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.
