High‐level recombinant protein production in CHO cells using lentiviral vectors and the cumate gene‐switch

Abstract

Fast and efficient production of recombinant proteins for structural and functional studies is a crucial issue for research and for industry. To this end, we have developed an efficient system to generate in less than 2 months, starting from the cDNA, pools of CHO cells stably expressing high-level of recombinant proteins. It is based on lentiviral vectors (LVs) for stable transduction coupled with the cumate gene-switch for inducible and efficient gene expression. Transcription is initiated upon binding of the cumate transactivator (cTA) or the reverse cTA (rcTA) to the CR5 promoter. Binding of cTA or rcTA is prevented or induced by addition of cumate respectively. We first validated the CHO/LV production system with an LV carrying the secreted alkaline phosphatase (SEAP), whose expression was linked to the green fluorescent protein (GFP) through an internal ribosome entry site (IRES). CHO cells stably expressing the cTA (CHO-cTA) were transduced at various multiplicity of infection (MOI). Pools of cells were incubated at 37 and 30 degrees C during 10 days. Optimal SEAP production (65 microg/mL) was achieved at 30 degrees C with a MOI of 200. The pool stability was demonstrated for 48 days of culture by GFP expression analysis. The system was also evaluated using LV expressing three typical therapeutic proteins (a protein made up of the extracellular domain of CD200 fused to IgG Fc region [CD200Fc], a chimeric antibody [chB43], and erythropoietin [EPO]). CHO cells expressing rcTA (CHO-Cum2) were transduced with these LVs at a MOI of 200 and production was tested at 30 degrees C. After 13 days of culture, 235, 160, and 206 microg/mL of CD200Fc, chB43, and EPO were produced, respectively. The ON/OFF ratio of these pools was equal to 6 for CD200Fc, 16 for chB43, and 74 for EPO. In conclusion, this system should be very useful to produce mg quantities of recombinant proteins in a timely manner in serum free suspension culture of CHO cells for preclinical studies.

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

“…Several systems with either repressor or activator configurations have been developed for mammalian cells to achieve tight control of gene expression. Among many, there are i) antibiotic-based regulation systems, such as the Tet-Off-On system regulated by tetracyclin (Gossen and Bujard, 1992;Mohan et al, 2007), the Pip system regulated by streptogramin (Fussenegger et al, 2000) and the E.REX system regulated by macrolides (Weber et al, 2002); ii) an aptamer-based regulation system (Werstuck and Green, 1998); and iii) the cumate gene-switch (Gaillet et al, 2010;Mullick et al, 2006). All these inducible systems have been successfully used in CHO cell lines, and a 0.24 g/L titer of an Fc-fusion protein has been reported for this system (Gaillet et al, 2010).…”

Utilization of non‐AUG initiation codons in a flow cytometric method for efficient selection of recombinant cell lines

“…To address the need for greater yields, several different strategies have been pursued. These include utilizing stronger promoters in expression vectors (Gaillet et al, 2010;Prentice et al, 2007;Running Deer and Allison, 2004;Xia et al, 2006), better post-transfection selection systems (Sautter and Enenkel, 2005;van Blokland et al, 2007), and more efficient strategies for selecting clones with high expression levels (Bailey et al, 2002;Bohm et al, 2005;Brezinsky et al, 2003;Browne and Al-Rubeai, 2007;Caron et al, 2009). With respect to clone selection strategies, we and others have shown that IRES mediated expression of a reporter in conjunction with flow cytometry or magnetic separation methods can effectively isolate high expressing clones without requiring an antibody specific for the recombinant protein of interest (DeMaria et al, 2007;Gaines and Wojchowski, 1999;Liu et al, 2000;Sleiman et al, 2008).…”

Interplay of Promoter Usage and Intragenic CpG Content: Impact on GFP Reporter Gene Expression

“…3 One subclass of retroviruses used for therapeutic protein expression are lentiviruses (LVs). 4,5 LVs have the ability to be actively transported through nuclear pores as a viral preintegration complex, which enables them to transduce nondividing cells. 6 Self-inactivating LVs (SIN-LVs), containing a deleted U3 region of the 3¢ long terminal repeat (LTR), additionally provide gene transfer with higher safety due to the reduced risk of enhancer-mediated mutagenesis.…”