RNA Interference Pathways and Therapeutic Exploitation - RNA Interference from Biology to Therapeutics - page 14

activation needs to be further established. Usually, however, only few precautions
are taken in typical cell culture experiment to avoid activation of PKR. Another
sensor of cytoplasmic dsRNA is RIG-I, which recognizes in part the poly-uridine
richness of dsRNA and in part the composition of dsRNA termini; 5¢ end triphos-
phates and blunt 21-27mer siRNA trigger RIG-I activation, whereas the standard
21mer siRNA design having two 2-nt 3¢ overhangs is tolerated [ 174 ] .

1.4.6 Reducing siRNA Off-Target Effects

The shared handling of exogenous siRNAs and endogenous miRNAs by RISC in the
cytoplasm inherently forces all siRNAs to behave as miRNAs and trigger unintended
downregulation (typically less than twofold) of hundreds of endogenous mRNAs
sharing sequence complementarity in their 3¢UTR to the siRNA seed region [ 62, 177,
178 ]. These “off-target” effects can result in toxic phenotypes [ 161 ] and compromise
the interpretation, outcome, and safety of the particular siRNA application. Although
siRNA sequences with low seed match frequency are predictable in silico, off-target-
ing cannot be fully avoided but significantly reduced by specific chemical modification
of the guide strand seed region; particularly, introducing a highly destabilizing UNA
modification at position 7 of the guide strand dramatically reduces off-targeting with
minimal loss of on-target activity [ 123 ] ; yet, also 2 ¢-OMe modification of position 2
[ 179 ] or substituting position 1-8 with DNA [ 180 ] reduces off-target albeit on-target
activity may concurrently be slightly decreased.
It is similarly important to minimize the contribution of the passenger strand to
off-targeting; most well-designed siRNAs will lead to preferential loading of the
guide strand into RISC; however, the passenger strand contribution cannot be fully
abrogated; chemically blocking the passenger 5¢ phosphate, e.g., by 5¢ -OMe
modi fi cation [ 181 ] or inclusion of an additional UNA residue [ 182 ] , abrogates
incorporation of the passenger strand into RISC. Another approach to avoid pas-
senger strand selection is the sisiRNA design in which the passenger strand is com-
posed of two shorter 10-12-nt RNA strands incapable of RNAi function [ 91 ] . It
should also be noted that excess RNAi substrates (such as various siRNA designs)
may also unintendedly impact endogenous gene regulation by competing with
endogenous miRNA for rate-limiting steps in the RNAi pathway [ 95 ] , thereby dis-
turbing endogenous miRNA production or function [ 29, 183 ] .

1.5 Vector-Borne RNAi

Vector-based RNAi is less widely used than synthetic siRNAs yet allows RNAi trig-
gers to be delivered via standard transgenic approaches [ 184, 185 ] and especially
viral vectors [ 29, 186- 198 ] to overcome delivery obstacles and allow longer-lasting
silencing and temporal-spatial control of gene silencing. In fact, engineered viruses