RNA Interference Pathways and Therapeutic Exploitation - RNA Interference from Biology to Therapeutics - page 7

to achieve gene knockdown. Yet, RNAi-based silencing strategies hold additional
benefits; exogenous RNAi substrates, such as synthetic siRNAs, are rapidly incorpo-
rated into RNAi protein complexes to protect them from nuclease degradation [ 48,
49 ] and, very importantly, to orchestrate their transport to their target mRNA in the
cytoplasm, thereby effectuating multiple rounds of target cleavage [ 10 ] . Furthermore,
the structural mimicry of endogenous miRNA species by artificial siRNA triggers
allows them, at least in theory, to remain undetected to cellular sensors of foreign
dsRNA, thereby preventing triggering of innate and adaptive immune responses.

1.3.2 Therapeutic Entry into the RNAi Pathways

Despite the overwhelming popularity of synthetic siRNAs, a wealth of strategies for
harnessing endogenous RNAi pathway as a gene silencing tool have been developed
to best suit the given experimental system or organism. Collectively, these strategies
exploit the fact that protein complexes in the RNAi pathways respond to the struc-
ture of their RNA substrates rather than their sequences and that miRNA pathways
and siRNA pathways intersect in the cytoplasm at the level of RISC loading and
shares degradation pathways (Fig. 1.1 ). Therefore, any desired guide strand can be
loaded into Ago2-RISC to effectuate mRNA target cleavage once embedded within
a suitably structured RNAi substrate mimic, e.g., primary miRNA transcripts, pre-
cursor miRNAs, miRNA duplexes, dsRNA shorter than 30 bp, or most popularly
21mer siRNAs (Fig. 1.2 ).
As discussed in greater detail in Sect. 1.4 , most experimenters typically use syn-
thetic 21mer siRNAs as triggers of RNAi: These have perfect structural identity to
natural Dicer cleavage products (typically two 21-nt RNA strands annealed to form a
19-bp dsRNA duplex stem and 2-nt 3¢ overhangs at both ends) and are upon introduc-
tion into the cell cytoplasm loaded into RISC by RLC to facilitate transient gene
knockdown. The siRNA sequence is typically designed to target the mRNA ORF, and
silencing effect persists for 2-7 days in typical cell-cultured siRNA. As described in
Sect. 1.4 , numerous successful siRNA designs utilizing shorter of longer RNA back-
bones and chemical modification have been developed to enhance silencing potency,
specificity, and longevity. Still, the lack of efficient means to achieve cytoplasmic
delivery in vivo is the major bottleneck for therapeutic applications, and reports of
siRNA immunogenicity and off-targeting also need immediate addressing.
As described in Sect. 1.5 , RNAi triggers may also be encoded by RNAi plasmids
or viral vectors, which are introduced into the cell nucleus and here utilize the cel-
lular transcription apparatus to ensure a continuous production of the intended RNAi
substrates. This potentially allows for long-lasting RNAi and can also ensure efficient
delivery of RNAi constructs into cells that are otherwise difficult to target. Especially
viral vectors hold therapeutic potential: Whereas retro- and lentiviral vectors may
have limited use in therapeutics due to their integration into the host genome (i.e.,
risk of insertional mutagenesis), the nonintegrating viral vectors such as adeno- and
adeno-associated viral vectors may hold greater therapeutic potential [ 50, 51 ] .