Manipulating Levels of Specific MicroRNAs in Mammalian Cells - MicroRNAs as Tools in Biopharmaceutical Production - page 118

can function as a safe-haven for transgene integration in these rodents as it does in
mice.
Some miRNAs may interfere at all stages of cell growth and protein production to
such an extent that complete and permanent knockout of the endogenous miRNA is a
better approach than partial knockdown with an inhibitor. Many individual miRNAs
have been knocked out in mice (Park et al. 2010 ) taking advantage of the well-
developed genetic methods and the relatively high HR rate in this model organism
(Capecchi 2005 ). In addition, a collection of miRNA targeting vectors to knockout
428 different mouse miRNAs are available from ES cell repositories (Prosser et al.
2011 ). However, miRNA knockout has not been reported in systems other than
mouse where HR is less efficient. In theory, ZFNs that target a miRNA's hairpin
precursor sequence could be used to disrupt its expression, but this has not been
published.

8.5 Selecting miRNAs for Manipulation

Muller et al. ( 2008 ) proposed a number of miRNAs involved in growth, apoptosis,
stress resistance, and translation in humans, mouse and/or rat, whose up or down-
regulation could potentially enhance protein production in CHO cells. Other relevant
miRNA targets can be identified by analyzing profile differences between cell lines,
as recently reported by several groups of biopharmaceutical researchers (Barron et al.
2011 ; Hernández Bort 2012 ; Druz et al. 2011 ; Hackl et al. 2011 ; Johnson et al. 2011 ;
Lin et al. 2011 ). Alternatively, libraries or panels of miRNA mimics and inhibitors
can be screened for those that trigger a phenotype conducive to recombinant protein
production, as mentioned above.

8.6 Use of miRNA Binding Sites

Binding sites for miRNAs that are highly expressed, either endogenously or ex-
ogenously, should be avoided in constructs used to express recombinant proteins.
miRNA target sites can be predicted using online tools such as FindTar3 (http://bio.sz.
tsinghua.edu.cn/).
Alternatively, a miRNA binding site can be included in constructs to prevent
recombinant protein expression during growth stages in which the miRNA is highly
expressed, or restrict expression to stages in which the miRNA is reduced or absent.
Brown et al. demonstrated proof of concept for this approach in human dendritic cells
using a GFP expression vector with four miR-155 target sites. GFP expression was
high in immature dendritic cells while miR-155 expression was low, but was reduced
10-fold as cells matured and miR-155 expression increased 1,000-fold. (Brown et al.
2007 ). Additional examples of the use of miRNA binding sites to restrict transgene
expression are reviewed in Brown and Naldini ( 2009 ).