RNA Interference Pathways and Therapeutic Exploitation - RNA Interference from Biology to Therapeutics - page 16

has been shown to induce cellular immune responses [ 176, 219 ] . Therefore, for
strategies utilizing pol III-dependent shRNA expression, great care should be taken
to ensure low, nontoxic shRNA expression levels, e.g., by keeping RNA vector copy
numbers low.

1.5.2 Second-Generation RNAi Vectors (Entering as

Pri-miRNA Mimics)

The observation that natural miRNAs are transcribed as parts of long pri-miRNA
transcripts, which are processed in the nucleus by the microprocessor into pre-
miRNA [ 25, 220, 221 ], inspired researches to deliver siRNA as part of pol
II-dependent transcriptional units. Importantly, this allows the use of well-charac-
terized tissue-specific promoters and tightly controlled inducible systems, such as
the Tet-On and Tet-Off technologies [ 222 ], to obtain temporal-spatial control of
siRNA production [ 223 ]. As the structural requirements for recognition by the
nuclear microprocessor are not fully established, researches have embedded siRNA
within a pol II-transcribed artificial pri-miRNA mimic simply by replacing the natu-
ral miRNA stem sequence [ 224 ], and a number of popular miRNA scaffolds cur-
rently exist building on the context of endogenous miRNA [ 225- 227 ] . A great
advantage of these second-generation designs is reduced toxicity and more reliable
knockdown phenotypes; expression levels can be fine-tuned to less toxic levels, and
pri-miRNA mimics are generally more efficiently processed [ 212, 228, 229 ] and
exported [ 230 ], resulting in more potent gene silencing phenotype, less saturation of
RNAi pathways, and cellular toxicity [ 217, 223, 231 ]. Finally, pol II-based designs
allow the inclusion of several pri-miRNA mimics and protein-coding sequences
(CDS) in a single polycistronic transcript [ 25, 226 ] also compatible with (lenti) viral
delivery [ 232 ] .

1.6 Future Perspectives

There is little doubt that manipulation of gene expression via RNAi is getting closer
to clinical successes, and much anticipated proof of therapeutic gene silencing by
siRNAs was recently established in humans [ 7 ]. So far, important progress has been
made in terms of engineering the synthetic RNAi triggers, such as siRNA, miRNAs,
and anti-miRs, to be more nuclease-resistant, potent, specific, and safe. Still, the
lack of efficient means of targeted delivery represents a bottleneck for broader clini-
cal application and the concurrent evaluation and refinement of RNAi triggers
in vivo. Consequently, researches may very well focus on implementing the pallet
of promising local delivery systems to achieve immediate successes in vivo, while
universal systemic delivery strategies are a more distant goal.