臺灣博碩士論文加值系統

自然殺手細胞是一種在免疫系統中可有效針對惡性腫瘤細胞進行毒殺的免疫細胞，在免疫治療中被視為具有相當潛力的細胞種類。自然殺手細胞的活化涉及一系列受體與目標細胞的配體互動所組成的複雜程序，該程序由活化型以及抑制型受體傳遞的訊息所控制並最終決定自然殺手細胞活化與否。精準且穩健的遺傳學工具可以有效地解析活化過程中的關鍵步驟與機制並可運用於強化自然殺手細胞針對惡性腫瘤細胞的毒殺能力。目前，廣泛運用於自然殺手細胞的遺傳學工具主要是慢病毒感染技術，然而該技術不僅耗時、低效率且具有隨機整合的風險。此論文旨在開發運用於自然殺手細胞之CRISPR-Cas9基因編輯平台。透過電轉染組裝好之Cas9：guide RNA核蛋白，NK-92細胞株的基因剔除效率在經過一系列的條件優化後可高達90%並同時維持80%細胞存活率。Cas9核蛋白方法亦可將attP39B重組序列精準插入細胞基因體特定位置中，該序列為phiC31重組酵素導引的基因重組平台。本研究建立了一個NK-92細胞株的基因編緝步驟準則，未來可應用於人類初代自然殺手細胞基因編輯以加速生物科學研究與醫療工程開發。

Natural Killer (NK) cell is an effective arsenal in immune system against malignant cells, and is highly regarded as a promising cell type for immunotherapy. Activation of NK cell is an intricate process that involves the interaction between a series of NK cell surface receptors and target cell ligands, providing positive and negative signaling to ultimately determine the activation status of NK cells. A robust and precise genetic tool will be useful to dissect this complex mechanism and to allow engineering of NK cells with enhanced cytotoxicity against malignant cells. Currently, the genetic modification of NK cells relies largely on retroviral transduction, which is time-consuming, inefficient and unsafe due to random viral integration. This thesis aimed to develop a CRISPR-Cas9 gene editing platform for NK cells. By electroporation of pre-assembled Cas9:guide RNA ribonucleoprotein (Cas9 RNP) complexes, the gene knockout process in NK-92 cell line was optimized to achieve editing efficiency up to 90% while maintaining the viability up to 80%. Cas9 RNP method also enabled site-specific knock-in of attP39B sequence, a common landing pad for phiC31-mediated recombination. Collectively, this thesis established a protocol for NK-92 genome editing that can be adopted for primary NK cells to facilitate biological research and therapeutic engineering.

Chapter 1. Introduction ......10
1.1 Natural killer cell ......10
1.1.1 Biological role of natural killer cell ......10
1.1.2 Roles of activating receptors and inhibitory receptors ......11
1.2 Off-the-shelf immune cell therapy ......14
1.2.1 The potential of NK-based adoptive transfer therapy ......14
1.2.2 NK-92 – an off-the-shelf cell therapy candidate ......15
1.3 CRISPR/Cas9 genome engineering system ......17
1.3.1 Introduction to CRISPR/Cas9 system ......17
1.3.2 CRISPR/Cas9 delivery method ......18
1.3.3 Current status in NK cell genome engineering ......19
1.4 Specific Aim ......20
Chapter 2. Result ......22
2.1 Optimizations for CRISPR-Cas9 mediated genome editing in NK-92 ......22
2.1.1 Genome editing pipeline of NK-92 cell ......23
2.1.2 Electroporation condition optimization for Cas9 RNP delivery ......23
2.1.3 Buffer X maintains high cell viability after electroporation ......23
2.1.4 Plasmid DNA is highly toxic to NK-92 cell ......24
2.1.5 Optimized condition enables efficient genome editing of NK-92 ......25
2.1.6 Supplement of fresh IL-2 is indispensable for NK-92 cell recovery ......25
2.2 Successful inhibitory receptors knock-out by CRISPR-Cas9 genome editing ......25
2.2.1 Screening of efficient guide RNA targeting CD96 gene ......25
2.2.2 Screening of efficient guide RNA targeting TIGIT gene ......26
2.2.3 Screening of efficient guide RNA targeting KLRC1 (NKG2A) gene ......26
2.2.4 Screening of efficient guide RNA targeting PDCD1 gene ......27
2.2.5 Double cut strategies enable higher editing efficiency at PDCD1 locus ......27
2.2.6 Co-electroporation of two Cas9 RNP allows successful multiplex inhibitory receptors knock-out ......28
2.3 attP39B recombination sequence can be knock-in at CD96 locus ......29
2.3.1 Development of recombinase system for large DNA fragment knock-in ......29
2.3.2 HindIII restriction site can be integrated into ROSA26 ......29
2.3.3 attP39B recombination sequence is successfully introduced into CD96 ......30
Chapter 3. Discussion ......31
Chapter 4. Material and Method ......34
4.1 Cell culture ......34
4.1.1 Culture medium ......34
4.1.2 Sub-culture method ......34
4.2 sgRNA production ......35
4.2.1 In vitro transcription ......35
4.2.2 Purification by Urea Polyacrylamide Gel (PAGE) electrophoresis ......35
4.2.3 Calf intestinal phosphatase (CIP) treatment .....37
4.3 HDR template production ......38
4.3.1 Short single-strand oligodeoxyribonucleotides (ssODN) template design ......38
4.3.2 Double-strand HDR template production ......38
4.3.3 Long single-strand ODN template production ......39
4.4 Cas9 RNP electroporation ......40
4.4.1 Buffer X preparation ......40
4.4.2 Cas9 RNP assembly ......40
4.4.3 Electroporation ......41
4.5 Genome editing analyses ......42
4.5.1 Genomic DNA extraction and PCR amplification of target region ......42
4.5.2 TIDE analysis ......42
4.5.3 T7E1 analysis ......43
4.5.4 HindIII digestion assay ......44
4.5.5 Agarose gel analysis ......44
4.6 Flow cytometry analyses ......45
4.6.1 Tracking cell viability by amine-reactive dye staining ......45
4.6.2 Surface marker staining ......45
4.6.3 Fluorescent-activated cell sorting ......46
4.7 WST-1 Viability assay ......46
4.8 NK-92 cytotoxicity assay ......47
Reference ......48
Figure 1. Multiplex signaling network that controls the activation of NK cells ......53
Figure 2. Specific aims of NK cell genome editing ......54
Figure 3. Genome editing pipeline of NK-92 cell ......55
Figure 4. Genotyping analyses – T7E1 endonuclease assay and TIDE analysis ......56
Figure 5. Work flow of NK-92 genome editing ......57
Figure 6. Electroporation of NK-92 cells in Buffer X yielded high viability ......58
Figure 7. Optimization of Lonza 4D electroporation program to enable efficient delivery of plasmid DNA and Cas9 RNP ......59
Figure 8. Plasmid is highly toxic to NK-92 ......60
Figure 9. Highest Cas9 editing efficiency was achieved by using electroporation program CM-189 ......61
Figure 10. Supplement of fresh IL-2 was crucial for recovery of NK-92 cells after Electroporation ......62
Figure 11. Screening of efficient guide RNA for knocking out CD96 gene ......63
Figure 12. Screening of efficient guide RNA for knocking out TIGIT gene ......64
Figure 13. Screening of efficient guide RNA for knocking out KLRC1 gene ......65
Figure 14. Screening of efficient guide RNA for knocking out PDCD1 gene ......66
Figure 15. Double-cut enabled higher knock-out efficiency at PDCD1 locus ......67
Figure 16. PDCD1 guide RNA 3 creates larger deletions ......68
Figure 17. Dosage titration of Cas9 RNP targeting at CD96 and KLRC1 loci ......70
Figure 18. Multiplex knock-out in one-shot electroporation ......71
Figure 19. Site-specific plug-in system combined with CRISPR-Cas9 and recombinase technology ......72
Figure 20. Cas9-mediated insertion of HindIII restriction site at ROSA26 ......73
Figure 21. attP39B recombination site is successfully knock-in at CD96 ......74
Supplementary Figure 1. CD96 KO didn’t enhance NK-92 cytotoxicity against K562 ......75
Supplementary Figure 2. GFP reporter knock-in at RAB11A locus in NK-92 and 293T ......76
Appendix ......77
Appendix A: Lonza 4D Nucleofector program optimization table ......77
Appendix B: Antibodies used in study ......78
Appendix C: sgRNA sequences ......79
Appendix D: Primer sequences and PCR conditions for genomic PCR ......82
Appendix E: Primer sequences and PCR conditions for HDR template production ......86
Appendix F: HDR template sequence ......88

1. Vivier, E. et al. Innate or adaptive immunity? The example of natural killer cells. Science (New York, N.Y.) 331, 44-49 (2011).
2. Lunemann, A., Lunemann, J.D. & Munz, C. Regulatory NK-cell functions in inflammation and autoimmunity. Molecular medicine (Cambridge, Mass.) 15, 352-358 (2009).
3. Raulet, D.H. Missing self recognition and self tolerance of natural killer (NK) cells. Seminars in immunology 18, 145-150 (2006).
4. Lodoen, M.B. & Lanier, L.L. Natural killer cells as an initial defense against pathogens. Current opinion in immunology 18, 391-398 (2006).
5. Morvan, M.G. & Lanier, L.L. NK cells and cancer: you can teach innate cells new tricks. Nature Reviews Cancer 16, 7-19 (2016).
6. Guillerey, C., Huntington, N.D. & Smyth, M.J. Targeting natural killer cells in cancer immunotherapy. Nature Immunology 17, 1025-1036 (2016).
7. Kruse, P.H., Matta, J., Ugolini, S. & Vivier, E. Natural cytotoxicity receptors and their ligands. Immunology and cell biology 92, 221-229 (2014).
8. Zhang, J., Basher, F. & Wu, J.D. NKG2D Ligands in Tumor Immunity: Two Sides of a Coin. Frontiers in immunology 6, 97-97 (2015).
9. Nimmerjahn, F. & Ravetch, J.V. Fcgamma receptors: old friends and new family members. Immunity 24, 19-28 (2006).
10. Chan, C.J. et al. The receptors CD96 and CD226 oppose each other in the regulation of natural killer cell functions. Nat Immunol 15, 431-438 (2014).
11. Zhang, Q. et al. Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity. Nature Immunology 19, 723-732 (2018).
12. Mahaweni, N.M. et al. NKG2A Expression Is Not per se Detrimental for the Anti-Multiple Myeloma Activity of Activated Natural Killer Cells in an In Vitro System Mimicking the Tumor Microenvironment. Frontiers in Immunology 9 (2018).
13. Hsu, J. et al. Contribution of NK cells to immunotherapy mediated by PD-1/PD-L1 blockade. Journal of Clinical Investigation 128, 4654-4668 (2018).
14. Gao, J., Zheng, Q., Xin, N., Wang, W. & Zhao, C. CD155, an onco-immunologic molecule in human tumors. Cancer science 108, 1934-1938 (2017).
15. Li, X.Y. et al. CD155 loss enhances tumor suppression via combined host and tumor-intrinsic mechanisms. The Journal of clinical investigation 128, 2613-2625 (2018).
16. Nishiwada, S. et al. Clinical significance of CD155 expression in human pancreatic cancer. Anticancer research 35, 2287-2297 (2015).
17. Zheng, Q. et al. CD155 knockdown promotes apoptosis via AKT/Bcl-2/Bax in colon cancer cells. Journal of cellular and molecular medicine 22, 131-140 (2018).
18. Iguchi-Manaka, A. et al. Increased Soluble CD155 in the Serum of Cancer Patients. PloS one 11, e0152982 (2016).
19. Kučan Brlić, P. et al. Targeting PVR (CD155) and its receptors in anti-tumor therapy. Cellular & Molecular Immunology 16, 40-52 (2019).
20. Mittal, D. et al. CD96 Is an Immune Checkpoint That Regulates CD8(+) T-cell Antitumor Function. Cancer immunology research 7, 559-571 (2019).
21. Pauken, K.E. & Wherry, E.J. TIGIT and CD226: tipping the balance between costimulatory and coinhibitory molecules to augment the cancer immunotherapy toolkit. Cancer cell 26, 785-787 (2014).
22. Kren, L. et al. Expression of immune-modulatory molecules HLA-G and HLA-E by tumor cells in glioblastomas: an unexpected prognostic significance? Neuropathology : official journal of the Japanese Society of Neuropathology 31, 129-134 (2011).
23. Spaans, V.M., Peters, A.A., Fleuren, G.J. & Jordanova, E.S. HLA-E expression in cervical adenocarcinomas: association with improved long-term survival. Journal of translational medicine 10, 184 (2012).
24. Sarkar, S. et al. Optimal selection of natural killer cells to kill myeloma: the role of HLA-E and NKG2A. Cancer immunology, immunotherapy : CII 64, 951-963 (2015).
25. Andre, P. et al. Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells. Cell 175, 1731-1743 e1713 (2018).
26. Ruggeri, L. et al. Effects of anti-NKG2A antibody administration on leukemia and normal hematopoietic cells. Haematologica 101, 626-633 (2016).
27. 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 11, 141-151 (1999).
28. Curran, M.A., Montalvo, W., Yagita, H. & Allison, J.P. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proceedings of the National Academy of Sciences 107, 4275-4280 (2010).
29. Topalian, S.L. et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. The New England journal of medicine 366, 2443-2454 (2012).
30. Veluchamy, J.P. et al. The Rise of Allogeneic Natural Killer Cells As a Platform for Cancer Immunotherapy: Recent Innovations and Future Developments. Frontiers in immunology 8, 631-631 (2017).
31. Granzin, M. et al. Shaping of Natural Killer Cell Antitumor Activity by Ex Vivo Cultivation. Front Immunol 8, 458 (2017).
32. Zeng, J., Tang, S.Y., Toh, L.L. & Wang, S. Generation of "Off-the-Shelf" Natural Killer Cells from Peripheral Blood Cell-Derived Induced Pluripotent Stem Cells. Stem Cell Reports 9, 1796-1812 (2017).
33. Li, Y., Hermanson, D.L., Moriarity, B.S. & Kaufman, D.S. Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Anti-tumor Activity. Cell stem cell 23, 181-192.e185 (2018).
34. Gong, J.H., Maki, G. & Klingemann, H.G. Characterization of a human cell line (NK-92) with phenotypical and functional characteristics of activated natural killer cells. Leukemia 8, 652-658 (1994).
35. Klingemann, H., Boissel, L. & Toneguzzo, F. Natural Killer Cells for Immunotherapy - Advantages of the NK-92 Cell Line over Blood NK Cells. Frontiers in immunology 7, 91-91 (2016).
36. Gomez-Lomeli, P. et al. Increase of IFN-gamma and TNF-alpha production in CD107a + NK-92 cells co-cultured with cervical cancer cell lines pre-treated with the HO-1 inhibitor. Cancer cell international 14, 100 (2014).
37. Maki, G., Klingemann, H.G., Martinson, J.A. & Tam, Y.K. Factors regulating the cytotoxic activity of the human natural killer cell line, NK-92. Journal of hematotherapy & stem cell research 10, 369-383 (2001).
38. Arai, S. et al. Infusion of the allogeneic cell line NK-92 in patients with advanced renal cell cancer or melanoma: a phase I trial. Cytotherapy 10, 625-632 (2008).
39. Williams, B.A. et al. A phase I trial of NK-92 cells for refractory hematological malignancies relapsing after autologous hematopoietic cell transplantation shows safety and evidence of efficacy. Oncotarget 8, 89256-89268 (2017).
40. Tang, X. et al. First-in-man clinical trial of CAR NK-92 cells: safety test of CD33-CAR NK-92 cells in patients with relapsed and refractory acute myeloid leukemia. American journal of cancer research 8, 1083-1089 (2018).
41. Han, J. et al. CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells. Scientific reports 5, 11483 (2015).
42. Romanski, A. et al. CD19-CAR engineered NK-92 cells are sufficient to overcome NK cell resistance in B-cell malignancies. Journal of cellular and molecular medicine 20, 1287-1294 (2016).
43. Schonfeld, K. et al. Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor. Molecular therapy : the journal of the American Society of Gene Therapy 23, 330-338 (2015).
44. Mace, E.M., Gunesch, J.T., Dixon, A. & Orange, J.S. Human NK cell development requires CD56-mediated motility and formation of the developmental synapse. Nature Communications 7, 12171 (2016).
45. Sevim, H., Kocaefe, Y.Ç., Onur, M.A., Uçkan-Çetinkaya, D. & Gürpınar, Ö.A. Bone marrow derived mesenchymal stem cells ameliorate inflammatory response in an in vitro model of familial hemophagocytic lymphohistiocytosis 2. Stem cell research & therapy 9, 198-198 (2018).
46. Horvath, P. & Barrangou, R. CRISPR/Cas, the Immune System of Bacteria and Archaea. Science 327, 167 (2010).
47. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013).
48. Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471-e00471 (2013).
49. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013).
50. Ceccaldi, R., Rondinelli, B. & D''Andrea, A.D. Repair Pathway Choices and Consequences at the Double-Strand Break. Trends in cell biology 26, 52-64 (2016).
51. Wang, Z. et al. Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation. Gene therapy 11, 711-721 (2004).
52. Kim, S., Kim, D., Cho, S.W., Kim, J. & Kim, J.S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome research 24, 1012-1019 (2014).
53. Yu, X. et al. Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX. Biotechnology letters 38, 919-929 (2016).
54. Waller, G. et al. High-Efficiency Lentiviral Genetic Modification of Primary Human Natural Killer Cells. Blood 130, 5566-5566 (2017).
55. Naeimi Kararoudi, M. et al. Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins. Journal of visualized experiments : JoVE (2018).
56. Rautela, J., Surgenor, E. & Huntington, N.D. Efficient genome editing of human natural killer cells by CRISPR RNP. bioRxiv, 406934 (2018).
57. Pomeroy, E.J. et al. A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy. bioRxiv, 430553 (2018).
58. Georgiev, H., Ravens, I., Papadogianni, G. & Bernhardt, G. Coming of Age: CD96 Emerges as Modulator of Immune Responses. Front Immunol 9, 1072 (2018).
59. Gaidukov, L. et al. A multi-landing pad DNA integration platform for mammalian cell engineering. Nucleic acids research 46, 4072-4086 (2018).
60. Zhu, F. et al. DICE, an efficient system for iterative genomic editing in human pluripotent stem cells. Nucleic acids research 42, e34 (2014).
61. Roth, T.L. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405-409 (2018).
62. Li, H. et al. Design and specificity of long ssDNA donors for CRISPR-based knock-in. bioRxiv, 178905 (2017).
63. Groth, A.C., Olivares, E.C., Thyagarajan, B. & Calos, M.P. A phage integrase directs efficient site-specific integration in human cells. Proceedings of the National Academy of Sciences of the United States of America 97, 5995-6000 (2000).