Genome-Wide RNAi Screening to Identify Human Host Factors Crucial for In fl uenza Virus Replication - RNA Interference from Biology to Therapeutics - page 243

Interestingly, these high-impact early studies describing RNAi in mammalians often
disregarded the fact that chemically synthesized and virally transcribed siRNAs are
still capable of activating components of the innate immune response in certain
in vitro culture systems, as demonstrated by several later reports [ 15- 19 ] . This
immune responsiveness for siRNA was shown to be highly cell type- and sequence-
specific, however. siRNAs capable of activating the mammalian immune response
do so primarily through endosomal activation of the Toll-like receptor 7/8 (TLR7/8)
pathway, resulting in induction of interferons (IFNs) and inflammatory cytokines
from immune cells [ 19, 20 ]. Cytokine production via siRNA was shown to be elimi-
nated by the selective incorporation of 2¢ - O -methyl (2 ¢ -OMe) modi fi ed ribonucle-
otides into the constituent RNA oligonucleotides [ 16, 21 ]. Furthermore, Judge et al.
were able to demonstrate that the innate immune system is able to recognize immu-
nostimulatory RNA motifs within both ssRNA and dsRNA via protein members of
the TLR family, a feature analogous to the recognition of CpG DNA motifs by
TLR9 [ 19, 22 ]. siRNA duplexes containing several 5¢ -UGU-3 ¢ motifs were found to
be highly immunostimulatory, and conversely, functional siRNA sequences lacking
these GU-rich regions were shown to have inherently low immunostimulatory
capacity [ 19 ] .
Chemically synthesized siRNA has become the method of choice to manipulate
gene expression in mammalian cell culture in low- and in high-throughput
approaches. The potential influence of siRNA-mediated immune response on key
readouts for in vivo RNAi studies is a critical consideration; however, responsibility
rests on the individual researcher to appropriately choose cell type and siRNA
library and to include prudent controls and hit identification criteria. Putatively
immunostimulatory motifs within siRNA should be considered when designing
synthetic siRNA to avoid or minimize immunotoxicity and to reduce potential off-
target gene effects.

12.2.2 Genome-Wide RNAi Screens

Cell-based RNAi screening technology allows for genome-wide loss-of-function
studies and is broadly used in the identification of genes associated with specific
biological phenotypes. The basic methodology of RNAi screening includes the fol-
lowing steps:

• Choice of an RNAi library, usually consisting of several thousand siRNAs

• Selection of a robust and suitable cell line, as well as of an adequate assay read-

out system
Transfection of cells with RNAi agents from the chosen RNAi library •

• Treatment and/or incubation of cells

• Signal detection and statistical and bioinformatical analysis

Five screens for host factors required for influenza virus replication were per-
formed in the manner described above and are presented and further discussed in
the following.