Nanomedicines for Systemic Delivery of RNAi Therapeutics - RNA Interference from Biology to Therapeutics - page 125

siRNA is a chemically synthesised dsRNA of 19-23 base pairs with 2 nucleotides
unpaired in the 5¢-phosphorylated ends and unphosphorylated 3¢ -ends [ 3, 4 ] . Within
the cell cytoplasm, siRNAs are incorporated into RNA-induced silencing complex
(RISC), a protein-RNA complex that separates the strands of the RNA duplex and
discards the sense strand. The antisense RNA strand then guides RISC to anneal and
cleave the homologous target mRNA or block its translation [ 2 ] . The RISC complex
incorporates the antisense strand and may show a therapeutic effect for up to 7 days
in dividing cells and for several weeks in nondividing cells. Furthermore, repeated
administration of siRNA can result in stable silencing of its target [ 5 ] .
The combination of knockdown in every gene of interest and the ability to treat
various diseases by addressing otherwise “undruggable” targets (i.e. molecules
without ligand-binding domains or those that have a structural homology with other
important molecules in the cell), and reduced toxicity, emphasises the potential of
siRNAs to serve as a new platform for therapy in personalised medicine, where an
individual genome will be sequenced and RNAi molecules could potentially be
designed to inhibit a specific defective mRNA.
Despite this promise, utilising siRNA as therapeutics is not a trivial task. For
example low cellular uptake across the plasma membrane associated with the high
molecular weight (~13 kD) and the net negative of naked siRNA is found [ 2, 6 ] .
When injected intravenously (one of the main routes of systemic administration), in
addition to rapid renal clearance and susceptibility to degradation by RNAses,
unmodified naked siRNAs are recognised by Toll-like receptors (TLRs). This often
stimulates the immune system provoking an interferon response, complement acti-
vation, cytokine induction and coagulation cascades. Beside the undesired immune
activation, those effects can globally suppress gene expression, generate off-target
effects and misinterpreted outcomes [ 6, 7 ]. There is a clear need, therefore, for
appropriate systems for extracellular delivery of siRNAs in addition to intracellular
mechanisms for internalisation, release (from the carriers) and escape (from the
endosomes), in addition to accumulation of siRNAs in the cell cytoplasm and RISC
activation.
Silencing of gene expression in vitro is a great tool for functional and validation
studies. Most of the methods commonly used for in vitro or ex vivo delivery of
RNAi molecules are conventional transfection methods. Studies with purely
physical methods such as microinjection and electroporation [ 8- 11 ], as well as the
use of calcium co-precipitation [ 12 ], commercial cationic polymers and lipids
[ 3, 13- 18 ] and cell penetrating peptides [ 19- 23 ], have demonstrated effective knock-
down of desired genes. Except for the physical methods (in which the cell is sub-
jected to an injection of small volumes of siRNAs directly into the cell cytoplasm or
to a burst of electricity that causes pores in the membrane, hence elevating the ability
of extracellular material to enter into the cell), all the methods share a main feature—
a positive (cationic) charge that enables complexation of the siRNAs and interact
with the negatively charged plasma membrane. In this manner, it is important to note
that there are evidences for toxicities of some commercial cationic lipids and poly-
mers ( [ 24 ] and reviewed in [ 25 ]). This emphasises the usefulness of the cell pene-
trating proteins (such as natural ligands, antibodies and their fragments), which are