利用CRISPR/Cas9双基因精准编辑和强化CHO抗体表达体系研究

Chinese Hamster Ovary (CHO) cells, as earliest mammalian cells used for foreign protein expression, had predominated in the bio-pharmaceutical industries, such as products of antibodies and vaccines. However the quality and efficient CHO strains can’t be purchased almost under the control of large foreign pharmaceutical companies as their core technology, which had seriously constrained the production quality and economic cost of biological drugs in China. In the past, due to the restriction of multi gene precise editing technology, the capacity optimization to CHO had focused on the middle or down steam technologies including screening of cultures, optimizing of protein expression, and scaling-up of products up to now. Basically the construction of excellent host cells is the critical and decisive for the qualified and high-yield expression of hetero-protein. But it has not been reported that the host cell was editing precisely with multi gene to improving the performance in production of protein expression. In the research, the genes of CHO cell will edited and modified precisely with human high active Survivin and QSOX1 genes replaced of rat source by CRISPR/Cas9 technology, and the positioning tag of nucleus, mitochondria and endoplasmic reticulum will be used for the location of the protein expression such as the model of fluorescent protein and PD-L1 antibody，so that capability of the CHO cells can be enhanced in anti-apoptosis and folding correctly the proteins, especially in forming disulfide bonds in the foreign proteins. Moreover, the interdependent relations between the growth of modified CHO, the quality of the hetero-protein and the unfolding protein response (UPR) in endoplasmic reticulum will be revealed and analyzed loosely. The study will lay important theoretical and technical foundation for the modification of the animal cell strain, and also provide the high quality and efficient CHO strains used for the production of biopharmacuetics.

中国仓鼠卵巢细胞(CHO)是最早来源于我国的哺乳动物细胞，其作为工程化抗体等重组蛋白的主要表达体系，在生物药品生产中可谓举足轻重。然而，目前生产抗体的优质细胞株，基本掌控在国外大的制药公司，作为核心技术而无法购得，这严重制约着我国生物制品的质量提升和经济成本。过去由于多基因编辑技术不够强，而难于对动物细胞从根上进行多基因精准编辑，强化其整体效能。本研究利用CRISPR/Cas9新技术以活性较鼠更强的人Survivin和QSOX1基因对CHO细胞基因组进行精准编辑，以期获得抗凋亡能力强、催化二硫键效率与抗体表达质量均高的、真正属于我国的优质“CHO种子”；在此基础上，将以荧光蛋白和抗肿瘤PD-L1抗体为示范，从培养环境、细胞和内在分子交互层面，揭示CHO细胞表达体系在内外源压力下，其抗凋亡生存能力、内质网应激和蛋白加工质量三者之间的内在规律，为从源头强化抗体高效细胞生产株提供理论与技术支撑。

CHO细胞在生物制药产业中应用广泛，是生产抗体的首选宿主，目前有70％以上的抗体蛋白是由CHO细胞生产的。目前CHO细胞在表达蛋白方面还存在产量低、蛋白质折叠氧化应激严重等问题，而且多数优良细胞株掌握在国外制药巨头公司的手中，其核心技术而无法购得，这严重制约着我国生物制品的生产质量和经济成本。本研究利用CRISPR技术获得QSOX、Survivin单基因编辑和双基因编辑的CHO细胞，这些细胞在抗凋亡、抗氧化和促进蛋白质表达中具有明显优势，双基因编辑的CHO-K1的细胞活力比对照提高26.14%，QSOX单基因编辑和 双基因编辑细胞株氧化还原能力提高了近 70%，survivin单基因编辑和双基因编辑的CHO细胞的抗凋亡能力比对照提高了1倍，能够促进蛋白质的表达，尤其是对复杂蛋白质提高活性，提升产量，具有重要意义，为未来产业化和工业应用奠定良好基础。同时本研究为从源头强化细胞株提供科学理论与可靠技术，发现新的内在关联机制，为加速CHO细胞改造探索新思路，对基础研究和工业应用提供借鉴和指导。