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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 凌嘉鴻(Steven Lin) | |
| dc.contributor.author | CHENG-YOU WANG | en |
| dc.contributor.author | 王承宥 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:03:23Z | - |
| dc.date.available | 2021-08-06 | |
| dc.date.available | 2022-11-24T03:03:23Z | - |
| dc.date.copyright | 2021-08-06 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-07-21 | |
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Nonnenmacher, M.E., et al., Syntaxin 5-dependent retrograde transport to the trans-Golgi network is required for adeno-associated virus transduction. Journal of virology, 2015. 89(3): p. 1673. 44. Sáenz, J.B., et al., Golgicide A reveals essential roles for GBF1 in Golgi assembly and function. Nature chemical biology, 2009. 5(3): p. 157-165. 45. Madigan, V.J., et al., The Golgi Calcium ATPase Pump Plays an Essential Role in Adeno-associated Virus Trafficking and Transduction. Journal of Virology, 2020. 94(21): p. e01604-20. 46. Mitchell, A.M. and R.J. Samulski, Mechanistic insights into the enhancement of adeno-associated virus transduction by proteasome inhibitors. Journal of virology, 2013. 87(23): p. 13035. 47. Monahan, P.E., et al., Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: a strategy for broad clinical application. Molecular therapy, 2010. 18(11): p. 1907-1916. 48. Johnson, J.S. and R.J. Samulski, Enhancement of adeno-associated virus infection by mobilizing capsids into and out of the nucleolus. Journal of virology, 2009. 83(6): p. 2632-2644. 49. Sonntag, F., et al., Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus. Journal of virology, 2006. 80(22): p. 11040-11054. 50. Kelich, J.M., et al., Super-resolution imaging of nuclear import of adeno-associated virus in live cells. Molecular Therapy-Methods Clinical Development, 2015. 2: p. 15047. 51. Nakai, H., T.A. Storm, and M.A. Kay, Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo. Journal of virology, 2000. 74(20): p. 9451-9463. 52. Schwartz, R.A., et al., The Mre11/Rad50/Nbs1 complex limits adeno-associated virus transduction and replication. Journal of virology, 2007. 81(23): p. 12936-12945. 53. Huang, R.S., et al., A robust platform for expansion and genome editing of primary human natural killer cells. J Exp Med, 2021. 218(3). 54. Huang, R.-S., et al., Enhanced NK-92 Cytotoxicity by CRISPR Genome Engineering Using Cas9 Ribonucleoproteins. Frontiers in Immunology, 2020. 11: p. 1008. 55. Aurnhammer, C., et al., Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences. Human Gene Therapy Methods, 2011. 23(1): p. 18-28. 56. Chandler, L.C., et al., Enhancement of Adeno-Associated Virus-Mediated Gene Therapy Using Hydroxychloroquine in Murine and Human Tissues. Molecular therapy. Methods clinical development, 2019. 14: p. 77-89. 57. Kararoudi, M.N., et al., CRISPR-Targeted CAR Gene Insertion Using Cas9/RNP and AAV6 Enhances Anti-AML Activity of Primary NK Cells. 2021, Cold Spring Harbor Laboratory. 58. 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Molecular Therapy, 2007. 15(7): p. 1331-1339. 64. Martino, A.T., et al., The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9–dependent innate immune responses in the liver. Blood, 2011. 117(24): p. 6459-6468. 65. Ding, W., et al., Intracellular trafficking of adeno-associated viral vectors. Gene Therapy, 2005. 12(11): p. 873-880. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80259 | - |
| dc.description.abstract | 在人體細胞中標準且有效傳遞轉基因的方式是利用病毒載體進行轉導。然而，由於自然殺手細胞對於病毒感染和外源DNA具有一定的抗性，導致病毒載體的轉導效率不佳且沒有一致性。逆轉錄病毒和慢病毒是僅有可以轉導自然殺手細胞表達轉基因的病毒，但需要高感染複數並且存在插入突變的風險。腺相關病毒是有前瞻性的替代品，因為它的安全性已在臨床環境中得到證實。一份文獻表明腺相關病毒血清型6在自然殺手細胞的高效使用，但我們實驗是和其他團隊無法重現他們的結果。在NK-92細胞株中，需要非常高的感染複數才能看到腺相關病毒血清型6轉導訊號；在初代自然殺手細胞則完全無法轉導。為了瞭解和克服腺相關病毒轉導的侷限性，我開始研究腺相關病毒附著和進入自然殺手細胞後可能引起的機制。我發現腺相關病毒受體在不同細胞型態有表達量和分子量的差異，以及我透過抑制劑研究了DNA傳感器的重要性，但是這兩種因素並非腺相關病毒無法轉導自然殺手細胞的主要機制。 最後，我證實腺相關病毒血清型9以及腺相關病毒血清型DJ能在初代自然殺手細胞有接近2%轉基因的表達，這是在先前研究中尚未有人報導的特性。探討腺相關病毒血清型適性以及細胞內抑制轉導的機制對於在具有臨床價值的自然殺手細胞實現強大的轉導是必要的。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:03:23Z (GMT). No. of bitstreams: 1 U0001-0407202121470200.pdf: 3763698 bytes, checksum: 3d82955f053e7e67f5b5b9dc967650dc (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "摘要 i Abstract ii 目錄 iv 1. Introduction 1 1.1 The physiological role and regulation of natural killer cells 1 1.2 Genome engineering of NK cells 2 1.3 Property and purification of adeno associated virus (AAV) 4 1.4 Factors affecting the efficiency of AAV infection 6 1.5 Current challenges in primary NK cells 9 1.6 Specific aim of this study 10 2. Result 12 2.1 Establish a quick and simple method of AAV purification 12 2.2 The low transduction efficiency of AAV6 in NK cells has no positive correlation between the expression of AAVR 13 2.3 AAV6 can be internalized into NK cells, but cannot be effectively used as an HDR template. 15 2.4 The effect of various transduction conditions on transfection efficiency. 17 2.5 Effects of various inhibitors on AAV6 transduction of NK cells 18 2.6 The potential AAV serotypes are more suitable for NK cells. 20 3. Discussion 22 4. Materials and Methods 28 4.1 Cell culture 28 4.2 Plasmid construct 30 4.3 Lipofectamine transfection 31 4.4 Synthesis of sgRNA by T7 in vitro transcription (IVT) 31 4.5 Calf intestinal alkaline phosphatase (CIP) treatment 35 4.6 Nucleofection 36 4.7 Transfection with polyethylenimine (PEI) 38 4.8 Collect cells and culture medium and break cells by freeze and thaw 39 4.9 PEG precipitation and Chloroform extraction 40 4.10 ATP partition, concentration and buffer exchange 41 4.11 Evaluating the titer of purified AAV by qPCR 42 4.12 SDS PAGE 43 4.13 Western blot 44 4.14 AAV transduction 44 4.15 Flow cytometry 48 4.16 AAV uptake test by semi-quantitative PCR 49 4.17 Nucleofection of Cas9 RNP and HDR transduction of AAV6 for KI 50 4.18 Gene-editing analysis by DNA sequencing 51 5. Reference 52 6. Figure and Table 57 圖目錄 Figure 1 : Flow chart of AAV purification 57 Figure 2 : Analysis of AAV purity, titration and transduction efficiency. 58 Figure 3 : Schematic diagram of AAV transduction pathway. 59 Figure 4 : Analysis of AAVR protein expression level in different cell lines and generate AAVR knock-out cells. 60 Figure 5 : AAV6 transduction efficiency in different cells type. 61 Figure 6 : Primary NK cells overexpress AAVR by electroporation and HeLa ∆AAVR was rescued. 63 Figure 7 : Semi quantitative PCR results of AAV internalization into various cells. 64 Figure 8 : Gene knock-in of primary NK cells using CRISPR and AAV6. 66 Figure 9 : Optimized AAV6 transduction conditions on NK-92. 67 Figure 10 : NK-92 treated with BAPTA-AM and DNA sensor inhibitors to improve AAV transduction efficiency. 69 Figure 11 : DNA sensor inhibitor G140 is not helpful for gene knock-in. 70 Figure 12 : AAV serotype kit and different source of AAV6 test on primary NK cells. 71 表目錄 Table 1 : Strains, plasmids, primers ,antibody and inhibitors used in this study. 72 Table 2 : PCR condition and program setting. 75 Table 3 : Commercial AAV serotype and source list 77" | |
| dc.language.iso | en | |
| dc.subject | 抗病毒機制 | zh_TW |
| dc.subject | 自然殺手細胞 | zh_TW |
| dc.subject | 腺相關病毒 | zh_TW |
| dc.subject | 腺相關病毒受體 | zh_TW |
| dc.subject | CRISPR/Cas9 | zh_TW |
| dc.subject | CRISPR/Cas9 | en |
| dc.subject | Antiviral mechanism | en |
| dc.subject | AAVR | en |
| dc.subject | NK cell | en |
| dc.subject | AAV | en |
| dc.title | 實現高效率的AAV感染在人類primary NK細胞上 | zh_TW |
| dc.title | Enabling robust AAV transduction in primary human NK cells | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張茂山(Hsin-Tsai Liu),許家維(Chih-Yang Tseng) | |
| dc.subject.keyword | 自然殺手細胞,腺相關病毒,腺相關病毒受體,CRISPR/Cas9,抗病毒機制, | zh_TW |
| dc.subject.keyword | NK cell,AAV,AAVR,CRISPR/Cas9,Antiviral mechanism, | en |
| dc.relation.page | 77 | |
| dc.identifier.doi | 10.6342/NTU202101267 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-07-21 | |
| 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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