PD-1 and PD-L1 targeting sgRNA Lentiviral Particles

产品介绍

GeneCopoeia™提供靶向PD-1和PD-L1的sgRNA慢病毒，用于原代细胞或癌细胞的CRISPR-Cas9基因编辑实验。

该即用型慢病毒与我司的Cas9克隆、Cas9慢病毒以及Cas9稳转株联用可有效地敲除多种细胞的PD-1和PD-L1基因，包括：难转染细胞、原代细胞、干细胞、非分裂细胞。

产品优势

所有的PD-1和PD-L1的sgRNA慢病毒都经过靶点切割验证
13个PD-1 sgRNA慢病毒和7个PD-L1 sgRNA慢病毒充分靶向PD-1和PD-L1的所有外显子
预制病毒发货快，直接用于CRISPR-Cas9基因编辑实验
滴度>10⁸ TU/ml，携带mCherry报告基因方便实验观察

该PD-1和PD-L1的sgRNA慢病毒载体由U6启动子启动，还包括一个mCherry报告基因和puromycin筛选基因，见图一。慢病毒感染情况可用荧光显微镜观察，见图二。

图一：sgRNA慢病毒载体

图二：sgRNA慢病毒转导HEK293细胞。A.PD-1 sgRNA慢病毒转导HEK293细胞; B.PD-L1 sgRNA慢病毒转导HEK293细胞

用IndelCheck™ T7内切酶I验证sgRNA慢病毒对PD-1和PD-L1的靶向性。

图三：IndelCheck™ T7内切酶I验证. A. PD-1的T7E1验证：PD-1的PCR片段大小为631bp，T7E1实验中切割出515bp和116bp的片段；B. PD-L1的T7E1验证：PD-L1的PCR片段大小为701bp，T7E1实验中切割出535bp和166bp的片段；

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产品列表

sgRNA Lentiviral Particles for PD-1

Catalog	Product	Description	Target Exon	Price(￥)
HCP001055-LvSG03-15-10-a-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-a-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-a-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000
HCP001055-LvSG03-15-10-b-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-b-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-b-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000
HCP001055-LvSG03-15-10-c-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-c-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-c-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000
HCP001055-LvSG03-15-10-d-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-d-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-d-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000
HCP001055-LvSG03-15-10-g-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-g-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-g-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000
HCP001055-LvSG03-15-10-h-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-h-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-h-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000
HCP001055-LvSG03-15-10-i-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-i-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-i-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000
HCP001055-LvSG03-15-10-j-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-j-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-j-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000
HCP001055-LvSG03-15-10-k-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-k-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-k-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000
HCP001055-LvSG03-15-10-l-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-l-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-l-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000
HCP001055-LvSG03-15-10-m-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-m-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-m-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000
HCP001055-LvSG03-15-10-n-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-n-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-n-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	3200
HCP001055-LvSG03-15-10-o-100	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-1，100 µl	100 µl	2000
HCP001055-LvSG03-15-10-o-200	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-1，200 µl	200 µl	3200
HCP001055-LvSG03-15-10-o-400	PD-1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-1，400 µl	400 µl	4000

sgRNA Lentiviral Particles for PD-L1

Catalog	Product	Description	Target Exon	Price(￥)
HCP001057-LvSG03-15-10-a-100	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-L1，100 µl	100 µl	2000
HCP001057-LvSG03-15-10-a-200	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-L1，200 µl	200 µl	3200
HCP001057-LvSG03-15-10-a-400	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-L1，400 µl	400 µl	4000
HCP001057-LvSG03-15-10-b-100	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-L1，100 µl	100 µl	2000
HCP001057-LvSG03-15-10-b-200	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-L1，200 µl	200 µl	3200
HCP001057-LvSG03-15-10-b-400	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-L1，400 µl	400 µl	4000
HCP001057-LvSG03-15-10-h-100	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-L1，100 µl	100 µl	2000
HCP001057-LvSG03-15-10-h-200	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-L1，200 µl	200 µl	3200
HCP001057-LvSG03-15-10-h-400	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-L1，400 µl	400 µl	4000
HCP001057-LvSG03-15-10-k-100	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-L1，100 µl	100 µl	2000
HCP001057-LvSG03-15-10-k-200	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-L1，200 µl	200 µl	3200
HCP001057-LvSG03-15-10-k-400	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-L1，400 µl	400 µl	4000
HCP001057-LvSG03-15-10-l-100	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-L1，100 µl	100 µl	2000
HCP001057-LvSG03-15-10-l-200	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-L1，200 µl	200 µl	3200
HCP001057-LvSG03-15-10-l-400	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-L1，400 µl	400 µl	4000
HCP001057-LvSG03-15-10-m-100	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-L1，100 µl	100 µl	2000
HCP001057-LvSG03-15-10-m-200	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-L1，200 µl	200 µl	3200
HCP001057-LvSG03-15-10-m-400	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-L1，400 µl	400 µl	4000
HCP001057-LvSG03-15-10-n-100	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×2 vials)	≥10⁸ TU/ml，PD-L1，100 µl	100 µl	2000
HCP001057-LvSG03-15-10-n-200	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×4 vials)	≥10⁸ TU/ml，PD-L1，200 µl	200 µl	3200
HCP001057-LvSG03-15-10-n-400	PD-L1-targeting sgRNA Lentifect™慢病毒颗粒 (50 µl×8 vials)	≥10⁸ TU/ml，PD-L1，400 µl	400 µl	4000

产品技术

PD-1和PD-L1通路

程序性死亡受体-1（PD-1），又名CD279，和程序性死亡配体-1（PD-L1）都是与免疫相关的蛋白。PD-1是分布在T细胞表面的一种免疫共抑制分子，其配体PD-L1表达于上皮细胞和内皮细胞，不同类型的免疫细胞，以及抗原递呈细胞（树突状细胞、巨噬细胞、B细胞）。PD-L1可分为组成型表达和炎症反应中干扰素上调型表达，如组织慢性感染的情况。在肿瘤和病毒感染时，细胞PD-L1表达上调，与T细胞表面的PD-1受体相互作用，能够抑制T细胞的活化、增殖及对肿瘤的杀伤，使其功能发生紊乱和枯竭。PD-1/PD-L1信号通路与肿瘤和病毒的免疫逃逸密切相关，是免疫治疗肿瘤和慢性传染病的重要新靶点。利用抗PD-1、PD-L1的单克隆抗体阻断PD-1/PD-L1信号通路，能够恢复T细胞的免疫杀伤功能。

PD-1，PD-L1与肿瘤治疗

当T细胞表面的PD-1与肿瘤细胞表面的PD-L1结合，则有利于细胞增殖、细胞因子的分泌以及肿瘤细胞的裂解。用单克隆抗体与PD-1和PD-L1结合的免疫治疗方法已取得初步成效[1]，包括恶性黑色素瘤、非小细胞肺癌、肾细胞癌和其他肿瘤类型[2]。然而，由于大多数组织依靠PD-L1的表达来限制T细胞应答，因此使用PD-L1和PD-1抗体来阻断PD-L1和PD-1需要解决外周耐受阻断的问题。

CRISPR和PD-1，PD-L1

CRISPR-Cas9系统是一种高效的基因编辑工具，经过引导RNA（sgRNA）引导，Cas9核算内切酶能够准确地靶向目的基因进行剪切，产生DNA双链切口(DSBs)，进而启动DNA修复程序，产生特定基因组修饰。该基因组编辑技术在2013第一次应用于哺乳动物细胞和动物，随之，基于CRISPR-Cas9的治疗方法在癌症建模和治疗研究中也快速建立起来了[3]。 CRISPR-Cas9系统可通过病毒或非病毒转导的方法直接应用于人类细胞[4]。敲除PD-1基因能够有效促进T细胞对肿瘤细胞的免疫杀伤力[5]。用Cas9-sgRNA和外源单链DNA模板替换靶核苷酸的方法可以成功敲除PD-1[6]，实验过程中T细胞表面PD-1敲除后，T细胞免疫应答增强，对癌细胞的杀伤力增大[7]。慢病毒CRISPR系统在Jurkat T细胞中的应用也促进了T细胞的功能研究[8]。

最近，NIH重组DNA咨询委员会批准了一项临床试验。在这个临床试验中，用CRISPR-Cas9的方法敲除了PD-1和NY-ESO-1受体，这是CRISPR-Cas9第一次在临床试验中得到应用[9]。类似的还有用CRISPR-Cas9敲除前列腺癌自体T细胞的PD-1(NCT02867345)，膀胱癌自体T细胞的PD-1(NCT02863913)，以及肾细胞癌自体T细胞的PD-1(NCT02867332)也在试验中。无论在体外水平还是动物模型中，用CRISPR基因编辑CAR T展现出强大的抗肿瘤活性[10]。肿瘤细胞表面的PD-L1会削弱CAR T细胞的功能，导致肿瘤细胞不能清除。用CRISPR/Cas9技术将抗CD19的CAR T细胞的PD-1敲除，在体外能够增强CAR T细胞对肿瘤细胞的杀伤能力，在体内移植肿瘤模型中能够增强CAR T细胞对肿瘤细胞的清除能力[11]。

参考文献：

1. Keir, M. E., Butte M. J., Freeman G. J. & Sharpe A. H. PD-1 and its ligands in tolerance and immunity. Annu. Rev. Immunol. 26, 677–704 (2008).

2. Suzanne, L. T. et al. Safety, activity, and immune correlates of anti–PD-1 antibody in cancer. N. Engl. J. Med. 366, 2443–2454 (2012).

3. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 339(6121):819-23 (2013).

4. H. Yin, C.-Q. Song, J.R. Dorkin, L.J. Zhu, Y. Li, Q. Wu, A. Park, J. Yang, S. Suresh, A. Bizhanova, Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo, Nat. Biotechnol. 34, 328-333 (2016).

5. S.L. Topalian, C.G. Drake, D.M. Pardoll, Immune checkpoint blockade: a common denominator approach to cancer therapy, Cancer Cell, 27, 450-461 (2015).

6. K. Schumann, S. Lin, E. Boyer, D.R. Simeonov, M. Subramaniam, R.E. Gate, G.E. Haliburton, J.Y. Chun, J.A. Bluestone, J.A. Doudna, Generation of knock-in primary human T cells using Cas9 ribonucleoproteins, Proc. Natl. Acad. Sci. U S A. 112, 10437-10442 (2015).

7. S. Su, B. Hu, J. Shao, B. Shen, J. Du, Y. Du, J. Zhou, L. Yu, L. Zhang, F. Chen, CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients, Sci. Rep. 6, 20070 (2016).

8. Chi S, Weiss A, Wang H. A CRISPR-Based Toolbox for Studying T Cell Signal Transduction. Biomed Res Int. 2016:5052369 (2016).

9. Cyranoski D, CRISPR gene-editing tested in a person for the first time. Nature 539:479 (2016).

10. Ren J, Liu X, Fang C, Jiang S, June CH, Zhao Y. Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition. Clin Cancer Res. 23(9):2255-2266 (2017).

11. Rupp LJ, Schumann K, Roybal KT, Gate RE, Ye CJ, Lim WA, Marson A. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep. 7(1):737 (2017).

PD-1 and PD-L1 targeting sgRNA Lentiviral Particles

产品优势

sgRNA Lentiviral Particles for PD-1

sgRNA Lentiviral Particles for PD-L1

PD-1和PD-L1通路

PD-1，PD-L1与肿瘤治疗

CRISPR和PD-1，PD-L1

参考文献：

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