Manipulation of Leukocytes Using Therapeutic RNAi Delivered by Targeted and Stabilized Nanoparticles - Nanotechnology for the Delivery of Therapeutic Nucleic Acids - page 181

exported to the cytoplasm, where RNase III Dicer generates the
mature double-stranded miRNAs of approximately 22 nucleotides.
The antisense strand of the mature miRNA is incorporated into the
miRNA-containing RNA-induced silencing complex (miRISC), while
the other strand is degraded. The miRISC usually hybridizes to
partially complementary binding sites on the 3

′

untranslated region
of target mRNAs. Thus, each individual miRNA can target many
different mRNAs, regulating their expression by interfering with
translation or by initiating their degradation [2-4].
In the nucleus, the pri-microRNA (miRNA) molecule is processed
by the RNase III enzyme Drosha into pre-miRNA. The pre-miRNA is
exported into the cytoplasm by Exportin-5, where it is processed by
the RNase III enzyme Dicer into the mature miRNA. From this step
on, the endogenous pathway is similar to the exogenous pathway;
synthetic RNAi molecules (e.g., siRNAs, miRNA mimetics, and
antagomirs) are delivered to the cell cytoplasm. The 19 to 23 dsRNA
molecule is then incorporated into the RNA-induced silencing
complex (RISC), or miRNA-containing RISC (miRISC), the sense
strand is released, and the antisense strand mediates the degradation
of the target mRNA, or inhibits its translation.
RNAi can be activated by exogenous strategy, for example, by
introducing viral vectors expressing shRNA and miRNA duplexes
into cells. Alternatively, double-stranded miRNA mimetic molecules,
anti-miRNA oligonucleotides (antagomirs), and the frequently used
synthetic siRNAs can be taken up directly into the cytoplasm of the
cell (Fig. 8.1) [5].
siRNAs are chemically synthesized dsRNAs of 19 to 23 bp with
2-bp nucleotide overhangs at the 3

′

ends of each strand. In the
cytoplasm of the cell, the siRNA is incorporated into the RISC and
undergoes the same process described for endogenous miRNAs. The
silencing effect of the RISC complex that incorporates the antisense
strand can range from seven days to several weeks in dividing and
non-dividing cells, respectively. Moreover, the stable silencing of a
compatible mRNA can be achieved by repeated administration of
the siRNA. The ability of siRNA to knockdown the expression of any
gene of interest makes addressing targets that are not candidates
for traditional drug design (e.g., molecules without ligand-binding
domains or those sharing structural homology with other molecules)
possible. Furthermore, siRNAs that enter a later stage of the
endogenous RNAi pathway are less likely to interfere with the gene