Intracellular Delivery Considerations for RNAi Therapeutics - RNA Interference from Biology to Therapeutics - page 80

4.2.1 siRNA

siRNA is a class of double-stranded RNA (dsRNA) molecules that are 20-25
nucleotides in length. siRNA conducts its silencing function in the cytoplasm by
first incorporating into RNA-induced silencing complex (RISC). siRNA duplex
then undergoes unwinding, and the sense strand is excluded from the RISC. RISC
then further pairs with the complementary mRNA and mRNA cleavage facilitated by
complementary base pairing with the antisense strand results in post-transcriptional
gene silencing. It is worth noting that a class of longer siRNA, the 27-mer Dicer-
substrate siRNA (DsiRNA), has been reported to exhibit more potent silencing than
standard 21-mer siRNA [ 3, 4 ]. In the cytoplasm, the 27-mer DsiRNA is first cleaved
by endonuclease Dicer to form standard 21-mer siRNA, which then proceeds
through the same silencing pathway as stated above. One possible explanation for
the greater potency of DsiRNA is that the process of Dicer-mediated cleavage may
facilitate the loading of siRNA into RISC [ 5 ], thus enhancing gene silencing. While
it is generally accepted that delivering siRNA into the cytoplasm is optimal, recent
findings suggest that siRNA can also induce transcriptional gene silencing through
DNA methylation and knockdown of transcripts restricted to the nucleus [ 6- 10 ] .
siRNA that engages in the nuclear RNAi pathways would require siRNA delivery
into the nucleus. This notion is supported by the observed increase in the transcrip-
tional gene silencing efficiency with delivery vectors containing nuclear localiza-
tion sequences [ 11 ] .

4.2.2 miRNA

The active mature miRNA is a single-stranded RNA generated during endogenous
transcription [ 12 ]. It is ~22 nucleotides in length, firstly transcribed in the nucleus
as long primary transcripts (pri-miRNA) with local hairpin structure and then pro-
cessed into precursor miRNAs (pre-miRNA) by Drosha enzyme, followed by export
to the cytoplasm where it is processed to form mature miRNA by Dicer [ 13 ] . Unlike
siRNA, which forms a perfect duplex with its target mRNA and directs the RISC-
mediated mRNA degradation, the action of miRNA depends on the level of comple-
mentarity. It may cause mRNA cleavage, or it may bind imperfectly with the
untranslated regions of mRNA, leading to translational repression. The endogenous
processing pathway of miRNA improves the silencing efficacy and avoids activa-
tion of the interferon system associated with externally introduced synthetic siRNA
[ 14 ]. Depending on the type of miRNA, the desired intracellular site of delivery
varies from cytoplasm to the nucleus. Commercially available miRNA mimics are
small dsRNA molecules mimicking endogenous mature miRNA and thus should be
delivered directly into the cytoplasm. Antagomir, a single-stranded oligonucleotide,
binds endogenous mature miRNA and blocks the function of miRNA-mediated
silencing [ 15, 16 ]. As the cytoplasm is the site for antagomir-miRNA binding,
antagomir success requires delivery into cytoplasm. In contrast, nuclear delivery