<meta charset="utf8"></head><body>מסכי CRISPR רחבי גנום בתאי CART לחקר עמידות לטיפול

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<table><tbody><tr><td>293T cells</td><td>ATCC</td><td>CRL-3216</td><td>Cells used for lentivirus production</td></tr><tr><td>Biotin ProteinL Antibody</td><td>GenScript</td><td>M00097</td><td>anti-kappa chain antibody for CAR detection</td></tr><tr><td>Bovine Serum Albumin</td><td>Millipore Sigma</td><td>A7906</td><td></td></tr><tr><td>Carbenicillin disodium salt</td><td>Millipore Sigma</td><td>C1389-1G</td><td>Carbenicillin antibiotic</td></tr><tr><td>CD4 Isolation Beads</td><td>Miltenyi Biotec</td><td>130-045-101&amp;nbsp;</td><td></td></tr><tr><td>CD8 Isolation Beads</td><td>Miltenyi Biotec</td><td>130-045-201</td><td></td></tr><tr><td>CTS (Cell Therapy Systems) Dynabeads CD3/CD28</td><td>Gibco</td><td>40203D</td><td></td></tr><tr><td>Cytoflex</td><td>Beckman Coulter</td><td>NC2279958</td><td></td></tr><tr><td>DNase-Free Water</td><td>Invitrogen</td><td>AM9937</td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle&#039;s medium (DMEM)</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>EasySep Human T Cell Isolation Kit</td><td>STEMCELL Technologies</td><td>17951RF</td><td>Negative isolation kit</td></tr><tr><td>Endura Electrocompetent Cells&amp;nbsp;</td><td>Biosearch Technologies</td><td>60242-1</td><td>Electrocompetent cells with recovery medium</td></tr><tr><td>Ethanol</td><td>Millipore Sigma</td><td>E7023</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>GeCKO v2 CRISPR Knockout Pooled Library A</td><td>AddGene</td><td>1000000048</td><td>CRISPR library plasmid</td></tr><tr><td>Gene Pulser II&amp;nbsp;</td><td>Bio-Rad</td><td>165-2105</td><td>Electroporator</td></tr><tr><td>Glycogen</td><td>Millipore Sigma</td><td>10901393001</td><td></td></tr><tr><td>JeKo-1</td><td>ATCC</td><td>CRL-3006</td><td>CD19&lt;sup&gt;+&lt;/sup&gt; target cells&amp;nbsp;</td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>ThermoFisher Scientific</td><td>L3000075</td><td>Transfection reagent kit with a transfection reagent (Lipofectamine 3000 Reagent) and a neutral co-lipid reagent (p3000)</td></tr><tr><td>LIVE/DEAD Aqua</td><td>Invitrogen</td><td>L34966</td><td></td></tr><tr><td>Lymphoprep</td><td>STEMCELL Technologies</td><td>7851</td><td>Density gradient medium</td></tr><tr><td>Machery-Nagel NucleoBond Xtra Maxi Kits</td><td>ThermoFisher Scientific</td><td>12748412</td><td>Maxi-prep kit</td></tr><tr><td>NEBNext High-Fidelity 2X PCR MasterMix</td><td>New England BioLabs</td><td>M0541S</td><td>High fidelity PCR mastermix</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985-070</td><td>Reduced serum medium</td></tr><tr><td>pCMVR8.74</td><td>AddGene</td><td>22036</td><td>Lentiviral packaging plasmid</td></tr><tr><td>Pennicillin-streptomycin-glutamine (100X)</td><td>Life Technologies</td><td>10378-016</td><td></td></tr><tr><td>pMD2.G</td><td>AddGene</td><td>12259</td><td>VSV-G envelope expressing plasmid</td></tr><tr><td>Pooled Human AB Serum&amp;nbsp;</td><td>Innovative Research</td><td>ISERABHI</td><td></td></tr><tr><td>Puromycin</td><td>Millipore Sigma</td><td>P8833</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>Gek extraction kit</td></tr><tr><td>Qucik-DNA Midiprep Plus Kit</td><td>Zymo Research</td><td>D4075</td><td>Kit used to isolate gDNA</td></tr><tr><td>RoboSep-S</td><td>STEMCELL Technologies</td><td>21000</td><td>Automated cell separator</td></tr><tr><td>Roswell Park Memorial Institute 1640 Medium (RPMI)</td><td>Gibco</td><td>21870092</td><td></td></tr><tr><td>SepMate-50</td><td>STEMCELL Technologies</td><td>85450</td><td>Density gradient separation tube</td></tr><tr><td>Sodium Acetate</td><td>Invitrogen</td><td>AM9740</td><td></td></tr><tr><td>Sodium Azide</td><td>Fisher Scientific</td><td>71448-16</td><td></td></tr><tr><td>Streptavidin Antibody (PE)</td><td>BioLegend</td><td>405203</td><td>Secondary antibody used for CAR detection</td></tr><tr><td>T100 Thermal Cycler</td><td>Bio-Rad</td><td>1861096</td><td></td></tr><tr><td>Ultracentrifuge (Optima XPN-80)</td><td>BeckmanCoulter</td><td>A99839</td><td></td></tr><tr><td>Vacuum Filter Systems, 0.22 &amp;micro;m</td><td>ThermoFisher Scientific</td><td>567-0020</td><td></td></tr><tr><td>Vacuum Filter Systems, 0.45 &amp;micro;m</td><td>ThermoFisher Scientific</td><td>165-0045</td><td></td></tr><tr><td>X-VIVO 15 Serum-Free Hematopoietic Cell Medium</td><td>Lonza</td><td>04-418Q</td><td>Hematopoietic cell medium</td></tr></tbody></table>

performing an in vitro genome-wide crispr knockout screen in chimeric antigen receptor t cells

<meta charset="utf8"></head><body>ביצוע מסך CRISPR רחב גנום במבחנה בתאי T של קולטן אנטיגן כימרי

ביצוע מסך CRISPR רחב גנום במבחנה בתאי T של קולטן אנטיגן כימרי

Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, ...

performing-an-vitro-genome-wide-crispr-knockout-screen-chimeric

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells

729 Views.  Mayo Clinic. המאמר מתאר פרוטוקול ליישום מודל in vitro לתשישות כדי להשלים מסך נוקאאוט CRISPR כלל גנומי בתאי T קולטן אנטיגן כימרי בריא מתורם. 

Video: ביצוע מסך CRISPR רחב גנום במבחנה בתאי T של קולטן אנטיגן כימרי

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

ברמת הגנום RNAi ההקרנה כדי לזהות גורמים מארח לווסת את הטיפול Oncolytic וירוס

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

ב- Rechallenge תא הגידול חוץ גופית להערכה חזוי של Chimeric אנטיגן קולטן תא T פונקציה Antitumor

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

הערכת הרג כמותי של תאי גידול על ידי תאי Jurkat המבטאים CAR

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

מחבר זרקור: סינון בתפוקה גבוהה של מבני תאי T CAR עבור ציטוטוקסיות משופרת וזיכרון חיסוני 

A protocol to evaluate quantitative tumor cell killing by Jurkat cells expressing chimeric antigen receptor (CAR) targeting single tumor antigen. This protocol can be used as a screening platform for rapid optimization of CAR hinge constructs prior to confirmation in peripheral blood-derived T cells.

הערכת ציטוטוקסיות מהירה במבחנה של קולטן אנטיגן כימרי Jurkat באמצעות הדמיה פלואורסצנטית

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable ...

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.

יעיל בידוד TCR תא בודד, דור של וקטורים Retroviral עבור במבחנה In Vivo הביטוי של האדם TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

Immunology and Infection

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

שיטת העוצמה בזמן אמת עבור שצ אנטיגן קולטן התאים T המיקוד תאים מוצק המטולוגי סרטן

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

פרוטוקול יעיל ליצירת תאי CAR-T בעכבר במודלים סינגניים

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

מחבר זרקור: שיפור תפקוד תאי CAR-T במודלים סינגניים של גידולים 

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

ייצור יעיל של תאי קולטן אנטיגן כימרי (CAR)-T

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

ביצוע מסך CRISPR רחב גנום במבחנה בתאי T של קולטן אנטיגן כימרי

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ברמת הגנום RNAi ההקרנה כדי לזהות גורמים מארח לווסת את הטיפול Oncolytic וירוס

ב- Rechallenge תא הגידול חוץ גופית להערכה חזוי של Chimeric אנטיגן קולטן תא T פונקציה Antitumor

הערכת ציטוטוקסיות מהירה במבחנה של קולטן אנטיגן כימרי Jurkat באמצעות הדמיה פלואורסצנטית

הערכת ציטוטוקסיות מהירה במבחנה של קולטן אנטיגן כימרי Jurkat באמצעות הדמיה פלואורסצנטית

הערכת ציטוטוקסיות מהירה במבחנה של קולטן אנטיגן כימרי Jurkat באמצעות הדמיה פלואורסצנטית

יעיל בידוד TCR תא בודד, דור של וקטורים Retroviral עבור במבחנה In Vivo הביטוי של האדם TCRs

שיטת העוצמה בזמן אמת עבור שצ אנטיגן קולטן התאים T המיקוד תאים מוצק המטולוגי סרטן

ייצור יעיל של תאי קולטן אנטיגן כימרי (CAR)-T

ייצור יעיל של תאי קולטן אנטיגן כימרי (CAR)-T

ייצור יעיל של תאי קולטן אנטיגן כימרי (CAR)-T