Development of a targeted integration Chinese hamster ovary host directly targeting either one or two vectors simultaneously to a single locus using the Cre/Lox recombinase‐mediated cassette exchange system

Abstract

Cell line development (CLD) by random integration (RI) can be labor intensive, inconsistent, and unpredictable due to uncontrolled gene integration after transfection.Unlike RI, targeted integration (TI) based CLD introduces the antibody-expressing cassette to a predetermined site by recombinase-mediated cassette exchange (RMCE). The key to success for the development of a TI host for therapeutic antibody production is to identify a transcriptionally active hotspot that enables highly efficient RMCE and antibody expression with good stability. In this study, a genome wide search for hotspots in the Chinese hamster ovary (CHO)-K1-M genome by either RI or PiggyBac (PB) transposase-based integration has been described. Two CHO-K1-M derived TI host cells were established with the Cre/Lox RMCE system and are described here. Both TI hosts contain a GFP-expressing landing pad flanked by two incompatible LoxP recombination sites (L3 and 2L). In addition, a third incompatible LoxP site (LoxFAS) is inserted in the GFP landing pad to enable an innovative two-plasmid based RMCE strategy, in which two separate vectors can be targeted to a single locus simultaneously. Cell lines generated by the TI system exhibit comparable or higher productivity, better stability and fewer sequence variant (SV) occurrences than the RI cell lines.

Streamlined Human Cell-Based Recombinase-Mediated Cassette Exchange Platform Enables Multigene Expression for the Production of Therapeutic Proteins

“…Several RMCE-based strategies have attempted to integrate multiple transgene copies to improve therapeutic protein production. Using incompatible loxP sequences, multiple genes can be integrated at a single genomic locus, either sequentially , or simultaneously. , Furthermore, a multi-landing pad cell line enables the production of therapeutic proteins from multiple genes. , To increase the productivity of recombinant proteins, recombinant cells are often engineered by expressing effector genes. However, since the ectopic expression of effector genes relies on random integration, it shows inconsistent results due to clonal variation .…”

Reshaping cell line development and CMC strategy for fast responses to pandemic outbreak

“…Site‐specific integration (SSI) technology has brought forward much greater efficiency in inserting genes of interest (GOIs) into the host cell genome than the classical random integration transfection methods. While SSI has improved the productivity of the stable cell lines, the much higher efficiency of recombinant gene integration into the genome of the host cells than the traditional random insertion is more attractive to the industry 26–33 . While the traditional method of plating and screening of stable cell pools usually takes 2 months, the SSI stable pool takes as short as 1–2 weeks by skipping the labor‐intensive mini‐pool screening, due to enhanced probability of genomic integration 34–37 .…”

“…While SSI has improved the productivity of the stable cell lines, the much higher efficiency of recombinant gene integration into the genome of the host cells than the traditional random insertion is more attractive to the industry. [26][27][28][29][30][31][32][33] While the traditional method of plating and screening of stable cell pools usually takes 2 months, the SSI stable pool takes as short as 1-2 weeks by skipping the laborintensive mini-pool screening, due to enhanced probability of genomic integration. [34][35][36][37] This important development, therefore, made us envisage a more aggressive CMC timeline for counter-pandemic projects like neutralizing-antibody productions.…”

Strategies and Considerations for Improving Recombinant Antibody Production and Quality in Chinese Hamster Ovary Cells

“…Some chromatin-modifying elements can prevent transgene silencing, including matrix attachment regions (MAR) ( Buceta et al, 2011 ; Mohammadian et al, 2019 ; Zhang et al, 2020 ), locus control regions (LCR) ( Sharma et al, 2019 ; Morgan et al, 2020 ), ubiquitous chromatin opening elements (UCOEs) ( Pfaff et al, 2013 ; Harraghy et al, 2015 ; Nematpour et al, 2017 ; Rocha-Pizaña et al, 2017 ), stabilizing anti-repressor elements (STAR) ( Kwaks et al, 2003 ; Otte et al, 2007 ; Van Blokland et al, 2007 ), and insulators ( Benabdellah et al, 2014 ; Chetverina et al, 2014 ; Naderi et al, 2018 ; Pérez-González and Caro, 2019 ). Moreover, artificial chromosome expression (ACE) and targeted integration technology, including Flp-In and recombinase-mediated cassette exchange system (RMCE), can overcome the shortcomings of random integration ( Kennard et al, 2009 ; Soler et al, 2018 ; Reinhart et al, 2019 ; Ng et al, 2021 ). The introduction of these functional elements into the vector construction can greatly increase the proportion of high-expression clones and shorten the construction cycle of engineered host cells ( Table 2 ).…”