Importance of Genetic Environment for Recombinant Gene Expression

Abstract

Production of biological (protein)-based therapeutics offers fundamental challenges. The ability to generate unique life-saving therapies has been engaged by many commercial concerns, mainly using eukaryotic (mammalian) cell culture platforms. Genes encoding the valuable biopharmaceuticals are introduced into the host cell where they integrate into the cellular genome. What goes on within the nucleus is no longer a black box but it has become clear over the last decade that we have not yet begun to fully appreciate the complexity of this sub-cellular compartment and there remains significant scope to further optimise this stage of commercial bioprocessing. This review highlights the current vision of the eukaryotic nucleus in relation to its role as the controller of expression of genes that are introduced for production of a desired product. The layers of interwoven complexity – eu- and hetero-chromatin, epigenetic marking of genes and genomes, nucleosomes, expression factories and chromosome territories – will be described. As this knowledge base has accumulated it has led to the use of approaches that seek to maximise the expression of introduced genes, using a rationalised understanding of nuclear architecture and higher level genome regulation.

The past decade has seen step-changes in our perception of the eukaryotic nucleus in terms of structural environments and, consequently, the potential for previously unconsidered modes of regulation of gene expression. Driven by technological developments, that have permitted increased understanding of nuclear structure, we perceive that there are layers of complexity in eukaryotic transcription control that may have the potential to either thwart or enhance cell gene engineering. The existence of regulation at the level of nuclear structure and genomic environment has relevance to approaches that utilise eukaryotic cells as hosts for expression of exogenous genes (as in the use of mammalian cells as “factories” for production of biopharmaceuticals). This level of regulation has consequences for the extent and stability of expression of genes introduced into cells (either genes that encode for the desired biopharmaceutical or genes encoding for proteins predicted to enhance the “factory” activity of the cell) and for endogenous cellular genes (for which expression may be modified in response to incorporation of foreign genes into specific areas of the genomic environment, with potential consequences for cellular function).

This review will highlight current understanding of the structural relationships between chromosomes, genes and the physical entity that comprises the eukaryotic nucleus. Current perceptions have been developed from information obtained from a number of experimental eukaryotic systems. After describing the generic model for relationships between nuclear structure and gene regulation, I will discuss the implications for production of biopharmaceuticals in relation to commercially-relevant eukaryotic cells (predominantly Chinese Hamster ovary, CHO, and NS0 myeloma). Within this context there are very clear linkages between our increasing understanding of genomic environment and current developments related to incorporation of specific DNA sequences within expression vectors for use with mammalian cell lines (Chapter 1, this volume). This will be discussed, as appropriate, along with the forward vision of how information on genomic environment may be used for further rationalised optimisation of future expression platforms.