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分钟, 并吸去中国人民银行的洗涤, 清洗恢复的 PBMCs 三次。NK 细胞可以在这个阶段被 RosettesSep13隔离。</li>
	</ol>
</li>
	<li>在0、7和14天, 用辐照的 10 x 106 mbIL21-expressing 馈线细胞刺激 NK 细胞。用新鲜的 RPMI 替换介质, 每隔一天 IL-2 10% 只血清、1% 谷氨酰胺、1% 青霉素链霉素和100毫升/mL。</li>
</ol>2. gRNA 设计与选型
<ol><li>使用在线工具,例如, NCBI, 合奏, 选择特定的基因组定位点。 例子。 说明: 转化生长因子β受体 2 (TGFBRR2) 胞外区 查看记录: PF08917 查看 InterPro: IPR015013 位置:49-157 aa 目标序列: TGFBR2 基因外显子 4 (ENSG00000163513)</li>
	<li>要设计 gRNAs, 请使用 CRISPR 设计 web 工具, 如 http://crispr.mit.edu 和 "Benchling"。<ol>
<li>输入在步骤 2.1中选择的 DNA 序列。选择人 (hg 19) 作为目标基因组。CRISPR 参考线 (20 核苷酸后跟 PAM 序列: NGG) 将从先前输入的序列扫描。它还显示了在选定的基因组中可能存在的非目标匹配。</li>
		<li>根据目标和非目标利率选择最佳的三 gRNAs, 得分最高。例如,表 1显示了设计的 CRISPR rna, 目标为 TGFBR2 基因的外显子 4, CRISPR 设计 web 工具建议。</li>
	</ol>
</li>
	<li>命令 CRISPR rna 作为合成序列特定的 crRNAs。</li>
	<li>命令一个守恒的 transactivating RNA (tracrRNA) 通过与 crRNA 的部分同源性进行交互。</li>
</ol>3. 设计删除筛选底漆
<ol><li>设计引物跨越 gRNA 裂点的 T7E1 突变试验。</li>
	<li>使用引物至少 100 bp 离预测解裂点, 以确保小插入-删除 (indels) 在 sgRNA 的目标站点将出现在1.5% 琼脂糖凝胶后, 突变检测。表 2显示了用于放大 TGFBR2 胞外区的引物。</li>
</ol>4. 人类初级和扩大 NK 细胞的转导
注: 采用4D 系统进行电穿孔, 将 Cas9/RNPs 元素传导到 NK 中。
<ol><li>
单元准备
	<ol>
<li>
对于原 nk 细胞, 在 RPMI 培养基中孵化新鲜的 nk 细胞, 在 IL-2 100 毫升/mL 的情况下, 在5天进行电穿孔。每隔一天更换介质, 如前述和转导前一天。</li>
		<li>
对于扩张的 NK 细胞, 在0天用辐照的馈线细胞刺激细胞的比例为 1:1, 并在5或6或7天进行电穿孔。每隔一天更换介质, 如前述和转导前一天。</li>
		<li>在电穿孔的日子, 准备一个 T25 瓶填充8毫升新鲜 RPMI 包含100毫升/mL IL-2 的细胞进行电穿孔和预孵化烧瓶在湿润37°c 和 5% CO2孵化器。 注:经过2次或 3路刺激的解冻细胞或细胞在恢复后的任何时间都可以转。</li>
		<li>3-4 x 106细胞每条件为26µL 的转导组合。 注:电穿孔液中 NK 细胞浓度极高, 提高了转导率。</li>
		<li>用 PBS 冲洗细胞3次以去除所有的血清, 通常含有 RNase 活性。每次在 300 x g 处旋转8分钟。 注:考虑7电穿孔条件为 Cas/RNPs 作为单 gRNA (gRNA1, gRNA2, gRNA3) 和两个 gRNAs (gRNA1+gRNA2, gRNA1+gRNA3, gRNA2+gRNA3) 和一个控制, 没有 Cas9/RNPs 的组合。</li>
	</ol>
</li>
	<li>
形成 crRNA: tracerRNA/复合体
	<ol>
<li>并用重悬 crRNAs (gRNA1, gRNA2, gRNA3) 和 tracerRNA 1x TE 溶液的最终浓度为200微米。混合 2.2 ul 每200微米 gRNA 与200微米 tracerRNA, 如表 3所示。</li>
		<li>加热样品在95°c 5 分钟, 并允许冷却的长凳上到室温 (15-25 °c)。存储悬浮 rna 和 crRNA: tracerRNA/复杂在-20 °c 为以后使用。</li>
	</ol>
</li>
	<li>
形成 RNP 情结 注意:为了节省时间, 在洗涤步骤 4.1.5中形成 RNP 复合体。
	<ol>
<li>对于单 crRNA: tracrRNA 双工反应, 稀释 Cas9 限制性到36微米, 如表 4中的示例所示。</li>
		<li>crRNA 的组合转导: tracrRNA 复式, 稀释 Cas9 限制性到36微米, 如表 5中的例子所示。
</li>
		<li>添加 Cas9 限制性到 crRNA: tracrRNA 复式缓慢, 而旋转吸管提示, 三十年代至1分钟。</li>
		<li>在室温下孵育 15-20 分钟的混合物。如果不准备在孵化后使用混合物, 保持在冰上的混合物, 直到使用。</li>
	</ol>
</li>
	<li>
穿孔
	<ol>
<li>将整个补充剂添加到电穿孔溶液 P3, 并保持室温。</li>
		<li>并用重悬细胞颗粒 (3-4 x 106细胞从步骤 4.1.5) 在 20 ul P3 主要4D 电穿孔溶液。吹打时避免气泡。 注意: 细胞在 P3 溶液中不应该长时间留着。</li>
		<li>立即添加5µL RNP 复合物 (步骤 4.3) 的细胞悬浮。</li>
		<li>添加 1 ul 100 微米 Cas9 电穿孔增强剂的 Cas9/RNPs/cell 混合。</li>
		<li>转移 Cas9/RNPs/cell 混合成 20 ul 电穿孔带。</li>
		<li>轻轻地点击小条, 以确保样品覆盖底部的小条。</li>
		<li>启动4D 电穿孔系统, 选择EN-138程序。</li>
	</ol>
</li>
</ol>5. 后转导
<ol><li>让细胞在小条中休息3分钟。</li>
	<li>将预平衡培养基的80µL 添加到试管中, 并将样品轻轻地转移到烧瓶中。</li>
	<li>转导48小时后, 从 5 x 105细胞中提取基因组 DNA, 用于筛选。</li>
	<li>使用 Taq DNA 聚合酶试剂盒在步骤3.2 中设计的引物放大感兴趣的基因。</li>
	<li>形成 PCR 扩增子 heteroduplexes T7EI 消化和孵化的产品 30-60 分钟与 T7EI 酶37°c。 注:T7EI 检测是首选的筛选, 因为它是快速, 简单, 并提供了清洁电泳结果相比, 使用测量。但是, 此方法无法检测在 Cas9 RNPs 实验14中由非同源端连接 (NHEJ) 活动生成的 < 2 基的插入和删除。</li>
	<li>将1.5% 琼脂糖凝胶上的消化 DNA 在110伏上运行 30-45 分钟, 每15分钟可视化凝胶。</li>
	<li>用 mbIL21-expressing 馈线细胞刺激其余细胞的比例为1:1。</li>
	<li>五天后, 刺激提取 RNA 的基因表达水平使用 qPCR。</li>
	<li>按先前报告的12进行 calcein 化验。简单地, 用 calcein AM 加载目标细胞 (在显示的例子中, 使用了3µg/mL/100万 DAOY 细胞)。在 IL2 (100 毫升/ml) 加或减10的可溶性 TGFβ中过夜, 准备 NK 细胞进行细胞毒性测定。与 NK 细胞在一夜间休息时, 在相同的细胞因子中进行 calcein 化验。</li>
</ol>

<ol>
	<li>Guven, H., 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+gene+transfer+into+primary+human+natural+killer+cells+by+retroviral+transduction.">Efficient gene transfer into primary human natural killer cells by retroviral transduction.</a> Experimental Hematology. 33 (11), 1320-1328 (2005).</li><li>Alici, E., Sutlu, T., Sirac Dilber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+gene+transfer+into+primary+human+natural+killer+cells.">Retroviral gene transfer into primary human natural killer cells.</a> Methods in Molecular Biology. 508, 127-137 (2009).</li><li>Carlsten, M., Childs, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Manipulation+of+NK+Cells+for+Cancer+Immunotherapy:+Techniques+and+Clinical+Implications.">Genetic Manipulation of NK Cells for Cancer Immunotherapy: Techniques and Clinical Implications.</a> Frontiers in Immunology. 6, 266 (2015).</li><li>Mehta, R. S., Rezvani, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+Antigen+Receptor+Expressing+Natural+Killer+Cells+for+the+Immunotherapy+of+Cancer.">Chimeric Antigen Receptor Expressing Natural Killer Cells for the Immunotherapy of Cancer.</a> Frontiers in Immunology. 283, 9 (2018).</li><li>Zuris, J. 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>

DAL 服务/已担任信使治疗, 黑曜石疗法, Intellia 疗法, 默克研究实验室和 Miltenyi Biotec 的顾问, 并在 CytoSen 疗法的公平/领导。

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 ...

generation-knock-out-primary-expanded-human-nk-cells-using-cas9

Research

JoVE Journal

Immunology and Infection

12.3K 次观看.  Nationwide Children's Hospital. 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)。

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

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

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

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

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

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

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

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

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

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

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

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

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

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

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

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

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

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

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

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, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

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