影响抗体药物在重组CHO细胞株中稳定高效表达的关键因子的研究

Chinese hamster ovary (CHO) cell is the mainstream cell line for antibody drug production. Establishing high producing CHO cell clones is a crucial step for commercial development of therapeutic antibodies. An ideal recombinant CHO cell clone requires not only a high protein expression level, but also high stability of protein expression during expansion of cell culture, allowing a process scalable to over 10K liter bioreactors. In CHO cell clones, antibody expression level and clone stability are affected by many factors and those clones with extremely high level expression and stability are rare events in a CHO cell pool. In our previous studies, through screening over thousand clones, we have obtained a panel of high producing clones with high stability. In this study, we will systematically analyze these clones to reveal how genetic and epigenetic variations determine protein expression levels and clone stabilities. Our studies will focus on: 1) Determining insertion sites of antibody expression cassette in CHO cell genome and establishing high antibody-producing expression system; 2) Revealing the characteristics of transcriptome in high producing/stable clones and identifying key genes that have great impacts on antibody expression and clone stability; 3) Analyzing methylation status in stable cell clones and how to devoid DNA methylation.

中国仓鼠卵巢细胞（Chinese hamster ovary cell, CHO cell）是用于抗体药物生产的主要工程细胞，高效表达CHO细胞株的建立是抗体药物产业化的关键。理想的细胞株需要高效、稳定的表达蛋白质，在扩大至上万升的生物反应器后仍然保持高水平的蛋白质表达。影响CHO细胞蛋白质表达高低和稳定性的因素极为复杂，高效稳定细胞克隆是细胞群体中的罕见事件。本项目的前期工作中，通过上千个CHO细胞克隆筛选，获得了一组能高效稳定地表达抗体的细胞克隆。在本研究中，我们将全面分析这些细胞株，揭示CHO细胞的遗传学和表观遗传学变化对蛋白质表达量和细胞稳定性的影响。本项目将重点研究：1) CHO细胞染色体基因高表达插入位点的确定及高表达系统的建立；2）高效稳定细胞株的基因转录组特征研究，鉴别对表达水平和稳定性具有影响的基因；3）稳定细胞株的DNA甲基化状态及如何避免DNA甲基化。

本项目通过筛选，得到了不同表型的CHO细胞株，之后使用RNA-seq技术对这些细胞株进行分析，得到了一些可能影响CHO细胞高效表达的相关基因，通过功能验证获得多个候选基因，同时初步检测到了影响CHO细胞稳定表达的相关基因，寻找到了与CHO细胞在培养过程中与结团相关的基因。研究了DNA甲基化对CHO细胞外源蛋白表达水平的影响。还使用CRISPR/dCas9-VP64系统提高载体启动子转录活性，此外，在使用CRISPR/dCas9库改造CHO细胞以提高表达量方面进行了探索。..项目资助已发表SCI论文四篇，后续两篇论文正在整理投稿中。培养博士生三名，其中两名已经取得学位，一名在读。项目投入经费80万元，支出79.1424万元，各项支出基本与预算相符。剩余经费0.8576万元，剩余经费计划用于本项目研究后续支出。

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