Original articles
Huan Deng, Songwei Tan, Xueqin Gao, Chenming Zou, Chenfeng Xu, Kun Tu, Qingle Song, Fengjuan Fan, Wei Huang, Zhiping Zhang. Cdk5 knocking out mediated by CRISPR-Cas9 genome editing for PD-L1 attenuation and enhanced antitumor immunity[J]. Acta Pharmaceutica Sinica B, 2020, 10(2): 358-373

Cdk5 knocking out mediated by CRISPR-Cas9 genome editing for PD-L1 attenuation and enhanced antitumor immunity
Huan Denga, Songwei Tana, Xueqin Gaoa, Chenming Zoua, Chenfeng Xua, Kun Tua, Qingle Songa, Fengjuan Fanb, Wei Huangc, Zhiping Zhanga,d,e
a School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;
b Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;
c Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;
d National Engineering Research Center for Nanomedicine, Huazhong University of Science and Technology, Wuhan 430030, China;
e Hubei Engineering Research Center for Novel Drug Delivery System, Huazhong University of Science and Technology, Wuhan 430030, China
Blocking the programmed death-ligand 1 (PD-L1) on tumor cells with monoclonal antibody therapy has emerged as powerful weapon in cancer immunotherapy. However, only a minority of patients presented immune responses in clinical trials. To develop an alternative treatment method based on immune checkpoint blockade, we designed a novel and efficient CRISPR-Cas9 genome editing system delivered by cationic copolymer aPBAE to downregulate PD-L1 expression on tumor cells via specifically knocking out Cyclin-dependent kinase 5 (Cdk5) gene in vivo. The expression of PD-L1 on tumor cells was significantly attenuated by knocking out Cdk5, leading to effective tumor growth inhibition in murine melanoma and lung metastasis suppression in triple-negative breast cancer. Importantly, we demonstrated that aPBAE/Cas9-Cdk5 treatment elicited strong T cell-mediated immune responses in tumor microenvironment that the population of CD8+ T cells was significantly increased while regulatory T cells (Tregs) was decreased. It may be the first case to exhibit direct in vivo PD-L1 downregulation via CRISPR-Cas9 genome editing technology for cancer therapy. It will provide promising strategy for preclinical antitumor treatment through the combination of nanotechnology and genome engineering.
Key words:    CRISPR-Cas9 genome editing system    Cyclin-dependent kinase 5 (Cdk5)    Programmed death-ligand 1 (PD-L1)    Antitumor immunity    Nanoparticles   
Received: 2019-04-22     Revised: 2019-06-29
DOI: 10.1016/j.apsb.2019.07.004
Funds: This work was supported by the National Natural Science Foundation of China (81872810, 81673374 and 81871473), Wuhan University of Science and Technology Plan for Applied Fundamental Research (2017060201010146, China), the Fundamental Research Funds for the Central Universities (2018KFYYXJJ019, 2019KFYRCPY049 and 2016YXMS138, China). We thank Weijun Feng, Ph.D, Professor, Fudan University, for professional advice.
Corresponding author: Zhiping Zhang     Email:zhipingzhang@hust.edu.cn
Author description:
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Huan Deng
Songwei Tan
Xueqin Gao
Chenming Zou
Chenfeng Xu
Kun Tu
Qingle Song
Fengjuan Fan
Wei Huang
Zhiping Zhang

1. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Files DB, et al. Tumor-associated B7-H1 promotes T-cell apoptosis:a potential mechanism of immune evasion. Nat Med 2002;8:793-800.
2. Keir ME, Butte MJ, Freeman GJ, Sharp AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 2008;26:677-704.
3. Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, et al. Colocalization of inflammatory response with B7-H1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med 2012;4:127ra37.
4. Spranger S, Spaapen RM, Zha Y, Williams J, Meng Y, Ha TT, et al. Up-regulation of PD-L1, IDO, and Tregs in the melanoma tumor microenvironment is driven by CD8+ T cells. Sci Transl Med 2013;5:200ra116.
5. Selby MJ, Engelhardt JJ, Quigley M, Henning KA, Chen T, Srinivasan M, et al. Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. Canc Immunol Res 2013;1:32-42.
6. Alsaab HO, Sau S, Alzhrani R, Tatiparti K, Bhise K, Kashaw SK, et al. PD-1 and PD-L1 checkpoint signaling inhibition for cancer immunotherapy:mechanism, combinations, and clinical outcome. Front Pharmacol 2017;8:561.
7. Zhao MX, Guo WJ, Wu YY, Yang CX, Zhong L, Deng GL, et al. SHP2 inhibition triggers anti-tumor immunity and synergizes with PD-1 blockade. Acta Pharm Sin B 2019;2:304-15.
8. Tompson RH, Gillett MD, Cheville JC, Lohse CM, Dong H, Webster WS, et al. Costimulatory molecule B7-H1 in primary and metastatic clear cell renal cell carcinoma. Cancer 2005;104:2084-91.
9. Bernstein MB, Garnett CT, Zhang H, Velcich A, Wattenberg MM, Gameiro SR, et al. Radiation-induced modulation of costimulatory and coinhibitory T-cell signaling molecules on human prostate carcinoma cells promotes productive antitumor immune interactions. Cancer Biother Radiopharm 2014;29:153-61.
10. He C, Duan X, Guo N, Chan C, Poon C, Weichselbaum RR, et al. Core-shell nanoscale coordination polymers combine chemotherapy and photodynamic therapy to potentiate checkpoint blockade cancer immunotherapy. Nat Commun 2016;7:12499.
11. Wang C, Wang J, Zhang X, Yu S, Wen D, Hu Q, et al. In situ formed reactive oxygen species-responsive scaffold with gemcitabine and checkpoint inhibitor for combination therapy. Sci Transl Med 2018;10:eaan3682.
12. Kuai R, Yuan W, Son S, Nam J, Xu Y, Fan Y, et al. Elimination of established tumors with nanodisc-based combination chemoimmunotherapy. Sci Adv 2018;4:eaao1736.
13. Wang D, Wang T, Liu J, Yu H, Jiao S, Feng B, et al. Acid-activatable versatile micelleplexes for PD-L1 blockade-enhanced cancer photodynamic immunotherapy. Nano Lett 2016;16:5503-13.
14. Dai L, Li K, Li M, Zhao X, Luo Z, Lu L, et al. Size/Charge changeable acidity-responsive micelleplex for photodynamic-improved PD-L1 immunotherapy with enhanced tumor penetration. Adv Funct Mater 2018;28:1707249.
15. Teo PY, Yang C, Whilding LM, Parente-Pereira AC, Maher J, George AJ, et al. Ovarian cancer immunotherapy using PD-L1 siRNA targeted delivery from folic acid-functionalized polyethylenimine:strategies to enhance T cell killing. Adv Healthc Mater 2015;4:1180-9.
16. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 2014;346:1258096.
17. Ledford H. CRISPR, the disruptor. Nature 2015;522:20-4.
18. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007;315:1709-12.
19. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 2011;471:602-7.
20. Dhavan R, Tsai LH. A decade of CDK5. Nat Rev Mol Cell Biol 2001; 2:749-59.
21. Contreras-Vallejos E, Ultreras E, Gonzalez-Billault C. Going out of the brain:non-nervous system physiological and pathological functions of Cdk5. Cell Signal 2012;24:44-52.
22. Arif A. Extraneuronal activities and regulatory mechanisms of the atypical cyclin-dependent kinase Cdk5. Biochem Pharmacol 2012;84:985-93.
23. Pareek TK, Lam E, Zheng X, Askew D, Kulkarni AB, Chance MR, et al. Cyclin-dependent kinase 5 activity is required for T cell activation and induction of experimental autoimmune encephalomyelitis. J Exp Med 2010;207:2507-19.
24. Dorand RD, Nthale J, Myers JT, Barkauskas DS, Avril S, Chirieleison SM, et al. Cdk5 disruption attenuates tumor PD-L1 expression and promotes antitumor immunity. Science 2016;353:399-403.
25. Lynn DM, Langer R. Degradable poly(b-amino esters):synthesis, characterization, and self-assembly with plasmid DNA. J Am Chem Soc 2000;122:10761-8.
26. Akinc A, Lynn DM, Anderson DG, Langer R. Parallel synthesis and biophysical characterization of a degradable polymer library for gene delivery. J Am Chem Soc 2003;125:5316-23.
27. Cheng W, Wu D, Liu Y. Michael addition polymerization of trifunctional amine and acrylic monomer:a versatile platform for development of biomaterials. Biomacromolecules 2016;17:3115-26.
28. Yin M, Bao Y, Gao X, Wu Y, Sun Y, Zhao X, et al. Redox/pH dualsensitive hybrid micelles for targeting delivery and overcoming multidrug resistance of cancer. J Mater Chem B 2017;5:2964-78.
29. Zhuang X, Wu T, Zhao Y, Hu X, Bao Y, Guo Y, et al. Lipid-enveloped zinc phosphate hybrid nanoparticles for codelivery of H-2Kb and H-2Dbrestricted antigenic peptides and monophosphoryl lipid A to induce antitumor immunity against melanoma. J Control Release 2016;228:26-37.
30. Kwak G, Kim D, Nam GH, Wang SY, Kim IS, Kim SH, et al. Programmed cell death protein ligand-1 silencing with polyethylenimineDermatan sulfate complex for dual inhibition of melanoma growth. ACS Nano 2017;11:10135-46.
31. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013;8:2281-308.
32. Li L, Song L, Liu X, Yang X, Li X, He T, et al. Artificial virus delivers CRISPR-Cas9 system for genome editing of cells in mice. ACS Nano 2017;11:95-111.
33. Kim SM, Yang Y, Oh SJ, Hong Y, Seo M, Jang M. Cancer-derived exosomes as a delivery platform of CRISPR/Cas9 confer cancer cell tropism-dependent targeting. J Control Release 2017;266:8-16.
34. Robert C, Schachter J, Long GV, Arance A, Grob JJ, Mortier L, et al. Pembrolizumab versus ipilimumab in advanced melanoma. N Engl J Med 2015;372:2521-32.
35. Sharma P, Allison JP. The future of immune checkpoint therapy. Science 2015;348:56-61.
36. He J, Hu Y, Hu M, Li B. Development of PD-1/PD-L1 pathway in tumor immune microenvironment and treatment for non-small cell lung cancer. Sci Rep 2015;5:13110.
37. Duan X, Chan C, Guo N, Han W, Weichselbaum RR, Lin W. Photodynamic therapy mediated by nontoxic core-shell nanoparticles synergizes with immune checkpoint blockade to elicit antitumor immunity and antimetastatic effect on breast cancer. J Am Chem Soc 2016;138:16686-95.
38. Wang C, Sun W, Ye Y, Hu Q, Bomba HN, Gu Z. In situ activation of platelets with checkpoint inhibitors for post-surgical cancer immunotherapy. Nat Biomed Eng 2017;1:0011.
39. Sagiv-Barfi I, Kohrt HEK, Czerwinski DK, Ng PP, Chang BY, Levy R. Therapeutic antitumor immunity by checkpoint blockade is enhanced by ibrutinib, an inhibitor of both BTK and ITK. Proc Natl Acad Sci U S A 2015;112:E966-72.
40. Pfirschke C, Engblom C, Rickelt S, Cortez-Retamozo V, Garris C, Pucci F, et al. Immunogenic chemotherapy sensitizes tumors to checkpoint blockade therapy. Immunity 2016;44:343-54.
41. Powles T, Eder JP, Fine GD, Braiteh FS, Loriot Y, Cruz C, et al. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature 2014;515:558-62.
42. Robert C, Long GV, Brady B, Dutriaux C, Maio M, Mortier L, et al. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med 2015;372:320-30.
43. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med 2015;373:23-34.
44. Rosenberg JE, Hoffman-Censits J, Powles T, van der Heijden MS, Balar AV, Necchi A, et al. Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy:a single-arm, multicentre, phase 2 trial. Lancet 2016;387:1909-20.
45. Uprichard SL. The therapeutic potential of RNA interference. FEBS Lett 2005;579:5996-6007.
46. Nishimura H, Nose M, Hiai H, Minato N, Honjo T. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999;11:141-51.
47. Astruc D, Boisseller E, Omelas C. Dendrimers designed for functions:from physical, photophysical, and supramolecular properties to applications in sensing, catalysis, molecular electronics, photonics, and nanomedicine. Chem Rev 2010;110:1857-959.
48. Ros XR, Vermeulen L. Turning cold tumors hot by blocking TGF-β. Trends Canc 2018;4:335-7.
49. Galluzzi L, Chan TA, Kroemer G, Wolchok JD, López-Soto A. The hallmarks of successful anticancer immunotherapy. Sci Transl Med 2018;10:eaat7807.