Effect of different UCOE-promoter combinations in creation of engineered cell lines for the production of Factor VIII

Abstract

BackgroundThe most common approach used in generating cell lines for the production of therapetic proteins relies on gene amplification induced by a drug resistance gene e. g., DHFR and glutamine synthetase. Practically, this results in screening large number of clones for the one that expresses high levels of the biologic in a stable manner. The inefficiency of mammalian vector systems to express proteins in a stable manner typically involves silencing of the exogenous gene resulting from modifications such as methylation of CpG DNA sequences, histone deacetylation and chromatin condensation. The use of un-methylated CpG island fragments from housekeeping genes referred to as UCOE (ubiquitous chromatin opening elements) in plasmid vectors is now well established for increased stability of transgene expression. However, few UCOE-promoter combinations have been studied to date and in this report we have tested 14 different combinations.FindingsIn this report we describe studies with two different UCOEs (the 1.5 Kb human RNP fragment and the 3.2 Kb mouse RPS3 fragment) in combination with various promoters to express a large protein (B domain deleted factor VIII; BDD-FVIII) in a production cell line, BHK21. We show here that there are differences in expression of BDD-FVIII by the different UCOE-promoter combinations in both attached and serum free suspension adapted cells. In all cases, the 1.5 Kb human RNP UCOE performed better in expressing BDD-FVIII than their corresponding 3.2 Kb mouse RPS3 UCOE. Surprisingly, in certain scenarios described here, expression from a number of promoters was equivalent or higher than the commonly used and industry standard human CMV promoter.ConclusionThis study indicates that certain UCOE-promoter combinations are better than others in expressing the BDD-FVIII protein in a stable manner in BHK21 cells. An empirical study such as this is required to determine the best combination of UCOE-promoter in a vector for a particular production cell line.

Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells

“…In contrast, a decrease in the cellspecific productivity was observed in two of the non-UCOE cell lines. These results are consistent with previous studies, where inclusion of UCOE in the vector construct resulted in higher levels of recombinant protein production [19,21,27,28,30] and improved the transgene expression in hematopoietic stem cells [25,26]. Otte et al compared the effect of different DNA elements, including UCOEs, S/MARs, cHS4 insulator and anti-repressor elements, on protein expression levels [37].…”

“…Anti-silencing activity was associated with strongly reduced DNA methylation of promoter, which strongly favours its application in PSC-based cell and gene therapy. In CHO and BHK21 cells, UCOE provided a higher level of transgene expression following stable transfection [19,21,[27][28][29] and maintained the stability of protein expression over 100 generations [30]. In this study, a UCOE vector was used in combination with MTX amplification to improve the frequency of positive clones and to achieve a high volumetric recombinant protein production.…”

“…The location of integrated plasmid DNA into the CHO cell chromosome [11] and silencing of the transgene as a result of epigenetic gene regulation such as DNA methylation [12][13][14] and histone deacetylation [15] are suspected to play a role in instability. In order to overcome these challenges, recent studies have focused on various regulatory elements that can be incorporated into the expression vectors to maintain transgenes in a transcriptionally active state including Locus control regions (LCRs) [16], Scaffold/Matrix Attachment Regions (S/MARs) [17,18] and Ubiquitous Chromatin Opening Elements (UCOEs) [14,[19][20][21]. UCOEs are derived from the heterogeneous nuclear ribonucleoproteins A2 B1/chromobox protein homologue 3 (HNRPA2B1/CBX3) housekeeping gene locus and are defined as being comprised of an extended methylation-free CpG-island and normally bi-directional promoters that are divergently transcribed [20,22].…”

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

“…UCOEs are genetic elements isolated from the upstream regions of housekeeper genes and include unmethylated CpG islands and other promoter-associated elements. Such elements are proposed to act by creating a local chromatin environment that is conducive to high level transcription and resistant to both position effects and silencing, although some UCOEs are superior in this regard to others (Nair et al 2011). When used to generate mammalian cell lines for manufacturing of a recombinant protein they have been shown to reduce the resource requirement during the cell line construction process by increasing the proportion of high expressing clones within the initial transfectant population (Benton et al 2002).…”

“…The stability of cell lines created using the UCOE system is also greatly enhanced relative to non-UCOE cell lines (Williams et al 2005). Interestingly the efficacy of specific UCOE elements is strongly influenced by the associated promoter, presumably because of the specific interactions between the UCOE and the promoter in determining the local chromatin architecture (Nair et al 2011).…”