The hCMV promoter has been by far the most commonly used promoter for high level transgene expression in mammalian cells, for a wide range of applications that includes the production of protein therapeutics in cultured cells, transgenic animals and gene therapies. Its utility in expressing transgenes for all these applications, however, has been hindered by its susceptibility to silencing, largely through effects involving adverse chromatin structure. For example, the isolation of clonal cell lines or transgenic animals showing stable, high level expression of transgenes is usually a slow and labour-intensive procedure because most integration events lead to silencing.
The results we report here show that incorporating a RNP CpG island fragment immediately upstream of the hCMV promoter gives major benefits in expression from the latter. These benefits include a substantial increase in the median level of expression observed in pools of transfectants, together with a substantial improvement in the proportion of cells in the pool that express. The CpG island fragments reproducibly gave 20 to 40-fold increases in the level of expression observed. With both EGFP and EPO these dramatically increased expression levels were maintained through at least 100 generations of subculture. The CpG island fragments also enabled the rapid and facile isolation of clonal cell lines showing stable, high level expression of EGFP and EPO.
Our previous work [9
] showed that a 16 kb DNA fragment containing the RNP
CpG island conferred resistance to heterochromatin-mediated silencing of expression from the endogenous RNP
promoter and reduced position effect variegation, giving very consistent expression levels in tissue culture cells. In the studies we report here, constructs with the 8.0 kb RNP
CpG island fragment preceding the hCMV
promoter gave detectable levels of transgene expression for all integration events in which the construct remained intact, together with substantial increases in the level of expression from the hCMV
promoter. Analysis of variance (by the single factor ANOVA method) showed these increases to be statistically significant (p = 8.13E-27). These constructs, however, gave much greater variability in levels of expression than those observed in our previous work with a larger CpG island fragment and the endogenous RNP
promoter. It is not clear whether the greater variability in expression level for the CpG island constructs in the studies we report here than our previous ones is due to the use of a different promoter or different cell line, or occurs because sequences giving more complete isolation from the effects of chromatin structure or transcriptional activity adjacent to the integration site are present on the 16 kb fragment but not on the 8 kb fragment.
The present results demonstrate two very important applications of this novel transgene expression technology. One is the rapid production of recombinant proteins, in the quantities required for basic research, drug discovery and preclinical studies, using stable pools of transfectants. The CpG island technology circumvents the need for slow, labour intensive screening of clonal lines for this purpose. The other is faster and easier identification of clonal cell lines that show stable, high level production as candidates for large scale manufacture of protein therapeutics. With the CpG island technology screening of only 20 clonal CHO cell lines was sufficient to identify lines showing very high yields of EPO, the latter being a blockbuster protein drug that is widely used to treat anaemia associated with renal failure and chemotherapy. It is noteworthy that the hCMV and CHO cells used in these experiments are the promoter and cells most commonly used for commercial manufacture of protein therapeutics.
Through their capacity to confer an increase in the proportion of integration events that are productive, together with improvements in the level of transgene expression, RNP CpG island vectors have many other potential applications, including use in mammalian cell-based in vitro screens for drug discovery, transgenic animals for basic research and drug discovery, and gene therapy. For some of these applications, notably those involving transgene delivery with integrating viral vectors, gene expression elements of small size are required. The data we present here suggest that the 1.5 kb RNP fragment confers major benefits in expression level from the hCMV promoter. Efforts to further characterise and minimise these elements are ongoing, but the 1.5 kb fragment should be small enough for incorporation into most viral vectors.
The mechanism by which the RNP
CpG island reduces silencing and improves transgene expression is under investigation in our laboratories. The data we report here show that the mechanism does not involve increasing the copy number of the integrated transgene. Our results to date, reported previously [9
] and in this paper, are consistent with these elements being able to establish and maintain a more open chromatin domain irrespective of the local chromosome environment. We propose that the promoter-containing CpG-islands of housekeeping genes possess a chromatin remodelling function and that this is designated a "Ubiquitously-acting Chromatin Opening Element", or "UCOE".