Delivery of Single siRNA Molecules - Nanotechnology for the Delivery of Therapeutic Nucleic Acids - page 96

(Alexis 2008). In addition, naked siRNA can trigger the immune

system through toll-like receptors (TLRs). Thus, several modifications
have been applied to the siRNA molecule in order to improve its
stability against nuclease degradation and to evade activation of the
immune response. For instance, introduction of phosphorothioates
on the backbone of the siRNA, as well as 2

et al.,

′

-O-methylpurines and

2 -fluoropyrimidines on the ribose molecule, results in resistance

against nuclease degradation (Czauderna

′

2003). Furthermore,
it has been shown that immune stimulation by synthetic siRNA can
be completely abrogated by selective incorporation of 2

et al.,

-O-methyl
uridine and guanosine on the ribose, into one strand of the siRNA
duplex (Morrissey

′

et al., 2005, Judge et al., 2006).

4.1.3 Off-Target Effects

Another major impediment in RNAi therapeutics has been off-target
effects. Both the guide and passenger strands can induce off-target
side effects that lead to measurable phenotypes in unwanted targets.
Initially, it was suggested that as few as 11 contiguous nucleotides
were enough to detect cross-reactions with non-targeted genes of
limited sequence similarity (Jackson

2003). However, more
recent studies have shown that a perfect match between the 3

et al.,

′

untranslated region (UTR) of the mRNA and the seed sequence
of the siRNA guide strand is required to induce off-target effects
(Birmingham 2006). However, if the seed sequence of the

guide strand matches the 5

et al.,

′

UTR or the open reading frame (ORF)
of the mRNA, the off-target effects do not seem to occur. Moreover,
by modifying a single nucleotide in the seed sequence to a 2

-O-
methyl, off-targeting activity can be reduced significantly (Robbins

′

et al., 2007). Up to date, no occurrence of seed-sequence-mediated

off-target effects has been reported in clinical trials of siRNA (Kim
and Rossi, 2007; Davidson and McCray, 2011). Although big progress
has been made with siRNA stability and toxicity, delivery is still the
biggest problem to be solved.

4.2 Delivery of siRNA

4.2.1 Peptide Transduction Domains

More than 20 years ago, during the development of an activity assay,
it was discovered that the HIV Trans-Activator of Transcription