MicroRNAs as Engineering Targets: Pathway Manipulation to Impact Bioprocess Phenotypes - MicroRNAs as Tools in Biopharmaceutical Production - page 74

erythroid progenitor cells by directly affecting NRF2 gene expression which induces
the expression of several antioxidant enzymes (Sangokoya et al. 2010 ). miR-451
protects the erythrocytes against oxidative stress and reverses differentiation de-
fect of erythroid cells by inhibiting the intracellular regulator of cytokine signaling,
14-3-3

gene (Patrick et al. 2010 ). (2) miR-34a and miR-93 were involved in the loss
of oxidative stress defense, and repress expression of genes associated with oxidative
stress regulation and defense mechanism such as Sp1, Sirt1, Mgst1, and Nrf2 (Li
et al. 2011 ).
Some miRNAs have been associated with other stress inducers in a bioreactor
such as osmostic pressure, shear stresses, and nutrient depletion /gradients: (1) miR-
200b and miR-717 are down-regulated by isotonic and hypertonic treatments in
renal medullary epithelial cells. However, when up-regulated, these miRs inhibit the
activity of transcriptional factor called osmotic response binding protein, OREBP,
a major cellular osmoregulator in kidney cells and T-lymphocytes (Huang et al.
2010 ); (2) miR-7b was over-expressed in hyperosmolar conditions to down-regulate
the protein levels of Fos. This reduces the activity of transcription factor activator
protein 1 (AP1), a regulator of cellular processes, which is formed by the dimerization
of Fos and Jun proteins (Lee et al. 2006 ); (3) miR-21 and miR-19a were induced by
shear stress in endothelial cells (Qin et al. 2010 ; Weber et al. 2010 ); (4) Members
of miR 297-669 cluster were up-regulated in response to nutrient depletion in CHO
cells (Druz et al. 2011 ).
The utilization of stress-related miRNAs should be considered for bioprocess-
relevant pathway engineering to eliminate negative effects on recombinant protein
production. The homologs for some stress-related miRNAs (miR-7b, 93, 107, 144,
200b, 210, 708) were reported in CHO cells via next-generation sequencing (Hackl
et al. 2011 ) and their activity can be examined in CHO cells.

ξ

5.2.4 Metabolic Disorders and Their Prevention

Accumulation of lactate and ammonia can significantly affect cell growth and pro-
duction capability (Ozturk et al. 1992 ). Lactate is a major by-product of glucose and
glutamine metabolism. When glucose is converted to lactate, glutamine metabolism
supports the TCA cycle by its conversion to glutamate and later to -ketoglutarate,

which generates excessive amounts of ammonia (Muller et al. 2008 ). Ammonia ac-
cumulation was shown to affect cell growth and the properties of secreted protein
via altered glycosylation pattern in CHO cells (Yang and Butler 2000 ).
The main approaches to optimize metabolic performance of mammalian cells in-
clude optimization of feeding strategies and genetic engineering of specific metabolic
pathways (Irani et al. 2002 ). Over-expression of cytosolic pyruvate carboxylase (PC)
enzyme in BHK cell cultures reduced lactate accumulation, and improved glucose
and glutamine metabolism and recombinant protein production in a broad range of
glucose concentrations in culture media (Elias et al. 2003 ; Irani et al. 2002 ). Knock-
down of lactate dehydrogenase A enzyme (LDH-A), which catalyzes conversion

α