Enhancing global and mRNA specific translation for improved recombinant protein expression in in vitro cultured mammalian cells

Many of the new drugs currently under development are based upon proteins rather than traditional small molecules (e.g. antibiotics). One of the type of protein molecules that is particularly challenging to make are antibodies e.g. herceptin. These protein drugs are produced for the treatment of diseases such as cancer by cells kept in culture under defined conditions. One problem with this is that the cells we use to make proteins for therapeutic uses are not as efficient as we would like them to be and therefore we may not be able to produce enough of these drugs and the cost and demand for them is high. Protein synthesis is the process by which the information in the genetic material in the cell, DNA is converted via an intermediary substrate mRNA, into proteins. For proteins to be synthesised the mRNA must interact with a large complex called the ribosome which consists of RNAs and proteins. Ribosomes are able to decode the genetic information that is held in the mRNA and carry out the synthesis of the proteins. There are two distinct mechanisms by which mRNAs can interact with the ribosomes. The most common mechanism requires the binding of a protein complex to the 5' end of the mRNA and this complex then recruits the ribosome. However, certain mRNAs contain 5' regions that do not code for sections of proteins (termed untranslated regions; UTRs) and these sequences of RNA harbour the information that is required to form a complex RNA structure. These RNA structures allow the ribosome to be recruited to the mRNA generally a considerable distance from the 5' end and so this method of ribosome recruitment has been termed internal ribosome entry. Interestingly, messages that use internal ribosome entry generally encode proteins that are used under situations of cell stress including under temperature reduction (cold-shock). This information is of industrial relevance since the production of commercially valuable proteins (e.g. antibodies) is hindered when cells become stressed later in culture and by the cold-shock that is commonly induced during fermentation. We aim to use the 5' UTRs of mRNAs that are translationally active during cold-shock to enhance the production of proteins that are important to industry. Achieving this is very important as it is expected that with an increasing number of protein 'drugs' being developed we will lack the capability of producing large enough amounts to meet the required demand for these new drugs for the majority, as opposed to for those who can afford what must currently remain prohibitively expensive, but very effective, medicines.

The ability of industrially relevant expression systems to produce recombinant protein (rP) has advanced considerably in recent years, however despite such advances our understanding of the cellular processes that determine/limit rP yield from in vitro cultured mammalian cells remains poor. Recent reports suggest that the limitations are at least partially linked to modulation of translation, the mechanism(s) by which mRNAs interact with/are loaded onto ribosomes, mRNA turnover and sequestration, and control elements within the non-coding regions (UTRs) of mRNAs. Despite such reports, whether these control mechanisms can be manipulated to enhance the translation of specific recombinant mRNAs and ultimately enhance rP synthesis under 'normal' (37degC) and sub-physiological (32degC) culture temperatures within the same fermentation in an industrially relevant manner remains open to question. The proposed programme of work will test the hypothesis that via modulation of global translation, over-expression of mRNA chaperones, and by utilising components of the 5' and 3'-UTRs of endogenous mRNAs, recombinant mRNA specific translation can be enhanced in mammalian cells in a controlled and predictable manner to increase rP yields. We will utilise a combination of molecular and protein based approaches to characterise the links between mRNA translation factors and the control of their activity, mRNA chaperones, mRNA UTRs, protein synthesis, and gene expression that are implicated in the cellular responses governing/limiting rP yield. The outcomes will be (i) an understanding of those mechanisms governing recombinant mRNA translation under biphasic temperature culturing conditions of in vitro cultured mammalian cells, (ii) the design of new approaches to modulate global mRNA expression in an industrial context, and (iii) the rational development of plasmid vectors to allow enhanced translation of recombinant mRNAs during bioprocessing.