Evaluation of the paired-Cas9 nickase and RNA-guided FokI genome editing tools in precise integration of an anti-CD52 bicistronic monoclonal antibody expression construct at Chinese hamster ovary cells 18S rDNA locus.

INTRODUCTION: The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus. METHODS: T7E1 and high-resolution melt (HRM) assays were used to compare the ability of mentioned systems in inducing double-strand break (DSB) at the target site. Moreover, 5'- and 3'-junction polymerase chain reactions (PCR) were used to verify the accuracy of the targeted integration of the mAb expression cassette into the 18S rDNA locus. Finally, anti-CD52 mAb gene copy number was measured and, its expression was analyzed using ELISA and western blot assays. RESULTS: Our results indicated that both paired-Cas9n and RFN induced DSB at the target site albeit RFN performance was slightly more efficient in HRM analysis. We also confirmed that the anti-CD52 mAb cassette was accurately integrated at the 18S rDNA locus and the mAb was expressed successfully in CHO cells. CONCLUSION: Taken together, our findings elucidated that both paired-Cas9n and RFN genome editing tools are promising in targeting the 18S rDNA locus. Site specific integration of the bicistronic anti-CD52 mAb expression cassette at this locus in the CHO-K1 cells was obtained, using RFN. Moreover, proper expression of the anti-CD52 mAb at the 18S rDNA target site can be achieved using the bicistronic internal ribosome entry site (IRES)-based vector system.

Development of an Expression Vector Engineering Strategy Based on tDNA Insulator Element for the Stable Expression of Vascular Endothelial Growth Factor Receptor-Fc Fusion Protein.

During the past decades, tremendous advances have occurred in manufacturing recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. Nevertheless, the production of stable high-producing cell lines has remained a major obstacle in the development process of the CHO cell line. It has been shown that genomic regulatory elements can promote cell line development efficiency by improving transgenes' productivity and stability. Such elements include insulators, ubiquitous chromatin opening elements, scaffold/matrix attachment regions, and antirepressors. In addition, tDNA elements are shown to act as insulators in mammalian cells. This study examines the effect of the tDNA insulator on stable expression of a vascular endothelial growth factor receptor-Fc fusion protein.

Overexpression of SIRT6 alleviates apoptosis and enhances cell viability and monoclonal antibody expression in CHO-K1 cells.

BACKGROUND: Chinese hamster ovary (CHO) cells are the most predominantly utilized host for the production of monoclonal antibodies (mAbs) and other complex glycoproteins. A major challenge in the process of CHO cell culture is the occurrence of cell death following different stressful conditions, which hinders the production yield. Engineering genes involved in pathways related to cell death is a remarkable strategy to delay apoptosis, improve cell viability and enhance productivity. SIRT6 is a stress-responsive protein that regulates DNA repair, maintains genome integrity, and is critical for longevity and cell survival in organisms. METHODS AND RESULTS: In this study, SIRT6 was stably overexpressed in CHO-K1 cells and the impact of its expression on apoptosis related gene expression profile, viability, apoptosis, and mAb productivity was investigated. While a significant increase was observed in Bcl-2 mRNA level, caspase-3 and Bax mRNA levels were decreased in the SIRT6 engineered cells compared to the parental CHO-K1 cells. Moreover, improved cell viability and decreased rate of apoptotic progression was observed in a SIRT6-derived clone in comparision to the CHO-K1 cells during 5 days of batch culture. anti-CD52 IgG1 mAb titers were improved up to 1.7- and 2.8-fold in SIRT6-derived clone during transient and stable expression, respectively. CONCLUSIONS: This study indicates the positive effects of SIRT6 overexpression on cell viability and anti-CD52 IgG1 mAb expression in CHO-K1 cells. Further studies are needed to examine the potential of SIRT6-engineered host cells for the production of recombinant biotherapeutics in industrial settings.