Page 15 - Read Online

Page 6 of 35 Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01

Figure 3. Mechanism of miRNA, siRNA, and shRNA action. The Center (blue arrows) represents the canonical miRNA maturation and
mechanism of action pathway. Transcription leads to a pri-miRNA, which folds into a small hairpin RNA with one or several
mismatches. The pri-miRNA is processed by Drosha and DGCR8 to a pre-miRNA. After association to exportin 5, the pre-miRNA is
transported through the nuclear envelope by a process dependent upon the small GTPase ran-GTP. The pre-miRNA is finally processed
by the Dicer endoribonuclease (a class III RNase), which deletes the hairpin loop. This generates an RNA duplex made of a passenger
and a guide strand. After miRNA homoduplex association with the RISC complex composed of protein TRBP and the argonaute 2
nuclease, the passenger strand is eliminated. The RISC/guide strand complex binds to the target mRNA, in the 5’ non-translated region,
inducing a steric blocking of ribosome entry and translation. In some instances, miRNA binding can also lead to mRNA cleavage and
degradation, but the mismatch generally make this less likely. Left side: siRNA mechanism. (green arrows), a double-strand siRNA with
no mismatch is administered to the cells. All action takes place in the cytosol. After siRNA binding to the RISC complex composed of
protein TRBP and the argonaute 2 nuclease, the passenger strand is eliminated. The RISC/guide strand complex binds to the target
mRNA. The target sequence is generally chosen within the translated region. Because of the complete siRNA matching with the mRNA
target sequence, argonaute 2 is now able to cleave the target mRNA with great efficiency. After mRNA cleavage and elimination, the
RISC/guide siRNA complex can bind and cleave another mRNA (grey arrow). Right side: (green arrows): shRNA mechanism. A gene
expression cassette coding a small hairpin RNA is administered either as a plasmid or by a viral gene delivery vector. The shRNA
transcript is then processed as for the miRNA. However, no mismatch is introduced, which leads to fully efficient mRNA degradation
and silencing.

The second RNAi therapeutic strategy uses exogenous siRNAs, which are made of synthetic 20-25 base
pairs. As shown in Figure 2B and Figure 3 left side, their mechanism of action also involves RISC. The
siRNAs are administered as two-strand perfect matching Watson-Crick homoduplexes. One “antisense”
strand, which is also called the “guide” strand, is complementary to a sequence on the targeted mRNA. In
addition, both 3’ ends possess two supplementary over-hanging non-hybridized nucleotides.

Such an siRNA homoduplex is recognized by the RISC nucleoprotein complex which dissociates the sense
siRNA strand. As with miRNAs, the RISC complex is then guided by the antisense strand to bind to the
complementary sequence on the targeted mRNA, leading to mRNA nucleolytic cleavage and degradation [