Eukaryotic genes are organised into chromatin domains which exist in either a “closed” state, in which the genes are tightly condensed with proteins and are transcriptionally silent, or in an “open” de-condensed state, a prerequisite for efficient gene expression.
When transfected genes integrate into mammalian chromosomes, the structure of the chromatin at the site of integration has a profound effect on expression of the transgene. Consequently, only relatively rare transfected clones where the expression vector has integrated into open chromatin show efficient expression of the transgene.
Ubiquitous Chromatin Opening Elements (UCOEs) have been isolated which ensure efficient expression in a wide range of cell types including CHO-K1, CHO-S, HeLa, and NSO cells. UCOE containing vectors have been shown to markedly enhance a wide variety of intracellular, secreted and membrane-bound proteins.
Inclusion of a UCOE (4-8kb in size) in a eukaryotic expression vector permits efficient expression in the vast majority of stable clones, whereas with conventional vectors, only a minor proportion of transfectants show high-level expression (Figure 1). There is therefore no need for amplification and as the vectors integrate at low copy number, they are stable genetically and expression has been demonstrated to be maintained over 130 generations. The size of the latest generation, optimised UCOEs has been reduced to >2kb.
Figure 1. CHO cells stably transfected with a human CMV-EGFP reporter gene combination with or without an upstream 8kb UCOE fragment. Median fluorescence indicates the level of expression of the reporter gene achieved in individual clonally derived transfectants
UCOEs allows the rapid isolation of high-yielding, stably expressing clones and have been evaluated with numerous therapeutic genes including erythroprotein (EPO) and antibodies. Typically a screen of 96 antibody expressing clones gives 5-10 clones yielding greater than 1g/L in shake flasks within 8 weeks of transfection. Expression of Antibody has been shown to be stable for at least 50 generations. When selected clones are grown in bioreactors,
yields of 1.5-2.0g/L can easily be achieved in non-optimised conditions.
Figure 2: Yields of IgGI from a CHO-S UCOE clone, enhanced by media optimisation.
A further benefit of UCOE vectors is the ability to rapidly produce gram quantities of material in pools that can be used for research purposes and in the shortening time scales associated with DSP optimisation. The presence of the UCOE in expression vectors increases yields in stable pools by 15-25 fold over conventional vectors, which enables expression levels of 800mg/L for both antibody and erythropoietin to be achieved at a 10L scale within 28 days of transfection (Fig.3). These yields are orders of magnitude higher than those routinely achieved with transient transfection whilst reducing the costs of transfection reagent, plasmid production and the operating scale. Another advantage of this system is the ability to produce frozen stable pools, which can be used in subsequent fermentations.
Figure 3: Rapid production of EPO and IgGI in CHO-S stably expressing pools in simulated 10L fermentations after 14 days selection and 14 days expression.
The technology represents a significant cost saving both in the production of research grade quantities of product and in the selection of high expressing clones for large-scale manufacturing.