Glycoprotein Targeted CAR-NK Cells for the Treatment of SARS-CoV-2 Infection

H84T-Banana Lectin (BanLec) CAR-NK cells bind high mannose glycosites that decorate the SARS-CoV-2 envelope, thereby decreasing cellular infection in a model of SARS-CoV-2. H84T-BanLec CAR-NK cells are innate effector cells, activated by virus. This novel cellular agent is a promising therapeutic, capable of clearing circulating SARS-CoV-2 virus and infected cells. Banana Lectin (BanLec) binds high mannose glycans on viral envelopes, exerting an anti-viral effect. A point mutation (H84T) divorces BanLec mitogenicity from antiviral activity. SARS-CoV-2 contains high mannose glycosites in proximity to the receptor binding domain of the envelope Spike (S) protein. We designed a chimeric antigen receptor (CAR) that incorporates H84T-BanLec as the extracellular moiety. Our H84T-BanLec CAR was devised to specifically direct NK cell binding of SARS-CoV-2 envelope glycosites to promote viral clearance. The H84T-BanLec CAR was stably expressed at high density on primary human NK cells during two weeks of ex vivo expansion. H84T-BanLec CAR-NK cells reduced S-protein pseudotyped lentiviral infection of 293T cells expressing ACE2, the receptor for SARS-CoV-2. NK cells were activated to secrete inflammatory cytokines when in culture with virally infected cells. H84T-BanLec CAR-NK cells are a promising cell therapy for further testing against wild-type SARS-CoV-2 virus in models of SARS-CoV-2 infection. They may represent a viable off-the-shelf immunotherapy for patients suffering from COVID-19.

Viral mediated knockdown of GATA6 in SMA iPSC-derived astrocytes prevents motor neuron loss and microglial activation

Spinal muscular atrophy (SMA), a pediatric genetic disorder, is characterized by the profound loss of spinal cord motor neurons and subsequent muscle atrophy and death. Although the mechanisms underlying motor neuron loss are not entirely clear, data from our work and others support the idea that glial cells contribute to disease pathology. GATA6, a transcription factor that we have previously shown to be upregulated in SMA astrocytes, is negatively regulated by SMN and can increase the expression of the inflammatory regulator NFκB. In this study, we identified upregulated GATA6 as a contributor to increased activation, pro-inflammatory ligand production, and neurotoxicity in spinal-cord patterned astrocytes differentiated from SMA patient induced pluripotent stem cells. Reducing GATA6 expression in SMA astrocytes via lentiviral infection ameliorated these effects to healthy control levels. Additionally, we found that SMA astrocytes contribute to SMA microglial phagocytosis, which was again decreased by lentiviral-mediated knockdown of GATA6. Together these data identify a role of GATA6 in SMA astrocyte pathology and further highlight glia as important targets of therapeutic intervention in SMA.

Identification the role of mono-ADP-ribosylation in colorectal cancer by integrated Transcriptome analysis

Purpose Our previous study has clarified the carcinogenic properties of arginine-specific mono-ADP ribosyltransferase 1(ART1), which is considered to be a critical post-translational modification that changes the structure and function of proteins and is widely involved in important processes. This study provides, for the first time, a comprehensive insight of transcriptomic analysis for colorectal cancer cells interfered with ART1 silencing by Illumina RNA-Seq and related verification experiments. Methods Lentiviral infection was used to construct a CT-26 cell line that stably knocks down the ART1 gene, a whole transcriptome sequencing technique was performed to identify differentially expressed genes (DEGs). GO and KEGG classification/enrichment analysis and verification experiments were performed to determine the role of ART1 in the progression of colorectal cancer. Results a total of 5552 DEGs, GO function and KEGG pathway with highest enrichment, forms of SNP and diverse splicing patterns were able to be identified. Importantly, knockdown of ART1 affected the occurrence of the splicing of certain key genes related to tumor cell growth, also down-regulated expression of the key gene PTBP1 for alternative splicing. The overall attenuation of the endoplasmic reticulum unfolded protein response (UPR) signaling pathway caused by ART1 inhibition would unbalance UPR signaling, leading to the occurrence of apoptosis to impede tumorigenesis. Conclusion ART1, which clustered in organelles, may promote the development of colorectal cancer by participating in a variety of new mechanisms including endoplasmic reticulum stress regulation, metabolic process or alternative splicing, which may provide a good clinical drug candidate closer to targeted therapy of CRC.

Upregulation of ETV2 Expression Promotes Endothelial Differentiation of Human Dental Pulp Stem Cells

The lack of vasculogenesis often hampers the survivability and integration of newly engineered tissue grafts within the host. Autologous endothelial cells (ECs) are an ideal cell source for neovascularization, but they are limited by their scarcity, lack of proliferative capacity, and donor site morbidity upon isolation. The objective of this study was to determine whether differentiation of human dental pulp stem cells (DPSCs) into the endothelial lineage can be enhanced by recombinant ETV2 overexpression. DPSCs were extracted from fresh dental pulp tissues. ETV2 overexpression in DPSCs was achieved by lentiviral infection and cellular morphological changes were evaluated. The mRNA and protein expression levels of endothelial-specific markers were assessed through quantitative real-time polymerase chain reaction, western blot, immunofluorescence staining, and flow cytometry. The tube formation assay and Matrigel plug assay were also performed to evaluate the angiogenic potential of the ETV2-transduced cells in vitro and in vivo, respectively. Additionally, proteomic analysis was performed to analyze global changes in protein expression following ETV2 overexpression. After lentiviral infection, ETV2-overexpressing DPSCs showed endothelial-like morphology. Compared with control DPSCs, significantly higher mRNA and protein expression levels of endothelial-specific genes, including CD31, VE-Cadherin, VEGFR1, and VEGFR2, were detected in ETV2-overexpressing DPSCs. Moreover, ETV2 overexpression enhanced capillary-like tube formation on Matrigel in vitro, as well as neovascularization in vivo. In addition, comparative proteomic profiling showed that ETV2 overexpression upregulated the expression of vascular endothelial growth factor (VEGF) receptors, which was indicative of increased VEGF signaling. Taken together, our results indicate that ETV2 overexpression significantly enhanced the endothelial differentiation of DPSCs. Thus, this study shows that DPSCs can be a promising candidate cell source for tissue engineering applications.

Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs

Canonical CRISPR screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene knockouts. These DSB-dependent methodologies may yield false-positive results by mistakenly assuming targeted loci as essential for cell viability, especially when high-copy-number sites are targeted. Here, we use CRISPR cytosine base editors for genome-scale knockout screens by perturbing gene start codons or splice sites, or by introducing premature termination codons (PTCs). Combining with iBAR strategy we have previously established, we realized an iBARed cytosine Base Editing-mediated gene KnockOut (BARBEKO) screening strategy at a genome-scale (targeting 17,501 genes) in multiple human cell lines. By constructing such a cell library through lentiviral infection at a high MOI (up to 10), we significantly reduced starting cells while producing screening results with improved efficiency and accuracy. More importantly, in comparison with Cas9-mediated cell fitness screens, BARBEKO screens are no longer affected by DNA-cleavage induced cytotoxicity in HeLa, K562, or DSB-sensitive RPE1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR screens in various settings.

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation

Background: Salmonella Enteritidis (SE) is one of the major foodborne zoonotic pathogens of worldwide importance which can induce activation of NLRC4 and NLRP3 inflammasomes during infection. Given that the inflammasomes play an essential role in resisting bacterial infection, Salmonella has evolved various strategies to regulate activation of the inflammasome, most of which largely remain unclear.Results: A transposon mutant library in SE strain C50336 was screened for the identification of the potential factors that regulate inflammasome activation. We found that T3SS-associated genes invC, prgH, and spaN were required for inflammasome activation in vitro. Interestingly, C50336 strains with deletion or overexpression of Dam were both defective in activation of caspase-1, secretion of IL-1β and phosphorylation of c-Jun N-terminal kinase (Jnk). Transcriptome sequencing (RNA-seq) results showed that most of the differentially expressed genes and enriched KEGG pathways between the C50336-VS-C50336Δdam and C50336-VS-C50336::dam groups overlapped, which includes multiple signaling pathways related to the inflammasome. C50336Δdam and C50336::dam were both found to be defective in suppressing the expression of several anti-inflammasome factors. Moreover, overexpression of Dam in macrophages by lentiviral infection could specifically enhance the activation of NLRP3 inflammasome independently via promoting the Jnk pathway.Conclusions: These data indicated that Dam was essential for modulating inflammasome activation during SE infection, there were complex and dynamic interplays between Dam and the inflammasome under different conditions. New insights were provided about the battle between SE and host innate immunological mechanisms.

Sirt1 Mediated High-Glucose-Induced Aβ Deposition and Cognitive Impairment through Activation of the TLR9/p53 Pathway

Background: Diabetic encephalopathy (DE) is a chronic central nervous system complication caused by diabetes mellitus (DM). β-amyloid (Aβ) deposition has been considered as the main cause of cognitive impairment in DE. Previous researches concerned the effect of canonical TLR9/Myd88 inflammatory pathway. However our study explored the function of non-inflammatory pathway of Toll-like receptor 9 (TLR9), acting on Sirt1 to influence Aβ deposition and cognitive function in DE.Results: We found that, compared with DM mice, TLR9-/-DM mice performed better learning ability and short-term memory, along with lower Aβ in hippocampi, but could be reversed by Sirt1 inhabition. Furthermore, in vitro, after intervention with high glucose and p53 over-expressed lentiviral infection, we observed the positive results of TLR9 inhibition, such as Sirt1 up-regulation, Aβ reduction or cognitive improvement, were altered (all P<0.05).Conclusions: we considered that TLR9/p53/Sirt1 signalling pathway induced by high glucose are one of molecular mechanisms underlying DE. These results not only confirm the importance of blood glucose management but also provide new insights for the diagnosis and treatment of DE.

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation

Background: Salmonella Enteritidis (SE) is one of the major foodborne zoonotic pathogens of worldwide importance which can induce activation of NLRC4 and NLRP3 inflammasomes during infection. Given that the inflammasomes play an essential role in resisting bacterial infection, Salmonella has evolved various strategies to regulate activation of the inflammasome, most of which largely remain unclear.Results: A transposon mutant library in SE strain C50336 was screened for the identification of the potential factors that regulate inflammasome activation. We found that T3SS-associated genes invC, prgH, and spaN were required for inflammasome activation in vitro. Interestingly, C50336 strains with deletion or overexpression of Dam were both defective in activation of caspase-1 and secretion of IL-1β. Transcriptome sequencing (RNA-seq) results suggest that the absence and overexpression of Dam had similar effects on infected cells, as high overlapping rates of differentially expressed genes and enriched pathways were found between the C50336-VS-C50336Δdam and C50336-VS-C50336::dam groups, including multiple signaling pathways related to the inflammasome. Moreover, overexpression of Dam in macrophages by lentiviral infection could specifically promote the activation of the NLRP3 inflammasome independently.Conclusions: These data indicate that Dam was essential for modulating inflammasome activation during SE infection, there are complex and dynamic interplays between Dam and the inflammasome under different conditions. New insights were provided about the battle between SE and host innate immunological mechanisms.

A192 DEPLETING ASCL2 IN ESOPHAGEAL ORGANOIDS TO INVESTIGATE ITS ROLE IN STEM CELLS MAINTENANCE

Background Recently, the first population of stem cells in the esophageal epithelium was identified with the help of the Keratin 15(Krt15) marker. However, little is known about the mechanisms underlying the expansion and the function of stem cells in the esophagus. It was shown that ASCL2, a transcription factor, is upregulated in Krt15+cells compared with Krt15- cells. ASCL2 is a gene target of the Wnt/β-catenin pathway, which act as a regulator of proliferation and maintenance of the stemness state. The ultimate goal of my research project is to determine the role of ASCL2 in the maintenance of esophageal stem cells and to identify his binding partners. Aims To investigate the role of ASCL2 in esophageal epithelial biology, we aim at establishingAscl2knockout esophageal organoid lines. Methods Lentiviral infection and CRISPR/Cas9 knockout approach were optimized in mouse esophageal organoids. ASCL2was invalidated with CRISPR/Cas9 and/or inducible specific shRNAs in primary mouse esophageal cell line and organoids. Antibodies directed against ASCL2 were also tested to validate cell lines. Results First, to optimize lentiviral infection in mouse esophageal organoids, we produced GFP-expressing lentiviruses. Viruses were then concentrated and incubated with a single cell suspension of mouse organoid cells under gentle activation. Infected cells were embedded in Matrigel and grown in organoids. GFP+ cells were then selected using an antibiotic resistance strategy. Second, we used our optimized lentiviral infection protocol to express specific gRNAs in mouse esophageal organoids, which were previously screened in a primary mouse esophageal cell line. Knockout were validated using Western Blot. Invalidation of ASCL2 was performed using CRISPR/Cas9 and inducible shRNAs. We also validated three commercially available ASCL2 antibodies in WB and IF. Conclusions ASCL2 is expressed in the mouse esophagus tissue and organoids, and it can be invalidated in cell lines and organoids. Funding Agencies NSERC, CRC Tier2

Loss of Stag2 cooperates with EWS-FLI1 to transform murine Mesenchymal stem cells

Background Ewing sarcoma is a malignancy of primitive cells, possibly of mesenchymal origin. It is probable that genetic perturbations other than EWS-FLI1 cooperate with it to produce the tumor. Sequencing studies identified STAG2 mutations in approximately 15% of cases in humans. In the present study, we hypothesize that loss of Stag2 cooperates with EWS-FLI1 in generating sarcomas derived from murine mesenchymal stem cells (MSCs). Methods Mice bearing an inducible EWS-FLI1 transgene were crossed to p53−/− mice in pure C57/Bl6 background. MSCs were derived from the bone marrow of the mice. EWS-FLI1 induction and Stag2 knockdown were achieved in vitro by adenovirus-Cre and shRNA-bearing pGIPZ lentiviral infection, respectively. The cells were then treated with ionizing radiation to 10 Gy. Anchorage independent growth in vitro was assessed by soft agar assays. Cellular migration and invasion were evaluated by transwell assays. Cells were injected with Matrigel intramuscularly into C57/Bl6 mice to test for tumor formation. Results Primary murine MSCs with the genotype EWS-FLI1 p53−/− were resistant to transformation and did not form tumors in syngeneic mice without irradiation. Stag2 inhibition increased the efficiency and speed of sarcoma formation significantly in irradiated EWS-FLI1 p53−/− MSCs. The efficiency of tumor formation was 91% for cells in mice injected with Stag2-repressed cells and 22% for mice receiving cells without Stag2 inhibition (p < .001). Stag2 knockdown reduced survival of mice in Kaplan-Meier analysis (p < .001). It also increased MSC migration and invasion in vitro but did not affect proliferation rate or aneuploidy. Conclusion Loss of Stag2 has a synergistic effect with EWS-FLI1 in the production of sarcomas from murine MSCs, but the mechanism may not relate to increased proliferation or chromosomal instability. Primary murine MSCs are resistant to transformation, and the combination of p53 null mutation, EWS-FLI1, and Stag2 inhibition does not confer immediate conversion of MSCs to sarcomas. Irradiation is necessary in this model, suggesting that perturbations of other genes beside Stag2 and p53 are likely to be essential in the development of EWS-FLI1-driven sarcomas from MSCs.