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.

The effect of Ccnb1ip1 insulator on monoclonal antibody expression in Chinese hamster ovary cells.

BACKGROUND: The increasing need for therapeutic monoclonal antibodies (mAbs) entails the development of innovative and improved expression strategies. Chromatin insulators have been utilized for the enhancement of the heterologous proteins in mammalian cells. METHODS AND RESULTS: In the current study the Ccnb1ip1 gene insulator element was utilized to construct a novel vector system for the expression of an anti-CD52 mAb in Chinese hamster ovary (CHO) cells. The insulator containing (pIns-mAb) and control (pmAb) vectors were generated and stable cell pools were established using these constructs. The expression level in the cells created with pIns-mAb vector was calculated to be 233 ng/mL, and the expression rate in the control vector was 210 ng/mL, which indicated a 10.9% increase in mAb expression in pIns-mAb pool. In addition, analysis of mAb expression in clonal cells established from each pool showed a 10% increase in antibody productivity in the highest mAb producing clone derived from the pIns-mAb pool compared to the clone isolated from pmAb pool. CONCLUSIONS: More studies are needed to fully elucidate the effects of Ccnb1ip1 gene insulator on recombinant therapeutic protein expression in mammalian cells. The combination of this element with other chromatin-modifying elements might improve its augmentation effect which could pave the way for efficient and cost-effective production of therapeutic drugs.

Development of an improved lentiviral based vector system for the stable expression of monoclonal antibody in CHO cells.

Therapeutic monoclonal antibodies (mAbs) have become the dominant products in biopharmaceutical industry. Mammalian cell expression systems including Chinese hamster ovary (CHO) cells are the most commonly used hosts for the production of complex recombinant proteins. However, development of stable, high producing CHO cell lines suffers from the low expression level and instability of the transgene. The increasing efforts in the development of novel therapeutic antibodies and the advent of biosimilars have revealed the necessity for the development of improved platforms for rapid production of products for initial characterization and testing. In line with this premise, vector design and engineering has been applied to improve the expression level and stability of the transgene. This study reports the application of an improved lentiviral vector system containing the human interferon-ß scaffold attachment region (IFN-SAR) for the development of antibody producing stable CHO cells. mAb expressing clones producing 1100 µg/L of IgG1 monoclonal antibody were isolated without extensive screening of a large number of clones. Our results here indicate the positive effects of IFN-SAR on stable mAb expression using lentiviral based expression vectors. We also observed that although IFN-SAR can improve light chain (LC) and heavy chain (HC) gene copy numbers in stable cell pools, mAb expression in single cell clones was not affected by the transgene copy number.

The Augmenting Effects of the tDNA Insulator on Stable Expression of Monoclonal Antibody in Chinese Hamster Ovary Cells.

Cell line development is one of the most critical steps in the production of complex recombinant therapeutic proteins such as monoclonal antibodies in mammalian cells. Generation of industrial cell lines is mainly based on the time-consuming and laborious process of selection and screening of a large number of clones. With the increasing demand for therapeutic proteins during the past years, more effort is invested to improve the efficiency of cell line development. In line with this premise, several studies employed expression vector engineering strategies based on incorporation of epigenetic regulatory elements, which can enhance the expression level and stability of the transgenes. Main examples of such elements include ubiquitous chromatin opening elements, scaffold or matrix attachment regions, stabilizing antirepressor elements, and insulators. This work evaluates the utility of the tDNA insulator element for stable expression of an IgG1 monoclonal antibody as well as the enhanced green fluorescent protein (EGFP) reporter gene in Chinese hamster ovary (CHO) cells. Initial analysis of EGFP transfected cells showed improved mean fluorescent intensity in cell pools and single cell clones when tDNA element was included in the expression vector. Our results also indicated up to nine- and sixfold enhancements in antibody titer and specific productivity of clones derived from tDNA containing vectors, respectively. Moreover, improved single cell cloning efficiency was observed for transfectants generated using tDNA harboring expression constructs. Our study clearly shows the beneficial effects of the tDNA insulator on monoclonal antibody expression in CHO cells.