MicroRNAs as Engineering Targets: Pathway Manipulation to Impact Bioprocess Phenotypes - MicroRNAs as Tools in Biopharmaceutical Production - page 67

recombinant protein, leading to increase of cell specific productivity. In addition, the
cell can respond faster to miRNA modulations, either naturally or by introduction of
exogenous mimics or inhibitors since there is no need for a translation step (Barron
et al. 2011a ; Muller et al. 2008 ).
Novel large scale transfection techniques allow relatively easy insertion of miRNA
mimics or inhibitors into CHO cells at some stage of the bioprocess (Barron
et al. 2011a ). Also, some efficient miRNA expression systems which contain drug-
inducible or heat-shock-inducible promoters that control the timing and expression
levels of chosen miRNAs have been recently reported (Weber and Fussenegger 2007 ;
Yang and Paschen 2008 ). Therefore, the investigation of miR expression profiles in
CHO cells under different physiological conditions will facilitate detection of spe-
cific miRNAs that can be manipulated to obtain desired properties in CHO cells.
Alteration of those miRNA expression profiles will introduce another dimension
to genetic engineering of CHO cell to generate more robust CHO cell lines better
suitable to production of biologics (Druz et al. 2011 ).

5.1.2 Current Strategies for CHO Cell Engineering

There is currently an effort to improve CHO cells' performance and productivity
through better understanding of the physiology of the cells and their response to the
physical and chemical environment in the bioreactor.
Current process optimization techniques focus on increasing integrated viable cell
density (IVCD) in the bioreactor by enhancing the specific growth rate, reducing
apoptosis, redirecting metabolic pathways to lower accumulation of toxic byprod-
ucts, and increasing nutrient utilization efficiency (Druz et al. 2011 ; Muller et al.
2008 ; Chen et al. 2001 ; Elias et al. 2003 ; Irani et al. 2002 ). Most cell line improve-
ment efforts are based on media manipulation or gene engineering approaches by
random integration in the cell. However, newly developed methods such as targeted
homologous integration, RNA interference techniques and zinc-finger nuclease dele-
tions should be evaluated as approaches for cell improvements (Kramer et al. 2010 ).
One of the main obstacles that researchers face while attempting to alter the CHO
cells performance is the complexity of the intended phenotype such as rapid cell
growth and resistance to various stresses in the bioreactor. Altering the expression
of a single gene or protein or even a single pathway may not be sufficient to produce
the desirable phenotype (Barron et al. 2011b ). Therefore in some instances, cells
have been “double engineered” to reach higher growth rates and increase apoptosis
resistance (Fussenegger and Bailey 1998 ; Ifandi and Al-Rubeai 2005 ). It should
be noted however that over-engineering of the cells may affect growth due to high
metabolic load on the cells (Yallop and Svendsen 2001 ).
A comprehensive understanding of cellular pathway control and interaction is
required for developing strategies to globally affect gene expression profiles for
the purpose of engineering CHO cells with improved production properties (Dinnis
and James 2005 ). Some success has been shown with the modification of the global
gene expression levels through over-expression of transcription factors and artificial