MicroRNAs as Engineering Targets: Pathway Manipulation to Impact Bioprocess Phenotypes - MicroRNAs as Tools in Biopharmaceutical Production - page 76

5.2.5 Protein Secretion Pathway Engineering

Recombinant protein production in mammalian cells is achieved by controlling
recombinant gene expression and optimizing protein secretion. Improving gene
expression has received considerable attention, and approaches such as codon opti-
mization and promoter and enhancer sequence design have been utilized (de Boer
et al. 2004 ; Kim and Lee 1997 ; Masuda et al. 2000 ). However, limited attention has
been given to protein secretion engineering (Muller et al. 2008 ).
One way to improve the secretion of the recombinant protein is over-expression
of the spliced form of X-box binding protein1 (Xbp-1S), a transcriptional activator
for a number of genes linked to protein secretion and endoplasmic reticulum (ER)
biosynthesis. Over-expression of Xbp-1S in mammalian cells increased recombinant
protein secretion in cases when protein accumulation exceeded the secretory capacity
of the cells (Ku et al. 2008 ).
Kantardjieff et al. showed direct correlation between enhanced protein production
and overall increase of ER and Golgi-related protein secretion system in CHO and
NS0 cells at low temperature and sodium butyrate treatment (Kantardjieff et al.
2010 ). Therefore, the genes that are closely engaged in the secretory pathway are
worth considering as potential targets for secretion pathway engineering. Suitable
candidates include coat protein complexes I and II, which recruit the cargo and direct
its traffic through the early secretory pathway between ER and Golgi (Muller et al.
2008 ; Barlowe 2000 ). Over-expression of Munc 18b, the regulator of the fusion
of secretory vesicles with the plasma membrane, increased heterologous protein
production in several mammalian cell lines (Peng 2010 ). In addition, the ectopic
expression of the synaptosome-associated protein (SNAP-23) and vesicle-associated
membrane protein 8 increased mammalian cell productivity (Peng et al. 2011 ).
Few miRNAs have been associated with controlling the secretory pathway in
mammalian cells: (1) miR-124a and miR-96 affected the genes involved in insulin
secretion in pancreatic

-cells (Lovis et al. 2008 ). miR-124a increased insulin exo-
cytosis at low glucose concentrations by indirectly increasing the levels of SNARE
protein SNAP25, GTPase Rab3A, and synapsin-1A and by directly targeting the
GTPase Rab27A. miR-96 increased the levels of granuphilin protein, a potent
inhibitor of insulin exocytosis, and decreased the expression of Rab27A-binding pro-
tein, Noc2 (Lovis et al. 2008 ); (2) miR-9 reduced glucose and potassium-dependent
insulin exocytosis by targeting transcription factor Onecut2 (OC2) which represses
Granuphilin/Slp4, that negatively affects the insulin secretion (Plaisance et al. 2006 );
(3) miR-375 has negative effects on the glucose-mediated insulin secretion (Gauthier
and Wollheim 2006 ).
Although most of the knowledge about miRNA involvement in protein secretion
comes from studies of pancreatic

β

-cells, there are some reports of miRNA con-
nection to the senescence -associated secretory phenotype, characterized by growth
arrest, increased gene expression and secretion of various proteins (Bhaumik et al.
2009 ). Bhamuik et al. showed up-regulation of miR-146a/b in senescent human fi-
broblasts. The ectopic expression of miR-146a/b was shown to target a component

β