Zen Bio (United States)

IntroductionGeneralized myasthenia gravis (gMG) is a rare, chronic, autoimmune disorder with a substantial disease burden. Oral glucocorticoids (GCs) are one of the common early-line treatments used globally, but there are growing concerns regarding risks associated with GC adverse effects, which can have serious medical consequences. In Japan, an important gMG treatment goal is minimal disease manifestation with a low target GC dose (≤5 mg/day) to minimize GC exposure. This study aimed to assess gMG treatment patterns in Japan, with a focus on GC exposure and incident comorbidities associated with long-term GC use.MethodsThis was a retrospective, observational database study of patient data from the Medical Data Vision (MDV) database (2008–2022) in Japan. Adults (aged ≥18 years) with ≥2 gMG diagnosis records were considered. First gMG diagnosis (ocular MG excluded) between 2018 and 2021 was considered the index date. All available follow-up was considered if the patient had ≥1 activity in the database annually after the index date.ResultsOf 9,687 patients with gMG (mean age: 65 years; 56% female; follow-up period: 3.2 years) included in the study, 3,696 (38.2%) were newly diagnosed (2018 onwards) and 5,991 (61.8%) were previously diagnosed (before 2018). Acetylcholinesterase inhibitor (AChEi) and GC monotherapy were the most common initial therapies after index, and most patients were treated with combinations of AChEi, GC, and/or nonsteroidal immunosuppressive therapies (NSISTs). The average daily GC dose was 8.5 mg/day (newly diagnosed: 10.6 mg/day; previously diagnosed: 7.5 mg/day). The target daily dose of 5 mg/day was exceeded by 70% of GC-treated patients, and 26% exceeded 10 mg/day (newly diagnosed: 35%; previously diagnosed: 21%). Compared to patients with no GC exposure, GC use was dose-dependently associated with osteoporosis, thrombosis, diabetes, and hyperlipidemia/hypercholesterolemia, even at GC doses ≤5 mg/day.ConclusionMost patients were treated with AChEi, GC, or NSIST monotherapy or combination therapies and received GCs exceeding 5 mg/day, which was associated with developing several GC-associated comorbidities in a dose-dependent manner. To achieve treatment goals, patients with gMG may benefit from additional treatment approaches to reduce GC usage.

Nav1.1 voltage-gated sodium (Nav) channels encoded by the SCN1A gene are critical to high frequency action potential generation in inhibitory interneurons and so play a crucial role in maintaining the excitatory-inhibitory balance in the brain. Rare, often loss-of-function, mutations in the SCN1A gene can lead to a spectrum of developmental and epileptic encephalopathies including Dravet syndrome with common SCN1A variants being risk factors for epilepsy, highlighting the potential for Nav1.1 as a therapeutic target for seizure disorders. Here, we describe a potent small molecule sodium channel potentiator ICA00600232 that exhibits >1000-fold selectivity for human Nav1.1 over all other Nav1.x family members except Nav1.3. Nav1.1 potentiation by ICA00600232 results from a slowing of channel inactivation. Employing both chimeras and single point mutations, we demonstrate that ICA00600232 interacts with the domain 4 voltage sensor region of Nav1.1. Three amino acid residues within the "extracellular" facing regions of the S2 and S3 transmembrane segments are major determinants of Nav1.1 potentiation and subtype selectivity. These same residues are also important for subtype selective inhibitor interactions with Nav1.1, albeit in a different structural conformation. Furthermore, we demonstrate that ICA00600232 at concentrations as low as 10 nM enhances firing frequency in parvalbumin positive fast-spiking interneurons recorded in brain slices from scn1a+/- mice. The current study shows that a small molecule Nav1.1 selective potentiator targeting the fourth voltage sensor can slow inactivation of Nav1.1, increase excitability of parvalbumin positive interneurons and inhibitory tone that could be beneficial in treating developmental and epileptic encephalopathies and other seizure disorders. SIGNIFICANCE STATEMENT: Loss-of-function mutations of Nav1.1 sodium channel genes in human inhibitory interneurons are associated with a spectrum of epilepsies including developmental and epileptic encephalopathies. Potential treatments include enhancement of Nav1.1 activity via administration of small molecule potentiating agents. This study describes a potent subtype selective Nav1.1 channel potentiator that interacts with a region that controls voltage sensitivity and enhances action potential firing in inhibitory interneurons, supporting its potential utility in treating developmental and epileptic encephalopathy associated seizure disorders.

Methylmalonic acidemia (MMA) is a severe metabolic disorder affecting multiple organs because of a distal block in branched-chain amino acid (BCAA) catabolism. Standard of care is limited to protein restriction and supportive care during metabolic decompensation. Severe cases require liver/kidney transplantation, and there is a clear need for better therapy. Here, we investigated the effects of a small molecule branched-chain amino acid transaminase (BCAT) inhibitor in human MMA hepatocytes and an MMA mouse model. Mitochondrial BCAT is the first step in BCAA catabolism, and reduction of flux through an early enzymatic step is successfully used in other amino acid metabolic disorders. Metabolic flux analyses confirmed robust BCAT inhibition, with reduction of labeling of proximal and distal BCAA-derived metabolites in MMA hepatocytes. In vivo experiments verified the BCAT inhibition, but total levels of distal BCAA catabolite disease markers and clinical symptoms were not normalized, indicating contributions of substrates other than BCAA to these distal metabolite pools. Our study demonstrates the importance of understanding the underlying pathology of metabolic disorders for identification of therapeutic targets and the use of multiple, complementary models to evaluate them.

Oral bioavailability is a crucial pharmacokinetic (PK) parameter optimized by drug developers working on new therapeutic compounds. The goal is to obtain adequate systemic exposure and to establish safe and effective therapeutic dosages. Critical to optimizing oral bioavailability is understanding a compound’s susceptibility to first-pass metabolism, determined by the intricate relationship between gut absorption, metabolism, and hepatic metabolism. Efforts to model this relationship in vitro have led to the emergence of microphysiological systems (MPSs) that consist of multiple, fluidically linked organs. Here, we describe an MPS that links the gut and liver, capable of simulating both oral and intravenous dosing routes and is made up of entirely primary human cells: a gut barrier tissue comprised of an intestinal epithelial monolayer derived from the human jejunum, and a liver microtissue, derived from primary human hepatocytes. Functionality of gut and liver tissues is maintained in coculture enabling the PK investigation of oral compounds. We combine the primary Gut/Liver MPS with a mechanistic mathematical model to generate organ-specific PK parameters and estimate human oral bioavailability and its components: the fraction absorbed (Fa), the fraction escaping gut wall elimination (Fg), and the fraction escaping hepatic elimination (Fh). We used the CYP3A-mediated compound midazolam, which is subjected to both intestinal and hepatic extraction, to demonstrate the transformative potential of the primary Gut/Liver MPS to mechanistically model the PKs of oral compounds in vitro.Significance StatementBioavailability underpins the success or when insufficient, can stall the development of oral therapeutics. The drug discovery process lacks in vitro assays that can profile the contribution to bioavailability by the gut and liver. The primary Gut/Liver MPS outlined in this study bridges this gap.

The USEPA issued the "Framework for Metal Risk Assessment" in 2007, recognizing that human and environmental exposure to metals and metalloids (MMEs) poses challenges risk assessment. Inhalation of aerosols containing MMEs is a primary pathway for exposure in the occupational setting, for consumer exposure, and to general population exposure associated with point-source emissions or ambient sources. The impacts of inhalation can be at the point of deposition (local exposure) or may manifest after uptake into the body (systemic exposure). Both local and systemic exposure can vary with factors that determine the regional deposition of MME-containing aerosols. Aerosol characteristics such as particle size combine with species-specific characteristics of airway morphology and lung function to modulate the deposition and clearance of MME particulates. In contrast to oral exposure, often monitored by measuring MME levels in blood or urine, inhalation exposure can produce local pulmonary impacts in the absence of significant systemic distribution. Exposure assessment for nutritionally essential MMEs can be further complicated by homeostatic controls that regulate systemic MME levels. Predictions of local exposure can be facilitated by computer models that estimate regional patterns of aerosol deposition, permitting calculation of exposure intensity in different regions of the respiratory tract. The utility of deposition modeling has been demonstrated in assessments of nutritionally essential MMEs regulated by homeostatic controls and in the comparison of results from inhalation studies in experimental animals. This facilitates extrapolation from animal data to humans and comparisons of exposures possessing mechanistic linkages to pulmonary toxicity and carcinogenesis. Pulmonary deposition models have significantly advanced and have been applied by USEPA in evaluations of particulate matter. However, regional deposition modeling has yet to be incorporated into the general guidance offered by the agency for evaluating inhalation exposure. Integr Environ Assess Manag 2024;20:952-964. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Environmental health research in the US has shown that racial and ethnic minorities and members of low-socioeconomic groups, are disproportionately burdened by harmful environmental exposures, in their homes, workplace, and neighborhood environments that impact their overall health and well-being. Systemic racism is a fundamental cause of these disproportionate exposures and associated health effects. To invigorate and inform current efforts on environmental justice and to raise awareness of environmental racism, the National Institute of Environmental Health Sciences (NIEHS) hosted a workshop where community leaders, academic researchers, and NIEHS staff shared perspectives and discussed ways to inform future work to address health disparities. To share best practices learned and experienced in partnerships between academic researchers and communities that are addressing environmental racism across the US; and to outline critical needs and future actions for NIEHS, other federal agencies, and anyone who is interested in conducting or funding research that addresses environmental racism and advances health equity for all communities. Through this workshop with community leaders and researchers funded by NIEHS, we learned that partnerships between academics and communities hold great promise for addressing environmental racism; however, there are still profound obstacles. To overcome these barriers, translation of research into plain language and health-protective interventions is needed. Structural changes are also needed in current funding mechanisms and training programs across federal agencies. We also learned the importance of leveraging advances in technology to develop creative solutions that can protect public health.

Abstract Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 minutes of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT).Graphical Abstract

Genomics Quality Assessment has provided external quality assessments (EQAs) for preimplantation genetic testing (PGT) for 12years for eight monogenic diseases to identify sub-optimal PGT strategies, testing and reporting of results, which can be shared with the genomics community to aid optimised standards of PGT services for couples. The EQAs were provided in two stages to mimic end-to-end protocols. Stage 1 involved DNA feasibility testing of a couple undergoing PGT and affected proband. Participants were required to report genotyping results and outline their embryo testing strategy. Lymphoblasts were distributed for mock embryo testing for stage 2. Submitted clinical reports and haplotyping results were assessed against peer-ratified criteria. Performance was monitored to identify poor performance. The most common testing methodology was short tandem repeat linkage analysis (59%); however, the adoption of single nucleotide polymorphism-based platforms was observed and a move from blastomere to trophectoderm testing. There was a variation in testing strategies, assigning marker informativity and understanding test limitations, some clinically unsafe. Critical errors were reported for genotyping and interpretation. EQA provides an overview of the standard of preimplantation genetic testing-M clinical testing and identifies areas of improvement for accurate detection of high-risk embryos.

Abstract Primary template-directed amplification (PTA) is an improved amplification technique for single-cell DNA sequencing. We generated whole-genome analysis of 76 single neurons and developed SCAN2, a computational method to accurately identify both clonal and non-clonal somatic (i.e., limited to a single neuron) single nucleotide variants (SNVs) and small insertions and deletions (indels) using PTA data. Our analysis confirms an increase in non-clonal somatic mutation in single neurons with age, but revises estimates for the rate of this accumulation to be 15 SNVs per year. We also identify artifacts in other amplification methods. Most importantly, we show that somatic indels also increase by at least 2 indels per year per neuron and that indels may have a larger impact on gene function than somatic SNVs in human neurons.

The National Institute of Environmental Health Sciences Superfund Research Program (SRP) funds diverse transdisciplinary research to understand how hazardous substances contribute to disease. SRP research focuses on how to prevent these exposures by promoting problem-based, solution-oriented research. SRP's mandate areas encompasses broad biomedical and environmental science and engineering research efforts and, when combined with research translation, community engagement, training, and data science, offers broad expertise and unique perspectives directed at a specific big picture question. The purpose of this commentary is to adapt a systems approach concept to SRP research to accommodate the complexity of a scientific problem. The SRP believes a systems approach offers a framework to understand how scientists can work together to integrate diverse fields of research to prevent or understand environmentally-influenced human disease by addressing specific questions that are part of a larger perspective. Specifically, within the context of the SRP, a systems approach can elucidate the complex interactions between factors that contribute to or protect against environmental insults. Leveraging a systems approach can continue to advance SRP science while building the foundation for researchers to address difficult emerging environmental health problems.