<meta charset="utf8"></head><body>在 CART 细胞中进行全基因组 CRISPR 筛选以研究治疗耐药性

<ol>
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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#39;s modified eagle&#39;s medium (DMEM)</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#160;</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&#160;</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+ target cells&#160;</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&#160;</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 &#181;m</td>
			<td>ThermoFisher Scientific</td>
			<td>567-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Filter Systems, 0.45 &#181;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>在嵌合抗原受体 T 细胞中进行体外全基因组 CRISPR 敲除筛选

在嵌合抗原受体 T 细胞中进行体外全基因组 CRISPR 敲除筛选

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

Research

JoVE Journal

Cancer Research

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

716 次观看.  Mayo Clinic. 本文介绍了一种应用 体外 耗竭模型的方案，以在健康供体嵌合抗原受体 T 细胞中完成全基因组 CRISPR 敲除筛选。 

视频: 在嵌合抗原受体 T 细胞中进行体外全基因组 CRISPR 敲除筛选

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.

全基因组的 rna 干扰筛查以确定调节溶瘤病毒治疗的宿主因素

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

科研

癌症研究

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of associated loci. In addition, the presence of specific modifications has been used to determine the position and identity non-coding functional elements such as enhancers. In recent years, chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) has become a powerful tool in determining the genome-wide profiles of individual histone modifications. However, it has become increasingly clear that the combinatorial patterns of chromatin modifications, referred to as Chromatin States, determine the identity and nature of the associated genomic locus. Therefore, workflows consisting of robust high-throughput (HT) methodologies for profiling a number of histone modification marks, as well as computational analyses pipelines capable of handling myriads of ChIP-Seq profiling datasets, are needed for comprehensive determination of epigenomic states in large number of samples. The HT-ChIP-Seq workflow presented here consists of two modules: 1) an experimental protocol for profiling several histone modifications from small amounts of tumor samples and cell lines in a 96-well format; and 2) a computational data analysis pipeline that combines existing tools to compute both individual mark occupancy and combinatorial chromatin state patterns. Together, these two modules facilitate easy processing of hundreds of ChIP-Seq samples in a fast and efficient manner. The workflow presented here is used to derive chromatin state patterns from 6 histone mark profiles in melanoma tumors and cell lines. Overall, we present a comprehensive ChIP-seq workflow that can be applied to dozens of human tumor samples and cancer cell lines to determine epigenomic aberrations in various malignancies.

Here, we describe an optimized high-throughput ChIP-sequencing protocol and computational analyses pipeline for the determination of genome-wide chromatin state patterns from frozen tumor tissues and cell lines.

肿瘤组织中高通量芯片测序的染色质状态全基因组图谱集成平台

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of ...

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.

体外肿瘤细胞恢复对 chimeric 抗原受体 t 细胞抗肿瘤功能的预测评价

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

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.

使用 CRISPR/Cas9 在 CAR-T 电池中敲除 GM-CSF

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

制造用于采用免疫疗法的奇美抗原受体（CAR）T细胞

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

胃肠癌DNA甲基化基因组全谱分析

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B cell malignancies or multiple myeloma (MM). However, numerous obstacles limit the efficacy and prohibit the widespread use of CAR T cell therapies due to poor trafficking and infiltration into tumor sites as well as lack of persistence in vivo. Moreover, life-threatening toxicities, such as cytokine release syndrome or neurotoxicity, are major concerns. Efficient and sensitive imaging and tracking of CAR T cells enables the evaluation of T cell trafficking, expansion, and in vivo characterization and allows the development of strategies to overcome the current limitations of CAR T cell therapy. This paper describes the methodology for incorporating the sodium iodide symporter (NIS) in CAR T cells and for CAR T cell imaging using [18F]tetrafluoroborate-positron emission tomography ([18F]TFB-PET) in preclinical models. The methods described in this protocol can be applied to other CAR constructs and target genes in addition to the ones used for this study.

Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography

This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform.

使用[18F]四氟硼酸盐正电子发射断层扫描/计算机断层扫描对嵌合抗原受体T细胞进行动态成像

T cells genetically engineered to express chimeric antigen receptors (CAR) have shown unprecedented results in pivotal clinical trials for patients with B ...