臺灣博碩士論文加值系統

愛滋病是由愛滋病毒(Human Immunodeficiency Virus, HIV)所引起的疾病，患者的免疫系統會被HIV攻擊，使患者抵抗力降低，進而導致患者得到各種疾病使病患死亡。目前主要的治療方式為雞尾酒療法(ART)，不過使用雞尾酒療法並不能完全消滅HIV，另一方面，因為柏林患者的產生，發現CCR5對於HIV感染的重要性，因此本研究計畫將選擇基因編輯技術來進行研究：利用CRISPR/Cas9將患者CCR5Δ32敲除，以現階段的研究成果顯示可以有效抑制CCR5表現，不過假使要應用在臨床方面，尚須優化方法步驟：使用免疫篩選法來替代使用puromysin進行篩選，使篩選的專一性大大的提升，剔除CCR5表現的效率也會跟著產生顯著性變化；另一方面挑選適當的剪切位置讓基因剔除的表現最佳，也讓脫靶效應降到最低，因此本研究以切割位點效率、篩選優化這兩大方向進行。在實驗結果中，標靶設計在CCR5 ECL-I與∆32區域突變的組別最後造成CCR5表現量降低最為顯著；而為了使用免疫篩選法，我們設計了只表現extracellular domain的truncated EGFR ，因此被篩選的免疫細胞不會因為被送入EGFR造成生長影響。之後我們便將tEGFR送入至CRISPR質體DNA，使有成功送入CRISPR系統的細胞也會在細胞表面上表現tEGFR。在轉染的測試中，確實有被送入CRISPR-tEGFR質體DNA的293T細胞表面也有表現著tEGFR，然而使用慢病毒系統將帶有CRISPR-tEGFR的慢病毒顆粒感染至免疫細胞THP-1後，在低病毒濃度組別(M.O.I 30)CCR5表現無明顯下調，而其細胞表面tEGFR表現不高，再經免疫篩選後則會觀察到經篩選的細胞CCR5表現是顯著下調的；另一方面使用高病毒濃度組別(M.O.I 50)則在未篩選前即可觀察到細胞CCR5表現受到調控，然而tEGFR表現也無提升現象，使用免疫篩選後細胞CCR5表現則未更進一步篩選出受調控的細胞。將篩選後之細胞利用偽HIV病毒感染後可以發現隨著CCR5表現的下調，偽HIV病毒的感染效率也跟著下調。從上述結果我們得知目前實驗室所設計之tEGFR能夠使細胞在低病毒濃度下藉由免疫篩選初步篩選出受調控之細胞，且篩選出來的細胞確實能有效抑制偽HIV的感染，而篩選效率的改善如增加tEGFR的表現是未來本研究能改進的目標。

AIDS is a disease caused by Human Immunodeficiency Virus (HIV). The patient's immune system is attacked by HIV, which reduces their resistance, and leads to death with various diseases. At present, the main treatment is Anti-Retroviral Therapy (ART), but the use of ART does not completely eliminate HIV virus. On the other hand, because of the born of Berlin patients, the importance of CCR5 for HIV infection is found. Therefore, this project will use gene editing technology for research. It has showed that CCR5 can be effectively inhibited by using CRISPR/Cas9 system to knockout CCR5Δ32 in patients. However, if it is to be applied in the clinical trial, it is necessary to optimize the method: using the immune-selection instead of puromycin selection, so that the specificity of screening is greatly improved, and the efficiency of eliminating CCR5 performance will also improve significantly. Therefore, this project is carried out in the two major directions of cutting site efficiency and screening optimization. In the results, the groups that targeted to the CCR5 ECL-I and ∆32 region caused the most significant reduction in CCR5 performance. In order to use the immune-selection, we designed a truncated EGFR (tEGFR) that only expresses the extracellular domain, and the selected cells will not be affected because of the effect of EGFR. In the transfection assay, the plasmid DNA that contained CRISPR-tEGFR had been transfected into 293T cells and had been detected the EGFR expression. However, after using the lentivirus system that contained the plasmid DNA of CRISPR-tEGFR to infect the immune cell THP-1, there was no significant down-regulation of CCR5 in the low-viral concentration group (MOI 30), and there was a little tEGFR expression on the cell surface. After immune-selection, it was observed that the CCR5 expression of the selected cells was significantly down-regulated. At last, after infecting the selected cells by pseudo-HIV virus, it was found that according to the down-regulation of CCR5, the infection efficiency of pseudo-HIV virus was also down-regulated. From all the above results, we can know that the current design of tEGFR in the laboratory enables cells to be selected by immune-selection, and the selected cells can effectively inhibit the infection of pseudo-HIV virus. On the other hand, the improvements such as increased the performance of tEGFR are the goal of this project.

摘 要 2
Abstract 3
緒 論 4
 前言 4
 愛滋病毒 5
 CCR5 6
 愛滋病成功治癒病例探討 6
 CRISPR/Cas9 8
 實驗目的 9
材 料 與 方 法 10
1. 細胞培養 10
2. 製備truncated EGFR 11
3. 將truncated EGFR送入pAll-Cas9.pPuro質體DNA中 12
4. 製作完整慢病毒顆粒 13
5. 將轉殖基因質體轉型至勝任細胞(competent cell) 15
6. 將含CRISPR/Cas9序列的慢病毒載體感染至THP-1 cell中 15
7. 以免疫篩選法篩選出轉染成功之細胞 16
8. 確認CCR5基因、蛋白與mRNA表現量有無發生改變 16
9. 生產pseudo HIV病毒顆粒 17
10. 確認野生型和CRISPR/Cas9改造的THP-1 cells被pseudo HIV感染情況 18
11. 數據分析 18
結 果 19
一、 偵測THP-1細胞內CD4、CCR5內生性表現 19
二、 確認慢病毒載體感染濃度與時間 19
三、 觀察THP-1細胞在被不同組別的慢病毒感染後其CCR5的表現量 20
四、 製備truncated EGFR 20
五、 將truncated EGFR送入質體pAll-Cs9.Ppuro裡 21
六、 生產CRISPR-tEGFR慢病毒 21
七、 觀察THP-1細胞在慢病毒感染與免疫篩選後CCR5的變化 22
八、 確認tEGFR表現不佳可能原因 23
九、 生產偽HIV病毒顆粒 23
十、 確認偽HIV病毒載體感染濃度與時間 24
十一、 觀察經改造之THP-1細胞是否可抑制偽HIV病毒感染 24
討 論 25
參 考 文 獻 28
圖 表 一 覽 31

Albanell, J., Codony, J., Rovira, A., Mellado, B., & Gascon, P. (2003). Mechanism of action of anti-HER2 monoclonal antibodies: scientific update on trastuzumab and 2C4. Adv Exp Med Biol, 532, 253-268. doi:10.1007/978-1-4615-0081-0_21
Bell, N. M., & Lever, A. M. (2013). HIV Gag polyprotein: processing and early viral particle assembly. Trends Microbiol, 21(3), 136-144. doi:10.1016/j.tim.2012.11.006
Berger, E. A., Murphy, P. M., & Farber, J. M. (1999). CHEMOKINE RECEPTORS AS HIV-1 CORECEPTORS: Roles in Viral Entry, Tropism, and Disease. Annual Review of Immunology, 17(1), 657-700. doi:10.1146/annurev.immunol.17.1.657
Brown, T. R. (2015). I am the Berlin patient: a personal reflection. AIDS research and human retroviruses, 31(1), 2-3. doi:10.1089/AID.2014.0224
Cestaro, G., De Rosa, M., Gentile, M., & Massa, S. (2015). A case of HPV and acquired genital lymphangioma: over-lapping clinical features. Ann Ital Chir, 86(ePub).
Clavel, F., & Hance, A. J. (2004). HIV drug resistance. N Engl J Med, 350(10), 1023-1035. doi:10.1056/NEJMra025195
De Clercq, E. (2003). The bicyclam AMD3100 story. Nat Rev Drug Discov, 2(7), 581-587. doi:10.1038/nrd1134
Devadas, K., Biswas, S., Haleyurgirisetty, M., Wood, O., Ragupathy, V., Lee, S., & Hewlett, I. (2016). Analysis of Host Gene Expression Profile in HIV-1 and HIV-2 Infected T-Cells. PLoS One, 11(1), e0147421. doi:10.1371/journal.pone.0147421
Didigu, C. A., & Doms, R. W. (2012). Novel approaches to inhibit HIV entry. Viruses, 4(2), 309-324. doi:10.3390/v4020309
Gupta, R. K., Abdul-Jawad, S., McCoy, L. E., Mok, H. P., Peppa, D., Salgado, M., . . . Olavarria, E. (2019). HIV-1 remission following CCR5Delta32/Delta32 haematopoietic stem-cell transplantation. Nature, 568(7751), 244-248. doi:10.1038/s41586-019-1027-4
Hunt, P. W., Harrigan, P. R., Huang, W., Bates, M., Williamson, D. W., McCune, J. M., . . . Deeks, S. G. (2006). Prevalence of CXCR4 tropism among antiretroviral-treated HIV-1-infected patients with detectable viremia. J Infect Dis, 194(7), 926-930. doi:10.1086/507312
Hutter, G., Bodor, J., Ledger, S., Boyd, M., Millington, M., Tsie, M., & Symonds, G. (2015). CCR5 Targeted Cell Therapy for HIV and Prevention of Viral Escape. Viruses, 7(8), 4186-4203. doi:10.3390/v7082816
Khalili, K., Kaminski, R., Gordon, J., Cosentino, L., & Hu, W. (2015). Genome editing strategies: potential tools for eradicating HIV-1/AIDS. J Neurovirol, 21(3), 310-321. doi:10.1007/s13365-014-0308-9
Li, S., Schmitz, K. R., Jeffrey, P. D., Wiltzius, J. J., Kussie, P., & Ferguson, K. M. (2005). Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell, 7(4), 301-311. doi:10.1016/j.ccr.2005.03.003
Liang, M., Kamata, M., Chen, K. N., Pariente, N., An, D. S., & Chen, I. S. (2010). Inhibition of HIV‐1 infection by a unique short hairpin RNA to chemokine receptor 5 delivered into macrophages through hematopoietic progenitor cell transduction. The journal of gene medicine, 12(3), 255-265.
Liu, R., Paxton, W. A., Choe, S., Ceradini, D., Martin, S. R., Horuk, R., . . . Landau, N. R. (1996). Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell, 86(3), 367-377.
Lodge, R., Gilmore, J. C., Ferreira Barbosa, J. A., Lombard-Vadnais, F., & Cohen, E. A. (2017). Regulation of CD4 Receptor and HIV-1 Entry by MicroRNAs-221 and -222 during Differentiation of THP-1 Cells. Viruses, 10(1). doi:10.3390/v10010013
Lopalco, L. (2010). CCR5: From Natural Resistance to a New Anti-HIV Strategy. Viruses, 2(2), 574-600. doi:10.3390/v2020574
Lopalco, L. (2011). Natural anti-CCR5 antibodies in HIV-infection and -exposure. Journal of translational medicine, 9 Suppl 1(Suppl 1), S4-S4. doi:10.1186/1479-5876-9-S1-S4
Lu, L., Yu, F., Cai, L., Debnath, A. K., & Jiang, S. (2016). Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41. Curr Top Med Chem, 16(10), 1074-1090.
Marmor, M., Hertzmark, K., Thomas, S. M., Halkitis, P. N., & Vogler, M. (2006). Resistance to HIV infection. J Urban Health, 83(1), 5-17. doi:10.1007/s11524-005-9003-8
McDermott, D. H., Zimmerman, P. A., Guignard, F., Kleeberger, C. A., Leitman, S. F., & Murphy, P. M. (1998). CCR5 promoter polymorphism and HIV-1 disease progression. Multicenter AIDS Cohort Study (MACS). Lancet, 352(9131), 866-870.
Nachega, J. B., Parienti, J. J., Uthman, O. A., Gross, R., Dowdy, D. W., Sax, P. E., . . . Giordano, T. P. (2014). Lower pill burden and once-daily antiretroviral treatment regimens for HIV infection: A meta-analysis of randomized controlled trials. Clin Infect Dis, 58(9), 1297-1307. doi:10.1093/cid/ciu046
Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., & Zhang, F. (2016). Rationally engineered Cas9 nucleases with improved specificity. Science, 351(6268), 84-88. doi:10.1126/science.aad5227
Wang, X., Chang, W. C., Wong, C. W., Colcher, D., Sherman, M., Ostberg, J. R., . . . Jensen, M. C. (2011). A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood, 118(5), 1255-1263. doi:10.1182/blood-2011-02-337360