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Furthermore, they are susceptible to serum nucleases and have poor pharmacoki-
netic properties as a consequence of e.g., rapid renal clearance upon intravenous
injection [
61
]. Moreover, siRNA silencing specificity and safety has been chal-
lenged by the general finding of siRNA off-targeting [
62,
63
] and potential immu-
nogenicity [
64,
65
] resulting in the realization that further clinical progress will
require greater investments than initially envisaged to overcome these issues.
Encouragingly, this process is well underway; synthetic siRNA technology is now
dramatically improved by chemical modification that will most likely fulfill its
potential if allowed to mature in a similar manner to development and the emerging
success of ASO technology [
66-
68
] .
1.4.1
Synthetic siRNA Types
The most simple and popular siRNA design today mimics natural Dicer cleavage
products and comprises a 21-nt guiding strand antisense to a given RNA target and
a complementary passenger strand annealed to form a siRNA duplex with a 19-bp
dsRNA stem and 2-nt 3¢ overhangs at both ends [
4,
5
]. Other siRNA designs mimic
Dicer substrates to enhance incorporation into RNAi pathways [
69
] and siRNA
potency [referred to as Dicer substrate siRNAs (DsiRNAs)] [
70
] ; especially syn-
thetic 27mer DsiRNAs [
71-
75
] can have very high activity; yet, concerns of unpre-
dictable efficiency and potential immunogenicity [
76
] need to be addressed [
73-
75
] .
A variety of alternative siRNA designs exhibiting different architectures have
been developed such as Dicer-independent short shRNAs with RNA stems £ 19 bp
[
77,
78
], blunt 19-bp siRNAs [
79,
80
] , blunt fork-siRNAs [
82,
83
] , single-stranded
siRNAs (ss-siRNAs) [
84-
86
] , dumbbell-shaped circular siRNAs [
87
] , asymmetric
siRNAs (aiRNA) and asymmetric shorter-duplex siRNA (asiRNA) harboring a
shortened SS [
78,
88,
89
] , bulge-siRNA [
90
] , and sisiRNAs [
91
] (Fig.
1.2
). Some of
these aim to enhance siRNA potency and specificity by ensuring the preferential
loading of the guide strand in RISC and/or rendering passenger strand nonfunc-
tional. Examples include the asymmetric siRNAs (asiRNAs) which utilize a 5¢ end
truncated passenger strand [
78,
88
] and the sisiRNAs which utilize two short 10-12-
nt passenger strands, all of which cannot contribute to siRNA off-targeting [
91
] .
Also, fork-siRNA contains mismatched bases in the 3¢ end of the passenger strand
which enhance loading of the siRNA guide strands due to a lower thermodynamic
stability of its 5¢ duplex end. Finally, the use of asymmetric siRNA overhangs also
ensures preferential guide strand loading and improved siRNA activity [
92
] . Other
designs aim to enhance siRNA nuclease resistance to prepare unmodified siRNA for
usage in vivo: Blunt 19-bp siRNAs and dumbbell-shaped circular siRNAs are both
reported to be more resistant to nuclease degradation, even when unmodified, as
they contain no free 3¢ overhang [
80,
87
]. Recently, siRNAs has also been incorpo-
rated in larger nucleic acid structures with the prospect of enhancing delivery and
bioavailability [
93,
94
] .
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