Biotherapeutic molecules, such as monoclonal antibodies (mAbs), represent a
sizeable proportion of newly approved therapeutic’s available for treatment of disease.
Reducing the production costs of these therapeutics, usually produced in massive
quantities from Chinese Hamster Ovary (CHO) cells, remains a key focus of the
pharmaceutical industry. The presence of introns in the expression cassettes of mAbs
are an effective way of achieving high therapeutic protein expression levels. However,
frequent mis-splicing events lead to the formation of aberrant protein structures,
lowering the yield of the therapeutic protein and requiring filtration from the final
product. This leads to increased production costs resulting in increased cost for the
consumer and thus lowering the availability of the drug.
To counteract this problem, in this project we set out to develop expression systems
with increased therapeutic protein yield from an intronless expression cassette. We
demonstrate that improving the GC content of an intronless sequence improves
protein expression through improved mRNA export via interactions with the mRNA
export factor ALYREF. We go on to show that inclusion of RNA-binding protein (RBPs)
recruitment motifs for SRSF1/SRSF7 and SF3b1 into the intronless sequence
improves therapeutic protein expression, showing that mRNA processing is a good
expression stage to target to in this process. We further demonstrate the potential for
both GC content and RBP recruitment motifs to work in tandem to improve therapeutic
protein expression and provide evidence that these improvements work in an industrial
setting.
Finally, we investigate the role of the HUSH complex in mediated silencing of intronless
expression sequences, showing that this HUSH complex may not play a key role in
reducing protein expression levels from intronless sequences. Additionally, we show
the potential of HNRNPU silencing as a method of improving therapeutic production,
but further development of this system is required to alleviate certain drawbacks
associated with HNRNPU silencing.
