合成生物學是一種將基因工程應用在生物系統的方法及工具，從基因體組成、調控、訊息傳遞延伸到細胞設計甚至是整個生物系統的改造。在這個研究裡，我們將合成生物技術應用於兩類生物系統-癌症治療基因載體的設計以及抗體生產細胞的改造。在第一部份-以合成開關控制的桿狀病毒載體用以調控外源基因表達及選擇性毒殺肝癌細胞: 桿狀病毒所發展的抗癌基因載體對於肝癌的治療有相當好的潛力，但應用到生物體內時，常無法避免抗癌基因也在正常細胞表現並產生毒性，為了提高安全性，我們設計了能夠感應細胞特有miRNA圖譜的miRNA感應器，並與RNA結合蛋白-L7Ae組裝成外源基因轉譯的開關，裝載到桿狀病毒載體，研究結果顯示重組的桿狀病毒載體能有效地進入肝細胞和正常細胞，但外源基因僅會在肝癌細胞中的啟動，當使用促凋亡的hBax作為外源基因時，桿狀病毒載體能在肝癌細胞和正常細胞共培養時具選擇性殺死肝癌細胞，這些結果證明了帶有合成開關元件的桿狀病毒載體，能辨識癌細胞執行專一性毒殺。在第二部分-利用CRISPR/Cas13系統進行CHO細胞株多基因調控增加去岩藻糖抗體的產量及活性: 中國倉鼠卵巢 (CHO)細胞是最廣泛使用在生產生物藥的細胞工廠，我們利用CRISPR-Cas13d RNA靶向系統進行CHO細胞系統的調控編輯，我們證實CRISPR-Cas13d有效地抑制LDHA, GFT, DDIT3以及CLU等基因，並造成功能上的改變，包含: 乳酸的產生、岩藻糖醣基化程度、氧化自由基的含量及細胞團塊的形成。再者，結合Sleeping Beauty system使CRISPR-Cas13d模組更穩定在CHO細胞株達到多重基因同時調控並證明整體抗體產量顯著提高，且降低抗體岩藻糖醣基化促使抗體依賴性細胞毒殺效果提升。展現Cas13d在CHO細胞系細胞工程中的應用潛能。綜合上述結果，在生物系統的架構裡，透過裝載合成的外源基因組件，我們優化病毒載體的效能及安全性應用於肝癌治療，並且編輯CHO細胞轉錄體增加抗體活性及產量。

Synthetic biology is a tool of applying genetic engineering to biological systems, which comprises genome manipulation, regulation, signal transduction, cell engineering or programming of entire biological systems. In this study, we applied synthetic biotechnology to cancer therapeutic vector design and engineering of antibody-producing cells. Part I-Synthetic switch-based baculovirus for transgene expression control and selective killing of hepatocellular carcinoma cells: Baculovirus (BV) holds promise as a vector for anticancer gene delivery to combat hepatocellular carcinoma (HCC). However, in vivo BV administration inevitably results in BV entry into non-HCC normal cells and leaky anticancer gene expression. To improve the safety, we employed a miRNA sensor and assembled it with RNA binding protein, L7Ae, for BV design. Our result showed the recombinant BV efficiently entered HCC and normal cells, enabled switching ON transgene exclusively in HCC cells. Using pro-apoptotic hBax as the transgene, the switch-based BV selectively killed HCC cells in mixed culture of HCC and normal cells. These data demonstrate the potential of synthetic switch-based BV to distinguish HCC and normal cells for selective killing of HCC cells. Part II-Enhancing the yield and activity of defucosylated antibody produced from CHO-K1 cells using Cas13-mediated multiplex gene targetin: Chinese hamster ovary (CHO) cells are the most widely used in biopharmaceutical cell factories. We have demonstrated CRISPR-Cas13d effectively modulate the expression of several endogenous gene such as LDHA, GFT, DDIT3 and CLU, which also gave rise to the inhibition of lactic acid production, fucosylation, the reactive oxygen species generation, and cell aggregation. Furthermore, exploitation of Sleeping Beauty system-mediated integration of CRISPR-Cas13d steadily suppressed multiplex gene expression in CHO cell and endow the cell with high productivity and trimming of antibody fucosylation relevant to the enhancement of antibody-dependent cell-mediated Cytotoxicity. Collectively, we already implanted the synthetic device into the vector or organism construction for optimizing the effectiveness of viral vectors in liver cancer treatment and engineering the CHO cell transcriptome to increase antibody yield and activity.

1. T. Tian et al., MiR-146a and miR-196a-2 polymorphisms are associated with hepatitis virus-related hepatocellular cancer risk: a meta-analysis. Aging (Albany NY) 9, 381-392 (2017).
2. C. L. Chen et al., Suppression of hepatocellular carcinoma by baculovirus-mediated expression of long non-coding RNA PTENP1 and MicroRNA regulation. Biomaterials 44, 71-81 (2015).
3. K. J. Airenne et al., Baculovirus: an insect-derived vector for diverse gene transfer applications. Mol Ther 21, 739-749 (2013).
4. J. Zeng, J. Du, Y. Zhao, N. Palanisamy, S. Wang, Baculoviral vector-mediated transient and stable transgene expression in human embryonic stem cells. Stem cells 25, 1055-1061 (2007).
5. W. Liu et al., MicroRNA-16 targets amyloid precursor protein to potentially modulate Alzheimer's-associated pathogenesis in SAMP8 mice. Neurobiology of aging 33, 522-534 (2012).
6. Y. H. Chen et al., Baculovirus-mediated production of HDV-like particles in BHK cells using a novel oscillating bioreactor. Journal of biotechnology 118, 135-147 (2005).
7. C. L. Chen et al., Development of hybrid baculovirus vectors for artificial MicroRNA delivery and prolonged gene suppression. Biotechnol Bioeng 108, 2958-2967 (2011).
8. S. Y. Lin, Y. C. Chung, Y. C. Hu, Update on baculovirus as an expression and/or delivery vehicle for vaccine antigens. Expert review of vaccines 13, 1501-1521 (2014).
9. C. H. Lu, Y. H. Chang, S. Y. Lin, K. C. Li, Y. C. Hu, Recent progresses in gene delivery-based bone tissue engineering. Biotechnology advances 31, 1695-1706 (2013).
10. S. Wang, G. Balasundaram, Potential cancer gene therapy by baculoviral transduction. Curr Gene Ther 10, 214-225 (2010).
11. T. J. Wickham, T. Davis, R. R. Granados, M. L. Shuler, H. A. Wood, Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system. Biotechnology progress 8, 391-396 (1992).
12. K. J. Airenne, O. H. Laitinen, A. J. Mahonen, S. Yla-Herttuala, Preparation of recombinant baculoviruses with the BVboost system. Cold Spring Harbor protocols 2009, pdb prot5181 (2009).
13. K. J. Airenne, E. Peltomaa, V. P. Hytonen, O. H. Laitinen, S. Yla-Herttuala, Improved generation of recombinant baculovirus genomes in Escherichia coli. Nucleic acids research 31, e101 (2003).
14. R. B. Hitchman et al., Genetic modification of a baculovirus vector for increased expression in insect cells. Cell biology and toxicology 26, 57-68 (2010).
15. S. Sokolenko et al., Co-expression vs. co-infection using baculovirus expression vectors in insect cell culture: Benefits and drawbacks. Biotechnology advances 30, 766-781 (2012).
16. C. Y. Chen et al., Biosafety assessment of human mesenchymal stem cells engineered by hybrid baculovirus vectors. Mol Pharm, 1505-1514 (2011).
17. B. Y. Hwang, D. V. Schaffer, Engineering a serum-resistant and thermostable vesicular stomatitis virus G glycoprotein for pseudotyping retroviral and lentiviral vectors. Gene therapy 20, 807-815 (2013).
18. A. Trabalza et al., Venezuelan equine encephalitis virus glycoprotein pseudotyping confers neurotropism to lentiviral vectors. Gene therapy 20, 723-732 (2013).
19. B. Balakrishnan, G. R. Jayandharan, Basic biology of adeno-associated virus (AAV) vectors used in gene therapy. Current gene therapy 14, 86-100 (2014).
20. D. J. Dismuke, L. Tenenbaum, R. J. Samulski, Biosafety of recombinant adeno-associated virus vectors. Current gene therapy 13, 434-452 (2013).
21. M. O'Reilly et al., NIH oversight of human gene transfer research involving retroviral, lentiviral, and adeno-associated virus vectors and the role of the NIH recombinant DNA advisory committee. Methods in enzymology 507, 313-335 (2012).
22. A. Baldo, E. van den Akker, H. E. Bergmans, F. Lim, K. Pauwels, General considerations on the biosafety of virus-derived vectors used in gene therapy and vaccination. Current gene therapy 13, 385-394 (2013).
23. T. A. Kost, J. P. Condreay, Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends in biotechnology 20, 173-180 (2002).
24. C. Wu et al., Combinatorial control of suicide gene expression by tissue-specific promoter and microRNA regulation for cancer therapy. Molecular therapy : the journal of the American Society of Gene Therapy 17, 2058-2066 (2009).
25. J. Lin, S. Teo, D. H. Lam, K. Jeyaseelan, S. Wang, MicroRNA-10b pleiotropically regulates invasion, angiogenicity and apoptosis of tumor cells resembling mesenchymal subtype of glioblastoma multiforme. Cell death & disease 3, e398 (2012).
26. Y. H. Liao et al., Osteogenic differentiation of adipose-derived stem cells and calvarial defect repair using baculovirus-mediated co-expression of BMP-2 and miR-148b. Biomaterials 35, 4901-4910 (2014).
27. X. Y. Bak, J. Yang, S. Wang, Baculovirus-transduced bone marrow mesenchymal stem cells for systemic cancer therapy. Cancer gene therapy 17, 721-729 (2010).
28. T. Suzuki, M. O. Chang, M. Kitajima, H. Takaku, Baculovirus activates murine dendritic cells and induces non-specific NK cell and T cell immune responses. Cellular immunology 262, 35-43 (2010).
29. C. Y. Chen, C. Y. Lin, G. Y. Chen, Y. C. Hu, Baculovirus as a gene delivery vector: recent understandings of molecular alterations in transduced cells and latest applications. Biotechnol Adv 29, 618-631 (2011).
30. M. C. Siomi, K. Sato, D. Pezic, A. A. Aravin, PIWI-interacting small RNAs: the vanguard of genome defence. Nature reviews. Molecular cell biology 12, 246-258 (2011).
31. J. Y. Yoo et al., Short hairpin RNA-expressing oncolytic adenovirus-mediated inhibition of IL-8: effects on antiangiogenesis and tumor growth inhibition. Gene therapy 15, 635-651 (2008).
32. M. Pihlmann et al., Adeno-associated virus-delivered polycistronic microRNA-clusters for knockdown of vascular endothelial growth factor in vivo. The journal of gene medicine 14, 328-338 (2012).
33. S. K. Singh, P. B. Hajeri, siRNAs: their potential as therapeutic agents--Part II. Methods of delivery. Drug Discov Today 14, 859-865 (2009).
34. S. Zacchigna, L. Zentilin, M. Giacca, Adeno-Associated Virus Vectors as Therapeutic and Investigational Tools in the Cardiovascular System. Circulation research 114, 1827-1846 (2014).
35. D. W. Trobaugh et al., RNA viruses can hijack vertebrate microRNAs to suppress innate immunity. Nature 506, 245-248 (2014).
36. H. Xia, L. L. Ooi, K. M. Hui, MicroRNA-216a/217-induced epithelial-mesenchymal transition targets PTEN and SMAD7 to promote drug resistance and recurrence of liver cancer. Hepatology 58, 629-641 (2013).
37. L. Bao et al., MicroRNA-21 suppresses PTEN and hSulf-1 expression and promotes hepatocellular carcinoma progression through AKT/ERK pathways. Cancer letters 337, 226-236 (2013).
38. M. S. Ebert, P. A. Sharp, MicroRNA sponges: progress and possibilities. RNA 16, 2043-2050 (2010).
39. E. van Rooij, A. L. Purcell, A. A. Levin, Developing microRNA therapeutics. Circulation research 110, 496-507 (2012).
40. R. E. Lanford et al., Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327, 198-201 (2010).
41. Y. P. Li, J. M. Gottwein, T. K. Scheel, T. B. Jensen, J. Bukh, MicroRNA-122 antagonism against hepatitis C virus genotypes 1-6 and reduced efficacy by host RNA insertion or mutations in the HCV 5' UTR. Proceedings of the National Academy of Sciences of the United States of America 108, 4991-4996 (2011).
42. M. Lindow, S. Kauppinen, Discovering the first microRNA-targeted drug. The Journal of cell biology 199, 407-412 (2012).
43. H. L. Janssen et al., Treatment of HCV infection by targeting microRNA. The New England journal of medicine 368, 1685-1694 (2013).
44. E. S. Hildebrandt-Eriksen et al., A locked nucleic acid oligonucleotide targeting microRNA 122 is well-tolerated in cynomolgus monkeys. Nucleic acid therapeutics 22, 152-161 (2012).
45. D. Na et al., Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature biotechnology 31, 170-174 (2013).
46. T. L. Deans, C. R. Cantor, J. J. Collins, A tunable genetic switch based on RNAi and repressor proteins for regulating gene expression in mammalian cells. Cell 130, 363-372 (2007).
47. C. Kemmer et al., Self-sufficient control of urate homeostasis in mice by a synthetic circuit. Nature biotechnology 28, 355-360 (2010).
48. L. Wroblewska et al., Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery. Nat Biotechnol 33, 839-841 (2015).
49. H. Saito et al., Synthetic translational regulation by an L7Ae-kink-turn RNP switch. Nat Chem Biol 6, 71-78 (2010).
50. D. Miyaki et al., Clinical outcome of sorafenib treatment in patients with advanced hepatocellular carcinoma refractory to hepatic arterial infusion chemotherapy. Journal of gastroenterology and hepatology 28, 1834-1841 (2013).
51. H. Y. Woo, J. Heo, Sorafenib in liver cancer. Expert opinion on pharmacotherapy 13, 1059-1067 (2012).
52. V. Ramakrishnan et al., Sorafenib, a multikinase inhibitor, is effective in vitro against non-Hodgkin lymphoma and synergizes with the mTOR inhibitor rapamycin. American journal of hematology 87, 277-283 (2012).
53. M. Scartozzi et al., VEGF and VEGFR genotyping in the prediction of clinical outcome for HCC patients receiving sorafenib: The ALICE-1 study. International journal of cancer. Journal international du cancer, (2014).
54. A. Villanueva, J. M. Llovet, Targeted therapies for hepatocellular carcinoma. Gastroenterology 140, 1410-1426 (2011).
55. T. Thurnherr et al., Differentially Expressed miRNAs in Hepatocellular Carcinoma Target Genes in the Genetic Information Processing and Metabolism Pathways. Scientific reports 6, 20065 (2016).
56. S. Meding et al., Tumor classification of six common cancer types based on proteomic profiling by MALDI imaging. Journal of proteome research 11, 1996-2003 (2012).
57. R. K. Sterling et al., Frequency of elevated hepatocellular carcinoma (HCC) biomarkers in patients with advanced hepatitis C. The American journal of gastroenterology 107, 64-74 (2012).
58. T. Kawaguchi et al., Lipiodol accumulation and transarterial chemoembolization efficacy for HCC patients. Hepato-gastroenterology 59, 219-223 (2012).
59. M. Peck-Radosavljevic, Back to basics: staging and prognosis in HCC for medical oncologist. Journal of hepatology 56, 488-489 (2012).
60. E. Callegari et al., MicroRNAs in liver cancer: a model for investigating pathogenesis and novel therapeutic approaches. Cell Death Differ 22, 46-57 (2015).
61. J. U. Marquardt, P. R. Galle, A. Teufel, Molecular diagnosis and therapy of hepatocellular carcinoma (HCC): an emerging field for advanced technologies. Journal of hepatology 56, 267-275 (2012).
62. E. van Rooij, W. S. Marshall, E. N. Olson, Toward microRNA-based therapeutics for heart disease: the sense in antisense. Circulation research 103, 919-928 (2008).
63. V. Villegas-Ruiz et al., Heterogeneity of microRNAs expression in cervical cancer cells: over-expression of miR-196a. Int J Clin Exp Pathol 7, 1389-1401 (2014).
64. J. Ge, Z. Chen, R. Li, T. Lu, G. Xiao, Upregulation of microRNA-196a and microRNA-196b cooperatively correlate with aggressive progression and unfavorable prognosis in patients with colorectal cancer. Cancer cell international 14, 128 (2014).
65. M. Sun et al., MiR-196a is upregulated in gastric cancer and promotes cell proliferation by downregulating p27(kip1). Mol Cancer Ther 11, 842-852 (2012).
66. F. Huang et al., MiR-196a promotes pancreatic cancer progression by targeting nuclear factor kappa-B-inhibitor alpha. PLoS One 9, e87897 (2014).
67. S. Shrivastava, R. Steele, R. Ray, R. B. Ray, MicroRNAs: Role in Hepatitis C Virus pathogenesis. Genes Dis 2, 35-45 (2015).
68. A. E. Hoffman et al., microRNA miR-196a-2 and breast cancer: a genetic and epigenetic association study and functional analysis. Cancer research 69, 5970-5977 (2009).
69. B. He et al., The association between four genetic variants in microRNAs (rs11614913, rs2910164, rs3746444, rs2292832) and cancer risk: evidence from published studies. PloS one 7, e49032 (2012).
70. M. S. Beg et al., Phase I study of MRX34, a liposomal miR-34a mimic, administered twice weekly in patients with advanced solid tumors. Investigational new drugs 35, 180-188 (2017).
71. C. L. Chen et al., Baculovirus-mediated miRNA regulation to suppress hepatocellular carcinoma tumorigenicity and metastasis. Mol Ther 23, 79-88 (2015).
72. H. Y. Murad et al., Phenotypic alterations in liver cancer cells induced by mechanochemical disruption. Sci Rep 9, 19538 (2019).
73. M. S. Ebert, J. R. Neilson, P. A. Sharp, MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods 4, 721-726 (2007).
74. T. Ishiguro et al., Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications. Cancer Sci 108, 283-289 (2017).
75. G. H. Qiu et al., Distinctive pharmacological differences between liver cancer cell lines HepG2 and Hep3B. Cytotechnology 67, 1-12 (2015).
76. T. Nomura et al., Expression of angiogenic factors in hepatocarcinogenesis: Identification by antibody arrays. Oncology reports 30, 2476-2480 (2013).
77. L. Liu et al., Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer research 66, 11851-11858 (2006).
78. L. Guo, B. Sun, Q. Wu, S. Yang, F. Chen, miRNA-miRNA interaction implicates for potential mutual regulatory pattern. Gene 511, 187-194 (2012).
79. L. D. Osellame, T. S. Blacker, M. R. Duchen, Cellular and molecular mechanisms of mitochondrial function. Best practice & research. Clinical endocrinology & metabolism 26, 711-723 (2012).
80. S. Wang et al., The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. Developmental cell 15, 261-271 (2008).
81. S. P. Guenther, S. Schrepfer, miR-126: a potential new key player in hypoxia and reperfusion? Annals of translational medicine 4, 377 (2016).
82. B. D. Brown, M. A. Venneri, A. Zingale, L. Sergi Sergi, L. Naldini, Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat Med 12, 585-591 (2006).
83. L. Huang, D. M. Lilley, The Kink Turn, a Key Architectural Element in RNA Structure. Journal of molecular biology 428, 790-801 (2016).
84. G. Walsh, Biopharmaceutical benchmarks 2018. Nat Biotechnol 36, 1136-1145 (2018).
85. S. Fischer, R. Handrick, K. Otte, The art of CHO cell engineering: A comprehensive retrospect and future perspectives. Biotechnol Adv 33, 1878-1896 (2015).
86. B. Yin et al., Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation. Biotechnol Bioeng 112, 2343-2351 (2015).
87. F. M. Wurm, Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22, 1393-1398 (2004).
88. G. J. Cost et al., BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cells. Biotechnol Bioeng 105, 330-340 (2010).
89. S. F. Lim et al., RNAi suppression of Bax and Bak enhances viability in fed-batch cultures of CHO cells. Metab Eng 8, 509-522 (2006).
90. N. Yamane-Ohnuki et al., Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng 87, 614-622 (2004).
91. S. S. Yip et al., Complete knockout of the lactate dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3 down-regulated CHO cells. Mol Biotechnol 56, 833-838 (2014).
92. S. Konermann et al., Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors. Cell 173, 665-676 e614 (2018).
93. J. Zhu, Mammalian cell protein expression for biopharmaceutical production. Biotechnol Adv 30, 1158-1170 (2012).
94. M. Hunter, P. Yuan, D. Vavilala, M. Fox, Optimization of Protein Expression in Mammalian Cells. Curr Protoc Protein Sci 95, e77 (2019).
95. M. Zhang, K. Koskie, J. S. Ross, K. J. Kayser, M. V. Caple, Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells. Biotechnol Bioeng 105, 1094-1105 (2010).
96. S. H. Kim, G. M. Lee, Down-regulation of lactate dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin. Appl Microbiol Biotechnol 74, 152-159 (2007).
97. W. Xia et al., High levels of protein expression using different mammalian CMV promoters in several cell lines. Protein Expr Purif 45, 115-124 (2006).
98. R. J. Kaufman, Overview of vector design for mammalian gene expression. Mol Biotechnol 16, 151-160 (2000).
99. J. J. Cacciatore, L. A. Chasin, E. F. Leonard, Gene amplification and vector engineering to achieve rapid and high-level therapeutic protein production using the Dhfr-based CHO cell selection system. Biotechnol Adv 28, 673-681 (2010).
100. M. M. St Amand, D. Radhakrishnan, A. S. Robinson, B. A. Ogunnaike, Identification of manipulated variables for a glycosylation control strategy. Biotechnol Bioeng 111, 1957-1970 (2014).
101. Y. Konno et al., Fucose content of monoclonal antibodies can be controlled by culture medium osmolality for high antibody-dependent cellular cytotoxicity. Cytotechnology 64, 249-265 (2012).
102. J. Ehret, M. Zimmermann, T. Eichhorn, A. Zimmer, Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells. Biotechnol Bioeng 116, 816-830 (2019).
103. Y. Fan et al., Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation. Biotechnol Bioeng 112, 521-535 (2015).
104. C. Zhang et al., Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d. Cell 175, 212-223 e217 (2018).
105. H. Morisaka et al., CRISPR-Cas3 induces broad and unidirectional genome editing in human cells. Nat Commun 10, 5302 (2019).
106. A. E. Dolan et al., Introducing a Spectrum of Long-Range Genomic Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas. Mol Cell 74, 936-950 e935 (2019).
107. A. Pickar-Oliver, C. A. Gersbach, The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol 20, 490-507 (2019).
108. S. Pereira, H. F. Kildegaard, M. R. Andersen, Impact of CHO Metabolism on Cell Growth and Protein Production: An Overview of Toxic and Inhibiting Metabolites and Nutrients. Biotechnol J 13, e1700499 (2018).
109. C. A. Wilkens, Z. P. Gerdtzen, Comparative metabolic analysis of CHO cell clones obtained through cell engineering, for IgG productivity, growth and cell longevity. PLoS One 10, e0119053 (2015).
110. C. Toussaint, O. Henry, Y. Durocher, Metabolic engineering of CHO cells to alter lactate metabolism during fed-batch cultures. J Biotechnol 217, 122-131 (2016).
111. K. Chen, Q. Liu, L. Xie, P. A. Sharp, D. I. Wang, Engineering of a mammalian cell line for reduction of lactate formation and high monoclonal antibody production. Biotechnol Bioeng 72, 55-61 (2001).
112. M. Zdralevic et al., Double genetic disruption of lactate dehydrogenases A and B is required to ablate the "Warburg effect" restricting tumor growth to oxidative metabolism. J Biol Chem 293, 15947-15961 (2018).
113. N. A. Pereira, K. F. Chan, P. C. Lin, Z. Song, The "less-is-more" in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity. MAbs 10, 693-711 (2018).
114. C. J. Blondel et al., CRISPR/Cas9 Screens Reveal Requirements for Host Cell Sulfation and Fucosylation in Bacterial Type III Secretion System-Mediated Cytotoxicity. Cell Host Microbe 20, 226-237 (2016).
115. H. Imai-Nishiya et al., Double knockdown of alpha1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC. BMC Biotechnol 7, 84 (2007).
116. Y. Yang et al., Transcription Factor C/EBP Homologous Protein in Health and Diseases. Front Immunol 8, 1612 (2017).
117. K. Prashad, S. Mehra, Dynamics of unfolded protein response in recombinant CHO cells. Cytotechnology 67, 237-254 (2015).
118. A. Le et al., Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc Natl Acad Sci U S A 107, 2037-2042 (2010).
119. F. Wang et al., Advances in CRISPR-Cas systems for RNA targeting, tracking and editing. Biotechnol Adv 37, 708-729 (2019).
120. M. R. O'Connell, Molecular Mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR-Cas Systems. J Mol Biol 431, 66-87 (2019).
121. O. O. Abudayyeh et al., C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016).
122. S. C. Brezinsky et al., A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. J Immunol Methods 277, 141-155 (2003).
123. C. Ronda et al., Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool. Biotechnol Bioeng 111, 1604-1616 (2014).
124. L. M. Grav et al., One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment. Biotechnol J 10, 1446-1456 (2015).
125. C. C. Shen, L. Y. Sung, S. Y. Lin, M. W. Lin, Y. C. Hu, Enhancing Protein Production Yield from Chinese Hamster Ovary Cells by CRISPR Interference. ACS Synth Biol 6, 1509-1519 (2017).
126. J. R. Silkensen et al., Clusterin promotes the aggregation and adhesion of renal porcine epithelial cells. J Clin Invest 96, 2646-2653 (1995).
127. I. P. Trougakos, A. So, B. Jansen, M. E. Gleave, E. S. Gonos, Silencing expression of the clusterin/apolipoprotein j gene in human cancer cells using small interfering RNA induces spontaneous apoptosis, reduced growth ability, and cell sensitization to genotoxic and oxidative stress. Cancer Res 64, 1834-1842 (2004).
128. W. X. Yan et al., Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein. Mol Cell 70, 327-339 e325 (2018).
129. H. Zhang, C. Dong, L. Li, G. A. Wasney, J. Min, Structural insights into the modulatory role of the accessory protein WYL1 in the Type VI-D CRISPR-Cas system. Nucleic Acids Res 47, 5420-5428 (2019).