An Epigenetic Understanding of Transcriptional Regulation in Chinese Hamster Ovary Cells

这项由美国国家科学基金会生物技术、生化和生物质工程项目颁发的奖项，支持对生产重组单克隆抗体的细胞系进行表征的工作，目的是确定高生产力细胞系的特征。单克隆抗体代表了最近批准的和正在开发的生物制药的很大一部分，为乳腺癌和类风湿性关节炎等疾病提供了重要的新疗法。然而，目前的细胞系开发需要筛选数百到数千个克隆，以确定具有高水平特定生产力和快速生长速度的细胞系，这限制了化合物进入临床试验和最终进入市场的速度。通过合理的方法提高生产力的能力将提高生物工艺开发的速度，这反过来又将使新的生物制药产品更快、更便宜地进入市场，从而改善人类健康。这一建议的智力价值在于分析高产细胞系和培养条件以及低生产力系统之间的表观遗传差异（不涉及DNA序列变化的可遗传性状），以了解转录调控。具体来说，我们将比较(i)亲代细胞克隆及其经甲氨蝶呤扩增的后代，以确定基因扩增后发生的表观遗传变化；（ii）由独立转染事件产生的细胞克隆，以确定转基因定位和表观遗传修饰之间的相互作用；（iii）丁酸盐处理和未处理的细胞克隆，以确定用已知可提高特定生产力的组蛋白去乙酰化酶抑制剂处理后发生的表观遗传变化。拟议工作的更广泛影响是：(i)为生物技术和生物制药业提供基础知识，从而加速生物工艺的发展，这反过来又将通过使新的生物制药产品更快、更便宜地进入市场，对人类健康产生重大影响；（ii）培养研究生和本科生，特别强调来自历史上少数民族院校的本科生，在哺乳动物细胞生物处理方面，满足生物技术/生物制药行业的关键需求；（iii）将拟议工作的结果纳入本科和研究生课程的教学材料，包括北卡罗来纳州立大学生物制造培训和教育中心的新研究生课程；继续开展外联活动，特别注重征聘年轻妇女从事科学事业。

This NSF award by the Biotechnology, Biochemical and Biomass Engineering program supports work to characterize cell lines producing recombinant monoclonal antibodies with an objective of identifying the characteristics of high productivity cell lines. Monoclonal antibodies represent a significant fraction of the recently approved and pipeline biopharmaceuticals, providing critical new therapies for diseases such as breast cancer and rheumatoid arthritis. However, cell-line development currently requires screening hundreds to thousands of clones to identify cell lines exhibiting high levels of specific productivity and rapid growth rates, limiting the rate at which compounds can be brought into clinical trials and ultimately, to market. The ability to improve productivity from a rational approach will improve the rates of bioprocess development, which will, in turn, improve human health by allowing novel biopharmaceutical products to reach the market more quickly and inexpensively.The intellectual merit of this proposal is to analyze the epigenetic differences (heritable traits that do not involve changes in the DNA sequence) between highly productive cell lines and culture conditions and lower productivity systems to understand regulation of transcription. Specifically, we will compare (i) parental cell clones and their methotrexate-amplified progeny, to identify epigenetic changes that occur upon gene amplification; (ii) cell clones arising from independent transfection events, to identify the interactions between transgene localization and epigenetic modifications, and (iii) butyrate-treated and untreated cell clones, to identify the epigenetic changes that occur upon treatment with a histone-deacetylase inhibitor known to increase specific productivity.The broader impacts of the proposed work are (i) to provide fundamental knowledge to the biotechnology and biopharmaceutical industry thus speeding bioprocess development, which will, in turn, have a significant impact on human health by allowing novel biopharmaceutical products to reach the market more quickly and inexpensively; (ii) to train graduate and undergraduate students, with a particular emphasis on undergraduates from historically minority institutions, in mammalian cell bioprocessing, meeting a critical need in the biotechnology/biopharmaceutical industry; (iii) to incorporate the results of the proposed work into instructional materials for undergraduate and graduate courses including a new graduate course at the Biomanufacturing Training and Education Center at NC State; (iv) to continue outreach activities, particularly focusing on recruiting young women into scientific careers.