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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (3): 72-81    DOI: 10.13523/j.cb.2107043
综述     
CHO细胞基因组中稳定hot spot位点研究进展
林健芬,罗顺**()
江西中医药大学 南昌 330004
Research Progress on Stable Hot Spot Sites in the Genome of CHO Cells
LIN Jian-fen,LUO Shun**()
Jiangxi University of Chinese Medicine, Nanchang 330004, China
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摘要：

近些年来,治疗性重组蛋白类药物是生物制药领域研究的热点。工业化生产中常用于重组蛋白表达的细胞系是中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞。传统CHO细胞系的表达大多数基于随机整合的方式,这可能会使目标基因整合到异染色质区域或者不稳定的染色质区域,导致CHO细胞表达不稳定,需要多轮筛选才能获得理想的表达细胞系。最新研究表明,外源基因在CHO细胞预测/特定的基因组位点中进行特异性整合,可以使重组CHO细胞的表达保持长期一致性和稳定性。CHO细胞基因组中高效稳定的转录整合位点被称为热点(hot spot)。阐述CHO细胞基因组稳定的hot spot位点近几年的研究进展,其中包括热门的hot spot位点,以及如何研究新的hot spot位点的方法。总结如何将外源基因高效定位于预测的CHO细胞hot spot位点,实现高水平稳定的表达重组蛋白,为发现新的有效的hot spot位点,构建稳定表达CHO细胞系提供参考。
关键词： CHO;  Hot spot;  位点特异性整合;  细胞系开发    
Abstract:

In recent years, therapeutic recombinant protein drugs have become a research hot topic in the field of biopharmaceuticals research. Chinese hamster ovary cell lines are commonly used for recombinant protein expression in industrial production. The traditional expression of CHO cell lines is mostly based on random integration. However, random integration may result in target genes targeted into heterochromatin regions or unstable chromatin regions, causing unstable expression in CHO cell lines, and in order to obtain the ideal expression cell line researchers have to do multiple rounds of screening. The latest research proves that GOI integrates specifically in the prediction or specific genomic sites of CHO cells, which can maintain long-term consistency and stability in the expression of recombinant CHO cells. The integration sites for efficient and stable transcription in the genome of CHO cells are called hot spots. This review describes the latest research progress of stable hot spot sites in the CHO cell genome, summarizes the research methods of these sites, and concludes how to locate the GOI at predicted gene locus efficiently, to achieve high-level and stable expression recombinant protein, and to help researchers discover new or effective hot spot sites and then construct stable expression CHO cell lines.
Key words: CHO    Hot spot    Site-specific integration    Cell line development
收稿日期: 2021-07-19 出版日期: 2022-04-07
ZTFLH:  Q819  
通讯作者: 罗顺     E-mail: shun.luo@tobiopharm.com
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引用本文:

林健芬, 罗顺. CHO细胞基因组中稳定hot spot位点研究进展[J]. 中国生物工程杂志, 2022, 42(3): 72-81.

LIN Jian-fen, LUO Shun. Research Progress on Stable Hot Spot Sites in the Genome of CHO Cells. China Biotechnology, 2022, 42(3): 72-81.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2107043    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I3/72

图1  重组CHO细胞系的构建
图2  与CHO细胞位点特异性整合主题密切相关的不同子主题的出版物数量
整合位点 细胞类型 目的基因 参考文献
Rosa26 CHO-K1 mAb-JUG444 (Pfizer) [25]
HEK293T GFP (Green fluorescent protein)gene [27]
Hprt CHO-K1 Red fluorescence protein gene [28-29]
CHO-K1 pFW (5.3 kb) [30]
CHO-K1 scFv-Fc antibody gene [31]
Fer1L4 CHO-S EGFP (Enhanced Green Fluorescent Protein) [32]
CHOK1SV IgG2 mAb Myo (Pfizer) gene [33]
C1GALT1C1(COSMC) CHO-S mCherry [34]
HEK293 EGFP [35]
CHO-S GFP gene [36]
NW_006880285.1 HEK-293T Zsgreen1 reporter gene [10]
C12orf35 CHO-K1 mCherry and anti-PD1 mAb gene [24]
Putative Sites T2 and T9 CHO-S Rituximab gene [37-38]
FRT site CHO-DHFR tissue plasminogen activator (tPA) gene [39]
FUT8 CHO-K1 mAb gene [40]
Hipp11 CHO-S GFP gene [27]
表1  CHO基因组中已证实的hot spot位点
图3  GOI位点特异性整合原理图
方法 优势 缺点 参考文献
重组酶介导的位点
特异性整合		 缩短细胞系开发的时间
保证外源基因的表达水平和稳定表达
有多个外源基因重组位点
不使用多组学数据确定hot spot位点
可构建重复靶向的主细胞系平台
基因框架在预定的靶点可以有效地表达		 靶向整合GOI前RTS需要提前构建
landing pads的随机整合
重组效率低
目标重组细胞系的获得需要多轮筛选		 [14,16,23,34,41]
核酸酶介导的位点
特异性整合		 外源基因的高保真整合
可介导单个基因组的整合进入多个宿主细胞基因组位点
外源基因整合入宿主基因组时具有方向性
多个外源基因组片段可同时靶向供体DNA		 同源重组效率低
可能会出现脱靶现象
双链断裂处基因重组是可能会出现异常		 [11, 37-38,41,46, 49]
表2  GOI位点特异性整合方法的比较
图4  CRISPR/Cas9和RMCE介导GOI位点特异性整合
[1] Rita Costa A, Elisa Rodrigues M, Henriques M, et al. Guidelines to cell engineering for monoclonal antibody production. European Journal of Pharmaceutics and Biopharmaceutics, 2010, 74(2):127-138.
doi: 10.1016/j.ejpb.2009.10.002 pmid: 19853660
[2] Wurm F M. Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnology, 2004, 22(11):1393-1398.
doi: 10.1038/nbt1026
[3] Kamionka M. Engineering of therapeutic proteins production in Escherichia coli. Current Pharmaceutical Biotechnology, 2011, 12(2):268-274.
pmid: 21050165
[4] Dumont J, Euwart D, Mei B S, et al. Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Critical Reviews in Biotechnology, 2016, 36(6):1110-1122.
doi: 10.3109/07388551.2015.1084266
[5] Kamachi Y, Omasa T. Development of hyper osmotic resistant CHO host cells for enhanced antibody production. Journal of Bioscience and Bioengineering, 2018, 125(4):470-478.
doi: 10.1016/j.jbiosc.2017.11.002
[6] Hacker D L, De Jesus M, Wurm F M. 25 years of recombinant proteins from reactor-grown cells-where do we go from here? Biotechnology Advances, 2009, 27(6):1023-1027.
doi: S0734-9750(09)00097-4 pmid: 19463938
[7] Lai T F, Yang Y S, Ng S K. Advances in mammalian cell line development technologies for recombinant protein production. Pharmaceuticals (Basel, Switzerland), 2013, 6(5):579-603.
[8] Mohan C Y, Kim Y G, Koo J, et al. Assessment of cell engineering strategies for improved therapeutic protein production in CHO cells. Biotechnology Journal: Healthcare Nutrition Technology, 2008, 3(5):624-630.
[9] Werner R G, Noé W, Kopp K, et al. Appropriate mammalian expression systems for biopharmaceuticals. Arzneimittel-Forschung, 1998, 48(8):870-880.
[10] Zhou S T, Ding X F, Yang L, et al. Discovery of a stable expression hot spot in the genome of Chinese hamster ovary cells using lentivirus-based random integration. Biotechnology & Biotechnological Equipment, 2019, 33(1):605-612.
[11] Shin S W, Lee J S. CHO cell line development and engineering via site-specific integration: challenges and opportunities. Biotechnology and Bioprocess Engineering, 2020, 25(5):633-645.
doi: 10.1007/s12257-020-0093-7
[12] Tihanyi B, Nyitray L. Recent advances in CHO cell line development for recombinant protein production. Drug Discovery Today: Technologies, 2020, 38:25-34.
doi: 10.1016/j.ddtec.2021.02.003
[13] Noh S M, Sathyamurthy M, Lee G M. Development of recombinant Chinese hamster ovary cell lines for therapeutic protein production. Current Opinion in Chemical Engineering, 2013, 2(4):391-397.
doi: 10.1016/j.coche.2013.08.002
[14] Lee J S, Kildegaard H F, Lewis N E, et al. Mitigating clonal variation in recombinant mammalian cell lines. Trends in Biotechnology, 2019, 37(9):931-942.
doi: 10.1016/j.tibtech.2019.02.007
[15] Sturtevant A H. The effects of unequal crossing over at the bar locus in Drosophila. Genetics, 1925, 10(2):117-147.
doi: 10.1093/genetics/10.2.117 pmid: 17246266
[16] Hamaker N K, Lee K H. Site-specific integration ushers in a new era of precise CHO cell line engineering. Current Opinion in Chemical Engineering, 2018, 22:152-160.
doi: 10.1016/j.coche.2018.09.011
[17] Kim S J, Kim N S, Ryu C J, et al. Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure. Biotechnology and Bioengineering, 1998, 58(1):73-84.
pmid: 10099263
[18] Gierman H J, Indemans M H G, Koster J, et al. Domain-wide regulation of gene expression in the human genome. Genome Research, 2007, 17(9):1286-1295.
pmid: 17693573
[19] Kim M O’Callaghan P M Droms K A, et al. A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies. Biotechnology and Bioengineering, 2011, 108(10):2434-2446.
doi: 10.1002/bit.v108.10
[20] Paredes V, Park J S, Jeong Y, et al. Unstable expression of recombinant antibody during long-term culture of CHO cells is accompanied by histone H3 hypoacetylation. Biotechnology Letters, 2013, 35(7):987-993.
doi: 10.1007/s10529-013-1168-8
[21] Moritz B, Woltering L, Becker P B, et al. High levels of histone H3 acetylation at the CMV promoter are predictive of stable expression in Chinese hamster ovary cells. Biotechnology Progress, 2016, 32(3):776-786.
doi: 10.1002/btpr.2271
[22] Osterlehner A, Simmeth S, Göpfert U. Promoter methylation and transgene copy numbers predict unstable protein production in recombinant Chinese hamster ovary cell lines. Biotechnology and Bioengineering, 2011, 108(11):2670-2681.
doi: 10.1002/bit.23216 pmid: 21618470
[23] Papapetrou E P, Schambach A. Gene insertion into genomic safe harbors for human gene therapy. Molecular Therapy, 2016, 24(4):678-684.
doi: 10.1038/mt.2016.38 pmid: 26867951
[24] Zhao M L, Wang J X, Luo M Y, et al. Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35. Applied Microbiology and Biotechnology, 2018, 102(14):6105-6117.
doi: 10.1007/s00253-018-9021-6
[25] Gaidukov L, Wroblewska L, Teague B, et al. A multi-landing pad DNA integration platform for mammalian cell engineering. Nucleic Acids Research, 2018, 46(8):4072-4086.
doi: 10.1093/nar/gky216 pmid: 29617873
[26] Zhao C P, Guo X, Chen S J, et al. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells. Scientific Reports, 2017, 7:42805.
doi: 10.1038/srep42805
[27] Chi X L, Zheng Q, Jiang R H, et al. A system for site-specific integration of transgenes in mammalian cells. PLoS One, 2019, 14(7):e0219842.
doi: 10.1371/journal.pone.0219842
[28] Iwao R, Kawabe Y, Murakami M, et al. Targeted knock-in of transgenes into the CHO cell genome using CRISPR-mediated integration systems. MATEC Web of Conferences, 2021, 333:07001.
doi: 10.1051/matecconf/202133307001
[29] Wang X, Kawabe Y, Kato R, et al. Accumulative scFv-Fc antibody gene integration into the hprt chromosomal locus of Chinese hamster ovary cells. Journal of Bioscience and Bioengineering, 2017, 124(5):583-590.
doi: S1389-1723(17)30257-8 pmid: 28662917
[30] Bachu R, Bergareche I, Chasin L A. CRISPR-Cas targeted plasmid integration into mammalian cells via non-homologous end joining. Biotechnology and Bioengineering, 2015, 112(10):2154-2162.
doi: 10.1002/bit.v112.10
[31] Kawabe Y, Komatsu S, Komatsu S, et al. Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems. Journal of Bioscience and Bioengineering, 2018, 125(5):599-605.
doi: 10.1016/j.jbiosc.2017.12.003
[32] Inniss M C, Bandara K, Jusiak B, et al. A novel Bxb1 integrase RMCE system for high fidelity site-specific integration of mAb expression cassette in CHO cells. Biotechnology and Bioengineering, 2017, 114(8):1837-1846.
doi: 10.1002/bit.26268 pmid: 28186334
[33] Zhang L, Inniss M C, Han S, et al. Recombinase-mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line. Biotechnology Progress, 2015, 31(6):1645-1656.
doi: 10.1002/btpr.2175 pmid: 26399954
[34] Lee J S, Kallehauge T B, Pedersen L E, et al. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Scientific Reports, 2015, 5:8572.
doi: 10.1038/srep08572
[35] Shin S W, Lee J S. Optimized CRISPR/Cas9 strategy for homology-directed multiple targeted integration of transgenes in CHO cells. Biotechnology and Bioengineering, 2020, 117(6):1895-1903.
doi: 10.1002/bit.v117.6
[36] Sergeeva D, Camacho-Zaragoza J M, Lee J S, et al. CRISPR/Cas9 as a genome editing tool for targeted gene integration in CHO cells. Methods in Molecular Biology, 2019, 1961:213-232.
doi: 10.1007/978-1-4939-9170-9_13 pmid: 30912048
[37] Pristovšek N, Hansen H G, Sergeeva D, et al. Using titer and titer normalized to confluence are complementary strategies for obtaining Chinese hamster ovary cell lines with high volumetric productivity of etanercept. Biotechnology Journal, 2018, 13(3):e1700216.
[38] Pristovšek N, Nallapareddy S, Grav L M, et al. Systematic evaluation of site-specific recombinant gene expression for programmable mammalian cell engineering. ACS Synthetic Biology, 2019, 8(4):758-774.
doi: 10.1021/acssynbio.8b00453 pmid: 30807689
[39] Zhou H, Liu Z G, Sun Z W, et al. Generation of stable cell lines by site-specific integration of transgenes into engineered Chinese hamster ovary strains using an FLP-FRT system. Journal of Biotechnology, 2010, 147(2):122-129.
doi: 10.1016/j.jbiotec.2010.03.020
[40] Cristea S, Freyvert Y, Santiago Y, et al. In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration. Biotechnology and Bioengineering, 2013, 110(3):871-880.
doi: 10.1002/bit.24733 pmid: 23042119
[41] Opdam S, Richelle A, Kellman B, et al. A systematic evaluation of methods for tailoring genome-scale metabolic models. Cell Systems, 2017, 4(3):318-329, e6.
doi: 10.1016/j.cels.2017.01.010
[42] Scarcelli J J, Shang T Q, Iskra T, et al. Strategic deployment of CHO expression platforms to deliver Pfizer’s monoclonal antibody portfolio. Biotechnology Progress, 2017, 33(6):1463-1467.
doi: 10.1002/btpr.2493 pmid: 28480558
[43] Balcerek J, Bednarek M, Sobieściak T D, et al. Toward shortened the time-to-market for biopharmaceutical proteins: improved fab protein expression stability using the cre/lox system in a multi-use clonal cell line. Journal of Pharmaceutical Sciences, 2021, 110(2):946-951.
doi: 10.1016/j.xphs.2020.10.005 pmid: 33058893
[44] Grav L M, Sergeeva D, Lee J S, et al. Minimizing clonal variation during mammalian cell line engineering for improved systems biology data generation. ACS Synthetic Biology, 2018, 7(9):2148-2159.
doi: 10.1021/acssynbio.8b00140
[45] Baser B, Spehr J, Büssow K, et al. A method for specifically targeting two independent genomic integration sites for co-expression of genes in CHO cells. Methods, 2016, 95:3-12.
doi: 10.1016/j.ymeth.2015.11.022
[46] Carroll D. Genome engineering with targetable nucleases. Annual Review of Biochemistry, 2014, 83:409-439.
doi: 10.1146/annurev-biochem-060713-035418 pmid: 24606144
[47] Banan M. Recent advances in CRISPR/Cas9-mediated knock-ins in mammalian cells. Journal of Biotechnology, 2020, 308:1-9.
doi: 10.1016/j.jbiotec.2019.11.010
[48] Hsu P D, Lander E S, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014, 157(6):1262-1278.
doi: 10.1016/j.cell.2014.05.010
[49] Lee J S, Park J H, Ha T K, et al. Revealing key determinants of clonal variation in transgene expression in recombinant CHO cells using targeted genome editing. ACS Synthetic Biology, 2018, 7(12):2867-2878.
doi: 10.1021/acssynbio.8b00290
[50] Baker O, Tsurkan S, Fu J, et al. The contribution of homology arms to nuclease-assisted genome engineering. Nucleic Acids Research, 2017, 45(13):8105-8115.
doi: 10.1093/nar/gkx497
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