatments for this debilitating disease.

Type 2 diabetes mellitus (T2D) represents a major public health challenge with significant effects on morbidity and mortality. Clinical guidelines provide treatment recommendations, but there is limited understanding of patients' preferences. This study aimed to elicit preferences for second-line drug treatments for T2D. A Discrete Choice Experiment with a partial-profile design was conducted from August to November 2023, involving German patients with experience in either monotherapy or second-line drug treatment. Participants completed 12 choice tasks, each presenting three alternatives described by attributes: risk of myocardial infarction, risk of stroke, risk of nerve damage, risk of nausea, risk of severe hypoglycemia, weight change, type and frequency of intake, and schedule of intake. Statistical analyses employed the Conditional Logit and Random Parameter Logit models to assess main effects and heterogeneity. The study encompassed 583 adult individuals with T2D, evenly divided between the two populations. Key factors influencing choice decisions included risk of nausea, risk of nerve damage, and weight change, with weekly type and frequency of intake risk of myocardial infarction followed. Less impactful but still relevant were risks of stroke, and severe hypoglycemia, while the intake schedule was least significant. Analysis of BMI categories revealed distinct preferences, particularly in weight change, with significant heterogeneity observed among respondents. This study highlights the importance of incorporating patient preferences into clinical decision-making. By quantifying preferences for second-line drug treatments, the study underscores the need for low-risk options that also consider weight change and intake conditions, aligning with the German National Health Care Guideline for T2D objectives for shared decision-making and treatment adherence. Recognizing individual sensitivities to risks and benefits is crucial for tailoring effective T2D treatment strategies. The study bridges clinical findings with patient perspectives, offering valuable insights into clinical practice, consideration for HTA processes, and design of clinical studies.

Establishing efficient cell factories involves a continuous process of trial and error due to the intricate nature of metabolism. This complexity makes predicting effective engineering targets a challenging task. Therefore, it is vital to learn from the accumulated successes of previous designs for advancing future cell factory development. In this study, we developed a method based on large language models (LLMs) to extract metabolic engineering strategies from research articles on a large scale. We created a database containing over 29006 metabolic engineering entries, 1210 products and 751 organisms. Using this extracted data, we trained a hybrid model combining deep learning and mechanistic approaches to predict engineering targets. Our model outperformed traditional metabolic engineering target prediction algorithms, excelled in predicting the effects of gene modifications, and generalized well to out-of-distribution products and multiple gene combinations. Our study provides a valuable dataset, a chatbot, and an engineering target prediction model for the metabolic engineering field and exemplifies an efficient method for leveraging existing knowledge for future predictions.

Abstract Atherosclerosis is a pervasive contributor to cardiovascular diseases including ischemic heart disease and stroke. Despite the advance and success of effective lipid lowering-therapies and hypertensive agents, the residual risk of an atherosclerotic event remains high and improving disease understanding and development of novel therapeutic strategies has proven to be challenging. This is largely due to the complexity of atherosclerosis with a spatial interplay of multiple cell types within the vascular wall. Here, we generated an integrative high-resolution map of human atherosclerotic plaques by combining single-cell RNA-seq from multiple studies and novel spatial transcriptomics data from 12 human specimens to gain insights into disease mechanisms. Comparative analyses revealed cell-type and atherosclerosis-specific expression changes and associated alterations in cell-cell communication. We highlight the possible recruitment of lymphocytes via different endothelial cells of the vasa vasorum, the migration of vascular smooth muscle cells towards the lumen to become fibromyocytes, and cell-cell communication in the plaque, indicating an intricate cellular interplay within the adventitia and the subendothelial space in human atherosclerosis.

<h3>Objective:</h3> We aimed to investigate the molecular effects of semaglutide on lipopolysaccharide (LPS) induced hippocampal neuroinflammation in mice and further to compare human gene expression signatures in Alzheimer’s disease (AD) brain tissue with those induced by LPS. <h3>Background:</h3> Neuroinflammation is part of the pathophysiology in AD. AD neuroimaging studies highlight increased inflammation, while genome wide association studies indicate that many AD-associated genes are expressed in glial cells. The glucagon-like peptide-1 receptor agonist semaglutide is currently being investigated in two phase 3a randomized, placebo-controlled trials in people with early Alzheimer’s disease (evoke/evoke+). <h3>Design/Methods:</h3> Mice were treated daily with subcutaneous semaglutide (30nmol/kg) or vehicle for 28 days, and LPS or vehicle was administered on days 15–17. RNA-sequencing and immunohistochemistry were performed to assess transcriptional and morphological changes in the hippocampus at Day 19 and Day 28 (Day 2 and 11 post-LPS, respectively). RNA-sequencing analyses included differentially expressed gene analysis, weighted gene co-regulated network analysis, pathway analysis and gene set enrichment analysis on published human AD datasets. <h3>Results:</h3> Semaglutide treatment significantly decreased the area of ionized calcium binding adaptor molecule 1 (Iba1) positive microglia (quantitative assessment) and attenuated co-expressed inflammatory genes vs vehicle in LPS-treated mice (p&lt;0.01) on Day 28. Pathway analysis was consistent with an overall dampening of inflammatory processes in mice treated with LPS and semaglutide. The mouse LPS model mirrored the transcriptional signatures identified in people with AD in data of human hippocampal subtype transcriptional signatures, suggesting that semaglutide could potentially alleviate homologous inflammatory molecular processes in humans. <h3>Conclusions:</h3> In an LPS-induced neuroinflammation mouse model, semaglutide reduced hippocampal neuroinflammation as measured by transcriptional changes and microglial area (Iba1). This could represent a novel mechanism whereby semaglutide may affect neuronal integrity and function, and thereby AD pathophysiology. <b>Disclosure:</b> Dr. Ludwig has nothing to disclose. Dr. Rausch has nothing to disclose. Dr. Secher has received personal compensation for serving as an employee of Novo Nordisk. An immediate family member of Dr. Secher has received personal compensation for serving as an employee of Novo Nordisk. Dr. Secher has stock in Novo Nordisk. Dr. Bentsen has received personal compensation for serving as an employee of Novo Nordisk. An immediate family member of Dr. Bentsen has stock in Novo Nordisk. Dr. Hansen has received personal compensation for serving as an employee of Neumirna Therapeutics. Dr. Hansen has received intellectual property interests from a discovery or technology relating to health care. Dr. Bjerregaard has received personal compensation for serving as an employee of Novo Nordisk A/S. Dr. Niss has nothing to disclose. Kristoffer L Egerod has nothing to disclose. Dr. Hansen has received personal compensation for serving as an employee of Novo Nordisk A/S. Dr. Hansen has stock in Novo Nordisk A/S. Dr. Dalgaard has received personal compensation for serving as an employee of Novo Nordisk. Dr. Merkestein has received personal compensation for serving as an employee of Novo Nordisk A/S. Dorte Holst has nothing to disclose. Charles Pyke has nothing to disclose. Mrs. Wichern has received personal compensation for serving as an employee of Gubra ApS. Dr. Polex-Wolf has received personal compensation for serving as an employee of Novo Nordisk A/S. Tune H Pers has nothing to disclose. Lotte B Knudsen has nothing to disclose.