MicroRNAs as Engineering Targets: Pathway Manipulation to Impact Bioprocess Phenotypes - MicroRNAs as Tools in Biopharmaceutical Production - page 72

Druz et al. recently showed the up-regulation of the large miR 297-669 cluster dur-
ing apoptotic conditions induced by nutrient depletion in CHO cells. One member of
this cluster, miR-466h was shown to alter the expression of five anti-apopototic genes
from different apoptosis-initiating pathways (bcl2l2, dad1, birc6, stat5a and smo).
Antisense knockdown of miR-466h delayed apoptosis onset in nutrient-depleted con-
ditions by decreasing caspase activation and increasing the cell viability (Druz et al.
2011 ). One other member of the miR 297-669 cluster, miR-669c, had been previ-
ously associated with impairments in glutathione metabolism which activated the
apoptosis cascade (Lanceta et al. 2010 ; Maes et al. 2008 ).
Application of apoptosis regulation-related miRNAs in the engineering of CHO
cells is possible; it should provide researchers with another tool for apoptosis inhi-
bition. The recent sequencing of the CHO cell genome and miRNA transcriptome
will provide information on gene down-regulation in CHO cells by CHO-specific
apoptosis-related miRNAs (Hackl et al. 2011 ; Johnson et al. 2011 ;Xuetal. 2011 ).
The sequencing of CHO-specific miRNAs has revealed a conserved sequence of the
apoptosis-regulating miRNAs such as the miR-15/16 cluster, let-7 family, miR-214,
miR-218, miR-1, and miR-10a. These miRNAs and the miR 297-669 cluster are
possible targets for apoptosis pathway engineering. Due to the complexity of the
PCD and the diversity of the apoptotic stimuli, it may be useful to investigate the
combined effects of several miRNAs affecting genes from different stages of the
apoptosis cascade, and the effects of miRNAs which seem to be involved in global
regulation of the pathway. In addition, it might be worthwhile to consider the engi-
neering of whole clusters of apoptosis-relevant miRNAs, since clustered miRNAs are
known to be transcribed together as polycistronic transcripts to regulate the mRNA of
genes with similar functions (Druz et al. 2011 ). More studies on CHO cell-specific
miRNAs, their biological role and their effects on CHO cell-specific gene targets
need to be conducted to suggest the most suitable miRNA candidates to be used in
industrial scale bioprocesses.

5.2.3 Role of MicroRNA in Engineering of Stress Response

Stress conditions in bioreactors do not only induce apoptosis but can also affect prod-
uct yield and properties (Muller et al. 2008 ). For example, high protein expression
and limited secretion can lead to accumulation of aggregated and misfolded pro-
teins, which can generate stress in the endoplasmic reticulum (ER). This stress was
shown to be reduced by ER expansion via ectopic expression of transcriptional factor
X-box-binding protein1, Xbp1 (Tigges and Fussenegger 2006 ), and over-expression
of the protein disulfide isomerase (PDI) (Borth et al. 2005 ). Over-expression of ER
chaperones, calreticulin and calnexin (induced by heat shock, amino acid deprivation
and perturbations of Ca 2 + levels) increased cell specific productivity of recombinant
thrombopoietin in CHO cells (Chung et al. 2004 ; Heal and McGivan 1998 ).
Dissolved oxygen gradients may affect growth, metabolism, and the recombi-
nant protein production. The gradients can be significant in bioreactors as a result of