RNAi as New Class of Nanomedicines - Harnessing Biomaterials in Nanomedicine: Preparation, Toxicity, and Applications - page 182

An additional source of potential toxicity is the saturation of
the RNAi machinery by excessive exogenously introduced silencing
RNA molecules [6, 55]. This has been shown to be the case for highly
expressed shRNAs. Similar to miRNA, shRNAs are expressed in the
nucleus and exported to the cytoplasm where they are processed
into active siRNAs. Cell culture experiments demonstrated that high-
level expression of shRNAs inhibited the maturation of endogenous
miRNAs but saturating the activity of Exportin 5 [159]. Similarly,
the sustained over-expression of shRNAs in the liver of adult mice
resulted in dose-dependent liver injury and death [55]. These toxic
side eff ects were correlated with decreased expression of liver-
derived miRNAs. The reduction of shRNA expression using weaker
promoters was found to greatly lower these cytotoxic eff ects.
Although siRNAs silence at such low concentrations that this source
of toxicity is unlikely in most cells, it may set a limit on how many
diff erent siRNAs can be used simultaneously in one target cell. In
fact, Bitko and colleagues found that the co-delivery of multiple
siRNAs leads to a reduced efficacy of silencing of each of the intended
targets [14, 70]. This suggests that siRNAs are able to saturate some
downstream component of the RNAi machinery (e.g., RISC).
6.6 Conclusions
In a very short period of time, RNAi-based gene-silencing approaches
have gone from a novel mechanism of gene expression regulation
in C. elegans to a potential therapeutic approach for the treatment
of a variety of intractable human diseases. This has necessitated
the development of a variety of novel delivery strategies that
seek to improve the pharmacokinetic properties of siRNAs. To be
eff ective, these delivery approaches need to optimize each step in
the delivery process from the initial administration of the siRNA to
the uptake into RISC and the cleavage of the target mRNA. This will
minimize the therapeutic dose required for eff ective silencing and,
as a consequence, reduce some the potential off -target toxicities.
These advances have included the incorporation of chemical
modifications into the siRNA to improve their stability, the use of
targeting agents to facilitate siRNA delivery to specific cell types and
tissues, the development of novel lipid formulations to maximize
intracellular trafficking and endosomal escape, and improvement in