RNA interference: research tool or therapeutic technique?

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RNA interference: research tool
or therapeutic technique?
Editorial
Correspondence:
Eleni Tzortzaki MD, PhD
Lecturer in Thoracic Medicine
Department of Thoracic Medicine,
University Hospital of Heraklion
Medical School, University of Crete
71110 Heraklion, Crete, Greece
Tel: (30) 2810 392433, Fax: (30) 2810542650
E-mail: tzortzaki@med.uoc.gr
RNA interference (RNAi) represents a fundamental biological process by
which cells regulate gene silencing post-transcriptionally. It is triggered by
double-stranded RNA (dsRNAs) precursors that vary in length and origin.
These dsRNAs are rapidly processed into short RNA duplexes of about 21 to
28 nucleotides in length, which then guide the recognition and ultimately
the cleavage or translational repression of complementary single-stranded
RNAs. RNAi targets include RNA from viruses and transposons (significant
for some forms of innate immune response), and they also play a role in
regulating development and genome maintenance (since short RNAs have
been implicated in guiding chromatin modification)1.
SMALL RNAs
Small interfering RNA strands (siRNA) are RNA sequences, roughly 23
nucleotides in length, which are crucial regulators of gene expression. They
have nucleotide sequences complementary to the targeted messenger RNA
strand, mediating mRNA degradation and or/ repressing the translation
of the mRNA into protein2. Specific RNAi pathway proteins are guided by
the siRNA to the targeted messenger RNA (mRNA), where they “cleave”
the target, breaking it down into smaller portions that can no longer be
translated into protein. A type of RNA transcribed from the genome itself,
microRNA (miRNA), works in the same way. In mammals, three major classes
of small regulatory RNAs have been identified so far: microRNAs (miRNAs),
short-interfering RNAs (shRNAs), and Piwi-assosiated RNAs (piRNAs). The
precursors of the miRNAs and piRNAs are endogenously encoded primary
RNA transcripts, while shRNAs are produced by the cleavage of exogenous
dsRNAs2.
RNAi PATHwAY
RNAi is an RNA-dependent gene silencing process that is controlled by
the RNA-induced silencing complex (RISC) and is initiated by short double-
stranded RNA molecules in the cytoplasm of a cell, where they interact with
the catalytic RISC component Αrgonaute1. Despite the differences in the
size and biogenesis of these small regulatory RNAs, their RNAi interference
Eleni G. Tzortzaki1,2,
Maria Psarrou2,
Nikolaos M. Siafakas1,2
1Department of Thoracic Medicine, Medical School,
University of Crete, Greece
2Laboratory of Molecular and Cellular Pneumonol-
ogy, Medical School, University of Crete, Greece
Key words:
siRNA,
miRNA,
RNAi,
Lung,
Gene-silencing
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pathways involve proteins that belong to the Argonaute
family
3
. These proteins make up a highly conserved
family whose members have been implicated in RNAi
and related phenomena in several organisms. In addition
to roles in RNAi-like mechanisms, Argonaute proteins
(AGO) influence development, and at least one subset
is involved in stem cell fate determination3.
The RNAi pathway is initiated by the enzyme Dicer,
which cleaves long, double-stranded dsRNA molecules
into short fragments of 20–25 base pairs. One of the
two strands of each fragment, known as the guide
strand, is then incorporated into the RISC and pairs with
complementary sequences
1,4
. More specifically, in an
initiation step, exogenous (e.g., viral) ds-RNA is digested
by the enzyme Dicer, a member of the RNase III family of
ds-RNA-specific ribonucleases, into siRNAs. In the effector
step, the antisense strand of siRNAs is incorporated into
the RISC, which then targets the mRNA complementary
to the siRNA within the complex, cleaves the target
mRNA, thus leading to inhibition of mRNA translation
(Figure 1A). In mammalian cells, RNAi can begin with
endogenously encoded primary micro-RNA transcripts
(pri-miRNA) that are transcribed by polymerase II (Pol II).
The precursor miRNA is bound in the cytoplasm by the
Dicer complex which processes it further for loading into
the AGO2-RISC complex. The AGO2-RISC-bound miRNA
then recognizes target sites in the mRNA, leading to
translation repression, hence halting production of the
corresponding protein (Figure 1B)4.
siRNA may be obtained by chemical synthesis or
expressed from a DNA-vector. In the latter process, a
DNA insert of about 70 bp encoding a short hairpin RNA
Figure 1. RNA interference (RNAi) pathways involve either small interfering RNA (siRNA) or micro-RNA (miRNA). A. The siRNA
pathway involves cleavage of long double-stranded RNA (dsRNA) by the Dicer enzyme complex to yield siRNA. These siRNA are
then loaded into Argonaute 2 (AGO2) and the RNAi-induced silencing complex (RISC). The active RISC with guide strand recog-
nizes target sites on mRNA and performs site-specific cleavage of the mRNA. B. The miRNA pathway begins with endogenously
encoded primary micro-RNA transcripts (pri-miRNA) that are transcribed by polymerase II (Pol II). The precursor miRNA is bound
in the cytoplasm by the Dicer complex which processes it further for loading into the AGO2-RISC complex. The AGO2-RISC-bound
miRNA then recognizes target sites in the mRNA, leading to translation repression. Modified from de Fougerolles A & Novobrantseva
T, Curr Opin Pharm 2008 (16).

237PNEUMON Number 3, Vol. 21, July - September 2008
(shRNA) targeting the gene of interest is cloned into an
expression vector. When the insert-containing vector is
transfected into the cell, it expresses the shRNA which is
rapidly processed by the cellular machinery into a 19–22
nucleotide ds-RNA5.
BIOLOGICAL APPLICATIONS
RNAi is viewed as one of the most important recent dis-
coveries in biology
6
. Because RNAi confers transient inter-
ference of gene expression in a sequence-specific manner,
it represents a new class of nucleic acid-based molecules
likely to have significant medical utility. Identification of
potentially potent lead siRNA candidates usually begins
with bioinformatic design and siRNA synthesis, followed
by in vitro characterization of siRNA for potency and
specificity, which ultimately results in the selection of
siRNA drug candidates There are two major considera-
tions with regard to siRNA specificity: ‘off-targeting’ due
to silencing of genes sharing partial homology with the
siRNA, and ‘immune stimulation’ due to the engage-
ment of components of the innate immune system by
the siRNA duplex7.
Having identified potentially optimized siRNA with
drug-like properties, the major hurdle remains of ef-
fective delivery to the target cell. The RNAi mechanism
has two important implications for drug discovery. First,
since RNAi-based gene silencing occurs in the cytoplasm,
where the RISC brings together the antisense strand of a
siRNA duplex and the corresponding mRNA molecule, any
method for delivery of siRNA-based therapeutics must be
capable of releasing the siRNA into the cytoplasm of the
relevant cells. This is the key challenge of siRNA delivery.
All other aspects of delivery, such as the targeting of
particular cell types, must be compatible with the basic
requirement that the cells must be able to take up the
siRNA and release it into the cytoplasm in an active form.
Second, since the process that leads to cleavage of the
mRNA strand does not consume the antisense strand of
the siRNA, the siRNA is able to act catalytically, and thus
can persist in silencing its gene for a significant period of
time without interfering with the genetic information8.
Several types of siRNA delivery are under active inves-
tigation, including: direct delivery of saline-formulated
siRNA; encapsulation into liposomes and lipoplexes;
conjugation to antibodies, peptides, aptamers, and other
molecules; and formation of complexes with chemical
and biological polymers. Each of these approaches has
potential advantages, but poses particular challenges8.
siRNA AND THE LUNG
As with any other nucleic acid, in the specific case of
delivery of siRNAs to the lung, the specific extracellular
barriers depend on the route of administration. Introduc-
tion of the complexes into the lung could be achieved,
in principle, by the intranasal, the intratracheal or the
systemic route8. The first two are particularly appealing
since they offer a unique opportunity for the delivery of
siRNAs to the airway and alveolar epithelium. However,
airway-directed gene delivery is not simple because the
lung has evolved both physical and immune barriers
that can hinder effective transduction of epithelial cells.
In addition to such physical phenomena as cilia beating
and mucociliary clearance, the possible interaction of the
complexes with the airway surface liquid (ASL) covering
the airway epithelial cells poses a further major barrier9.
First, negatively charged ASL constituents could bind
directly to positively charged complexes, altering their
size and switching their overall charge to negative, and
hence affecting their diffusion to the target cells or cel-
lular uptake. Additionally, binding of negatively charged
ASL components might displace the nucleic acid from
the complexes and consequently lower their delivery ef-
ficiency. Considering the foregoing difficulties, systemic
administration may be perceived as an alternative to
intranasal and intratracheal administration of the com-
plexes
8,9
. Owing to the similarities between siRNA and
DNA, considering the case of the complexes of DNA would
be instructive, the biodistribution of which is a complex
process dependent on their colloidal properties, as well
as their interaction with blood components. Furthermore,
scavenger cells usually absorb complexes bearing strong
anionic charge, which would result in elimination of the
genetic material from the body. A strong positive charge
on the complexes can also be deleterious. The distribu-
tion of transgene expression following administration of
DNA–polycation complexes is primarily observed in the
lungs and to a much lesser extent in other major organs,
such as the spleen, liver and heart. Transgene expression
is significantly altered when plasmid DNA is formulated
with polycations grafted with hydrophilic polymers, such
as pluronic. In this case, the expression of the reporter
gene is evenly distributed among the aforementioned
major organs. Similarly to the case of DNA, intravenous
injection of siRNA with PEG-containing liposomes has
also been reported to result in preferential delivery of
siRNA to the liver, implying that the surface properties
of the polyplexes is an important factor in determining

238 ΠΝΕΥΜΩΝ Τεύχος 3ο, Τόμος 21ος, Ιούλιος - Σεπτέμβριος 2008
their in vivo distribution9.
SiRNA could be rapidly designed to combat viral
targets. Local RNAi can protect against respiratory viral
infections including RSV
10
, PIV
10
, SARS-SCV
11
and influ-
enza
12,13
. Moreover, many endogenous lung targets have
been silenced in the context of acute lung injury (ALI)
models14,15. In summary, these reports suggest that, un-
like for systemic delivery, the direct administration of
saline-formulated siRNA under certain conditions is able
to silence specifically both viral and endogenous gene
targets expressed in lung epithelial cells10-15.
Delivery of siRNA directly to the lung has the advantage
that, as for any drug, the dose of siRNA required for efficacy
is substantially lower when administered near the target
cell type. In addition, direct delivery might also reduce
any theoretical undesired systemic side effects. Lastly,
and most important in the context of treating respiratory
disease, instillation of siRNA (for instance by intranasal or
intratracheal administration) could allow direct access to
lung epithelial cells in a variety of pulmonary disorders,
apart from viral infections, such as cystic fibrosis, chronic
obstructive pulmonary disease, asthma, and pulmonary
fibrosis16.
However, research is needed in order to formulate ways
of improving cellular uptake and cytoplasmic localization
of siRNA, to broaden the lung cell types to which siRNA
can be delivered, and to improve the pharmacokinetic
and lung distribution profile of siRNA. The rapid preclinical
progress and encouraging initial clinical results, suggest
that siRNA therapeutics may have a useful role in the
treatment of respiratory disease.
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