Abstract: Background: Extended T-cell culture periods in vitro deplete the CAR-T final product of naive and stem cell memory T-cell (T scm) subpopulations that are associated with improved antitumor efficacy. YTB323 is an autologous CD19-directed CAR-T cell therapy with dramatically simplified manufacturing, which eliminates complexities such as long culture periods. This improved T-Charge™ process preserves T-cell stemness, an important characteristic closely tied to therapeutic potential, which leads to enhanced expansion ability and greater antitumor activity of CAR-T cells. Methods: The new T-Charge TM manufacturing platform, which reduces ex vivo culture time to about 24 hours and takes & lt;2 days to manufacture the final product, was evaluated in a preclinical setting. T cells were enriched from healthy donor leukapheresis, followed by activation and transduction with a lentiviral vector encoding for the same CAR used for tisagenlecleucel. After ≈24 hours of culture, cells were harvested, washed, and formulated (YTB323). In parallel, CAR-T cells (CTL*019) were generated using a traditional ex vivo expansion CAR-T manufacturing protocol (TM process) from the same healthy donor T cells and identical lentiviral vector. Post manufacturing, CAR-T products were assessed in T-cell functional assays in vitro and in vivo, in immunodeficient NSG mice (NOD-scid IL2Rg-null) inoculated with a pre-B-ALL cell line (NALM6) or a DLBCL cell line (TMD-8) to evaluate antitumor activity and CAR-T expansion. Initial data from the dose escalation portion of the Phase 1 study will be reported separately. Results: YTB323 CAR-T products, generated via this novel expansionless manufacturing process, retained the immunophenotype of the input leukapheresis; specifically, naive/T scm cells (CD45RO -/CCR7 +) were retained as shown by flow cytometry. In contrast, the TM process with ex vivo expansion generated a final product consisting mainly of central memory T cells (T cm) (CD45RO +/CCR7 +) (Fig A). Further evidence to support the preservation of the initial phenotype is illustrated by bulk and single-cell RNA sequencing experiments, comparing leukapheresis and final products from CAR-Ts generated using the T-Charge™ and TM protocols. YTB323 CAR-T cell potency was assessed in vitro using a cytokine secretion assay and a tumor repeat stimulation assay, designed to test the persistence and exhaustion of the cell product. YTB323 T cells exhibited 10- to 17-fold higher levels of IL-2 and IFN-γ secretion upon CD19-specific activation compared with CTL*019. Moreover, YTB323 cells were able to control the tumor at a 30-fold lower Effector:Tumor cell ratio and for a minimum of 7 more stimulations in the repeat stimulation assay. Both assays clearly demonstrated enhanced potency of the YTB323 CAR-T cells in vitro. The ultimate preclinical assessment of the YTB323 cell potency was through comparison with CTL*019 regarding in vivo expansion and antitumor efficacy against B-cell tumors in immunodeficient NSG mouse models at multiple doses. Expansion of CD3+/CAR+ T-cells in blood was analyzed weekly by flow cytometry for up to 4 weeks postinfusion. Dose-dependent expansion (C max and AUC 0-21d) was observed for both YTB323 and CTL*019. C max was ≈40-times higher and AUC 0-21d was ≈33-times higher for YTB323 compared with CTL*019 across multiple doses. Delayed peak expansion (T max) of YTB323 by at least 1 week compared with CTL*019 was observed, supporting that increased expansion was driven by the less differentiated T-cell phenotype of YTB323. YTB323 controlled NALM6 B-ALL tumor growth at a lower dose of 0.1×10 6 CAR+ cells compared to 0.5×10 6 CAR+ cells required for CTL*019 (Fig B). In the DLBCL model TMD-8, only YTB323 was able to control the tumors while CTL*019 led to tumor progression at the respective dose groups. This ability of YTB323 cells to control the tumor at lower doses confirms their robustness and potency. Conclusions: The novel manufacturing platform T-Charge™ used for YTB323 is simplified, shortened, and expansionless. It thereby preserves T-cell stemness, associated with improved in vivo CAR-T expansion and antitumor efficacy. Compared to approved CAR-T therapies, YTB323 has the potential to achieve higher clinical efficacy at its respective lower doses. T-Charge™ is aiming to substantially revolutionize CAR-T manufacturing, with concomitant higher likelihood of long-term deep responses. Figure 1 Figure 1. Disclosures Engels: Novartis: Current Employment, Current equity holder in publicly-traded company. Zhu: Novartis: Current Employment, Current equity holder in publicly-traded company. Yang: Novartis: Current Employment, Patents & Royalties. Price: Novartis: Current Employment. Sohoni: Novartis: Current Employment. Stein: Novartis: Current Employment. Parent: Novartis: Ended employment in the past 24 months; iVexSol, Inc: Current Employment. Greene: iVexSol, Inc: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Niederst: Novartis: Current Employment, Current equity holder in publicly-traded company. Whalen: Novartis: Current Employment. Orlando: Novartis: Current Employment. Treanor: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties: no royalties as company-held patents. Brogdon: Novartis Institutes for Biomedical Research: Current Employment.

Abstract: Newly arrived species on young or remote islands are likely to encounter less predation and competition than source populations on continental landmasses. The associated ecological release might facilitate divergence and speciation as colonizing lineages fill previously unoccupied niche space. Characterizing the sequence and timing of colonization on islands represents the first step in determining the relative contributions of geographical isolation and ecological factors in lineage diversification. Herein, we use genome-scale data to estimate timing of colonization in Naesiotus snails to the Galápagos islands from mainland South America. We test inter-island patterns of colonization and within-island radiations to understand their contribution to community assembly. Partly contradicting previously published topologies, phylogenetic reconstructions suggest that most Naesiotus species form island-specific clades, with within-island speciation dominating cladogenesis. Galápagos Naesiotus also adhere to the island progression rule, with colonization proceeding from old to young islands and within-island diversification occurring earlier on older islands. Our work provides a framework for evaluating the contribution of colonization and in situ speciation to the diversity of other Galápagos lineages.

Abstract: Phages Cassita and Fransoyer were isolated from soil in northwestern Wisconsin using Microbacterium paraoxydans as the host. The genomes of Cassita and Fransoyer are 61,868 bp and 62,277 bp, respectively, with direct terminal repeats. Both phages exhibit siphoviral morphology and are predicted to have lytic life cycles.

Abstract: Missense variants in the SCN8A voltage-gated sodium channel gene are linked to early-infantile epileptic encephalopathy type 13, also known as SCN8A-related epilepsy. These patients exhibit a wide spectrum of intractable seizure types, severe developmental delay, movement disorders, and elevated risk of sudden unexpected death in epilepsy. The mechanisms by which SCN8A variants lead to epilepsy are poorly understood, although heterologous expression systems and mouse models have demonstrated altered sodium current properties. To investigate these mechanisms using a patient-specific model, we generated induced pluripotent stem cells from three patients with missense variants in SCN8A: p.R1872 & gt;L (Patient 1); p.V1592 & gt;L (Patient 2); and p.N1759 & gt;S (Patient 3). Using small molecule differentiation into excitatory neurons, induced pluripotent stem cell-derived neurons from all three patients displayed altered sodium currents. Patients 1 and 2 had elevated persistent current, while Patient 3 had increased resurgent current compared to controls. Neurons from all three patients displayed shorter axon initial segment lengths compared to controls. Further analyses focused on one of the patients with increased persistent sodium current (Patient 1) and the patient with increased resurgent current (Patient 3). Excitatory cortical neurons from both patients had prolonged action potential repolarization. Using doxycycline-inducible expression of the neuronal transcription factors neurogenin 1 and 2 to synchronize differentiation of induced excitatory cortical-like neurons, we investigated network activity and response to pharmacotherapies. Both small molecule differentiated and induced patient neurons displayed similar abnormalities in action potential repolarization. Patient induced neurons showed increased burstiness that was sensitive to phenytoin, currently a standard treatment for SCN8A-related epilepsy patients, or riluzole, an FDA-approved drug used in amyotrophic lateral sclerosis and known to block persistent and resurgent sodium currents, at pharmacologically relevant concentrations. Patch-clamp recordings showed that riluzole suppressed spontaneous firing and increased the action potential firing threshold of patient-derived neurons to more depolarized potentials. Two of the patients in this study were prescribed riluzole off-label. Patient 1 had a 50% reduction in seizure frequency. Patient 3 experienced an immediate and dramatic seizure reduction with months of seizure freedom. An additional patient with a SCN8A variant in domain IV of Nav1.6 (p.V1757 & gt;I) had a dramatic reduction in seizure frequency for several months after starting riluzole treatment, but then seizures recurred. Our results indicate that patient-specific neurons are useful for modelling SCN8A-related epilepsy and demonstrate SCN8A variant-specific mechanisms. Moreover, these findings suggest that patient-specific neuronal disease modelling offers a useful platform for discovering precision epilepsy therapies.

Abstract: New terrigenous grain size and provenance records from Integrated Ocean Drilling Program Sites U1406 and U1411, Newfoundland ridges contourite drifts, North Atlantic Sortable silt abundance data reveals long‐term coarsening at both sites over the studied interval (35–26 Ma) Deep North Atlantic circulation intensified prior to the Eocene‐Oligocene Transition and continued to intensify into the mid‐Oligocene

Abstract: Human induced pluripotent stem cell (iPSC) models of epilepsy are becoming a revolutionary platform for mechanistic studies and drug discovery. The skyrocketing pace of epilepsy gene discovery is vastly outstripping the development of in vivo animal models. Currently, antiepileptic drug prescribing to patients with specific genetic epilepsies is based on small-scale clinical trials and empiricism; however, rapid production of patient-derived iPSC models will allow for precision therapy. We review iPSC-based studies that have already afforded novel discoveries in diseases with epileptic phenotypes, as well as challenges to using iPSC-based neurological disease models. We also discuss iPSC-derived cardiomyocyte studies of arrhythmia-inducing ion channelopathies that exemplify novel drug discovery and use of multielectrode array technology that can be translated to epilepsy research. Beyond initial studies of Rett, Timothy, Phelan-McDermid, and Dravet syndromes, the stage is set for groundbreaking iPSC-based mechanistic and therapeutic discoveries in genetic epilepsies with the potential to impact patient treatment and quality of life.

Abstract: Neural tube defects (NTDs), including anencephaly and spina bifida, are common major malformations of fetal development resulting from incomplete closure of the neural tube. These conditions lead to either universal death (anencephaly) or severe lifelong complications (spina bifida). Despite hundreds of genetic mouse models of neural tube defect phenotypes, the genetics of human NTDs are poorly understood. Furthermore, pharmaceuticals, such as antiseizure medications, have been found clinically to increase the risk of NTDs when administered during pregnancy. Therefore, a model that recapitulates human neurodevelopment would be of immense benefit to understand the genetics underlying NTDs and identify teratogenic mechanisms. Using our self-organizing single rosette cortical organoid (SOSR-COs) system, we have developed a high-throughput image analysis pipeline for evaluating the SOSR-CO structure for NTD-like phenotypes. Similar to small molecule inhibition of apical constriction, the antiseizure medication valproic acid (VPA), a known cause of NTDs, increases the apical lumen size and apical cell surface area in a dose-responsive manner. GSK3β and HDAC inhibitors caused similar lumen expansion; however, RNA sequencing suggests VPA does not inhibit GSK3β at these concentrations. The knockout of SHROOM3, a well-known NTD-related gene, also caused expansion of the lumen, as well as reduced f-actin polarization. The increased lumen sizes were caused by reduced cell apical constriction, suggesting that impingement of this process is a shared mechanism for VPA treatment and SHROOM3-KO, two well-known causes of NTDs. Our system allows the rapid identification of NTD-like phenotypes for both compounds and genetic variants and should prove useful for understanding specific NTD mechanisms and predicting drug teratogenicity.