MicroRNAs as Engineering Targets: Pathway Manipulation to Impact Bioprocess Phenotypes - MicroRNAs as Tools in Biopharmaceutical Production - page 70

and increased cell productivity, suggesting its possible role in CHO cells engineer-
ing via decoupling cell growth from growth arrest to enhance recombinant protein
production (Barron et al. 2011a ; Kefas et al. 2008 ).
The use of miRNAs for engineering CHO cells better suitable for growth and
production needs to be evaluated carefully since their effects may be tissue or cell-
specific (Gammel 2007 ). miR-21 and miR-24 have already been reported in CHO
cells and seem to have growth-inhibitory properties (Gammel 2007 ). Recent work
on sequencing the CHO microRNA transcriptome will serve as a valuable resource
for identification of specific miRNAs involved in control of cell proliferation and cell
cycle. Next-generation sequencing of miRNAs across different industrially-relevant
conditions such as temperature shift and sodium butyrate treatment, helped to iden-
tify and annotate 387 mature miRNAs conserved in CHO including the members of
miR 17-92 polycistronic cluster, miR-221/222 cluster, let-7 family, miR-34 family,
miR-7, miR-125b, miR-143, and miR-31 (Hackl et al. 2011 ; Johnson et al. 2011 ).
The sequencing of cDNA of miR 17-92 cluster validated targets confirmed the con-
servation of respective miRNA binding sites in 19 genes in CHO. The latter serves
as a confirmation of the conserved biological role of this cluster in CHO cells (Hackl
et al. 2011 ). Further studies of biological functionality of the reported CHO-specific
miRNAs as compared to their role in other mammalian cells will facilitate identifi-
cation of key miRNAs for growth and cell cycle engineering in CHO cell factories.

5.2.2 Engineering of Apoptosis-Resistant Cell Lines

to Increase IVC

Apoptosis or programmed cell death (PCD) is a necessary physiological function
in multicellular organisms that presents difficulty for maintaining high viable cell
densities in mammalian bioprocess applications (Muller et al. 2008 ). Different stress
conditions in bioreactors such as nutrient limitation, byproduct accumulation, shear
and oxidative stresses, pH, osmolality and hypoxia can trigger apoptosis during CHO
cell cultures. Onset of apoptosis results in a lowered IVC which affects product yield
and properties (Druz et al. 2011 ; Gammel 2007 ). As a result, apoptosis prevention
is one of the most investigated techniques in CHO cells engineering.
There are two main approaches to inhibit or slow the apoptotic cascade activation.
The first involves the manipulation of the outer cellular environment by media
supplementation with growth factors, limiting nutrients, and hydrolysates (Majors
et al. 2007 ; Zanghi et al. 1999 ). The second employs genetic engineering methods
to reallocate the tightly regulated balance of pro- and anti-apoptotic factors in favor
of the anti-apoptotic proteins (Chiang and Sisk 2005 ; Lim et al. 2006 ; Wong et al.
2006 ). Anti-apoptotic proteins such as Bcl-x L , Bcl-2 and Mcl-1 are known to protect
the cells from apoptosis by maintaining the integrity of mitochondrial membrane.
Exogenous expression of these proteins increases cell density and viability and
protects the culture from apoptosis induction by different stimuli in mammalian
cells (Fassnacht et al. 1999 ; Fussenegger et al. 2000 ; Mastrangelo et al. 2000 ;