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constitute the majority of vectors used in clinical trials due to their high efficiency
in vivo, thereby making vector-based RNAi highly relevant for RNAi therapeutics.
Compared to chemically synthesized siRNAs, vector-borne RNAi does not allow
chemical modification of the siRNA itself to enhance its biogenesis and activity or
reduce siRNA off-target effects and immunogenicity (see Sect.
1.4
). Therefore,
careful considerations have to be made to ensure adequate expression levels without
altering endogenous miRNA function.
1.5.1
First-Generation RNAi Vectors
(Producing pre-miRNA Mimics)
First-generation RNAi vectors aimed to encode artificial small dsRNA resembling
either 21-27mer siRNA or short hairpin RNA (shRNAs) mimicking pre-miRNA
structures by using pol III promoter-based transcription units, most often designed
using H1 or U6 promoters [
199-
207
]. These promoters are optimal for transcribing
short and defined RNAs; transcription is initiated at a single position and terminates
after the second T of a templated “TTTTT” stretch, thereby leaving two terminal Us
in the resulting RNA transcript. The conceptually most simple setups employ a
“tandem design” in which siRNA strands are transcribed from individual units [
200,
201
] or convergent transcription of a single siRNA sequence flanked by pol III pro-
moters [
208,
209
]. The transcribed strands anneal to form siRNA, which is exported
to the cytoplasm and loaded into RISC. Most researchers prefer a more efficient
short hairpin RNA (shRNA) design in which the 19-21mer siRNA strands are teth-
ered by a short 5-12-nt loop sequence in a single transcript, which somewhat mim-
ics nuclear pre-miRNAs [
199,
202,
203,
205
]. General design rules for the shRNA
stem are as for siRNAs (Sect.
1.5
); yet, U6 and H1 promoters favor a G or A residue,
respectively, at the first position of the transcribed sequence [
210,
211
] . Also, the
shRNA structure and particularly the length of the shRNA duplex stem and choice
of loop affect shRNA processing by RNAi proteins and consequently their silencing
efficiencies. Early designs encoded 19-21-bp RNA stems connected by 5-9-nt
artificial loops; however, slightly longer stems and natural miRNA loops are both
shown to improve shRNA processing [
49,
212
] .
Pol III promoters were initially considered ideal for expression of shRNA/pre-
miRNA mimics as they are highly and ubiquitously expressed [
213,
214
] and allow
for the use of very small transcriptional units (<200 bps including promoters) well
suited for, e.g., retroviral delivery strategies [
186-
193
] . Indeed, successful RNAi
using pol III-based transcriptional units has been reported, e.g., in genetically
modi fi ed mice [
184-
186,
215,
216
]; however, the number of successful applications
is still relatively low. Notably, the high transcriptional activity of pol III promoters
may flood endogenous RNAi pathways with artificial and poorly processable shR-
NAs, and pol III-transcribed shRNA can block endogenous miRNA production by
saturating limiting amounts of XPO5 in mouse livers [
29
] , an observation subse-
quently confirmed in other tissues [
196,
217,
218
]. Also, the use of shRNA vectors
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