GFP-positive Cells Research Articles

Establishment of a 3D organoid culture model for the investigation of adult slow-cycling putative intestinal stem cells.

The study of intestinal stem cells is a prerequisite for the development of therapies aimed at regenerating the gut. To enable investigation of adult slow-cycling H2B-GFP-retaining putative small intestinal (SI) stem cells in vitro, we have developed a three-dimensional (3D) SI organoid culture model based on the Tet-Op histone 2 B (H2B)-green fluorescent protein (GFP) fusion protein (Tet-Op-H2B-GFP) transgenic mouse. SI crypts were isolated from 6- to 12-week-old Tet-Op-H2B-GFP transgenic mice and cultured with appropriate growth factors and an animal-derived matrix (Matrigel). For in vitro transgene expression, doxycycline was added to the culture medium for 24h. By pulse-chase experiments, H2B-GFP expression and retention were assessed through direct GFP fluorescence observations, both by confocal and fluorescence microscopy and by immunohistochemistry. The percentages of H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring relevant GFP-positive cells. Our results indicate that 24 h exposure to doxycycline (pulse) induced ubiquitous expression of H2B-GFP in the SI organoids. During subsequent culture, in the absence of doxycycline (chase), there was a gradual loss (due to cell division) of H2B-GFP. At 6-day chase, slow-cycling H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells were detected in the SI organoids. The developed culture model allows detection of slow-cycling H2B-GFP-retaining putative SI stem cells and will enable the study of self-renewal and regeneration for further characterization of these cells.

B-094 Assessment of a Live Cell-based Flow Cytometry Assay for MuSK-IgG4 Testing in Myasthenia Gravis

Abstract Background Myasthenia gravis (MG) is caused by disrupting the function of postsynaptic acetylcholine receptors (AChRs) at the neuromuscular junction. 85% of MG patients have autoantibodies targeting AChRs, while 6% of MG patients have autoantibodies targeting the muscle-specific receptor tyrosine kinase (MuSK). MuSK is located on the muscle membrane and is activated by the low-density lipoprotein receptor-related protein 4 (LRP4) upon the binding with ligand Agrin. Activated MuSK undergoes autophosphorylation and stimulates downstream signaling to cluster AChRs, which is important for the formation and maintenance of neuromuscular synapses. IgG4 antibodies that bind MuSK are thought to directly disrupt the ability of MuSK to cluster AChR. The gold standard methodology for MuSK antibody testing is radioimmunoprecipitation assay (RIA) using anti-human IgG secondary antibodies. Performing RIA methods in the clinical laboratory is a burden due to radioactive biohazard concerns and the analytical limitations of these assays. This study is to assess the analytical and clinical performance of a live cell-based flow cytometry assay as a potential replacement for the MuSK RIA. Methods HEK293T cells were transiently transfected with a vector co-expressing human MuSK protein and GFP. Patient sera were incubated with a mix of HEK293 cells (GFP-positive cells that express MuSK protein on the cell surface and GFP-negative cells that do no express MuSK) followed by incubation with anti-human IgG4 secondary antibody conjugated with alexa fluor 647 (AF647). An IgG binding index (IBI) was calculated for each sample as the ratio of the median AF647 fluorescence intensity (MFI) of the MuSK-expressing cells (GFP+) to the MFI of the non-MuSK expressing cells (GFP-). An arbitrary positive IBI cut-off of 2.0 was selected. A preliminary assessment of assay imprecision was performed for the flow cytometry assay, as well as a method comparison study to compare this assay to RIA utilizing a cohort of 130 (34 positive and 96 negative) residual samples previously tested by the gold-standard assay. 133 disease control samples were also tested to assess the clinical specificity of the flow cytometry-based assay. Results To assess the imprecision, eight samples with varying IBIs were tested with 5 replicates in each assay over five days. The coefficient of variation (CV) for intra-assay replicates ranged from 1% to 7% for all samples, while CV for inter-assay replicates ranged from 2% to 15%. Based on the 130 samples previously tested by RIA, the Positive Percentage Agreement (PPA) between these two assays was 76.5% and the negative Percentage Agreement (NPA) was 100%. Of the discordant samples (n=8), clinical information was available for only 1 sample (positive by RIA, negative by flow). This patient had a final non-MG diagnosis (ALS). All control samples were negative on the flow cytometry-based assay. Conclusions The live cell-based flow cytometry MuSK IgG4 assay demonstrated encouraging preliminary analytical performance that warrants further development and validation.

A live single-cell reporter system reveals drug-induced plasticity of a cancer stem cell-like population in cholangiocarcinoma.

Cancer stem cells (CSC) play an important role in carcinogenesis and are acknowledged to be responsible for chemoresistance in cholangiocarcinoma (CCA). Studying CCA CSC has been challenging, due to lack of consensus CSC markers, and to their plastic nature. Since dual expression of the core pluripotent factors SOX2/OCT4 has been shown to correlate with poor outcome in CCA patients, we selected the SOX2/OCT4 activating short half-life GFP-based live reporter (SORE6-dsCopGFP) to study CSC dynamics at the single-cell level. Transduction of five human CCA cell lines resulted in the expression of 1.8-13.1% GFP-positive (SORE6POS) cells. By live imaging, we found that SORE6POS CCA cells possess self-renewal capacity and that they can be induced to differentiate. Significantly, the SORE6POS cells were highly tumorigenic, both in vitro and in vivo, thus implicating the characteristics of primary CSCs. When we then analyzed for selected CSC-related markers, we found that the majority of both CD133+/CD44+, and CD133+/LGR5+ CCA cells were SORE6POS cells. Exposing transduced cells to standard CCA chemotherapy revealed higher growth rate inhibition at 50% (GR50s) for SORE6POS cells compared to GFP-negative (SORE6NEG) ones indicating that these CSC-like cells were more resistant to the treatment. Moreover, the chemotherapy induced SORE6POS from SORE6NEG cells, while retaining the existing SORE6POS population. Finally, treatment of transduced cells with CDK4/6 inhibitors in vitro for 3 days resulted in a lowered CSC number in the culture. Thus, applying a live reporter system allowed us to elucidate the stem cell diversity and drug-induced plasticity of CCA CSCs. These findings have clear implications for future management of such patients.

Bi-layered Adipose Mesenchymal Cell Sheets Improve Bladder Compliance in Spinal Cord-Injured Rats.

To improve bladder compliance in patients with low-compliance bladders, augmentation cystoplasty with the intestinal tract is performed. However, the use of the intestinal tract often leads to serious surgical complications. Tissue engineering technologies have the potential to improve bladder compliance without using the intestinal tract. In this study, we fabricated bi-layered adipose-derived mesenchymal cell (AMC) sheets and then determined whether the bi-layered AMC sheets could improve bladder compliance in rats with spinal cord injury (SCI). The abdominal adipose tissues of green fluorescence protein (GFP)-transfected Sprague-Dawley (SD) rats were harvested, and the attached and proliferating cells on type I collagen were used as AMCs. The AMCs were then cultured on temperature-responsive culture dishes. After reaching over-confluence, the AMCs that maintained cell-cell contacts were detached from the dishes and applied to a gelatin hydrogel sheet. Then, another detached AMC monolayer was accumulated on the AMC monolayer-applied gelatin. Prior to 4 weeks of transplantation, the levels of T8-9 in the spinal cords of recipient SD rats were partially transected. After producing the bi-layered AMC sheets and the rats with SCI, the detrusor muscles of the anterior bladder walls of the rats with SCI were incised, and the bi-layered AMC sheet was patch-transplanted onto the exposed bladder epithelium (n = 8). As a control, the sham operation was performed (n = 7). Four weeks after the transplantation, bladder capacity and bladder compliance in AMC sheet-transplanted SCI rats were significantly higher than those in sham-operated control SCI rats. The smooth muscle layers in AMC sheet-transplanted bladders were significantly larger than those in control bladders. In addition, the collagen fibers in the AMC sheet-transplanted bladders were significantly smaller than those in the control bladders. Some GFP-positive transplanted AMCs differentiated into smooth muscle actin- or desmin-positive cells. Furthermore, GFP-positive cells secreted transforming growth factor-β1 or vascular endothelial growth factor. Therefore, this study showed that bi-layered AMC sheets could improve bladder compliance and bladder tissues in SCI rats.

Morphological characteristics of the cerebral cortex of a mini-pig under conditions of gene therapy after experimental stroke

Aim. To study the effectiveness of preventive gene therapy (within 2 days) and gene therapy in the acute phase (after 4 hours) of ischemic stroke in mini-pigs using an autologous leucoconcentrate (AutoLeuc) enriched with recombinant genes of vascular endothelial growth factor (VEGF165), glial cell line-derived neurotrophic factor (GDNF) and neural cell adhesion molecule 1 (NCAM1), as well as the migration of leukocytes transduced with a chimeric adenoviral vector serotype 5 with fiber 35 serotype (Ad5/F35) and the green fluorescent protein (GFP) genome into immune defense organs.Materials and methods. The experiment was conducted on 8-month-old Vietnamese lop-bellied mini-pigs (n=16). An ischemic stroke was created by occlusion of the distal branches of the left middle cerebral artery and the right common carotid artery. Genetically modified AutoLeuc was administered preventively intravenously 2 days before or in the acute phase 4 hours after stroke modelling; the control group was injected with 30 ml of saline solution. The morphology of the cerebral cortex was assessed using histological methods in the areas bordering the infarction and peri-infarction after 21 days. The migration of genetically modified Ad5/F35-GFP leukocytes into the brain, spleen, and submandibular lymph nodes was studied a week after stroke modelling.Results. In the peri-infarction zone, the content of pyknotic neurons in control animals was higher, while the number of capillaries was lower than in the gene therapy groups. In the latter, neurons had a typical morphology with preserved outgrowths; in the control group, the outgrowths were tortuous and fragmented. Fluorescence microscopy after injection of AutoLeuc with Ad5/F35-GFP revealed GFP-positive cells in the spleen and submandibular lymph nodes.Conclusion. 21 days after modeling a stroke in mini-pigs against the background of preventive gene therapy or gene therapy in the acute phase using VEGF165/GDNF/NCAM1 AutoLeuc, greater preservation of neurons and a higher density of capillaries in the peri-infarction zone of ischemic brain damage were established. Leukocytes with Ad5/ F35-GFP were found in the spleen and submandibular lymph nodes.

HIV latency-reversing activity of latex from Euphorbia umbellata (Pax) Bruyns and three diterpenes isolated from this species

Two unusual phorbol esters, namely 20-deoxyphorbol-3,4,12-triacetate-13-phenylacetate (1) and phorbol-3,4,12,13-tetraacetate-20-phenylacetate (2) plus ingol-3,8,12-triacetate-7-phenylacetate (3) were isolated from the latex of Euphorbia umbellata and identified by HRESIMS and 2D NMR. Compound 1 is herein described for the first time. Assignment of the phenylacetyl group at C-7 in compound 3 was suggested by the HMBC and NOESY spectra obtained in pyridine-d5 . In addition to the latex and its distinct terpenoid fractions, the isolated compounds were tested as latent reversal agents against HIV-1-infected J-Lat cells, with reference to phorbol-12-myristate-13-acetate and ingenol-B. Compound 2 reverted 75-80% the viral latency on the GFP-positive cells, resulting EC50 3.70 μg/mL (SI 6.7), while 1 induced 34-40% reactivation at the same concentration range (4-20 µg/mL). The ingol derivative 3 was ineffective. Phorbol esters were confirmed as effective constituents in the latex since the fraction containing them was 2.4-fold more active than the lyophilised latex at the lowest concentration assayed.

Abstract 244: Patient-derived organoids: A pre-clinical model to assess infection and killing capacity of viral vectors for cancer therapy

Abstract HUB’s patient-derived organoids (PDOs) are in vitro models that preserve tissue morphology and original cellular complexity. Additionally, PDOs are compatible with (high) throughput screening methods, enabling informed decision-making early in the drug development pipeline and significantly improving the odds of positive outcomes in clinical trials. Akamis Bio has developed experimental viral vector-based tumour gene therapies that have the potential to treat a variety of solid tumours by driving the expression of immunologically active biomolecules and therapeutic proteins hampering tumour immune evasion. These vectors are based on a replication competent, chimeric group B adenovirus backbone that selectively replicates and expresses transgene payloads in tumor cells, and additionally exerts oncolytic activity. To evaluate the efficacy of such a virus-based gene delivery strategy, HUB set up a proof-of-concept study to assess the propagation capability of Akamis Bio’s viral vectors in tumour PDOs. Intact PDOs from primary pancreatic tumours were infected with several viral loads, ranging from 0.1 viral particles per cell (PPC) to 100 PPC. The viral vector encoded a GFP reporter gene to identify infected cells using fluorescence imaging. The cultures were imaged daily for 14 days, allowing the assessment of cytotoxicity and infection efficiency and dynamics. Independently of PPC, infections always started in the exterior of PDOs in a few GFP-positive cells. The gradual increase over time in GFP-signal toward the interior of PDOs suggests the high likelihood of secondary infection events. Crucially, GFP intensity was correlated with cell death in PDOs, as evidenced by morphological features detected in brightfield images. Moreover, the infection dynamics were dose-dependent, as the GFP intensity peak arose later with decreasing PPC. These results show the capacity of HUB PDOs to study the infection dynamics of Akamis viral vectors. This pre-clinical platform permits rapid and efficient screening and dose evaluation for oncolytic virotherapies. Unique access to extensive tumour PDO biobanks with characterized genomic landscapes allows best-in-class stratification of treatment response based on tumour type and genetic heterogeneity. Citation Format: Francisco Morales-Rodriguez, Maria Stella Sasso, Taya Bezhaeva, Samantha Bailey-Bucktrout, Ka Wai Lai, Foteini Gkogkou, Sylvia Boj. Patient-derived organoids: A pre-clinical model to assess infection and killing capacity of viral vectors for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 244.

Abstract 5606: The role of KMT2C in the homologous recombination repair in epithelial ovarian cancer

Abstract Background: Epithelial ovarian cancer (EOC) with homologous recombination (HR) deficiency is susceptible to PARP inhibitor therapy due to synthetic lethality. Identification of the patient population for PARP inhibitor therapy is imperative to reduce recurrence of EOC and improve the survival outcomes of patients. Current biomarkers for PARP inhibitor therapy consist of an array of genes involved in the HR repair pathway in EOC, including BRCA1, BRCA2 and PALB2. Through TCGA analysis, we found that the lysine (K)-specific methyltransferase 2C (KMT2C) gene is mutated in 10.3% of all EOC patients and considerably enriched in BRCA1 mutated EOC patients. However, the role of KMT2C in HR repair and PARP inhibitor sensitivity of EOC remains largely undefined. Methods: BRCA2-mutated PEO1 and BRCA2-wild type PEO1-NR cells were transfected with KMT2C siRNA and assayed for KMT2C protein expression by western blotting. DR-GFP (HR) and EJ5-GFP (NHEJ)-SKOV3 cell lines were co-transfected with the ISceI-expressing plasmid pCBA-ISceI and KMT2C siRNA. GFP-positive cells were analyzed by flow cytometry. SKOV3 cells were transfected with KMT2C siRNA and the expression of BRCA2 and Rad51 mRNA was determined using quantitative RT-PCR analysis. Results: siRNA-mediated knockdown of KMT2C resulted in marked down-regulation of KMT2C protein in PEO1 and PEO1-NR cells. KMT2C knockdown caused a significant decrease in ISceI-induced HR activity in DR-GFP-SKOV3 cells (p < 0.05). In contrast, KMT2C knockdown had no effects on ISceI-induced NHEJ activity in EJ5-GFP-SKOV3 cells. As a control, Rad51 knockdown caused complete suppression of HR activity and had no effects on NHEJ activity. Furthermore, quantitative RT-PCR analysis of key HR genes showed that KMT2C knockdown resulted in a moderate but significant down-regulation of BRCA2 (p < 0.05) and Rad51 (p < 0.01) expression in SKOV3 cells. Conclusion: Our results suggest that KMT2C partakes in the HR repair pathway in EOC. However, the contribution of KMT2C to HR activity is not rate-limiting. Based on the known function of KMT2C in histone modification, we speculate that KMT2C serves to positively regulate the transcription of HR genes and therefore promote HR activity. Future investigation into the exact roles of KMT2C in HR repair, etiology of EOC, and diagnostic biomarkers for the PARP inhibitor sensitivity of EOC is warranted. Citation Format: Mirielle C. Ma, Z. Ping Lin, Elena S. Ratner. The role of KMT2C in the homologous recombination repair in epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5606.

Abstract 6317: Developing a fully human heavy chain only Claudin-18.2-specific CAR-T cell therapy

Abstract Camelid VHH/nanobodies have been widely applied as the antigen binding domain of CAR-T cell therapies due to a smaller size and simpler structure compared to the traditionally used single chain variable fragment (scFv). Additionally, camelid VHH/nanobodies provide greater flexibility for dual-targeting CAR-T cell therapies. However, camelid VHH/nanobodies require laborious and costly humanization and still have a significant risk of inducing anti-drug antibodies when administered to patients. Transgenic mice exclusively harboring the most stable human VH genes (HCAb Harbour Mice®) provide one of the best solutions to get fully human heavy chain only antibodies (HCAbs). To efficiently identify lead HCAb VHs for Claudin-18.2-targeting CAR-T cells, we developed an innovative direct CAR-function based HCAb library screening platform. From Claudin-18.2-immunized HCAb Harbour Mice®, we constructed a total VH library in a CAR lentiviral vector that we developed in-house. After producing the lentiviral particle library, we transduced a Jurkat-NFAT-GFP reporter cell line and stimulated with Claudin-18.2 overexpression cell line. Subsequently, GFP positive reporter cells, representing Claudin-18.2-specific CARs, were isolated through FACS sorting, expanded, and further stimulated with Claudin-18.2-positive NUGC-4 tumor cells. Then, Next Generation Sequencing and abundance ranking was performed on VHs recovered from sorted CAR-reporter cells. Lead VH candidates were cloned into the CAR-P2A-RFP lentiviral vector, and many of these VH clones exhibited superior Claudin-18.2-specific functionality compared to a clinically-validated reference scFv in both reporter and primary CAR-T cells. Many lead VH clones also exhibited excellent developability as soluble HCAbs. These data suggest that this innovative method of combining our fully human HCAb and CAR-based library screening platform will provide new avenues for CAR-T cell therapies. Citation Format: Jason Noon, Yao Lu, Itishree Kaushik, Oyku Ay, Adam Luk, Bradley Delaney, David DeMeritt, Musheng Bao. Developing a fully human heavy chain only Claudin-18.2-specific CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6317.

Abstract 4659: A genome-wide CRISPR screen identifies ARID1A as a potential resistance marker of IDH1 inhibitor in IDH1-mutant cholangiocarcinoma cell

Abstract Background: Isocitrate dehydrogenase 1 (IDH1) mutations have been identified in approximately 20% of intrahepatic cholangiocarcinoma cases. These predominant gain-of-function mutations at codon 132 lead to abnormal production of the oncometabolite D-2-hydroxyglutarate. Targeted pharmacological disruption of the IDH1 mutation demonstrates tumor growth inhibition in IDH1-mutated cholangiocarcinoma. However, the efficacy of an IDH1 inhibitor (AG-120) as monotherapy for IDH1-mutated cholangiocarcinoma has been proven clinically insufficient, suggesting a need for investigating predictive biomarkers. In this study, we employed a genome-wide CRISPR screen to pursue resistance mechanisms to pharmacological IDH1 inhibition. Methods: The screening utilized SNU1079 cells harboring the IDH1 R132C mutation and the Brunello genome-wide CRISPR knockout (KO) pooled library (Addgene #73178, Broad Institute GPP, 4 guides/gene). The read couns of sgRNAs were normalized to reads per million, and then log2 was transformed. The log fold change (LFC) of each sgRNA of the AG-120-treated group was determined relative to that of the DMSO-treated group. Hypergeometric distribution was used to determine statistical significance. P values per gene were calculated as the average -log10(P value) of individual sgRNAs. We validated the CRISPR screening outcomes in vitro using both IDH1R132C mutant (SNU1079) and IDH1R132S mutant (RBE) cells by WST-1 assays, clonogenic assay and competition assay. (Tsujino T et al. Nat Commun. 2023) In competition assay, GFP was constitutively expressed in ARID1A-knockout cells and cultured together with ARID1A intact cells, under treatment with 1 µM AG120 or DMSO for 21 days. Relative cell numbers of GFP-positive (ARID1A-KO) cells in comparison with GFP-negative (ARID1A intact) cells were quantified using a BD LSRFortessa Analyzer (BD Biosciences). Results: Based on a volcano plot analysis, we identified three genes (SMCHD1, ARID1A, and PGP) with LFC > 1.5. Following a recent report of frequent ARID1A alteration (20%; 84/412) in a clinical study (Boerner, et al. Hepatology. 2021), we focused our research on ARID1A (LFC = 1.531; −log10 P = 3.75). WST-1 assays and clonogenic assays as short-term (3-7 days) assays failed to validate the results of CRISPR screening; however, in competition assays under treatment with AG120, cells with ARID1A-KO exhibited significantly greater proliferation in comparison with ARID1A intact cells in two different cell lines (SNU1079, RBE). Conclusions: The results demonstrated that ARID1A disruption reduced the efficacy of the IDH1 inhibitor against IDH1-mutatedcholangiocarcinoma cells. Based on these findings, we are planning comprehensive analyses employing in vivo models and clinical data to substantiate these observations. Citation Format: Tomohiro Kondo, Osamu Kikuchi, Yafeng Wang, Yukie Nakai, Tomomi Ida, Yang Cao, Trang H. Vu, Yuki Kondo, Tomohiko Sunami, Yoshihiro Yamamoto, Tomoki Saito, Shigeki Kataoka, Atsushi Yamada, Masashi Kanai, Shinya Ohashi, Manabu Muto. A genome-wide CRISPR screen identifies ARID1A as a potential resistance marker of IDH1 inhibitor in IDH1-mutant cholangiocarcinoma cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4659.

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells using GFP Based Reporter Systems.

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.

Engineered AAV2.7m8 Serotype Shows Significantly Higher Transduction Efficiency of ARPE-19 and HEK293 Cell Lines Compared to AAV5, AAV8 and AAV9 Serotypes.

The level of transduction efficiency of the target retinal cells affects the choice of AAV serotype and the outcome of gene replacement therapy for inherited retinal diseases. This study focused on the tropism and transduction efficiency of AAV2.7m8-, AAV5-, AAV8-, and AAV9-GFP in ARPE-19 and HEK293 cells. Fluorescence intensity was assessed bi-hourly by means of IncuCyte S3 live imaging microscopy. Within 12 h, AAV2.7m8 demonstrated the highest transduction efficiency at four viral concentrations of 1-, 3-, 6-, and 8 × 104 VG/cell in a dose-dependent manner, followed by AAV5 in ARPE-19 and AAV9 in HEK293 cells. The transduction efficiency of AAV2.7m8 at a dose of 6 × 104 VG/cell was 21, 202, and 323 times higher in ARPE-19 cells and 324, 100, and 52 times higher in HEK293 cells compared to AAV5, AAV8, and AAV9, respectively. This trend remained for 4 days at all viral concentrations, as additionally shown by flow cytometry. At a dose of 6 × 104 VG/cell, AAV2.7m8 (97% GFP-positive cells, GFP +) was nearly two and 10 times as efficient as AAV5 (52% GFP+) and AAV9 or AAV8 (both 9%), respectively, in ARPE-19 cells. In HEK293 cells, 95% of AAV2.7m8-, 26% of AAV9-, 17% of AAV8-, and 12% of AAV5-transduced cells were GFP-positive.

NXPE2 Is the Target of Ter-119 When Complexed with Gypa in Mice

Background: Red blood cells (RBCs) have a complex biology that allows regulation of metabolism, cellular rigidity, ion exchange, pH, and molecular communication with the vasculature. A central regulator of these biologies are macrocomplexes of integral membrane proteins that are both linked to cytoskeletal proteins and also differentially bind to metabolic enzymes based upon oxygenation status of the RBC. RBC surface staining, co-immunoprecipitation, and cross-linking proteomic approaches have generated a model of the macrocomplex encompassing Band 3, Rh, RhAG, CD47, glycophorin A, glycophorin B, and LW. TER-119 is a monoclonal antibody with exquisite specificity for the erythroid lineage in mice. However, the molecular target of TER-119 has remained unknown. TER-119 immunoprecipitates 4 specific protein bands, two of which are glycophorin A (GYPA) monomers or homodimers respectively; however, TER-119 is non-reactive to GYPA when assayed through Western blots and flow cytometry with erythroid cell lines expressing GYPA. These data led the originators of TER-119 to conclude it binds to a GYPA associated protein, but not GYPA itself. In contrast, others have concluded that TER-119 binds GYPA directly, and have inferred GYPA biology based upon TER-119 reactivity. Methods: TER-119 reactivity with peripheral blood RBCs from mice or from transfected HEK cells was determined by flow cytometry. Mice were genotyped using a dense array of 143,000 markers. Quantitative trait loci (QTL) analysis was carried out using TER-119 staining as a trait. cDNA for the open reading frames of the variants of interest in candidate genes or GYPA were cloned into eukaryotic expression vectors drive by the CMV promoter. HEK cells were transfected with expression vectors, in combination with a GFP expression plasmid. Cells were stained with TER-119 48 hours post transfection and analyzed by flow cytometry, gating on GFP positive cells to isolate the transfected population. Hematopoietic lineage expression of mRNA was obtained from the BloodSpot database. Results: The Diversity Outbred mice (DO mice) are an outbred population descended from 8 parental strains (A/J, C57BL/6J, 129S1/SvlmJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). While analyzing RBC biology phenotypes in DO mice, using TER-119 as a marker of RBCs, we serendipitously observed TER-119 non-reactivity in peripheral blood of 5 out of 550 DO mice. QTL analysis using TER-119 reactivity as a trait identified a highly significant QTL (genome-wide adjusted p-value <1.11e-16) driven by alleles specific to WSB/EiJ mice. Analysis of each of the parental strains confirmed that only peripheral blood from WSB/EiJ mice was non-reactive with TER-119. The QTL region contained 4 genes with missense mutations specific to the WSB/EiJ strain (HTR3a, HTR3b, NXPE2, and NXPE4). TER-119 was weakly reactive with HEK cells transfected with NXPE2 (but not the other genes). However, TER-119 became strongly reactive with HEK cells co-transfected with NXPE2 and GYPA (but not the other genes co-transfected with GYPA). There is no GYPA coding variation present in 8 DO parental strains. NXPE2 mRNA is highly expressed in the murine erythroid lineage with lower expression in B cells and granulocytes. Discussion: These data demonstrate that TER-119 reactivity is conferred by co-expression of NXPE2 and GYPA. NXPE2 is a single pass transmembrane protein and the weak reactivity of TER-119 with NXPE2 alone suggests that TER-119 recognizes an epitope on NXPE2 that is allosterically enhanced by complexing with GYPA. However, other binding scenarios cannot be ruled out. These data are important for three reasons. First, they identify NXPE2 as a hitherto unrecognized surface protein specific to the erythroid lineage in mice. Second, they raise the possibility that the models of the macrocomplex are incomplete. Third, these findings may allow re-interpretation of existing data that assumed TER-119 recognized GYPA. Additional studies are required to determine what function (if any) NXPE2 plays in erythropoiesis in mice and whether this function translates to human NXPE2 or a homologous gene.

Chimeric Antigen-Receptor (CAR)-Engineered Natural Killer (NK) Cells Targeting Chronic Myeloid Leukemia (CML) Blast Crisis

Background The use of tyrosine kinase inhibitors (TKIs) has dramatically modified the therapy of chronic myeloid leukemia (CML), generating durable remissions and prolonging survival in TKI-responders. However, progression to blast crisis (BC) still occurs especially in TKI-resistant patients and represents a clinical challenge. We and others have identified IL2RA/CD25 as a typical cell surface marker of BC-CML and reported that its overexpression is correlated with the progression of CML from CP-CML to BC-CML (Imeri et al, Cells 2023). Here we show the experimental development of a third-generation CAR-NK therapy strategy against the CD25 based on the scFV of the clinically approved monoclonal humanized antibody, Basiliximab. Methods: As NK cell model, we have used the NK92 cell line which has a well-established and clinically demonstrated NK cell activity. We have lentivirally transduced NK92 cells with the CAR construct containing a selectable gene (GFP). After FACS-sorting of GFP-positive cells, phenotypical characterization was performed by FACS. The expression of the CAR-CD25 at the surface of the cells was demonstrated an anti-Fab antibody and double-positive (Fab/GFP) cells were further purified. The functionality of the cells was evaluated using CD107a degranulation assay after 3h co-culture and ELISA for IFN-gamma release. We have in parallel engineered K562 cells expressing CD25 by lentiviral transduction (K562-CD25) as well as also a second target cell line (RAJI) using the same strategy. Annexin V staining of target K562 cells was used for in vitro cytotoxicity assessments. For in vivo assays, NSG mice were intraperitoneally (IP) injected with K562-CD25 cells expressing Luciferase at Day-3 (3.10 6 cells/mouse, n = 13). At Days 0, 3, and 7, mice were treated by IP injection of either irradiated CD25 CAR-NK92 cells (10 .10 6/mouse n=6) or irradiated Wild-type (WT)-NK92 cells (n=5). The clinical evolution of mice transplanted mice was followed weekly by luminescence (IVIS 200). Results: After cell sorting, we obtained more than 90% of double-positive NK92 CAR+/GFP+ cells. Lentiviral transduction did not affect the activatory or inhibitory signals of NK92 cells. No statistical differences were observed between CD25 CAR-NK92 and WT- NK92 cells for the expression of NKp30, NKp46, KirDl2-3, TIGIT, and DNAM. However, we observed a strong increase in the Granzyme B and Perforin in CD25 CAR-NK92 cells after co-culture with K562-CD25 as compared to WT NK92 cells (p<0.001). Importantly, we have found increased levels of degranulation after co-culture of target K562-CD25 with CD25-CAR-NK92 cells (40%) as compared to cells co-cultured with WT NK92 alone (20% ) suggesting strongly the occurrence of an additional specific effect due to CAR-CD25. IFN-gamma levels after co-culture of CAR CD25 NK92 cells were also found to be significantly increased ( 400 pg/ml) in as compared to co-cultures of target cells with WT-NK92 (200 pg/ml)(p<0.0001). Similarly,in vitro cytotoxicity assays showed induction of higher levels of apoptosis in target cells (K562-CD25 and Raji-CD25) when co-cultured with CD25 CAR-NK92 as compared to NK92 WT (p<0.0001). In in vivo experiments, we have analyzed K562-CD25 leukemia-bearing mice treated with CAR-NK92 cells (n=6) or WT-NK92 cells (n=5). These experiments analyzed at D+30 post-transplant showed stronger anti-leukemia effect of CAR-NK therapy by IVIS imaging with a survival rate of 84% for mice treated with CD25 CAR-NK92 versus 40% for those treated with WT-NK92. All control mice transplanted with K562-CD25 cells and left untreated died by D+20. Conclusion: We show here for the first time the potential use of an NK cell-mediated CAR therapy strategy targeting CD25 which has been shown to be upregulated in CML blast crisis. The experimental data show a significantly increased and selective in vitro and in vivo cytotoxicity of CD25 CAR-NK92 cells against CD25-expressing leukemia cells as compared to WT-NK92 cells. These results suggest that targeting CD25 by a CD25 CAR based on Basilixiamb's scFV might be an interesting tool in BC-CML and in all acute leukemias overexpressing CD25. In order to translate these findings to NK cells derived from induced pluripotent stem cells (iPSCs), we have produced iPSCs expressing CAR-CD25 constructs and experiments are underway to evaluate the therapeutic potential of iPSC-derived CD25 CAR-NK cells in CML blast crisis or AML models.

Identification of Epigenetic Modifiers Essential for Growth and Survival of AML1/ETO-Positive Leukemia

Introduction Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with direct or indirect dysregulation of epigenetic modifiers. Previously, we and others have shown that the AML1/ETO (RUNX1/MTG8) fusion protein, encoded by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes (Rejeski et al., Oncogene 2021) Aim We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Materials and methods In the customized shRNA library screen, 2009 shRNAs developed to target 671 genes were pool-cloned into a retroviral vector allowing doxycycline-inducible expression to generate a plasmid shRNA library (Antonova et al., Cell Reports 2019). We transduced the AML1/ETO-positive Kasumi-1 and SKNO-1 cell lines, selected with blasticidine, and the expression vectors were induced with doxycycline. After 12 days of doxycycline treatment, double-positive dsRed/GFP cells were FACS-sorted, sequenced and compared with FACS-sorted single GFP-positive cells from day 0. Single shRNAs against two selected genes of interest (DNMT1, ATR), were cloned and validated following the same protocol used for shRNA library screen. The 9/10/17-LacZ and 9/14/18-AML1/ETO clones were described previously (Duque-Afonso et al., 2011). Briefly, individual U937 clones were stably transduced with an ecdysone-inducible system for the expression of AML1/ETO (9/14/18-AML1/ETO) or LacZ gene (9/10/7-LacZ as control cells). Gene expression was induced by 5 uM ponasterone A (PonA). RNA was isolated, RNA libraries were prepared and sequenced. Bioinformatic analysis were performed on the European Galaxy instance platform. Results Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. Among them, several epigenetic regulators as DNMT1, ATR, DMAP1, SMYD1, SMYD2, BRD4, SETD8, SETD1A and HDAC8 were depleted. We validated, genetically and pharmacologically, DNMT1 and ATR using several AML1/ETO-positive (SKNO-1, Kasumi-1) and AML1/ETO-negative cell lines (OCI-AML3, HL60) with median IC50 for decitabine 74 nM (range 44-219), azacytidine 146 nM (range 14-267), berzosertib 219 nM (range 16-460) and ceralasertib 85 nM (range 42-328). We also demonstrated in vivo differentiation of myeloblasts after treatment with the DNMT1-inhibitor decitabine in a patient with an AML1/ETO-positive AML. To address the regulation of epigenetic modifiyer genes by AML1/ETO in an unbiased fashion, bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 1319 differentially expressed genes (DEGs) (822 up- and 497 downregulated). Four genes (DNMT1, PARP2, SMARCA4, and PRKCD) were both downregulated after AML1/ETO induction, and also detected in the survival shRNA screens of both cell lines as decreasing survival and proliferation. Conclusions To our knowledge we show here the first shRNA knock-down library screen for epigenetic modifiyer genes in AML1/ETO-positive cells. Using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. Some of these epigenetic regulators, such as DNMT1 and ATR, are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy, and confirmation studies (Baeten et al., ASH meeting 2022) appear warranted.

Identification of target cells of human papillomavirus 18 using squamocolumnar junction organoids.

Human papillomavirus 18 (HPV18) is a highly malignant HPV genotype among high-risk HPVs, characterized by the difficulty of detecting it in precancerous lesions and its high prevalence in adenocarcinomas. The cellular targets and molecular mechanisms underlying its infection remain unclear. In this study, we aimed to identify the cells targeted by HPV18 and elucidate the molecular mechanisms underlying HPV18 replication. Initially, we established a lentiviral vector (HPV18LCR-GFP vector) containing the HPV18 long control region promoter located upstream of EGFP. Subsequently, HPV18LCR-GFP vectors were transduced into patient-derived squamocolumnar junction organoids, and the presence of GFP-positive cells was evaluated. Single-cell RNA sequencing of GFP-positive and GFP-negative cells was conducted. Differentially expressed gene analysis revealed that 169 and 484 genes were significantly upregulated in GFP-positive and GFP-negative cells, respectively. Pathway analysis showed that pathways associated with cell cycle and viral carcinogenesis were upregulated in GFP-positive cells, whereas keratinization and mitophagy/autophagy-related pathways were upregulated in GFP-negative cells. siRNA-mediated luciferase reporter assay and HPV18 genome replication assay validated that, among the upregulated genes, ADNP, FHL2, and NPM3 were significantly associated with the activation of the HPV18 early promoter and maintenance of the HPV18 genome. Among them, NPM3 showed substantially higher expression in HPV-related cervical adenocarcinomas than in squamous cell carcinomas, and NPM3 knockdown of HPV18-infected cells downregulated stem cell-related genes. Our new experimental model allows us to identify novel genes involved in HPV18 early promoter activities. These molecules might serve as therapeutic targets in HPV18-infected cervical lesions.

Impact of rising seawater temperature on a phagocytic cell population during V. parahaemolyticus infection in the sea anemone E. pallida.

Climate change is increasing ocean temperatures and consequently impacts marine life (e.g., bacterial communities). In this context, studying host-pathogen interactions in marine organisms is becoming increasingly important, not only for ecological conservation, but also to reduce economic loss due to mass mortalities in cultured species. In this study, we used Exaiptasia pallida (E. pallida), an anemone, as an emerging marine model to better understand the effect of rising temperatures on the infection induced by the pathogenic marine bacterium Vibrio parahaemolyticus. The effect of temperature on E. pallida was examined at 6, 24, or 30 h after bath inoculation with 108 CFU of V. parahaemolyticus expressing GFP (Vp-GFP) at 27°C (husbandry temperature) or 31°C (heat stress). Morphological observations of E. pallida and their Hsps expression demonstrated heat stress induced increasing damage to anemones. The kinetics of the infections revealed that Vp-GFP were localized on the surface of the ectoderm and in the mucus during the first hours of infection and in the mesenterial filaments thereafter. To better identify the E. pallida cells targeted by Vp-GFP infection, we used spectral flow cytometry. E. pallida cell types were identified based on their autofluorescent properties. corresponding to different cell types (algae and cnidocytes). We identified an AF10 population whose autofluorescent spectrum was identical to that of human monocytes/macrophage, suggesting that this spectral print could be the hallmark of phagocytic cells called "amebocytes''. AF10 autofluorescent cells had a high capacity to phagocytize Vp-GFP, suggesting their possible role in fighting infection. This was confirmed by microscopy using sorted AF10 and GFP-positive cells (AF10+/GFP+). The number of AF10+/GFP+ cells were reduced at 31°C, demonstrating that increased temperature not only damages tissue but also affects the immune response of E. pallida. In conclusion, our study provides a springboard for more comprehensive studies of immune defense in marine organisms and paves the way for future studies of the dynamics, activation patterns, and functional responses of immune cells when encountering pathogens.

Cell uptake and intracellular trafficking of bioreducible poly(amidoamine) nanoparticles for efficient mRNA translation in chondrocytes.

Disulfide-containing poly(amidoamine) (PAA) is a cationic and bioreducible polymer, with potential use as a nanocarrier for mRNA delivery in the treatment of several diseases including osteoarthritis (OA). Successful transfection of joint cells with PAA-based nanoparticles (NPs) was shown previously, but cell uptake, endosomal escape and nanoparticle biodegradation were not studied in detail. In this study, C28/I2 human chondrocytes were transfected with NPs co-formulated with a PEG-polymer coating and loaded with EGFP mRNA for confocal imaging of intracellular trafficking and evaluation of transfection efficiency. Compared with uncoated NPs, PEG-coated NPs showed smaller particle size, neutral surface charge, higher colloidal stability and superior transfection efficiency. Furthermore, endosomal entrapment of these PEG-coated NPs decreased over time and mRNA release could be visualized both in vitro and in live cells. Importantly, cell treatment with modulators of the intracellular reducing environment showed that glutathione (GSH) concentrations affect translation of the mRNA payload. Finally, we applied a D-optimal experimental design to test different polymer-to-RNA loading ratios and dosages, thus obtaining an optimal formulation with up to ≈80% of GFP-positive cells and without toxic effects. Together, the biocompatibility and high transfection efficiency of this system may be a promising tool for intra-articular delivery of therapeutical mRNA in OA treatment.

Early revascularization activates quiescent dental pulp stem cells following tooth replantation in mice

IntroductionThe intentional perforation of the pulp chamber floor before tooth replantation promotes pulpal healing by facilitating the revascularization of the pulp cavity. This study aimed to elucidate the effects of this method on the dynamics of quiescent dental pulp stem cells (DPSCs). MethodsThe right and left maxillary first molars of Crlj:CD1 mice and TetOP-histone 2B (H2B)-green fluorescent protein (GFP) mice were extracted. The left molars were immediately replanted as the control group (CG), whereas the pulp chamber floor of the right molars were perforated before the tooth was replanted as the experimental group (EG). Immunohistochemistry for Nestin and GFP, and quantitative RT-PCR for Nestin, Opn, CD11c, and Oct3/4 mRNA were performed. ResultsThe rate of Nestin-positive perimeter along the pulp–dentin border in the EG tended to be higher than that of the CG at days 5 and 7 and was significantly increased between days 3 and 7. The rate of GFP-positive cells in the EG was significantly higher than that of the CG at days 5 and/or 7 in the mesial and middle coronal pulp. CD11c mRNA in the EG at day 5 was significantly higher than that of the CG and tended to be higher than that of the CG during the observation period. Oct3/4 mRNA expression in the EG was significantly higher than that of the CG at day 7. ConclusionsThe current experimental model demonstrated the promotion of the survival of DPSCs and their differentiation into odontoblast-like cells (OBLCs). Thus, the use of this model is expected to clarify the crosstalk mechanism between immune cells, including macrophages and dendritic cells, and DPSCs with regards to pulpal healing after tooth replantation. It also provides insight into the differentiation process of DPSCs into OBLCs.

Inhibition of Topors Ubiquitin Ligase Augments the Efficacy of DNA Hypomethylating Agents through DNMT1 Stabilization

DNA hypomethylating agents (HMAs) are commonly used to treat myeloid malignancies including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, their therapeutic effects are not satisfactory. We performed CRISPR-Cas9 knockout (KO) screening to identify molecular targets that enhance the efficacy of HMAs. MDS-L and MOLM-13 cells expressing Cas9 were infected with an sgRNA lentiviral library containing 12,409 sgRNAs targeting 1,383 epigenetic factors and exposed to low-dose HMAs, decitabine (DAC) or azacitidine (AZA), for 14 days. The read counts of sgRNA against TOPORS which encodes a ubiquitin/SUMO E3 ligase, were reproducibly decreased during culture specifically in the presence of HMAs in both screening. To validate the screening results, we performed competitive growth assays by co-culturing parental cells and TOPORS-KO cells using MOLM-13, MDS-L, SKK-1, and SKM-1 cells. In all cell lines, GFP-positive TOPORS-KO cells decreased over time only in the presence of HMAs. We established single-cell clones of TOPORS-KO MOLM-13 and MDS-L cells using two different sgRNAs. The cell growth of all TOPORS-KO clones was dramatically suppressed only in the presence of DAC. Mice transplanted with TOPORS-KO cells showed significantly longer survival when treated with DAC. Next, we analyzed Topors-deficient mice and no significant changes were observed in the peripheral blood or in the percentage of bone marrow (BM) hematopoietic stem and progenitor cells. Topors−/− BM cells successfully restored hematopoiesis in lethally irradiated recipient mice at levels comparable to those of WT BM cells after transplantation, suggesting that Topors is dispensable for normal hematopoiesis. Topors−/−MLL/AF9 leukemic cells showed comparable growth in the absence of DAC but significantly inferior growth in the presence of DAC compared to WT MLL/AF9 leukemic cells. We transplanted them with support BM cells into lethally irradiated recipient mice and treated with DAC. Topors−/− leukemic cells significantly decreased during treatment compared with WT leukemic cells. Enhanced cytotoxic effects of TOPORS KO combined with HMAs was closely associated with increased apoptosis. Cell cycle analysis revealed that among TOPORS-KO cells, those in the G2/M phase accumulate and those in the S phase are reduced upon DAC treatment. RNA sequencing on cells treated with DAC showed that gene sets associated with chromosome segregation and mitosis are significantly downregulated in TOPORS-KO cells. Addback experiment revealed that TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. To identify TOPORS ubiquitination substrates, we performed second CRISPR-Cas9 screening with the same library used as the first screening, using TOPORS-KO MDS-L and MOLM-13 clone cells. Both screening showed that UHRF1-DNMT1 axis is implicated in the augmented sensitivity to DAC. Consistently, DNMT1 remained more stable in TOPORS-KO cells than WT cells after DAC exposure not only in human MDS/AML cell lines but also in murine leukemic cells. HMAs incorporated into DNA trap DNMT1 and form DNA-DNMT1 crosslinks, which causes apoptosis and mitotic defects. DNMT1-DNA crosslink formation triggers the prominent SUMOylation of DNMT1 followed by ubiquitination, which promotes the resolution of crosslinks via proteasomal degradation and cell fitness. Mass analysis of ubiquitinated peptides and immunoprecipitation with Nanobody against DNMT1 demonstrated that TOPORS loss causes accumulation of SUMOylated DNMT1 due to inefficient ubiquitination, suggesting that TOPORS works as SUMO-targeted ubiquitin ligase for DNMT1. In patient samples treated with AZA (n=23), treatment resistance significantly correlated with TOPORS expression levels. Furthermore, an ubiquitination inhibitor TAK-243 as well as a SUMOylation inhibitor TAK-981 showed synergistic effect with HMAs through DNMT1 stabilization. These results suggested that they are likely to be promising therapeutic agents in clinical practice. Our findings unveil a novel mechanism of resistance to HMAs and provide an attractive therapeutic strategy for myeloid malignancies that interferes with resolution of DNA-DNMT1 crosslinks by targeting DNMT1 post-translational modification.