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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 凌嘉鴻(Steven Lin) | |
| dc.contributor.author | Min-Chi Lai | en |
| dc.contributor.author | 賴敏碁 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:03:23Z | - |
| dc.date.available | 2021-07-20 | |
| dc.date.available | 2022-11-24T03:03:23Z | - |
| dc.date.copyright | 2021-07-20 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-07-05 | |
| dc.identifier.citation | 1. Vivier, E., J.A. Nunès, and F. Vély, Natural killer cell signaling pathways. Science, 2004. 306(5701): p. 1517-1519. 2. Lanier, L.L., Natural killer cell receptor signaling. Current opinion in immunology, 2003. 15(3): p. 308-314. 3. Pegram, H.J., et al., Activating and inhibitory receptors of natural killer cells. Immunol Cell Biol, 2011. 89(2): p. 216-24. 4. Stetson, D.B., et al., Constitutive cytokine mRNAs mark natural killer (NK) and NK T cells poised for rapid effector function. The Journal of experimental medicine, 2003. 198(7): p. 1069-1076. 5. Raulet, D.H. Missing self recognition and self tolerance of natural killer (NK) cells. in Seminars in immunology. 2006. Elsevier. 6. Prager, I. and C. Watzl, Mechanisms of natural killer cell‐mediated cellular cytotoxicity. Journal of leukocyte biology, 2019. 105(6): p. 1319-1329. 7. Romee, R., et al., NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17). Blood, 2013. 121(18): p. 3599-3608. 8. Myers, J.A. and J.S. Miller, Exploring the NK cell platform for cancer immunotherapy. Nature Reviews Clinical Oncology, 2021. 18(2): p. 85-100. 9. 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, 2017. 8: p. 631. 10. Parkhurst, M.R., et al., Adoptive transfer of autologous natural killer cells leads to high levels of circulating natural killer cells but does not mediate tumor regression. Clinical Cancer Research, 2011. 17(19): p. 6287-6297. 11. Ruggeri, L., et al., Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science, 2002. 295(5562): p. 2097-2100. 12. Liu, E., et al., Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. New England Journal of Medicine, 2020. 382(6): p. 545-553. 13. Ruggeri, L., et al., Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value. Blood, 2007. 110(1): p. 433-440. 14. Lupo, K.B. and S. Matosevic, Natural killer cells as allogeneic effectors in adoptive cancer immunotherapy. Cancers, 2019. 11(6): p. 769. 15. Oh, S., et al., Natural killer cell therapy: a new treatment paradigm for solid tumors. Cancers, 2019. 11(10): p. 1534. 16. Steinhauer, E., et al., Defective natural cytotoxicity in patients with cancer: normal number of effector cells but decreased recycling capacity in patients with advanced disease. The Journal of Immunology, 1982. 129(5): p. 2255-2259. 17. Yang, Y., et al., Superior expansion and cytotoxicity of human primary NK and CAR-NK cells from various sources via enriched metabolic pathways. Molecular Therapy-Methods Clinical Development, 2020. 18: p. 428-445. 18. Ojo, E.O., et al., Membrane bound IL-21 based NK cell feeder cells drive robust expansion and metabolic activation of NK cells. Scientific reports, 2019. 9(1): p. 1-12. 19. Bae, D.S. and J.K. Lee, Development of NK cell expansion methods using feeder cells from human myelogenous leukemia cell line. Blood research, 2014. 49(3): p. 154. 20. Denman, C.J., et al., Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells. PloS one, 2012. 7(1): p. e30264. 21. Nagai, K., et al., Highly Activated Ex Vivo-expanded Natural Killer Cells in Patients With Solid Tumors in a Phase I/IIa Clinical Study. Anticancer research, 2020. 40(10): p. 5687-5700. 22. Tanaka, J., et al., Phase I study of cellular therapy using ex vivo expanded natural killer cells from autologous peripheral blood mononuclear cells combined with rituximab-containing chemotherapy for relapsed CD20-positive malignant lymphoma patients. haematologica, 2020. 105(4): p. e190. 23. Bailey, S.R. and M.V. Maus, Gene editing for immune cell therapies. Nature biotechnology, 2019. 37(12): p. 1425-1434. 24. Roth, T.L., et al., Reprogramming human T cell function and specificity with non-viral genome targeting. Nature, 2018. 559(7714): p. 405-409. 25. Rautela, J., E. Surgenor, and N.D. Huntington, Efficient genome editing of human natural killer cells by CRISPR RNP. BioRxiv, 2018: p. 406934. 26. Ingegnere, T., et al., Human CAR NK cells: a new non-viral method allowing high efficient transfection and strong tumor cell killing. Frontiers in immunology, 2019. 10: p. 957. 27. Hacein-Bey-Abina, S., et al., Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. The Journal of clinical investigation, 2008. 118(9): p. 3132-3142. 28. Imai, C., S. Iwamoto, and D. Campana, Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood, 2005. 106(1): p. 376-383. 29. Pomeroy, E.J., et al., A genetically engineered primary human natural killer cell platform for cancer immunotherapy. Molecular Therapy, 2020. 28(1): p. 52-63. 30. Kararoudi, M.N., et al., Generation of knock-out primary and expanded human NK cells using Cas9 ribonucleoproteins. JoVE (Journal of Visualized Experiments), 2018(136): p. e58237. 31. Kwang, T.W., X. Zeng, and S. Wang, Manufacturing of AcMNPV baculovirus vectors to enable gene therapy trials. Molecular Therapy-Methods Clinical Development, 2016. 3: p. 15050. 32. Lu, H.-Y., Y.-H. Chen, and H.-J. Liu, Baculovirus as a vaccine vector. Bioengineered, 2012. 3(5): p. 271-274. 33. Lewis, L., R. Lynch, and J. Jackson, Pathology of a baculovirus of the alfalfa looper, Autographa californica, in the European corn borer, Ostrinia nubilalis. Environmental Entomology, 1977. 6(4): p. 535-538. 34. Boyce, F.M. and N. Bucher, Baculovirus-mediated gene transfer into mammalian cells. Proceedings of the National Academy of Sciences, 1996. 93(6): p. 2348-2352. 35. Kataoka, C., et al., Baculovirus GP64-mediated entry into mammalian cells. Journal of virology, 2012. 86(5): p. 2610-2620. 36. Mansouri, M., et al., Highly efficient baculovirus-mediated multigene delivery in primary cells. Nature communications, 2016. 7(1): p. 1-13. 37. Sanchez, L., Using Vesicular Stomatitis Virus Glycoprotein to Pseudotype BacMam Viruses for Enhancement of Transduction Efficiency. 2011. 38. Alter, G., J.M. Malenfant, and M. Altfeld, CD107a as a functional marker for the identification of natural killer cell activity. Journal of immunological methods, 2004. 294(1-2): p. 15-22. 39. Wang, R., et al., Natural killer cell‐produced IFN‐γ and TNF‐α induce target cell cytolysis through up‐regulation of ICAM‐1. Journal of leukocyte biology, 2012. 91(2): p. 299-309. 40. Chauvin, J.-M. and H.M. Zarour, TIGIT in cancer immunotherapy. Journal for immunotherapy of cancer, 2020. 8(2). 41. Bottino, C., et al., Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule. The Journal of experimental medicine, 2003. 198(4): p. 557-567. 42. Liu, F., et al., CD96, a new immune checkpoint, correlates with immune profile and clinical outcome of glioma. Scientific reports, 2020. 10(1): p. 1-10. 43. Makkonen, K.-E., et al., 6-O-and N-sulfated syndecan-1 promotes baculovirus binding and entry into mammalian cells. Journal of virology, 2013. 87(20): p. 11148-11159. 44. Finkelshtein, D., et al., LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proceedings of the National Academy of Sciences, 2013. 110(18): p. 7306-7311. 45. Leung, W., Infusions of allogeneic natural killer cells as cancer therapy. Clinical cancer research, 2014. 20(13): p. 3390-3400. 46. Carson, W.E., et al., Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. Journal of Experimental Medicine, 1994. 180(4): p. 1395-1403. 47. Truong, V.A., et al., CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration. Nucleic acids research, 2019. 47(13): p. e74-e74. 48. Amalfi, S., et al., Baculovirus Transduction in Mammalian Cells Is Affected by the Production of Type I and III Interferons, Which Is Mediated Mainly by the cGAS-STING Pathway. Journal of Virology, 2020. 94(21). 49. Lin, S., et al., Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. elife, 2014. 3: p. e04766. 50. Kim, S., et al., CRISPR RNAs trigger innate immune responses in human cells. Genome research, 2018. 28(3): p. 367-373. 51. Jeong, J., et al., High-efficiency CRISPR induction of t (9; 11) chromosomal translocations and acute leukemias in human blood stem cells. Blood advances, 2019. 3(19): p. 2825-2835. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80258 | - |
| dc.description.abstract | 自然殺手細胞是在癌症免疫治療中非常有潛力的先天性免疫細胞。為了研究和增強自然殺手細胞的功能，強大且完整的基因編輯工具是必須的。但是，自然殺手細胞對於傳統的基因編輯方式例如質體轉染和病毒轉導都是很困難的，所以為了克服這些限制，我在這篇論文中為了自然殺手細胞建立一個強大的CRISPR基因編輯的平台並且是利用電轉染組裝好之Cas9: guide RNA核蛋白送入細胞並進行基因剔除。為了要送入大片段的轉殖基因到自然殺手細胞中，我們選擇了擁有不會嵌入宿主基因體和大轉殖基因乘載量(38kb)的桿狀病毒，這能讓我們送入許多不同的基因。然而我們發現野生型的桿狀病毒病無法感染自然殺手細胞。在這篇論文當中，我觀察並編輯了自然殺手細胞表面受體使其能被野生型的桿狀病毒感染，我也利用了細胞內先天免疫反應的抑制藥物，希望能避免桿狀病毒感染細胞後產生的免疫反應。這篇論文當中的發現提供了桿狀病毒感染自然殺手細胞後重要的現象，也為未來以桿狀病毒為基礎的自然殺手細胞基因編輯平台奠定了重要的基礎。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:03:23Z (GMT). No. of bitstreams: 1 U0001-0507202100173400.pdf: 3281252 bytes, checksum: 4a541eef780f8b2aaed61141713cbf8a (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | Chapter 1. Introduction 10 1.1 Natural killer cell 10 1.1.1 Immunology of human natural killer cells 10 1.1.2 Therapeutic potential of NK cells 11 1.2 Current limitations of NK cell immunotherapy 12 1.2.1 Ex vivo culture and expansion of NK cells 12 1.2.2 Current status of genome editing in NK cells 14 1.2.3 Baculoviral vector for transgene delivery in NK cells 15 1.3 Specific aims 17 Chapter 2. Result 18 2.1 Ex vivo expansion of primary NK cells 18 2.2 Optimization for CRISPR-Cas9 genome engineering in primary NK cells 19 2.2.1 Optimized nucleofection condition enables efficient delivery of Cas9 RNP and DNA 19 2.2.2 NK cells can re-expand and sustain comparable degranulation ability 21 2.2.3 High dosages of Cas9 RNP and the 5’ phosphate moiety of the in vitro transcribed sgRNA are toxic to NK cells 22 2.2.4 Multiple KO by Cas9 RNP nucleofection is highly efficient 22 2.2.5 Multiple KO induces chromosome translocation 23 2.3 Optimization of baculoviral transduction in NK cells 24 2.3.1 Large-scale baculoviral production using Sf9 with serum-free medium 24 2.3.2 Overexpression of CD138 receptor in NK cells does not improve baculoviral transduction 25 2.3.3 The modified BacMam baculovirus enters NK-92 cell line after transduction 26 2.3.4 Baculoviral transduction triggers cGAS-dependent intracellular innate immunity responses 27 Chapter 3. Discussion 30 Chapter 4. Materials Methods 36 4.1 Ex vivo expansion of primary human NK cells 36 4.2 Cryopreservation and thawing of expanded NK cells 38 4.3 Flow cytometry analyses and cell sorting 39 4.4 Precision beads viability assay 41 4.5 Calcein-AM cytotoxic assay 41 4.6 Degranulation assay 43 4.7 Cell culture 43 4.8 Synthesis of single-guide RNA 44 4.9 Cas9 RNP and DNA nucleofection 46 4.10 Chromosome translocation analysis 46 4.11 Baculovirus production 47 4.11.1 Transfer plasmid construction 47 4.11.2 Bacmid recombination 48 4.11.3 Sf9 bacmid transfection 50 4.11.4 Virus amplification 51 4.11.5 Virus concentration 51 4.12 baculoviral DNA extraction and titer titration by quantitative-PCR 52 4.13 CD138 cDNA construction and precipitation 53 4.14 Detection of intracellular baculoviral DNA by quantitative-PCR 54 4.15 Detection of IFNβ and IL-6 mRNA by quantitative reverse transcription PCR 54 4.16 Blocking of intracellular innate immunity responses by small molecule 55 inhibitors 55 References 56 Figure 1 Specific aims of primary NK cell CRISPR genome engineering 60 Figure 2 Optimized feeder-free ex vivo expansion from cryopreserved human primary NK cells 62 Figure 3 The NK cell expanded in NK MACS and EL837 showed similar surface marker and cytotoxicity. 63 Figure 4 Nucleofection optimization identified conditions for efficient and viable delivery of Cas9 RNP and DNA. 65 Figure 5 The engineered and parental NK cell showed comparable expansion and degranulation ability. 66 Figure 6 Cas9 RNP dosage and sgRNA preparation approach were important for cell viability after nucleofection. 67 Figure 7 Multiple KO was highly efficient by Cas9 RNP nucleofection. 69 Figure 8 Multiplex KO induced chromosomal translocation. 70 Figure 9 Large-scale baculovirus production in Sf9 suspension cell culture. 71 Figure 10 CD138 receptor could be overexpressed by dsDNA nucleofection but failed to improve baculovirus transduction. 73 Figure 11 VSV-G modified baculovirus (BacMam) could transduce NK-92 cell line. 75 Figure 12 Baculovirus triggered cGAS-relative innate immune cellular responses. 77 Appendix 78 Appendix A: Lonza 4D Nucleofector program optimization table 78 Appendix B: Antibodies list 79 Appendix C: sgRNA sequences 80 Appendix D: PCR primer list 81 Appendix E: PCR program 83 | |
| dc.language.iso | en | |
| dc.subject | 免疫療法 | zh_TW |
| dc.subject | 自然殺手細胞 | zh_TW |
| dc.subject | CRISPR | zh_TW |
| dc.subject | 電轉染 | zh_TW |
| dc.subject | 桿狀病毒 | zh_TW |
| dc.subject | CRISPR | en |
| dc.subject | Immunotherapy | en |
| dc.subject | Baculovirus | en |
| dc.subject | Nucleofection | en |
| dc.subject | Natural killer cell | en |
| dc.title | 為初代人類自然殺手細胞建立CRISPR 基因編輯平台 | zh_TW |
| dc.title | CRISPR genome engineering platform for primary human natural killer cells | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.author-orcid | 0000-0002-5937-116X | |
| dc.contributor.oralexamcommittee | 張茂山(Hsin-Tsai Liu),趙裕展(Chih-Yang Tseng) | |
| dc.subject.keyword | 自然殺手細胞,CRISPR,電轉染,桿狀病毒,免疫療法, | zh_TW |
| dc.subject.keyword | Natural killer cell,CRISPR,Nucleofection,Baculovirus,Immunotherapy, | en |
| dc.relation.page | 83 | |
| dc.identifier.doi | 10.6342/NTU202101268 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-07-06 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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