MicroRNAs as Engineering Targets: Pathway Manipulation to Impact Bioprocess Phenotypes - MicroRNAs as Tools in Biopharmaceutical Production - page 68

Zn-finger proteins (ZFP). Over-expression of ATF4 and Xbp1 transcription factors
and the artificial ZFP-TF derived from a synthetic library increased recombinant
protein production capacity of CHO cells (Kwon et al. 2006 ;Ohyaetal. 2008 ;
Tigges and Fussenegger 2006 ). Dinnis and James ( 2005 ) suggested an approach
based on imitating the molecular events of differentiation of B-lymphocytes
into high producing plasma cells. This inverse engineering strategy enabled the
synchronized expansion and maintenance of high levels of metabolic and secretory
cellular machinery to increase cell-specific production rate in CHO cells.
Below we describe the industrially-relevant phenotypes and pathways with the
existing strategies to improve performance of CHO cells, together with reports on
miRNA involvement in the control of respective pathways. We concentrate on the
possible manipulation of selected microRNAs in CHO cells to globally affect gene
regulation and improve cell performance.

5.2 Applications of MicroRNA for Pathways Engineering

in CHO Cells

5.2.1 Engineering of Growth and Growth Arrest

Volumetric production of mammalian cell culture is a function of cell specific pro-
ductivity and IVC. Higher growth rates increase the rate of biomass accumulation
and the number of viable cells, but suppressing cell growth can increase cell spe-
cific productivity by redirecting the metabolic energy from growth to recombinant
protein production and secretion (Muller et al. 2008 ; Dinnis and James 2005 ). This
conflicting relationship is managed by a “biphasic” cell culture strategy where in the
first phase the cell growth is not limited, and in the second phase the cells growth is
arrested to form high-producing cells. The challenge in this approach is to find a way
to arrest cell division without promoting cell death or interfering with recombinant
protein production (Dinnis and James 2005 ).
Techniques to induce growth arrest include manipulation of growth parameters
(temperature, pH, hyperosmotic pressure), alteration of extracellular environment by
adding specific metabolites or DNA synthesis inhibitors, and addition of cell-cycle
regulators such as nucleotides or nucleosides (Altamirano et al. 2001 ;Bietal. 2004 ;
Carvalhal et al. 2003 ; Kim and Lee 2002 ). Each of these methods may affect multiple
cellular processes simultaneously. For example, reduction of culture temperature
was reported to suppress cell growth in a biphasic process which increased product
titer in CHO cells; at the same time, lowered temperature induces changes in gene
expression, protein phosphorylation, nucleotide pools, and consequently a reduction
in cell metabolism (Kaufmann et al. 1999 ; Yoon et al. 2003 ). In addition, the effects
of reduced temperature may be cell or product-specific and cannot be generalized
(Dinnis and James 2005 ; Yoon et al. 2003 ).
Genetic control of cell cycle progression is another strategy to induce growth
arrest. This can be done by activation of intrinsic cell cycle modulators including