Transient Transfection Of Cells Research Articles

High-throughput cell optoporation system based on Au nanoparticle layers mediated by resonant irradiation for precise and controllable gene delivery

The development of approaches based on genetically modified cells is accompanied by a constant intensive search for new effective and safe delivery systems and the study of existing ones. Recently, we developed a new plasmonic nanoparticle layers-mediated optoporation system that can be proposed for precisely controlled, high-performance laser transfection compatible with broad types of cells and delivered objects of interest. The main goal of the present study is to demonstrate the broad possibilities and advantages of our system for optoporation of several mammalian cells, classified as "easy-to-transfect" cells, namely HeLa and CHO lines, and "hard-to-transfect" cells, namely A431 and RAW 264.7 cells. We show the efficient delivery of various sized cargo molecules: from small molecular dyes propidium iodide (PI) with molecular mass 700 Da, control plasmids (3–10 kb) to fluorophore-labeled dextranes with masses ranging from 10 kDa up to 100 kDa. The performance of optoporation was investigated for two types of laser sources, 800-nm continuous-wave laser, and 1064-nm ns pulsed laser. We provided a comparative study between our system and commercial agent Lipofectamine for transient transfection and stable transfection of HeLa cells with plasmids encoding fluorescent proteins. The quantitative data analysis using flow cytometry, Alamar blue viability assay, and direct fluorescence microscopy revealed higher optoporation efficacy for hard-to-transfect A431 cells and Raw 264.7 cells than lipofection efficacy. Finally, we demonstrated the optoporation performance at the single-cell level by successful delivering PI to the individual CHO cells with revealed high viability for at least 72 h post-irradiation.

Improved Manufacturing Methods of Extracellular Vesicles Pseudotyped with the Vesicular Stomatitis Virus Glycoprotein.

Extracellular vesicles (EV), which expose the vesicular stomatitis virus glycoprotein (VSVG) on their surface, are used for delivery of nucleic acids and proteins in human cell lines. These particles are biomanufactured using methods that are difficult to scale up. Here, we describe the development of the first EV-VSVG production process in serum-free media using polyethylenimine (PEI)-based transient transfection of HEK293 suspension cells, as well as the first EV-VSVG purification process to utilize both ultracentrifugation and chromatography. Three parameters were investigated for EV-VSVG production: cell density, DNA concentration, and DNA:PEI ratio. The best production titer was obtained with 3 × 106cells/mL, a plasmid concentration of 2µg/mL, and a DNA:PEI ratio of 1:4. The production kinetics of VSVG was performed and showed that the highest amount of VSVG was obtained 3days after transfection. Addition of cell culture supplements during the transfection resulted in an increase in VSVG production, with a maximum yield obtained with 2mM of sodium butyrate added 18h after transfection. Moreover, the absence of EV-VSVG during cell transfection with a GFP-coding plasmid revealed to be ineffective, with no fluorescent cells. An efficient EV-VSVG purification procedure consisting of a two-step concentration by low-speed centrifugation and sucrose cushion ultracentrifugation followed by a heparin affinity chromatography purification was also developed. Purified bioactive EV-VSVG preparations were characterized and revealed that EV-VSVG are spherical particles of 176.4 ± 88.32nm with 91.4% of protein similarity to exosomes.

Activation and characterization of G protein-coupled receptors for CHHs in the mud crab, Scylla paramamosain

Crustacean hyperglycemic hormone (CHH) superfamily peptides constitute a group of neurohormones, including the crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH), which reportedly play an essential role in regulating various biological activities by binding to their receptors in crustaceans. Although bioinformatics analyses have identified G protein-coupled receptors (GPCRs) as potential CHH receptors, no validation through binding experiments has been carried out. This study employed a eukaryotic expression system, HEK293T cell transient transfection, and ligand-receptor interaction tests to identify the GPCRs of CHHs in the mud crab Scylla paramamosain. We found that four GPCRs (Sp-GPCR-A34-A37) were activated by their corresponding CHHs (Sp-CHH1-v1, Sp-MIH, Sp-VIH) in a dose-dependent manner. Of these, Sp-GPCR-A34 was exclusively activated by Sp-VIH; Sp-GPCR-A35 was activated by Sp-CHH1-v1 and Sp-VIH, respectively; Sp-GPCR-A36 was activated by Sp-CHH1-v1 and Sp-MIH; Sp-GPCR-A37 was exclusively activated by Sp-MIH. The half-maximal effective concentration (EC50) values for all CHHs/GPCRs pairs (both Ca2+ and cAMP signaling) were in the nanomolar range. Overall, our study provided hitherto undocumented evidence of the presence of G protein-coupled receptors of CHH in crustaceans, providing the foothold for further studies on the signaling pathways of CHHs and their corresponding GPCRs.

Quantitative Analysis of Siglec Ligands by Flow Cytometry.

Siglecs (sialic acid-binding, immunoglobulin superfamily, lectins) are a family of transmembrane receptor-type glycan recognition proteins in vertebrates that are primarily expressed on leukocytes and regulate immune responses. Siglecs are involved in several diseases, such as cancer and neurodegenerative diseases. Most Siglecs suppress the activation of leukocytes by recognizing ligands containing sialic acid, a group of acidic sugars commonly found in vertebrate glycans, but rare among microbes. Siglec ligands are critical in the interaction between leukocytes and target cells. The abundance of the Siglec ligand is influenced by both the abundance of the glycoconjugate carrier (glycoprotein or glycolipid) and that of the terminal glycan epitope directly recognized by the Siglec. Therefore, a direct approach to evaluate the expression level of a Siglec ligand on cells of interest is to analyze the binding of recombinant Siglec protein to these cells. In this article, we describe a protocol for semi-quantitatively analyzing the expression level of Siglec ligands via flow cytometry using recombinant Siglec-Fc fusion protein. Support protocols describe how to remove sialic acids from the cell surface with sialidase under mild conditions to demonstrate the sialic acid dependence of Siglec binding, and the preparation of recombinant Siglec-Fc fusion proteins by transient transfection of mammalian cells. © 2023 Wiley Periodicals LLC. Basic Protocol: Quantitative analysis of Siglec ligands on mammalian cells via flow cytometry with recombinant Siglec-Fc fusion protein Support Protocol 1: Sialidase treatment of mammalian cells Support Protocol 2: Preparation of recombinant Siglec-Fc fusion protein via transient transfection of mammalian cells.

Replication and Expression of the Consensus Genome of Hepatitis B Virus Genotype C from the Chinese Population.

Hepatitis B virus (HBV) genotype C is a prevalent HBV genotype in the Chinese population. Although genotype C shows higher sequence heterogeneity and more severe liver disease than other genotypes, its pathogenesis and immunological traits are not yet fully elucidated. In this study, we first established and chemically synthesized the consensus sequence based on representative 138 full-length HBV genotype C genomes from the Chinese population. The pHBV1.3C plasmid system, containing a 1.3-fold full-length HBV genotype C consensus sequence, was constructed for subsequent validation. Next, we performed functional assays to investigate the replicative competence of pHBV1.3C in vitro through the transient transfection of HepG2 and Huh7 cells and validated the in vivo function via a hydrodynamic injection to BALB/c recipient mice. The in vitro investigation revealed that the extracellular HBV DNA and intracellular replicative intermediate (i.e., pregenomic RNA, pgRNA) were apparently measurable at 48 h, and the HBsAg and HBcAg were still positive in hepatoma cells at 96 h. We also found that HBsAg and HBeAg accumulated at the extracellular and intracellular levels in a time-dependent manner. The in vivo validation demonstrated that pHBV1.3C plasmids induced HBV viremia, triggered morphological changes and HBsAg- or HBcAg- positivity of hepatocytes, and ultimately caused inflammatory infiltration and focal or piecemeal necrosis in the livers of the murine recipients. HBV protein (HBsAg) colocalized with CD8+ T cells or CD4+ T cells in the liver. F4/80+ Kupffer cells were abundantly recruited around the altered murine hepatocytes. Taken together, our results indicate that the synthetic consensus sequence of HBV genotype C is replication-competent in vitro and in vivo. This genotype C consensus genome supports the full HBV life cycle, which is conducive to studying its pathogenesis and immune response, screening novel antiviral agents, and further optimizing testing and therapeutics.

Erratum: High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension.

An erratum was issued for:High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension. The Authors section was updated. from: Ganesh P. Subedi1 Roy W. Johnson2 Heather A. Moniz2 Kelley W. Moremen2 Adam Barb1 1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 2Complex Carbohydrate Research Center, University of Georgia to Ganesh P. Subedi1 Roy W. Johnson2 Heather A. Moniz2 Kelley W. Moremen2 Adam W. Barb1 1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 2Complex Carbohydrate Research Center, University of Georgia.

Schwann cell derived-peroxiredoxin protects motor neurons against hydrogen peroxide-induced cell death in mouse motor neuron cell line NSC-34

Schwann cells and oligodendrocytes secrete proteins that promote neuron survival, but their role in amyotrophic lateral sclerosis (ALS) is unclear. To address this question, we evaluated the effect of molecules secreted by Schwann cells on reactive oxygen species (ROS)-induced motor neuronal cell death. We observed that in motor neuron cell line NSC-34 cultures, the conditioned medium (CM) from Schwann cell line YST-1 (YST-1 CM) cultures had a protective effect against hydrogen peroxide-induced cell death. However, this protective effect of YST-1 CM was abolished by removing peroxiredoxin 1–4 (PRDX1–4) from the CM. We found that the expression of PRDX1 mRNA was markedly downregulated in the lumbar spinal cord of the superoxide dismutase 1 (SOD1)G93A mouse model of ALS. We also found that transient transfection of YST-1 cells with G93A SOD1 resulted in reduced PRDX1 mRNA expression. Additionally, in the mutant transfected cells, YST-1 CM showed decreased neuroprotective effect against hydrogen peroxide-induced NSC-34 cell death compared to those transfected with WT SOD1. Our results suggest that Schwann cells protect motor neurons from oxidative stress by secreting PRDX1 and that the reduction of PRDX secreted from Schwann cells contributes to increased ROS and associated motor neuronal death in ALS.

Efficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM

CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor templates that enable efficient tagging of endogenous proteins with affinity tags by transient transfection and selection of genome-edited cells in various human cell lines. Combined with technological advancements in single-particle cryogenic electron microscopy, this strategy allows efficient structural studies of endogenous proteins captured in their native cellular environment and during different cellular processes. We demonstrated this strategy by tagging six different human proteins in both HEK293T and Jurkat cells. Moreover, analysis of endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293T cells allowed us to follow its behavior spatially and temporally in response to prolonged oxidative stress, correlating the increased number of oxidation-induced inactive catalytic sites in GAPDH with its translocation from cytosol to nucleus.

Transcriptional activity of the long control region in human papillomavirus type 33 intratype variants

BackgroundHigh-risk human papillomaviruses (HPVs) are responsible for the development of cervical and other anogenital cancers. Intratype sequence variants of certain high-risk HPV types (e.g. 16, 18 and 31) are thought to have different oncogenic potential, partly due to nucleotide sequence variation in the viral long control region (LCR). The LCR has an important role in the regulation of viral replication and transcription. The purpose of this study was to explore sequence variation in the LCR of HPV 33 intratype variants in Hungary and to see whether there are differences in the transcriptional activities of the variants.MethodsThe complete HPV 33 LCR was amplified from HPV 33 positive cervical samples. After sequencing the LCR variants, multiple sequence alignment and phylogenetic analyses were carried out. Representative HPV 33 LCR sequence variants were selected for cloning and functional analysis. After transient transfection of HeLa cells, luciferase reporter assays were used to analyse the transcriptional activities of different LCR variants.ResultsAltogether 10 different variants were identified by sequence analysis of the HPV 33 LCR. The results of phylogenetic analysis showed that 3 variants belonged to sublineage A1, while the other 7 variants clustered with sublineage A2. Variants belonging to sublineage A2 had significantly lower transcriptional activities than variants belonging to sublineage A1. Within sublineage A2, the two variants analysed had significantly different transcriptional activities, which was shown to be caused by the A7879G variation.ConclusionsNucleotide variation in the HPV 33 LCR can result in altered transcriptional activity of the intratype variants. Our results can help to understand the correlation between LCR polymorphism and the oncogenic potential of HPV 33 variants.

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix.

BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aquaporin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic analyses suggested that the sequences 434-452 were α-helical and amphipathic. By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an intrinsically disordered to a more α-helical conformation for the residues 434-467. Recombinantly produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as determined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non-lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compartments, such as the nuclear and mitochondrial membranes, were negative. The N-terminal myristoylation of the amphipathic helix appeared not to change either the lipid affinity or membrane localisation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it did appear to enhance its helical content. These data support the conclusion that C-terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane binding properties via an adjacent amphipathic helix.

Serum-free lentiviral vector production is compatible with medium-resident nuclease activity arising from adherent HEK293T host cells engineered with a nuclease-encoding transgene

At present lentiviral vector production for cell and gene therapy commonly involves transient plasmid transfection of mammalian cells cultivated in serum-containing media and addition of exogenous nuclease to reduce host cell and plasmid DNA impurities. Switching from serum-containing media to chemically-defined, serum free media, and minimising the number of process additions, are both increasingly regarded as necessary steps for simplifying and potentially automating lentiviral vector bioprocessing in future. Here we adapted human embryonic kidney 293T (HEK293T) cells to grow in serum-free media and also modified these cells with transgenes designed to encode a secreted nuclease activity. Stable transfection of HEK293T cells with transgenes encoding the Staphylococcus aureus nuclease B (NucB) open reading frame with either its native secretion signal peptide, the murine Igκ chain leader sequence or a novel viral transport fusion protein, all resulted in qualitatively detectable nuclease activity in serum-free media. Serum-free transient transfection of human embryonic kidney HEK293T cells stably harbouring the transgene for NucB with its native secretion signal produced active lentivirus in the presence of medium-resident nuclease activity. This lentivirus material was able to transduce the AGF-T immortal T cell line with a green fluorescent protein reporter payload at a level of 2.05 × 105 TU/mL (±3.34 × 104 TU/mL). Sufficient nuclease activity was present in 10 μL of this unconcentrated lentivirus material to degrade 1.5 μg DNA within 2 h at 37 °C, without agitation - conditions compatible with lentivirus production. These observations demonstrate that lentiviral vector production, by transient transfection, is compatible with host cells harbouring a nuclease transgene and evidencing nuclease activity in their surrounding growth media. This work provides a solid basis for future investigations, beyond the scope of this present study, in which commercial and academic groups can apply this approach to therapeutic payloads and potentially omit exogenous nuclease bioprocess additions.

Germline C1GALT1C1 mutation causes a multisystem chaperonopathy

Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase (C1GALT1), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal C1GALT1C1. Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in C1GALT1C1. They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type C1GALT1C1 partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients.

A novel dual-plasmid platform provides scalable transfection yielding improved productivity and packaging across multiple AAV serotypes and genomes

Transient transfection of mammalian cells using plasmid DNA is a standard method to produce adeno-associated virus (AAV) vectors allowing for flexible and scalable manufacture. Typically, three plasmids are used to encode the necessary components to facilitate vector production; however, a dual-plasmid system, termed pDG, was introduced over 2 decades ago demonstrating two components could be combined resulting in comparable productivity to triple transfection. We have developed a novel dual-plasmid system, pOXB, with an alternative arrangement of sequences that results in significantly increased AAV vector productivity and percentage of full capsids packaged in comparison to the pDG dual design and triple transfection. Here, we demonstrate the reproducibility of these findings across seven recombinant AAV genomes and multiple capsid serotypes as well as the scalability of the pOXB dual-plasmid transfection at 50-L bioreactor scale. Purified drug substance showed a consistent product quality profile in line with triple-transfected vectors, except for a substantial improvement in intact genomes packaged using the pOXB dual- transfection system. Furthermore, pOXB dual- and triple-transfection-based vectors performed consistently invivo. The pOXB dual plasmid represents an innovation in AAV manufacturing resulting in significant process gains while maintaining the flexibility of a transient transfection platform.

MiR‑4732‑5p promotes ovarian cancer mobility by targeting MCUR1.

MicroRNAs (miRNAs/miRs) play critical roles in tumor progression. However, the role of miR-4732 and its underlying molecular mechanism in ovarian cancer (OC) remain unclear. In the present study, the high expression of miR-4732 was confirmed to be associated with the mortality of patients with OC following surgery, according to The Cancer Genome Atlas Ovarian Cancer database (TCGA-OV). Additionally, the expression of miR-4732 was positively associated with an increased tendency to exhibit an early TNM stage (IIA, IIB and IIC) of OC, indicating its promotional role in the early stages of tumorigenesis. By performing in vitro gain-of-function experiments, the transient transfection of IGROV1 cells with miR-4732-5p mimics enhanced cell viability according to Cell Counting Kit-8 assay, and cell migration and invasion in Transwell assays. However, though the application of loss-of-function experiments, the transient transfection of IGROV1 cells with miR-4732-5p inhibitors hindered cell viability, cell migration and invasion in vitro. Mitochondrial calcium uniporter regulator 1 (MCUR1) was validated as a downstream direct target of miR-4732-5p through bioinformatics analysis, western blotting and luciferase assays. Therefore, the results of the present study provide evidence that miR-4732-5p may promote OC cell mobility through the direct targeting of the tumor suppressor, MCUR1.

Abstract 3916: role of her3 v104l mutation on tumor growth and her3 stabilization

Abstract We aimed to determine if naturally occurring HER3 mutations could drive oncogenic activity of HER2+ HCC1569 cells in which endogenous HER3 was knocked out using CRISPR-Cas9 technology. A series of HER3 mutations found in breast cancer patients (F94L, V104L, G284R, D297Y, T355I, and E928G) were introduced via lentiviral transduction and stable cell lines were generated in HER3 knock out (KO) HCC1569 cells and were maintained in 0.5 mg/mL puromycin. Our data indicated that cells stably transduced with HER3V104L mutation had significantly higher cell growth with higher p-HER3 expression versus wild-type (wt) and empty-vector (EV) HER3. We found that HCC1569HER3KO cells with WT and V104L were sensitive to increasing concentration of neratinib (0.1-0.5 µM) as indicated by crystal violet assay. Next we examined if V104L mutation rendered resistance to another FDA- approved irreversible HER2 tyrosine kinase inhibitor, tucatinib. Our results showed that V104L cells were sensitive to higher concentration of tucatinib compared to neratinib. In other experiments, we used COS7 cells to examine the signaling pathways of HER3V104L mutation. Our western blot data demonstrated that transient transfection of COS7 cells with HER3V104L mutant significantly upregulated p-HER3 and p-HER2 expression versus WT HER3 in a NRG- dependent manner. In addition, we found that the V104L mutation stabilizes HER3 protein expression independent of ligand stimulation. Our western blot data showed that V104L induced HER3 stabilization is independent on HER3 binding partners HER1, HER2 and HER4. Our cycloheximide chase data indicated that V104L mutation stabilizes HER3 expression in COS7 cells as well as low HER3 expressing colon cancer cell line, SNUC5. Our western blot data indicates that V104L mutation activates ERK signaling in NRG dependent manner over AKT pathway in COS7 cells. In addition, we are using various PDXs with different HER3 mutations (V104M and E928D) to determine the role of these driver HER3 mutations in cancer and how this can be targeted in the clinic using HER targeted therapy. Citation Format: Rosalin Mishra, Mary Kate Kilroy, Wasim Feroz, Hima Patel, Samar Alanazi, Joan T. Garrett. role of her3 v104l mutation on tumor growth and her3 stabilization. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3916.

Induced expression of miR-1250-5p exerts tumor suppressive role in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and it has a prevalence rate of 15%-20% among all breast cancer cases in younger women. Still, the underlying molecular mechanisms of its pathogenesis are not entirely understood. In the previous study, we identified that microRNA (miR)-1250-5p is significantly down-expressed in TNBC cells. Thus, in the present study, we explore the functional anticancer role of miR-1250-5p in the transient mimic transfected TNBC cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to examine the effect of miR-1250-5p on cell viability of TNBC (MDA-MB-231 and MDA-MB-453) cells. The confocal microscopy, quantitative real-time polymerase chain reaction, and western blot analysis techniques were used to assess the effect of miR-1250-5p on cancer hallmarks in test cells. Induced miR-1250-5p expression in MDA-MB-231 and MDA-MB-453 cells decreased cell viability in a time-dependent manner. Increased miR-1250-5p expression levels significantly decreased cell cycle G1/S phase transition markers (Cyclin D1 and CDK4) at messenger RNA (mRNA) and protein levels in TNBC cells compared to scrambled sequence transfected cells. Transient transfection of TNBC cells with miR-1250-5p mimic increased apoptosis in TNBC cells by increasing the level of active caspase (Caspase 8 and Caspase 3) of the intrinsic pathway. Apoptosis-related morphological changes were also observed in the test cells. Further, the induced expression of miR-1250-5p significantly decreased epithelial-mesenchymal transition (EMT) by altering the mRNA and protein levels of E-cadherin and Vimentin. Moreover, results of confocal microscopy revealed increased reactive oxygen species generation, and decreased mitochondria membrane potential in miR-1250-5p mimic transient transfected TNBC cells. In conclusion, miR-1250-5p acts as tumor suppressor in TNBC cells and its induction by therapeutics might be a novel strategy for the disease treatment.

In Vivo Crispr Screening Identifies Novel Vulnerabilities in ETO2/GLIS2-rearranged Leukemia

Background: CRISPR screening has allowed for massively parallel interrogation of the genome for vulnerabilities in a variety of cancer models. However, these experiments are largely performed in vitro due to the scale and conditions required to effectively conduct these screens. Furthermore, there still remain genetic subtypes of acute leukemia which are poorly represented among existing cell lines. We sought to determine the feasibility of conducting screens with focused libraries in patient-derived leukemia xenografts. Methods: We utilized a patient-derived acute megakaryoblastic leukemia (AMKL) bearing an ETO2/GLIS2 fusion, representing an aggressive subtype of pediatric acute leukemia with limited comparable cell lines (Gruber TA, et al. Cancer Cell 2012.) Based on its transplantability in NOD.scid.Il2Rγcnull-SGM3 (NSGS) mice vs culture in vitro, we performed all subsequent experiments in vivo. We first sought to establish stable Cas9 expression, which we successfully achieved by lentiviral transduction followed by FACS-enriched transplant, followed by a second round of FACS enrichment with limiting dilution transplant. As judged by mCherry flow cytometry, we achieved highly pure (>90%) Cas9-expressing populations, which we verified by western blotting. We further verified functionality of these cells by verifying cutting at the safe harbor locus AAVS1. In order to identify molecular vulnerabilities in this system, we identified the top 100 differentially expressed genes (DEG) between ETO2/GLIS2-rearranged leukemias versus other leukemia PDXs which we previously characterized. We included eight transcription factors with motifs enriched in our DEG analysis, as well as eight pan-essential positive controls (such as MYC, BRD4). Four guide RNAs (gRNA) per gene were selected from the Broad Brunello genome-wide library (Doench JG, et al. Nat Biotechnol 2016) as well as 50 "safe targeting" control guides (Morgens DW, et al. Nat Commun 2017) directed against non-essential genomic regions for a total of 518 guides. Guides were ordered as pooled oligonucleotides, cloned into a lentiviral gRNA backbone using standard techniques, then used to prepare lentivirus via transient transfection of 293T cells followed by titration to achieve an MOI of 0.3. With this MOI and an approximate engraftment frequency of 2% following intrafemoral injection, we transplanted 8.7e7 cells across eight NSGS mice to achieve adequate starting representation of our pooled library. Mice were sacrificed at humane endpoints, and spleens and bone marrow underwent PCR amplification for gRNA sequences followed by Illumina deep sequencing, and analysis with MAGeCK-VISPR (Wei L, et al. Genome Biol 2015). Results: One mouse per cohort was sacrificed at 24 hours post-transplant and sequenced for gRNA representation, which confirmed that library representation was largely preserved with our approach. At the time of sacrifice, spleen and bone marrow samples were independently sequenced, but no major bias in guide dropouts was observed between these compartments. Subsequent analyses were performed on the marrow alone. We observed the dropout of 38 genes with a false discovery rate < 0.25, including all positive control gRNAs as well as targets previously described to be essential through our own orthogonal experiments (BCL2) or previously published findings (GLIS2, GATA3) (Thirant C et al. Cancer Cell 2017). Among transcription factors found to be essential, we identified several which had not previously been strongly associated with acute leukemia (FOXD4L1, ZNF740). Conclusion: Patient-derived xenografts represent a unique platform for targeted functional genomic interrogation of acute leukemias. These screens can be performed with a tractable number of animals and modest sequencing requirements. Additional experiments are ongoing to identify the mechanistic implications of the vulnerabilities described here in ETO2/GLIS2-rearranged AMKL.

Transfection reagent artefact likely accounts for some reports of extracellular vesicle function.

Extracellular vesicles (EV) are important mediators of cell communication and physiology. EVs are frequently investigated by transiently transfecting cells with plasmid DNA to produce EVs modified with protein(s) or nucleic acid(s) of interest. DNA‐transfection reagent complexes (DTC) are approximately the same size as EVs, raising the possibility that some purification procedures may fail to separate these two species and activity arising from carryover DTC may be improperly attributed to EVs. We find that differential ultracentrifugation, a commonly employed EV isolation procedure, does not separate EVs from DTC present in the cell culture supernatant of transiently transfected cells. We demonstrate that the biological activity of an EV‐directed Cre recombinase is due to contaminating plasmid DNA and not EV‐mediated delivery of Cre protein. Moreover, steps commonly taken to remove plasmid DNA from EV samples, such as media exchanges and treatment with nucleases, are ineffective at avoiding this artefact. Due to the pernicious nature of plasmid DNA in these cellular assays, some reports of EV function are likely artefacts produced by contaminating DTC. EVs and DTC can be separated by density gradient ultracentrifugation, highlighting the importance of validating elimination of DTC when using transient transfection of EV‐producing cells to interrogate EV function.

Circulating extracellular vesicle-encapsulated microRNA-557 induces a proinflammatory immune response and serves as a diagnostic or relapse marker in autoimmune hepatitis.

This study investigated serum microRNAs (miRNAs/miRs) in autoimmune hepatitis (AIH) and the relationship of these molecules with diagnostic and relapse markers. Initially, extracellular vesicle-encapsulated miRNAs (EV-miRNAs) in serum with altered expression in AIH relative to healthy control (HC) samples were identified using microarray analysis. To validate the microarray results, the expression levels of selected EV-miRNAs were determined. Among the 2569 mature miRNAs evaluated in the microarray, EV-miR-557 discriminated patients with AIH from healthy controls (HCs). Validation by digital polymerase chain reaction indicated that serum EV-miR-557 levels were higher in patients with AIH (7.75 copies/μl) than in patients with non-alcoholic steatohepatitis (1.60 copies/μl; p<0.001), patients with primary biliary cholangitis (2.16 copies/μl; p<0.005), and HCs (1.86 copies/μl; p<0.005). The area under the receiver operating characteristic curve values for the probability of AIH using serum EV-miR-557 between the AIH and non-alcoholic steatohepatitis, AIH and primary biliary cholangitis, and AIH and HC groups were 0.81, 0.78, and 0.79, respectively. In addition, serum EV-miR-557 levels >7.69 copies/μl were associated with a significantly higher risk of relapse in patients with AIH (7-year incidence rate: 11.1 vs. 35.4%, log-rank test, p<0.05). Interestingly, gene expression analysis revealed that increased miR-557 expression following transient transfection of peripheral blood mononuclear cells with a miR-557 mimic resulted in enhanced expression of proinflammatory cytokine-related genes such as interleukin-6, interferon-γ, and tumor necrosis factor. Moreover, miR-557 induced significant tumor necrosis factor-α production (mean: 313.5 vs. 10642.3 pg/ml, p<0.05). EV-miR-557 may play an important role as a potential biomarker of AIH and may be a promising therapeutic target for AIH.

Abstract 5412: Role of her3 mutations on breast cancer oncogenesis

Abstract We sought to determine if naturally occurring mutations in HER3 could drive oncogenic growth of HER3KO HER2+ HCC1569 cells in which endogenous HER3 has been eliminated via CRISPR-Cas9. A series of HER3 mutations identified in breast cancer patients (F94L, V104L, G284R, D297Y, T355I, and E928G) were introduced using lentiviral transduction and stable cell lines were generated in HER3KOHCC1569 cells via puromycin selection. We identified HER3V104L mutation to have higher cell proliferation and higher p-HER3 expression compared to wild-type (wt) and empty-vector (EV) HER3. We observed that HCC1569HER3KO cells stably expressing WT and V104L were sensitive to increasing doses of neratinib (0.1-0.5 µM) concentration. Next we analyzed if this mutation rendered resistance to the recently FDA- approved irreversible HER2 tyrosine kinase inhibitor, tucatinib. Our data indicated that both that V104L cells were sensitive to higher concentration of tucatinib compared to neratinib. In parallel experiments, we utilized COS7 cells to examine the signaling properties of HER3V104L. Our data indicated that transient transfection of COS7 cells with HER3V104L mutant significantly induces p-HER3/p-AKT and p-HER2 expression compared to WT HER3 in a ligand dependent manner. In addition, we observed that the V104L mutation stabilizes HER3 protein expression independent of HER2 and ligand stimulation. Experiments are ongoing to determine whether V104L induced HER3 stabilization and downstream signaling activation is dependent on HER3 binding partners including EGFR, HER2 or HER4. We also aim to understand how the V104L mutation stabilizes HER3 expression. Structural modeling of V104L mutation will provide insight about the mechanism of stabilization of the HER3 protein. We will use MCF10A and HEK293 cells to determine the effect of the V104L mutation on HER3 expression and downstream signaling. We also aim to decipher the signaling mechanism that drives the oncogenic potential of V104L mutation. In addition, we are using various PDXs with different HER3 mutations to determine other driver HER3 mutations in breast cancer and how this can be targeted in the clinic using HER targeted therapy. Citation Format: Rosalin Mishra, Mary Kate Kilroy, Hima Patel, Samar Alanazi, Joan T. Garrett. Role of her3 mutations on breast cancer oncogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5412.