Platform technology for the enhanced manufacture and utilisation of synthetic mRNA

The use of synthetic messenger RNA (mRNA) in the fields of medicine and
biotechnology is becoming increasingly prevalent, due to the well characterised
advantages of the molecule as a means of transferring genetic information. The rapid
expansion of synthetic mRNA related applications necessitates the development of
new technologies for the manufacturing of the molecule in a more cost efficient and
less resource intensive manner. Presented in this thesis are two approaches for
improving the manufacture of synthetic mRNA, in addition to a novel usage of the
synthetic mRNA molecule itself.

Firstly, a new method is described for identifying RNA polymerases that could be used
in existing in vitro transcription processes. Current manufacturing methods suffer from
poor product related impurity profiles, as a result of off-target activity from T7 RNA
polymerase. Using a novel cell free coupled transcription-translation system, a library
of 10 new RNA polymerases is introduced, with diverse yield profiles. The nature of
active polymerase and promoter pairs discovered in this study allow for conclusions
to be drawn on which RNA polymerases are optimal targets for future characterisation.

Secondly, a new manufacturing platform for the production of synthetic mRNA is
introduced, with E. coli being used as a cellular chassis for the accumulation of
recombinant mRNA. The use of a cell as a factory for mRNA production reduces raw
material costs, due to the ability of E. coli to synthesise the components of the
transcription reaction endogenously from inexpensive media. Coordinated mRNA,
DNA, cell and media engineering, primarily focussed on disrupting interactions
between synthetic mRNA molecules and host cell RNA degradation machinery,
increases product yields 40-fold compared to standard ‘unengineered’ E. coli
expression systems.