RNA Interference Pathways and Therapeutic Exploitation - RNA Interference from Biology to Therapeutics - page 6

RISC instead destabilizes mRNA or represses its translation [ 23, 44, 45 ] in a process
much less potent than siRNA-mediated slicing. Of the four closely related Ago
proteins, Ago1-4, only Argonaute2 has endonucleolytic mRNA cleavage potential,
whereas the three other closely related Ago proteins (Ago1, 3, and 4) are devoid of
endonucleolytic activity [ 41 ]. The vast majority of characterized miRNA-mediated
effects are facilitated by seed match target sites in the mRNA 3¢UTR leading to
mRNA destabilization or translational repression by mechanisms that are still
debated but likely involve recruitment of a deadenylation complex
(CAF1:CCR4:NOT1) and mRNA destabilization through deadenylation. Due to
their functional identity, however, siRNAs also behave as miRNAs to destabilize
hundreds of RNAs in addition to the intended target upon base pairing of the siRNA
seed region to 3¢UTRs, a process known as off-targeting (discussed in Sect. 1.4.6 ).

1.3 Harnessing RNAi Pathways for Gene Silencing

Therapeutics

1.3.1 The Bene fi ts of RNAi-Based Therapeutics

The breakthrough for RNAi as a potential human therapeutic was the observation of
potent, sequence-specific, and seemingly safe knockdown of endogenous gene
function in cell culture upon introduction of synthetic 21mer siRNAs with perfect
sequence complementarity toward its target [ 5 ]. The importance of this observation
for RNAi therapeutics cannot be underestimated, and synthetic siRNAs are the pre-
ferred gene silencing tool in vitro, a success that emanates from their general con-
sistency, high efficiency, and ease of use. Today, it is a simple matter to order
designed commercial synthetic siRNAs to obtain the desired gene knockdown in
short-term cell culture experiments using commercial transfection reagents.
Hitherto, synthetic siRNAs have provided the bedrock for the successful RNAi-
based therapeutics: The first knockdown of an endogenous gene, apolipoprotein B
(ApoB) using a clinically relevant formulation and administration route, was
observed in mouse livers after standard intravenous injections of a chemically
modified, but naked (non-formulated), siRNA in 2004 [ 46 ] . Also, the fi rst success-
ful knockdown of a cancer target gene in humans, the M2 subunit of ribonucleotide
reductase (RRM2), was achieved in a clinical phase I trial in tumors from mela-
noma patients upon systemic delivery of siRNA nanoparticles [ 7 ] (for more details
on ongoing clinical trials, refer to http://clinicaltrials.gov ).
There are several contributing components to the great success of RNAi-based
gene silencing as compared to the competing antisense oligonucleotide (ASOs) tech-
nology developed in the 1970-1980s [ 47 ]. Similarly to ASO technology, the high
predictability and specificity of nucleic acid base pairing provides fully program-
mable and specific gene silencing which in practice renders all genes “druggable”:
Researchers need only to produce short dsRNA species with a structure recognizable
to RNAi proteins and perfect sequence complementarity to a particular mRNA target