RNA Interference Pathways and Therapeutic Exploitation - RNA Interference from Biology to Therapeutics - page 9

In addition to silencing mRNA by custom siRNAs, recent RNAi strategies have
focused on modulating the levels of endogenous miRNAs as these are often found
to be up- or downregulated in human diseases such as cancer [ 52 ] . The so-called
miRNA replacement therapy aims to introduce mimics of natural miRNAs into the
RNAi pathway [ 53 ]. Here they will exert the functions of their natural counterpart
to simultaneously target multiple mRNAs through seed-mediated base pairing to
their 3¢UTR (rather than cleaving a single target as intended for siRNAs). miRNA
mimics are very similar to siRNAs and can be introduced as synthetic dsRNA mol-
ecules or via DNA vectors (described in the following sections). In practice, syn-
thetic miRNA mimics are often designed as perfectly base-pairing siRNA-like
molecules where the guiding strand is identical to a given endogenous miRNA.
Therefore, delivery and chemical optimization strategies are basically similar to
siRNA design; however, modifications that reduce siRNA off-targeting (i.e., miRNA
effects) should obviously not be applied in miRNA mimic design.
Finally, endogenous miRNA function can be inhibited by introducing 12-25-nt
single-stranded oligonucleotides designed antisense to the given miRNA. Upon
introduction into the cell cytoplasm, such molecules, referred to as anti-miRs or
antagomirs [ 54, 55 ], will bind to RISC-loaded miRNAs and block their natural
functions. Being single-stranded, anti-miR typically needs extensive chemical
modification both to resist degradation by nucleases and also to enhance their bind-
ing affinity to target miRNAs (e.g., by locked nucleic acids (LNA) [ 56 ] ), to trap the
RISC-loaded miRNAs in a nonfunctional state or even promote their degradation
[ 57 ]. Significant progress has already been made using anti-miR designs in primates
[ 56, 58 ]. As an alternative, the so-called miRNA sponges transcribed from DNA
vectors introduced into the cell nucleus have allowed long-lasting inhibition of
endogenous miRNA function [ 59 ]. In practice, such sponges are mimicking the
structure of natural miRNA targets as RNA transcripts expressed from strong pol II
promoters designed to contain multiple binding sites, often heptameric sequences
complementary to the target miRNA seed. Notably, emerging evidence suggests
that the unknown function of human pseudogenes or long noncoding RNAs may
indeed be to function as natural sponges for endogenous miRNAs [ 60 ] .

1.4 The Application of Synthetic siRNA as RNAi-Based

Therapeutics

Synthetic siRNAs are by far the most widely used triggers of RNAi in cell culture
in which they are introduced into the cell cytoplasm and directly loaded into RISC.
Although efficient transfection can be achieved in most cell culture systems by the
use of commercial transfection reagents, in vivo delivery of synthetic siRNAs into
the target cells is far more challenging. Synthetic siRNA suffers from a number of
drawbacks compared to most small-molecule drugs; namely, they are macromole-
cules (~14,000 Da) and hydrophilic due to their anionic phosphodiester backbone
that restricts entry across the cellular membrane required for target interaction.