RNA Interference Pathways and Therapeutic Exploitation - RNA Interference from Biology to Therapeutics - page 8

RNAi vectors

1 2

Pri-miRNA mimic
Drosha

shRNA

Synthetic DsiRNA

DGCR8

shRNA

3

27mer siRNA

shRNA

XPO5

Pre-miRNA mimic Pre-miRNA mimic

Dumbbell siRNA

XPO5

4

4

Synthetic siRNA / miRNA

5

5

RLC

Dicer Dicer

TRBP

TRBP

21mer siRNA 16 nt siRNA sisiRNA

Ago Ago

asiRNA aiRNA

Fork siRNA

6

ss-siRNA

pre-RISC

miRNA-mimic

Ago

TRBP

Dicer

Synthetic anti-miR

7

RISC

Ago

9 8

ORF

AAAA AAAA

Ago1-4 Ago2

Target destabilisation
”miRNA effect”

Target cleavage
”siRNA effect ”

1
2

Transcription by Pol-II 4

5

7
8

RISC activation

Nuclear export by XPO5

Uptake into RLC Ago2-mediated cleavage

Transcription by Pol-III

mRNA destabilisation/
translational inhibition

3 Microprocessor cleavage 6 Dicer cleavage 9

Fig. 1.2 Harnessing endogenous RNAi pathways for gene silencing therapeutics. Artificial trig-
gers of RNAi can be introduced via RNAi vectors that are transcribed in the nucleus; pol III-
transcribed cassettes will typically encode shRNAs that are directly transported to the cytoplasm
and loaded into RLC ( right side ). Alternatively, RNAi triggers are delivered as pol II-transcribed
pri-miRNA mimics, which are cleaved by the microprocessor prior to loading into RLC in the
cytoplasm. Synthetic triggers of RNAi are instead introduced directly into the cell cytoplasm;
Dicer substrate siRNAs (DsiRNAs), either in the form of shRNAs, pre-miRNA mimics, or 27mer
dsRNA, can enter the RLC and are subsequently loaded into pre-RISC upon Dicer cleavage.
Various design versions of synthetic 21-23mer siRNAs, and notably also miRNA mimics, are
instead loaded into pre-RISC without Dicer cleavage and can subsequently guide Ago2-mediated
target cleavage (referred to as on-targeting for siRNAs) or Ago-mediated RNA destabilization/
translational inhibition of targets sharing perfect seed matching (the natural miRNA function
which is referred to as of-targeting for siRNAs). Endogenous miRNAs can also be inhibited by
introducing 12-25-nt chemically engineered antisense oligonucleotides, named anti-miRs or