RNA Interference Pathways and Therapeutic Exploitation - RNA Interference from Biology to Therapeutics - page 11

1.4.2 Maximizing siRNA Activity

Maximizing siRNA silencing activity minimizes the siRNA dose required for efficient
gene silencing which is important when considering the difficulties of siRNA deliv-
ery in vivo. The siRNA sequence is a primary determinant of siRNA activity [ 36, 95,
96 ], and large screens have identified several design rules [ 97- 103 ] such as a low
GC-content of 30-50% [ 99, 104 ], especially in the guide strand seed region [ 99, 102 ]
and preferences for low internal thermostability [ 99, 101, 102 ] . Importantly, siRNA
sequences should be chosen to render the siRNA duplex thermodynamically asym-
metric as the siRNA strand having the 5¢ end engaged in the thermodynamically least
stable part of the duplex will, as noted above, be preferentially utilized as guiding
strand in RISC [ 36, 37 ]. Furthermore, siRNA target sites should be accessible and not
hidden in stable secondary structures [ 105- 109 ] nor occupied by RNA-binding pro-
teins [ 110 ], and in effect, efficient target sites are often found in AU-rich regions
[ 111 ]. SiRNA chemical modification screens show that careful chemical engineering
of siRNA can enhance their activity beyond unmodified siRNAs [ 112 ] ; a few exam-
ples of dramatic potency improvements have been reported [ 148 ] ; yet, typically, only
modest (less than twofold) improvements in siRNA potency are seen, for example,
upon moderate modi fi cation of siRNA strand 3 ¢ ends [ 112- 118 ] . Instead, modi fi cations
that favor loading of the guide strand into Ago2-RISC seem to be a reliable, sequence-
unspecific strategy to enhance siRNA potency either by introducing optimal siRNA
thermodynamic asymmetry using stabilizing modifications (e.g., LNA [ 119 ] , 2-thio-
uracil [ 120 ] , 2 ¢-F) in the passenger strand 5¢ end or by introducing destabilizing
modifications (such as OXE, ethylamino, UNA, dihydrouracil, or PS [ 112, 119, 121 ] )
in its 3¢ end. Furthermore, chemically modified 3¢ overhangs that are favored and
unfavored during strand selection by RISC have been identified and can easily be
incorporated into the guide and passenger strands of the siRNA, respectively [ 92,
112 ]. As a note of caution, the industry standard and popular 2-nt DNA overhang
dTdT has been suggested to reduce silencing longevity significantly [ 122 ] .

1.4.3 Motivation for Chemically Modi fi ed siRNA

Unmodified synthetic siRNAs seem most suited for short-term gene silencing
experiments in cell culture, where adverse side effects such as siRNA immunoge-
nicity and off-targeting (see below) go unnoticed or are disregarded. Higher stan-
dards for siRNA performance and safety are required to establish siRNA-based
therapeutics. These concerns can be resolved by chemical modification [ 81, 91, 112,
123- 126 ]. Unmodified, naked siRNAs are highly labile in biological fluids due to
their degradation by ribonucleases, and their poor pharmacokinetic properties
in vivo reduce intracellular delivery [ 46, 61, 127 ]. Furthermore, siRNAs may be
immunogenic [ 64, 65, 128- 130 ] and inherently trigger non-intended off-target
regulation of genes harboring seed matches of either siRNA strand (i.e., each siRNA