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

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

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.

Technical Summary

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.

Publications

10 25 50
 
Description Key findings are
i. The finding that the decrease in translation rate (protein synthesis) following hypothermia is mediated via phosphorylation of a specific protein eIF2alpha (subsequent grants have shown a second factor eEF2 is also key in its phosphorylation). This is accompanied by a selective decrease in the number of 40S ribosomal subunits and changes to the synthesis/turnover rates of other small ribosomal subunit associated proteins.
ii. Confirmation that upon coldshock there is an increased association with polysomes of specific mRNAs that are required for the stress response and that elements within the 5' of these mRNAs allow their selective synthesis in the cold.
iii. That exposure of CHOK1 cells to hypothermia activates a specific signalling pathway (the ATR-p53-p21 signalling pathway) and is a key control mechanism upon hypothermia and that cold-induced changes in membrane lipid composition are correlated with the activation of this pathway. Further, that the molecular chaperonin CCT is central to the control of gene expression via the folding of translation eIF3 subunits.
Exploitation Route The identification of specific parts of mRNAs that are responsive to cold are being developed by ourselves for the potential to improved therapeutic recombinant protein production of high cost biological based drugs (e.g. antibodies) from cultured mammalian cells. These may be used by others as well to try and improve the amounts of these proteins that may be made and possibly to also improve the quality of such proteins for use in the clinic in the treatment of a variety of diseases and conditions.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description We have used these findings to understand how cultured mammalian cells engineered to express biotherapeutic recombinant proteins respond to changes in temperature and translation efficiency (how efficient they are are making the target recombinant protein) during culture. This understanding is then utilised to develop both the cells themselves, the culture/bioprocess conditions and the mRNAs for the target molecules to improve the biotherapeutic yields from mammalian cells and potentially to enhance quality of the product as well.
First Year Of Impact 2011
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Royal Society Industrial Fellowship
Amount £173,850 (GBP)
Funding ID IF130004 
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 11/2013 
End 10/2017
 
Description Wellcome Trust Collaborative Award
Amount £1,877,553 (GBP)
Funding ID UNS16981 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2016 
End 08/2021
 
Description Lonza Biologics Mass Spec Fingerprinting Project 
Organisation Lonza Group
Country Global 
Sector Private 
PI Contribution Development of a mass spectrometry fingerprinting technology for the identification of highly productive recombinant CHO cell lines.
Collaborator Contribution Cell line development, fermentation and bioreactor runs and validation, provision of reagents and expertise.
Impact IP/Patents, publications.
Start Year 2008
 
Description Open Days At University 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This activity is take take parents and secondary students around my research laboratories to explain the research undertaken and to demonstrate some of the research that we do in the laboratory.

Parents and students asked about engineering of cell lines and therapeutic recombinant protein drugs and how these are made, cost implications and on-going research.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011
 
Description Turkey Public Biotechnology Talk 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Presentation to public audience in Turkey on behalf of the British Council around biotechnology and its application. Event was filmed and followed by a question and answer session, answering questions sent in before the talk by social media and then from the audience. The event was filmed and shown on national TV in Turkey. Large range of topics discussed around the application of biotechnology to every day life and issues with long discussion/debate.
Year(s) Of Engagement Activity 2016
URL http://www.britishcouncil.org.tr/en/programmes/education/science-innovation-talks/biotechnology