Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis
Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis
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DOI：
10.1002/bit.27599
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发表时间：
2020-10-24
影响因子：
3.8
通讯作者：
Lee, Kelvin H.
中科院分区：
文献类型：
--
作者：
Hilliard, William;Lee, Kelvin H.
用于生产商业量治疗性蛋白质的中国仓鼠卵巢 (CHO) 细胞系通常会随着培养时间的推移而表现出生产力下降，这种现象称为生产不稳定。将编码目的蛋白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影响的位置，通常会因拷贝数丢失和表达沉默而导致生产不稳定。最近的几篇出版物表明，可以通过使用位点特异性整合（SSI）技术将转基因插入基因组基因座（通常称为“热点”）来克服这些细胞系开发挑战，这些基因座是转录许可的并且相对于基因组其余部分具有增强的稳定性。然而，需要对 CHO 表观基因组进行广泛的表征，以确定在工业生物过程环境中保持其所需表观遗传特性的热点，并最大限度地提高单个整合转基因副本的转录。为此，使用高通量染色体构象捕获和RNAseq对两种关系较远的细胞系（一种工业相关的单克隆抗体产生细胞系及其亲代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。总共 10.9% 的 CHO 基因组包含转录允许的三维染色质结构，相对于基因组的其余部分，其遗传和表观遗传稳定性增强。这些安全港区域也与已发表的 CHO 表观基因组数据表现出良好的一致性，表明该方法适合寻找具有活跃和稳定基因表达的表观遗传标记的基因组区域。这些区域在寻找具有广泛适用性的 CHO 热点时显着减少了基因组搜索空间，并且可以指导未来的研究，以最大限度地发挥 SSI 技术在工业生产 CHO 细胞系中的潜力。
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.