Name: generation of knock-out primary and expanded human nk cells using cas9 ribonucleoproteins
Uploaded: 2018-06-14T15:00:00
Description: Watch this Scientific Journal Video about Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins at JoVE.com

我们感谢布赖恩. 西塞罗在编辑手稿方面的帮助。

健康捐赠者巴菲外套被获取了作为来源材料从中央俄亥俄区域美国红十字。本研究决定由全国儿童医院机构审查委员会进行豁免研究。
1. 人类 NK 细胞的纯化和扩展
<ol><li>隔离 PBMCs 从巴菲大衣12。<ol>
<li>35毫升的巴菲涂层样品在15毫升的 Ficoll-Paque。</li>
		<li>离心机在 400 x g 20 分钟没有刹车和收集 PBMC 从接口。</li>
		<li>通过增加至少一个同等体积的 PBS, 离心5?...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipid-mediated+delivery+of+proteins+enables+efficient+protein-based+genome+editing+in+vitro+and+in+vivo.">Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.</a> Nature Biotechnology. 33 (1), 73-80 (2015).</li><li>Wang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+delivery+of+genome-editing+proteins+using+bioreducible+lipid+nanoparticles.">Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles.</a> PNAS. 113 (11), 2868-2873 (2016).</li><li>Liang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+DNA-free+genome+editing+of+bread+wheat+using+CRISPR/Cas9+ribonucleoprotein+complexes.">Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes.</a> Nature Communications. 8, 14261 (2017).</li><li>DeWitt, M. A., Corn, J. E., Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+via+delivery+of+Cas9+ribonucleoprotein.">Genome editing via delivery of Cas9 ribonucleoprotein.</a> Methods. , 9-15 (2017).</li><li>Rezvani, K., Rouce, R., Liu, E., Shpall, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Natural+Killer+Cells+for+Cancer+Immunotherapy.">Engineering Natural Killer Cells for Cancer Immunotherapy.</a> Molecular Therapy. 25 (8), 1769-1781 (2017).</li><li>Sutlu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+intracellular+antiviral+defense+mechanisms+augments+lentiviral+transduction+of+human+natural+killer+cells:+implications+for+gene+therapy.">Inhibition of intracellular antiviral defense mechanisms augments lentiviral transduction of human natural killer cells: implications for gene therapy.</a> Human Gene Therapy. 23 (10), 1090-1100 (2012).</li><li>Viel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+inhibits+the+activation+and+functions+of+NK+cells+by+repressing+the+mTOR+pathway.">TGF-beta inhibits the activation and functions of NK cells by repressing the mTOR pathway.</a> Science Signaling. 9 (415), (2016).</li><li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion,+purification,+and+functional+assessment+of+human+peripheral+blood+NK+cells.">Expansion, purification, and functional assessment of human peripheral blood NK cells.</a> JoVE. (48), (2011).</li><li>Lee, D. A., Verneris, M. R., Campana, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition,+preparation,+and+functional+assessment+of+human+NK+cells+for+adoptive+immunotherapy.">Acquisition, preparation, and functional assessment of human NK cells for adoptive immunotherapy.</a> Methods in Molecular Biology. , 61-77 (2010).</li><li>Vouillot, L., Thelie, A., Pollet, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+T7E1+and+surveyor+mismatch+cleavage+assays+to+detect+mutations+triggered+by+engineered+nucleases.">Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.</a> G3. 5 (3), 407-415 (2015).</li><li>Eyquem, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+a+CAR+to+the+TRAC+locus+with+CRISPR/Cas9+enhances+tumour+rejection.">Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.</a> Nature. 543 (7643), 113-117 (2017).</li><li>Liang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+highly+efficient+mammalian+cell+engineering+via+Cas9+protein+transfection.">Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.</a> Journal of Biotechnology. 208, 44-53 (2015).</li><li>Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+RNA-guided+genome+editing+in+human+cells+via+delivery+of+purified+Cas9+ribonucleoproteins.">Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.</a> Genome Research. 24 (6), 1012-1019 (2014).</li><li>Chmielecki, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Profiling+of+a+Large+Set+of+Diverse+Pediatric+Cancers+Identifies+Known+and+Novel+Mutations+across+Tumor+Spectra.">Genomic Profiling of a Large Set of Diverse Pediatric Cancers Identifies Known and Novel Mutations across Tumor Spectra.</a> Cancer Research. 77 (2), 509-519 (2017).</li><li>Schultz, L. M., Majzner, R., Davis, K. L., Mackall, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+immunotherapy+for+pediatric+solid+tumors.">New developments in immunotherapy for pediatric solid tumors.</a> Current Opinion in Pediatrics. 30 (1), 30-39 (2018).</li></ol>

对 NK 细胞的 DNA 依赖性修饰已经挑战了4,9。因此, 我们直接介绍了一个综合预制 ribonucleoprotein (RNPs) 复合体和 Cas9 蛋白作为纯化蛋白成初级和扩大 NK 细胞8。这种方法使我们能够消除由 RNA 聚合酶 II 开始的封盖、尾矿和其他转录和平移过程, 这可能会导致 NK 细胞凋亡与 DNA 相关的转导方法有关。
此外, 这里报告的方?...

癌症免疫治疗近年来取得了进展。基因修饰的嵌合体抗原受体 (车载) T 细胞是成功地应用于癌症免疫治疗的工程免疫细胞的一个很好的例子。这些细胞最近被 FDA 批准用于治疗 CD19 + B 细胞恶性肿瘤, 但迄今为止成功仅限于携带少量多弹头抗原的疾病, 而针对这种有限的抗原曲目容易发生免疫逃逸的失败。此外, 由于异基因 t 细胞引起的移植物抗宿主病的风险, 汽车 t 细胞一直关注自体 t 细胞的使用。相反, NK 细胞能够以抗原无关的方式杀死肿瘤靶点, 不会引起 GvHD, 这使他们成为癌症免疫治疗6、7、8、9的好候选者。
CRISPR/Cas9 技术最近在工程免疫细胞中得到了应用, 但用质粒对 NK 细胞进行基因重组一直是很有挑战性的。这是由于在 DNA 依赖的情况下, 转基因分娩的困难, 如慢病毒载体和逆转录病毒转导导致大量程序相关的 nk 细胞凋亡和有限的基因工程 nk 细胞的生产4,9。
许多先天免疫细胞表达了与病原体相关的分子模式的高水平的受体, 如维甲酸诱导基因 i (钻机 i), 这使更多的识别外国 DNA。在使用基于 DNA 的基因修饰方法10时, 抑制这些通路使 NK 细胞的转导效率提高。
本文介绍了利用 Cas9 ribonucleoprotein 配合物 (Cas9/RNPs) 对原发性和扩张的人 NK 细胞进行无 DNA 基因组编辑的方法。Cas9/RNPs 由三组分组成, 重组 Cas9 蛋白与合成的单导 RNA 组成的复合 crRNA 和 tracerRNA。这些 Cas9/RNPs 能够分裂基因组目标, 与国外 DNA 依赖的方法相比, 由于其作为功能性复合物的交付。此外, 快速清除细胞中的 Cas9/RNPs 可减少诱导凋亡等非靶向效应。因此, 它们可以用来产生敲出, 或与 DNA 结合, 以同源重组6,7。结果表明, Cas9/RNPs 电穿孔是克服 NK 细胞遗传修饰的早期约束的一种简便、相对有效的方法。
TGFβ是抑制 NK 细胞活化和功能的主要免疫抑制因子。有人建议, 靶向 TGFβ通路可以增加免疫细胞功能。我们针对的区域编码 TGBR2 胞外区绑定 TGFβ11.结果表明, 该基因 mRNA 表达水平显著下降, 进一步证明改良后的 NK 细胞对 TGFβ有抵抗力。此外, 改良后的细胞保持生存能力和增殖潜能, 因为它们能够在电穿孔后使用辐照的馈线细胞扩大.因此, 下面的方法是一个有希望的方法, 基因操作 NK 细胞为任何进一步的临床或研究目的。

<table>
	<tbody>
		<tr>
			<td>RosetteSep&#8482; Human NK Cell Enrichment Cocktail</td>
			<td>STEMCELL Technologies</td>
			<td>15065</td>
			<td>The RosetteSep&#8482; Human NK Cell Enrichment Cocktail is designed to isolate NK cells from whole blood by negative selection.</td>
		</tr>
		<tr>
			<td>Ficoll-Paque&#174; PLUS</td>
			<td>GE Healthcare - Life Sciences</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; CRISPR-Cas9 tracrRNA</td>
			<td>Integrated DNA Technologies</td>
			<td>1072532</td>
			<td>TracrRNA that contains proprietary chemical modifications conferring increased nuclease resistance. Hybridizes to crRNA to activate the Cas9 enzyme</td>
		</tr>
		<tr>
			<td>Alt-R&#174; CRISPR-Cas9 crRNA</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>Synthetically produced as Alt-R&#174; CRISPR-Cas9 crRNA, based on the sequence of designed gRNA targeting the gene of intrest</td>
		</tr>
		<tr>
			<td>Alt-R&#174; Genome Editing Detection Kit</td>
			<td>Integrated DNA Technologies</td>
			<td>1075931</td>
			<td>Each kit contains T7EI endonuclease, T7EI reaction buffer, and T7EI assay controls.</td>
		</tr>
		<tr>
			<td>Platinum&#174; Taq DNA Polymerase High Fidelity</td>
			<td>Invitrogen</td>
			<td>11304-011</td>
			<td></td>
		</tr>
		<tr>
			<td>4D-Nucleofector&#8482; System</td>
			<td>Lonza</td>
			<td>AAF-1002B</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant IL-2 Protein</td>
			<td>Novartis</td>
			<td>65483-0116-07</td>
			<td></td>
		</tr>
		<tr>
			<td>P3 Primary Cell 4D-Nucleofector&#8482; X Kit</td>
			<td>Lonza</td>
			<td>V4XP-3032</td>
			<td>Contains pmaxGFP&#8482; Vector, Nucleofector&#8482; Solution, Supplement, 16-well Nucleocuvette&#8482; Strips</td>
		</tr>
		<tr>
			<td>Non-targeting: Custom siRNA, Standard 0.05 2mol ON-TARGETplus</td>
			<td>Dharmacon</td>
			<td>CTM-360019</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; S.p. Cas9 Nuclease 3NLS</td>
			<td>Integrated DNA Technologies</td>
			<td>1074181</td>
			<td>Cas9 Nuclease</td>
		</tr>
		<tr>
			<td>DNeasy&#174; Blood &#38; Tissue Handbook</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>ThermoFisher</td>
			<td>C3099</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946;</td>
			<td>Biolegend</td>
			<td>580706</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; CRISPR-Cas9 Control Kit, Human</td>
			<td>Integrated DNA Technologies</td>
			<td>1072554</td>
			<td>Includes tracrRNA, HPRT positive control crRNA, negative control crRNA#1, HPRT Primer Mix, and Nuclease-Free Duplex Buffer.</td>
		</tr>
		<tr>
			<td>IDTE pH 7.5 (1X TE Solution)</td>
			<td>Integrated DNA Technologies</td>
			<td>11-01-02-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R&#174; Cas9 Electroporation Enhancer</td>
			<td>Integrated DNA Technologies</td>
			<td>1075915</td>
			<td>Cas9 Electroporation Enhancer</td>
		</tr>
	</tbody>
</table>

generation of knock-out primary and expanded human nk cells using cas9 ribonucleoproteins

CRISPR/Cas9 技术正在加速许多细胞类型的基因组工程, 但到目前为止, 基因传递和稳定的基因修饰在原 NK 细胞中具有挑战性。例如, 使用慢病毒载体或逆转录病毒转导的转基因分娩导致转基因 nk 细胞的产量有限, 这是由于大量程序相关的 nk 细胞凋亡所致。我们这里描述了一个无 DNA 的方法, 基因组编辑的人原发性和扩大 NK 细胞使用 Cas9 ribonucleoprotein 配合物 (Cas9/RNPs)。这种方法可以有效地剔除 NK 细胞中的 TGFBR2和HPRT1 基因。rt-pcr 数据显示, 基因表达水平显著下降, 一个代表性细胞产品的细胞毒性试验表明, RNP 修饰 NK 细胞对 TGFβ的敏感性较低。经辐照 mbIL21-expressing 馈线细胞刺激后, 基因修饰细胞可扩大电穿孔。

电穿孔效率
为了优化 Cas9/RNPs 的电穿孔, 我们对16种不同的方案进行了实验, 将 GFP 非靶向 siRNA 和 DNA 质粒转导到 NK 细胞。流式细胞仪检测表明, EN-138的细胞活力和转导效率的百分比最高 (35% 活 GFP 阳性细胞) 为两个粒子 (图 1 &图 2)。有趣的是, 使用这个程序 Cas9/RNPs 电穿孔的...

Watch this Scientific Journal Video about Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins at JoVE.com

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

在这里, 我们提出了一个协议, 基因改造主要或扩大人类自然杀手 (NK) 细胞使用Cas9 Ribonucleoproteins (Cas9/RNPs)。通过使用该协议, 我们生成的人 NK 细胞缺乏转化生长 factor–b 受体 2 (TGFBR2) 和嘌呤 phosphoribosyltransferase 1 (HPRT1)。

利用 Cas9 Ribonucleoproteins 生成人 NK 细胞的研究

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Research

JoVE Journal

Immunology and Infection

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

从小学的人角质形成细胞器官筏文化的代

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human NaÃƒÆ’Ã‚Â¯ve and Memory T Cells

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

小RNA转染原代人Th17细胞通过下一代电穿孔

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...