Sequential intravenous and intracerebroventricular GD2-CAR T-cell therapy for H3K27M-mutated diffuse midline gliomas.

H3K27M-mutant diffuse midline gliomas (DMGs) express high levels of the GD2 disialoganglioside and chimeric antigen receptor modified T-cells targeting GD2 (GD2-CART) eradicate DMGs in preclinical models. Arm A of the Phase I trial NCT04196413 administered one IV dose of autologous GD2-CART to patients with H3K27M-mutant pontine (DIPG) or spinal (sDMG) diffuse midline glioma at two dose levels (DL1=1e6/kg; DL2=3e6/kg) following lymphodepleting (LD) chemotherapy. Patients with clinical or imaging benefit were eligible for subsequent intracerebroventricular (ICV) GD2-CART infusions (10-30e6 GD2-CART). Primary objectives were manufacturing feasibility, tolerability, and identification of a maximally tolerated dose of IV GD2-CART. Secondary objectives included preliminary assessments of benefit. Thirteen patients enrolled and 11 received IV GD2-CART on study [n=3 DL1(3 DIPG); n=8 DL2(6 DIPG/2 sDMG). GD2-CART manufacturing was successful for all patients. No dose-limiting toxicities (DLTs) occurred on DL1, but three patients experienced DLT on DL2 due to grade 4 cytokine release syndrome (CRS). Nine patients received ICV infusions, which were not associated with DLTs. All patients exhibited tumor inflammation-associated neurotoxicity (TIAN). Four patients demonstrated major volumetric tumor reductions (52%, 54%, 91% and 100%). One patient exhibited a complete response ongoing for >30 months since enrollment. Eight patients demonstrated neurological benefit based upon a protocol-directed Clinical Improvement Score. Sequential IV followed by ICV GD2-CART induced tumor regressions and neurological improvements in patients with DIPG and sDMG. DL1 was established as the maximally tolerated IV GD2-CART dose. Neurotoxicity was safely managed with intensive monitoring and close adherence to a management algorithm.

CD22 CAR T cells demonstrate high response rates and safety in pediatric and adult B-ALL: Phase 1b results.

Chimeric antigen receptor (CAR) T cells targeting CD22 (CD22-CAR) provide a therapeutic option for patients with CD22+ malignancies with progression after CD19-directed therapies. Using on-site, automated, closed-loop manufacturing, we conducted parallel Phase 1b clinical trials investigating a humanized CD22-CAR with 41BB costimulatory domain in children and adults with heavily treated, relapsed/refractory (r/r) B-ALL. Of 19 patients enrolled, 18 had successful CD22-CAR manufacturing, and 16 patients were infused. High grade (3-4) cytokine release syndrome (CRS) and immune effector-cell-associated neurotoxicity syndrome (ICANS) each occurred in only one patient; however, three patients experienced immune-effector-cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS). Twelve of 16 patients (75%) achieved CR with an overall 56% MRD-negative CR rate. Duration of response was overall limited (median 77 days), and CD22 expression was downregulated in 4/12 (33%) available samples at relapse. In summary, we demonstrate that closed-loop manufacturing of CD22-CAR T cells is feasible and is associated with a favorable safety profile and high CR rates in pediatric and adult r/r B-ALL, a cohort with limited CD22-CAR reporting.

Homology-independent targeted insertion (HITI) enables guided CAR knock-in and efficient clinical scale CAR-T cell manufacturing.

BACKGROUND: Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA). METHODS: We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach. RESULTS: Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 108-3.6 × 109 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity. CONCLUSIONS: Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.

Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake.

BACKGROUND: Human cell-secreted extracellular vesicles (EVs) are versatile nanomaterials suitable for disease-targeted drug delivery and therapy. Native EVs, however, usually do not interact specifically with target cells or harbor therapeutic drugs, which limits their potential for clinical applications. These functions can be introduced to EVs by genetic manipulation of membrane protein scaffolds, although the efficiency of these manipulations and the impacts they have on the properties of EVs are for the most part unknown. In this study, we quantify the effects of genetic manipulations of different membrane scaffolds on the physicochemical properties, molecular profiles, and cell uptake of the EVs. METHODS: Using a combination of gene fusion, molecular imaging, and immuno-based on-chip analysis, we examined the effects of various protein scaffolds, including endogenous tetraspanins (CD9, CD63, and CD81) and exogenous vesicular stomatitis virus glycoprotein (VSVG), on the efficiency of integration in EV membranes, the physicochemical properties of EVs, and EV uptake by recipient cells. RESULTS: Fluorescence imaging and live cell monitoring showed each scaffold type was integrated into EVs either in membranes of the endocytic compartment, the plasma membrane, or both. Analysis of vesicle size revealed that the incorporation of each scaffold increased the average diameter of vesicles compared to unmodified EVs. Molecular profiling of surface markers in engineered EVs using on-chip assays showed the CD63-GFP scaffold decreased expression of CD81 on the membrane surface compared to control EVs, whereas its expression was mostly unchanged in EVs bearing CD9-, CD81-, or VSVG-GFP. The results from cell uptake studies demonstrated that VSVG-engineered EVs were taken up by recipient cells to a greater degree than control EVs. CONCLUSION: We found that the incorporation of different molecular scaffolds in EVs altered their physicochemical properties, surface protein profiles, and cell-uptake functions. Scaffold-induced changes in the physical and functional properties of engineered EVs should therefore be considered in engineering EVs for the targeted delivery and uptake of therapeutics to diseased cells.

Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1.

BACKGROUND: Advancement in genome engineering enables rapid and targeted disruption of any coding sequences to study gene functions or establish human disease models. We explored whether this approach can be used to study Gaucher disease, one of the most common types of lysosomal storage diseases (LSDs) in a near-haploid human cell line (HAP1). RESULTS: CRISPR-Cas9 targeting to coding sequences of ß-glucocerebrosidase (GBA), the causative gene of Gaucher disease, resulted in an insertional mutation and premature termination of GBA. We confirmed the GBA knockout at both the gene and enzyme levels by genotyping and GBA enzymatic assay. Characterization of the knockout line showed no significant changes in cell morphology and growth. Lysosomal staining revealed more granular lysosomes in the cytosol of the GBA-knockout line compared to its parental control. Flow cytometry analysis further confirmed that more lysosomes accumulated in the cytosol of the knockout line, recapturing the disease phenotype. Finally, we showed that this knockout cell line could be used to evaluate a replacement therapy by recombinant human GBA. CONCLUSIONS: Targeted gene disruption in human HAP1 cells enables rapid establishment of the Gaucher model to capture the key pathology and to test replacement therapy. We expect that this streamlined method can be used to generate human disease models of other LSDs, most of which are still lacking both appropriate human disease models and specific treatments to date.

Orchestrating Extracellular Vesicle With Dual Reporters for Imaging and Capturing in Mammalian Cell Culture.

Background: Recent technological advancements have enabled live-cell imaging of intracellular organelles to monitor their biogenesis in mammalian cells. However, applying this method to gain insight into extracellular organelles, such as extracellular vesicles (EVs), presents unique challenges that require special considerations in design and engineering. Results: We have developed a dual-reporter system that combines genetic fusion, fluorescence microcopy and magnetic beads capture of EVs to study the biogenesis of EVs in mammalian cell cultures. First, we genetically produced a series of reporters by fusing a green fluorescent protein (GFP) and an affinity peptide (6xHis), with either the endogenous transmembrane protein, CD63, or EVs targeting vesicular stomatitis viral glycoprotein (VSVG). Transfection of these reporters into human 293T cells resulted in expression and integration of these reporters into pre-exosome compartments, which were subsequently released into the culture medium. Confocal imaging and nano-particle tracking analysis demonstrated that EVs were appropriately labeled and exhibited a single dominant peak in the 80-110 nm size range, indicating that isolated EVs were comprised of micro-vesicles and/or exosome subpopulations. Incubation of isolated EVs with nickel-coated magnetic beads resulted in successful capture of GFP-positive EVs. Finally, addition of EVs into culture medium was able to reveal the cellular uptake of GFP-labeled EVs by recipient cells. Taken together, our dual-reporter system provides a powerful method for both monitoring and capturing of EVs in mammalian cell culture systems. Conclusion: A dual-reporter system provides a robust tool to study the life cycle of EVs in mammalian cells from biogenesis and excretion to cellular uptake.

Sequential deletion of CD63 identifies topologically distinct scaffolds for surface engineering of exosomes in living human cells.

Exosomes are cell-derived extracellular vesicles that have great potential in the field of nano-medicine. However, a fundamental challenge in the engineering of exosomes is the design of biocompatible molecular scaffolds on their surface to enable cell targeting and therapeutic functions. CD63 is a hallmark protein of natural exosomes that is highly enriched on the external surface of the membrane. We have previously described engineering of CD63 for use as a molecular scaffold in order to introduce cell-targeting features to the exosome surface. Despite this initial success, the restrictive M-shaped topology of full-length CD63 may hinder specific applications that require N- or C-terminal display of cell-targeting moieties on the outer surface of the exosome. In this study, we describe new and topologically distinct CD63 scaffolds that enable robust and flexible surface engineering of exosomes. In particular, we conducted sequential deletions of the transmembrane helix of CD63 to generate a series of CD63 truncates, each genetically-fused to a fluorescent protein. Molecular and cellular characterization studies showed truncates of CD63 harboring the transmembrane helix 3 (TM3) correctly targeted and anchored to the exosome membrane and exhibited distinct n-, N-, Ω-, or I-shaped membrane topologies in the exosomal membrane. We further established that these truncates retained robust membrane-anchoring and exosome-targeting activities when stably expressed in the HEK293 cells. Moreover, HEK293 cells produced engineered exosomes in similar quantities to cells expressing full-length CD63. Based on the results of our systematic sequential deletion studies, we propose a model to understand molecular mechanisms that underlie membrane-anchoring and exosome targeting features of CD63. In summary, we have established new and topologically distinct scaffolds based on engineering of CD63 that enables flexible engineering of the exosome surface for applications in disease-targeted drug delivery and therapy.

Genetic labeling of extracellular vesicles for studying biogenesis and uptake in living mammalian cells.

Molecular imaging methods are powerful tools for gaining insight into the cellular organization of living cells. To understand the biogenesis and uptake of extracellular vesicles (EVs) as well as to engineer cell-derived vesicles for targeted drug delivery and therapy, genetic labeling with fluorescent proteins has increasingly been used to determine the structures, locations, and dynamics of EVs in vitro and in vivo. Here, we report a genetic method for the stable labeling of EVs to study their biogenesis and uptake in living human cells. Fusing a green fluorescent protein (GFP) with either the endogenous CD63 (CD63-GFP) or a vesicular stomatitis virus envelope glycoprotein, VSVG (VSVG-GFP), we successfully obtained distinct fluorescence signals in the cytoplasm, revealing the biogenesis of EVs in post-transfected cells. We describe experimental procedures in detail for EV isolation, imaging, and cellular uptake using both confocal microscopy and flow cytometry. We also provide a perspective on how genetic labeling methods can be used to study EV biology, characterization of engineered EVs, and development of EV-based nano-medicine.

Targeted delivery of lysosomal enzymes to the endocytic compartment in human cells using engineered extracellular vesicles.

Targeted delivery of lysosomal enzymes to the endocytic compartment of human cells represents a transformative technology for treating a large family of lysosomal storage diseases (LSDs). Gaucher disease is one of the most common types of LSDs caused by mutations to the lysosomal ß-glucocerebrosidase (GBA). Here, we describe a genetic strategy to produce engineered exosomes loaded with GBA in two different spatial configurations for targeted delivery to the endocytic compartment of recipient cells. By fusing human GBA to an exosome-anchoring protein: vesicular stomatitis virus glycoprotein (VSVG), we demonstrate that the chimeric proteins were successfully integrated into exosomes which were secreted as extracellular vesicles (EVs) by producer cells. Isolation and molecular characterization of EVs confirmed that the fusion proteins were loaded onto exosomes without altering their surface markers, particle size or distribution. Further, enzyme-loaded exosomes/EVs added to cultured medium were taken up by recipient cells. Further, the endocytosed exosomes/EVs targeted to endocytic compartments exhibited a significant increase in GBA activity. Together, we have developed a novel method for targeting and delivery of lysosomal enzymes to their natural location: the endocytic compartment of recipient cells. Since exosomes/EVs have an intrinsic ability to cross the blood-brain-barrier, our technology may provide a new approach to treat severe types of LSDs, including Gaucher disease with neurological complications.

Decoy exosomes as a novel biologic reagent to antagonize inflammation.

Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases. Methods: We engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Results: Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. We developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. We characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. We demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line. Conclusions: Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα.

Racism and the Christian Church in America: Caught between the Knowledge of Good and Evil.

Those persons with church membership or professing a faith or relationship with God represent a microcosm of society. Therefore, the demons of racism, bigotry, and prejudice found in society at large are found in the church. Despite the very nature of Christianity that calls on Christians to be a countervoice in the world against evil, many have capitulated to various strains of racism in the world and in the Church. Some Christian denominations have begun to explore racism in the church and have developed responses to addressing the issues in the church and in the world. This article examines the historical context of race and religion in the Christian church in the United States, and addresses the current efforts of some Christian denominations to become proactive in the struggle against racism.

Correction: Earliest evidence for equid bit wear in the ancient Near East: The "ass" from Early Bronze Age Tell es-Sâfi/Gath, Israel.

[This corrects the article DOI: 10.1371/journal.pone.0196335.].

Earliest evidence for equid bit wear in the ancient Near East: The "ass" from Early Bronze Age Tell es-Sâfi/Gath, Israel.

Analysis of a sacrificed and interred domestic donkey from an Early Bronze Age (EB) IIIB (c. 2800-2600 BCE) domestic residential neighborhood at Tell es-Sâfi/Gath, Israel, indicate the presence of bit wear on the Lower Premolar 2 (LPM2). This is the earliest evidence for the use of a bit among early domestic equids, and in particular donkeys, in the Near East. The mesial enamel surfaces on both the right and left LPM2 of the particular donkey in question are slightly worn in a fashion that suggests that a dental bit (metal, bone, wood, etc.) was used to control the animal. Given the secure chronological context of the burial (beneath the floor of an EB IIIB house), it is suggested that this animal provides the earliest evidence for the use of a bit on an early domestic equid from the Near East.

Mobile soak pits improve spray team mobility, productivity and safety of PMI malaria control programs.

In the President's Malaria Initiative (PMI)-funded Africa Indoor Residual Spraying Project (AIRS), end-of-day clean-up operations require the safe disposal of wash water resulting from washing the exterior of spray tanks and spray operators' personal protective equipment. Indoor residual spraying (IRS) programs typically use soak pits - large, in-ground filters - to adsorb, filter and then safely degrade the traces of insecticide found in the wash water. Usually these soak pits are permanent installations serving 30 or more operators, located in a central area that is accessible to multiple spray teams at the end of their workday. However, in remote areas, it is often impractical for teams to return to a central soak pit location for cleanup. To increase operational efficiency and improve environmental compliance, the PMI AIRS Project developed and tested mobile soak pits (MSP) in the laboratory and in field applications in Madagascar, Mali, Senegal, and Ethiopia where the distance between villages can be substantial and the road conditions poor. Laboratory testing confirmed the ability of the easily-assembled MSP to reduce effluent concentrations of two insecticides (Actellic 300-CS and Ficam VC) used by the PMI AIRS Project, and to generate the minimal practicable environmental "footprint" in these remote areas. Field testing in the Mali 2014 IRS campaign demonstrated ease of installation and use, resulted in improved and more consistent standards of clean-up, decreased transportation requirements, improved spray team working conditions, and reduced potential for operator exposure to insecticide.

The state of mental health services for children and adolescents: an examination of programs, practices and policies.

This article presents an overview of the state of mental health services for children and adolescents. It provides a brief historical review of policies affecting mental health services for children and adolescents with emotional and behavioral disorders. It discusses the roles of various systems in the provision services for emotionally and behaviorally disordered children and adolescents, and the need for cross-systems collaboration and funding. A model psycho-educational day treatment program. City Lights, is highlighted as an approach to serving inner-city African American adolescents with a profile of services and of the types of youth best served by such a program.