Hilliard William, Lee Kelvin H
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, USA.
Biotechnol Bioeng. 2021 Feb;118(2):659-675. doi: 10.1002/bit.27599. Epub 2020 Oct 24.
The Chinese hamster ovary (CHO) cell lines that are used to produce commercial quantities of therapeutic proteins commonly exhibit a decrease in productivity over time in culture, a phenomenon termed production instability. Random integration of the transgenes encoding the protein of interest into locations in the CHO genome that are vulnerable to genetic and epigenetic instability often causes production instability through copy number loss and silencing of expression. Several recent publications have shown that these cell line development challenges can be overcome by using site-specific integration (SSI) technology to insert the transgenes at genomic loci, often called "hotspots," that are transcriptionally permissive and have enhanced stability relative to the rest of the genome. However, extensive characterization of the CHO epigenome is needed to identify hotspots that maintain their desirable epigenetic properties in an industrial bioprocess environment and maximize transcription from a single integrated transgene copy. To this end, the epigenomes and transcriptomes of two distantly related cell lines, an industrially relevant monoclonal antibody-producing cell line and its parental CHO-K1 host, were characterized using high throughput chromosome conformation capture and RNAseq to analyze changes in the epigenome that occur during cell line development and associated changes in system-wide gene expression. In total, 10.9% of the CHO genome contained transcriptionally permissive three-dimensional chromatin structures with enhanced genetic and epigenetic stability relative to the rest of the genome. These safe harbor regions also showed good agreement with published CHO epigenome data, demonstrating that this method was suitable for finding genomic regions with epigenetic markers of active and stable gene expression. These regions significantly reduce the genomic search space when looking for CHO hotspots with widespread applicability and can guide future studies with the goal of maximizing the potential of SSI technology in industrial production CHO cell lines.
用于生产商业数量治疗性蛋白质的中国仓鼠卵巢（CHO）细胞系在培养过程中通常会随着时间的推移而出现生产力下降的现象，这种现象被称为生产不稳定性。将编码目标蛋白质的转基因随机整合到CHO基因组中易受遗传和表观遗传不稳定性影响的位置，常常会通过拷贝数丢失和表达沉默导致生产不稳定性。最近的几篇出版物表明，通过使用位点特异性整合（SSI）技术将转基因插入基因组位点（通常称为“热点”），可以克服这些细胞系开发挑战，这些位点在转录上是允许的，并且相对于基因组的其他部分具有更高的稳定性。然而，需要对CHO表观基因组进行广泛的表征，以识别在工业生物工艺环境中保持其理想表观遗传特性并使单个整合转基因拷贝的转录最大化的热点。为此，使用高通量染色体构象捕获和RNA测序对两个远缘相关细胞系（一个与工业相关的单克隆抗体生产细胞系及其亲本CHO-K1宿主）的表观基因组和转录组进行了表征，以分析细胞系发育过程中发生的表观基因组变化以及全系统基因表达的相关变化。总的来说，10.9%的CHO基因组包含转录允许的三维染色质结构相对于基因组的其他部分具有更高的遗传和表观遗传稳定性。这些安全港区域也与已发表的数据显示出良好的一致性，表明该方法适用于寻找具有活跃和稳定基因表达表观遗传标记的基因组区域。在寻找具有广泛适用性的CHO热点时，这些区域显著减少了基因组搜索空间，并可以指导未来的研究，目标是在工业生产CHO细胞系中最大化SSI技术的潜力。
