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Title: Manipulation of RNA and RNA-binding proteins for control of biopharmaceutical titre
Author: Baker, James Alexander
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2019
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The biopharmaceutical industry is at a crossroads, where changing industry and ethical demands means it must invest in research and development (R&D) to maintain a diverse pipeline of novel drugs, and simultaneously reduce the substantial risks and costs associated with this R&D. In the future, a complete toolkit of synthetic expression elements could be used to create a streamlined, rationalised next generation cell line development (CLD) process, maximising the benefits offered by synthetic biology. However, synthetic, predictable, titratable molecular biology tools have not yet been developed for all the desired steps of biopharmaceutical expression. In this thesis, I will present work endeavouring to expand this toolbox, by investigating synthetic control of glycoprotein expression through messenger RNA (mRNA) engineering. The tethering of various RNA-binding proteins to recombinant mRNA was tested, as a method of controlling mRNA processing, and stimulating intronless mRNA export. C1orf35 and HuR were identified as target effector genes to increase transient protein production. However, inconsistency in their effect disqualified them as effective molecular biology tools. Two families of 3’ untranslated region (UTR) RNA elements were screened for their ability to control productivity through enhancement of mRNA stability. Though triple helices failed to increase expression, a stability element was discovered to increase transient SEAP productivity by 1.28-fold, via a mechanism of extension of SEAP mRNA half-life from 0.68h to 4.04h, compared to an industry-standard vector. 5’ terminal oligo-pyrimidine (TOP) motifs were investigated for their ability to control productivity through translation initiation. Enhancement of transient titre was demonstrated with various recombinant proteins and culture conditions, chemical supplements screened for their ability to specifically affect 5’TOP activation, mechanism of titre control investigated, and 5’TOP motifs integrated with synthetic proximal and core promoters. Different 5’TOP motifs were shown to control titre of a biotherapeutic fusion protein in an industry transient production process, up to a 2.12-fold increase compared to an industry-standard vector. These synthetic elements were screened together for their modularity, demonstrating titratable control of SEAP titre, from a 0.82-fold decrease to a 5.23-fold increase compared to an industry-standard vector. Further steps were then recommended, to render these tools truly predictable, and contribute maximally towards a rationalised, next-generation CLD process.
Supervisor: James, David ; Wilson, Stuart ; Gibson, Suzanne Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available